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dissertation entitled

USE OF LIGHTING AND TEMPERATURE STRATEGIES TO
CONTROL FLOWERING AND ARCHITECTURE
OF SELECT HERBECEOUS PLANTS

presented by

ERIK SANFORD RUNKLE

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USE OF LIGHTING AND TEMPERATURE STRATEGIES
To CONTROL FLOWERING AND ARCHITECTURE
0F SELECT HERBACEOUS PLANTS
By

Erik Sanford Runkle

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

2000

ABSTRACT
USE OF LIGHTING AND TEMPERATURE STRATEGIES
To CONTROL FLOWERING AND ARCHITECTURE
OF SELECT HERBACEOUS PLANTS
By

Erik Sanford Runkle

Successful production of many floricultural crops requires precise tinting of
flowering and a final plant height that meets preset Specifications. To meet height
requirements, one method of suppressing internode extension is to provide a day
temperature cooler than that during the night. In addition to temperature fluctuations, the
incident distribution of spectral radiation influences stem extension and flowering of
many plants. In particular, light with a high red (R, 600 to 700 nm) to far-red (FR, 700 to
800 nm) ratio suppresses extension growth, but it can also delay flowering in some long-
day plants. Experiments were performed to determine: 1) if phytochrome A mediated the
reduction in stem extension from cool day-temperature treatments, 2) how flowering and
stem extension of Echinacea purpurea L. were mediated by lighting duration and quality,
and 3) extension growth and flowering responses of a variety of herbaceous species,
particularly long-day plants (LDP), in environments deficient in blue (B, 400 to 500 nm),
R, or FR light.

Transgenic potato over- or under-expressing phytochrome A (phyA) and tomato
phyA mutants were grown at one of three temperature regimens with a daily mean of 20
°C. Compared with that under a constant 20 °C, an 8 0C temperature depression at the

onset of the photoperiod or a 16 / 24 °C day / night temperature suppressed extension

growth of tomato, potato, or both, regardless of phyA level. Therefore, it appears that
phyA does not control extension growth in relation to cool temperature treatments.

Experiments were performed to determine how light regulates growth and
development of Echinacea purpurea, a herbaceous Asteraceae perennial grown for its
reported medicinal properties and its aesthetic value in the landscape. Plants were
exposed to a variety of photoperiods (9 to 24 h), night-interruption (NI) lighting durations
(7.5 to 240 min), and photoperiods deficient in B, R, or FR light. Flowering was most
complete and rapid under photoperiods of 13 to 15 h, which indicates that Echinacea
purpurea is an intermediate-day plant. Plants flowered when 15-h dark periods were
interrupted with low-intensity lighting for 7.5 min, but flowering was most rapid when
lighted for 30 to 60 min. A model composed of two distinct mechanisms is proposed to
explain the flowering behavior of intermediate day plants such as E. purpurea.

Finally, experiments with photoselective plastic filters were performed to
determine how photoperiods deficient in B, R, and especially FR light influenced stem
extension and flowering in a variety of herbaceous annual and perennial plants.
Photoperiods deficient in B or R generally promoted extension growth but had little or no
effect on time to flower. However, an FR-deficient photoperiod inhibited extension
growth and flowering in some LDP, such as pansy (Viola Xwittrockiana). Further
experiments were performed to determine if lighting strategies could be used to produce
short, compact plants without a concomitant delay in flowering. Results with pansy

indicate that extension grth and flowering can not readily be separated with lighting
strategies. Results from these studies and their applications to the floriculture industry

are discussed.

ACKNOWLEDGMENTS

I wholeheartedly thank my major professor, Dr. Royal Heins, for his
encouragement, advice, friendship, and support throughout my program. I would also
like to express gratitude to Dr. Art Cameron, Dr. Simon Pearson, and Dr. William
Carlson, whose guidance and camaraderie broadened my graduate experience. Thanks to
Dr. Jan Zeevaart and Dr. Joe Ritchie for their valuable input to my dissertation and for
serving on my Ph.D. committee. I thank the numerous greenhouse growers supportive of
Michigan State University Floriculture, project GREEEN, 3M, and British Visqueen for
funding this research.

Thanks to the many past and present graduate students of the Horticulture
Department at Michigan State University, whose names are too numerous to list. A
special thank you to those in Floriculture that provided me with valuable support and
friendship. I owe many thanks to Dan Tschirhart, David Joeright, Ron Wik, and a helpful
crew of undergraduate students for assisting with the experiments. I am also appreciative
of the graduate students and staff at the University of Reading for making my four-month
visit productive and stimulating. Finally, I thank my family and Sabrina Lall for their

continual love and blessing.

iv

NOTE TO GUIDANCE COMMITTEE

The paper format was adopted for this dissertation in accordance with
departmental and university regulations. As of November 10, 2000, Chapter I
(Phytochrome A does not mediate stem extension in relation to cool-temperature
treatments) was published in Physiologia Plantarum (104:596-602). Chapter II
(Photocontrol of flowering and stem extension of the intermediate-day plant Echinacea
purpurea Moench.) has been submitted to Physiologia Plantarum. Chapter 111 (Specific
functions of red, far-red, and blue light in flowering and stem extension of long-day
plants) has been accepted for publication in the Journal of American Society for
Horticultural Science. Chapter IV (Stem extension and subsequent flowering of

seedlings grown under a film creating a far-red deficient environment) is to be submitted

to HortScience. Chapter V (Photoeontrol of flowering and extension growth of the long-
day plant pansy) is to be submitted to the Journal of American Society for Horticultural

Science.

TABLE OF CONTENTS

LIST OF TABLES ..................................................... viii
LIST OF FIGURES ..................................................... x
CHAPTER I. Phytochrome A does not mediate stem extension in relation to cool-
temperature treatments ...................................... 1
Abstract ......................................................... 2
Introduction ...................................................... 3
Materials and Methods .............................................. 7
Results. . ....................................................... l 1
Discussion ...................................................... 12
Literature Cited .................................................. 16

CHAPTER II. Photocontrol of flowering and stem extension of the intermediate-day

plant Echinacea purpurea Moench. ........................... 25
Abstract ........................................................ 26
Introduction ..................................................... 27
Materials and Methods ............................................. 30
Results. . ....................................................... 35
Discussion ...................................................... 38
Literature Cited .................................................. 43

CHAPTER 111. Specific functions of red, far-red, and blue light in flowering and stem

extension of long—day plants ................................. 54
Abstract ........................................................ 55
Introduction ..................................................... 56
Materials and Methods ............................................. 59
Results. . ....................................................... 64
Discussion ...................................................... 67
Literature Cited .................................................. 72

CHAPTER IV. Stem extension and subsequent flowering of seedlings grown under a

film creating a far-red deficient environment .................... 91
Abstract ........................................................ 92
Introduction ..................................................... 93
Materials and Methods ............................................. 95
Results. . ....................................................... 98
Discussion ...................................................... 99
Literature Cited ................................................. 102

vi

CHAPTER V. Photocontrol of flowering and extension growth of the long-day plant

pansy .................................................. 1 10
Abstract ....................................................... 1 l 1
Introduction .................................................... 1 12
Materials and Methods ............................................ 115
Results. . ...................................................... l 18
Discussion ..................................................... 121
Literature Cited ................................................. 126

vii

LIST OF TABLES

CHAPTER II.

. Seed, shipping, and forcing dates, initial node counts, and average air

temperatures from date of forcing to average date of flowering (Expts. l and 2)

or visible bud (Expt. 3) for each photoperiod and cold treatment of Echinacea
purpurea. The average air temperature during 100 days of forcing is provided

when no plants flowered within a treatment. N1 = 4-h night interruption;

— (long dash) = not included in experiment. .............................. 46

. Light quantity, quantum transmission percentage (from 400 to 800 nm) of red

(R), far-red (FR), and blue (B) light, and calculated phytochrome

photoequilibria (Pf/P) (Sager et al. 1988) under filters with sun or high-pressure
sodium (HPS) lamps as the sole light source. The average daily light integral

was measured under each filter and calculated from date of forcing to average

time to visible bud for each filter treatment. B = 400 to 500 nm; FR = 700 to

800 nm; FR“ = 725 to 735 nm; R = 600 to 700 nm; R" = 655 to 665 nm. ......... 47

CHAPTER III.

. Seed, shipping, and forcing dates, initial node counts, and average air

temperature and photosynthetic daily light integral during experiments.
Environmental data were calculated from date of forcing to average date of

visible bud (Experiment I), average date of flowering (Experiment III), or 144

d from forcing (Experiment II) under each treatment. N = neutral-density; R¢I

= red (600 to 700 nm) deficient; Bd = blue (400 to 500 nm) deficient; FRd =

far-red (700 to 800 nm) deficient; na = not applicable; — (long dash) =

incomplete data. ..................................................... 79

. Quantum ratios of red (R), far-red (FR), and blue (B) light, calculated

phytochrome photoequilibria (Pf/P), and relative quantum efficiency (RQE)

under filters with sun or high-pressure sodium (HPS) lamps as the sole light

source (McCree, 1972; Sager et al., 1988). B = 400 to 500 nm; FR = 700 to

800 nm; FRn = FR narrow band width (725 to 735 nm); R = 600 to 700 nm;

R11 = R narrow band width (655 to 665 nm). ............................... 80

. Flowering characteristics of Viola Xwittrockiana under the neutral (N) or far-

red deficient (FRd) filter. Plants were transferred from the N to the F Rd filter,

or vice versa, following 5, 10, 15, 20, 25, 30, 35, or 40 d. Data were pooled by

filter type because transfer time and filter x transfer time interaction had no
significant effect on any measured characteristics. .......................... 81

viii

. Flowering responses of Coreopsis Xgrandiflora transferred from an unfiltered
16-h photoperiod with supplemental high pressure sodium lamps [delivering a

high daily light integral (DLI)] to a neutral (N) or far-red deficient (FRd) filter. . . .

CHAPTER V.

. Propagation and forcing dates, initial node counts, and average air temperatures
and photosynthetic daily light integrals under treatments during experiments.

.82

N = neutral-density; FRd = far-red (700 to 800 nm) deficient. ................ 130

. Spectral radiation and estimated phytochrome photoequilibria (Pfi/P; Sager et al.

1988) under neutral (N) or far-red deficient (FRd) filters and of incandescent
(INC) and soft-white fluorescent (SWF) lamps alone or combined. All lamps
were surrounded with a filter to reduce the transmission of blue light (400 to
500 nm) and an N filter surrounded the combined INC + SWF lamps to provide
a more similar PPF among light quality treatments. R = red light (600 to 700

nm); FR = far-red light (700 to 800 nm). ................................. 131

. Analysis of variance (ANOVA) for various flowering and extension growth
parameters of pansy as affected by filter treatment during the photoperiod and

night interruption (NI) quality and duration. .............................. 132

ix

LIST OF FIGURES

CHAPTER I.

. Air temperature regimens at plant canopy level. Actual temperatures measured
at plant height were within i0.2°C of the indicated settings. The dark and light
periods are indicated by closed and open bars, respectively .................... 22

. Intemode length (Fig. 2A) and developed stem length (Fig. 2B) of tomato WT
and fii— and tri-mutant plants after 10 days of temperature treatments. Error bars
are 95% confidence intervals (11 = 22 to 25). ............................... 23

. Average internode length (Fig. 3A) and developed stem length (Fig. 3B) of WT

(n = 10) and four lines of transgenic potato: 15-9 and 15-11 are antisense phyA

(n = 9); PS2 and PS4 are with overexpressed phyA (n = 8). Measurements were
taken after 14 days of temperature treatments. Error bars are 95% confidence
intervals. . . ........................................................ 24

CHAPTER II.

. Spectral transmissions relative to that under a neutral-density filter (N) under
sunlight. Rd, red (600 to 700 nm) deficient filter; Bd, blue (400 to 500 nm)

deficient filter; FRd, far-red (700 to 800 nm) deficient filter. See Table 2 for

light wave band ratios. ................................................ 48

. Flowering of Echinacea purpurea ‘Bravado’ and ‘Magnus’ under continual
photoperiods consisting in 9-h natural days extended with light from

incandescent lamps (N I = 4-h night interruption). At first open flower, the

number of nodes on the main stem below the inflorescence was counted and

total plant height was measured. Data for noncooled and cooled plants were

pooled, since cold treatment effects were insignificant. Legend in D applies to

all figures. Values with the same letter (‘Bravado’ in uppercase, ‘Magnus’ in
lowercase) are not statistically different at P = 0.05. ......................... 50

. Flowering of Echinacea purpurea ‘Bravado’ and ‘Magnus’ under various

durations of night interruption lighting or with a 5-h day extension (l4-h
photoperiod). Photoperiods consisted in 9-h natural days with light from
incandescent lamps during the middle of the dark period (night interruption) or
following the natural photoperiod for 5 h (14-h photoperiod). Error bars are

95% confidence intervals. Legend in D applies to all figures. Nonlinear

regression analysis was used to describe plant height (cm) as a function of

night interruption duration (minutes) with the equation in the form y =

a/(l + exp (- (x - x0)/b)). For ‘Bravado’ and ‘Magnus’, respectively, a = 74.87

and 62.13; b = 17.37 and 9.06; x0 = 15.01 and 14.73; and R2 = 0.994 and 0.989. .. .52

. Flowering of Echinacea purpurea ‘Bravado’ under a neutral-density filter or

filters that selectively reduced transmission of red (Rd, 600 to 700 nm), blue

(Bd, 400 to 500 nm), or far-red (FRd, 700 to 800 nm) light. See Fig. 1 and

Table 2 for filter transmission properties. Photoperiods consisted in day

lengths extended with light from supplemental high-pressure sodium lamps
positioned above filters from 0600 to 2200 HR. Values with the same letter

are not statistically different at P = 0.05. .................................. 53

CHAPTER III.

. Spectral transmissions of photoselective filters relative to sunlight (A) or

relative to that under the neutral-density filter treatment with an equal

photosynthetic photon flux (B). Rd, red (600 to 700 nm) deficient filter; Bd,

blue (400 to 500 nm) deficient filter; FRd, far-red (700 to 800 nm) deficient

filter. See Table 2 for light wave band ratios. .............................. 83

. Plant height at visible bud of Campanula carpatica, Coreopsis xgrandiflora,

Lobelia Xspeciosa, and Pisum sativum under a neutral filter or a light

environment deficient in red (Rd, 600 to 700 nm), far red (FRd, 700 to 800 nm),

or blue (Bd, 400 to 500 nm). A 16-h photoperiod was delivered with a

combination of sunlight and high-pressure sodium lamps positioned above

filters. Values with the same letter within species are not statistically different
atP=0.05.... ..................................................... 84

. Stern and inflorescence elongation from visible bud to flowering of Coreopsis
Xgrandiflora, Lobelia Xspeciosa, and Viola Xwittrockiana under a neutral

filter or a light environment deficient in red (Rd, 600 to 700 nm), far red (FRd,

700 to 800 nm), or blue (Bd, 400 to 500 nm). A 16-h photoperiod was

delivered with a combination of sunlight and high-pressure sodium lamps

positioned above filters. Values with the same letter within species and

measurement are not statistically different at P = 0.05. ....................... 85

. Days to visible bud (A), node count increase to first open flower (B), and

flower number (C) or dry weight (D) of Campanula carpatica, Coreopsis
Xgrandiflora, Lobelia Xspeciosa, and Pisum sativum under a neutral filter or

a light environment deficient in red (Rd, 600 to 700 nm), far red (FRd, 700 to

800 nm), or blue (Bd, 400 to 500 nm). A l6-h photoperiod was delivered with

a combination of sunlight and high-pressure sodium lamps positioned above

filters. Values with the same letter within species are not statistically different

at P = 0.05. Legend in A applies to all figures. NS = not significant. ........... 87

. Flowering percentage of Viola Xwittrockiana under a neutral filter or a light
environment deficient in red [Rd, 600 to 700 nm, (A)], far red [FRd, 700 to 800
nm, (B)], or blue [Bd, 400 to 500 mm, (C)] light. Plants were held under the
light treatments until visible bud (until VB), continually, or after visible bud
(after VB). Plants were under the neutral filter at all other times. A l6—h

xi

photoperiod was delivered with a combination of sunlight and high-pressure
sodium lamps positioned above filters. Legend in A applies to all figures. ....... 89

. Stem extension of Campanula carpatica, Coreopsis Xgrandiflora, Pisum

sativum, and Viola Xwittrockiana relative to that under the far-red (FR)

deficient filter. Stem length was related to red (R) : FR ratios and the

estimated phytochrome photoequilibria (Pf/P) under the filter treatments. The

R : FR ratios were determined using narrow [10 nm (A)] and wide [100 nm

(B)] band widths. See Fig. l and Table 2 for spectral data. Open symbols

represent plants exposed to a neutral filter or light deficient in R or FR.

Closed symbols represent plants exposed to light deficient in blue (400 to

500 nm). Legend in A applies to all figures ................................ 90

CHAPTER IV.

. Spectral transmission of sunlight under the far-red (700 to 800 nm) deficient

filter relative to a filter that reduced the transmission of all wavelengths equally.

The photosynthetic daily light integrals under the two filters were similar (see

text) ............................................................. 107

. Percentage reduction in stem length (or longest petiole length for pansy) of
seedlings transferred from the neutral (N) to far-red deficient (F Rd) filter when
leaves of each species began to touch within the plug tray ( D ), or held
continually under the F Rd filter ( I ), relative to that continually under the N
filter. Seedlings were under filter treatments for 26, 31, 32, 35, and 35 d for
tomato, impatiens, snapdragon, petunia, and pansy, respectively.
Measurements were taken of seedlings from the outer two rows (outside), from
the next inner two rows (middle), and from the innermost rows (inside) from
each plug tray. Bars represent means with n = 40. Asterisks indicate that
height was significantly (P = 0.05) less than of plants that were continually
under the N filter. Spectral transmission of sunlight under the far-red (700 to
800 nm) deficient filter relative to a filter that reduced the transmission of all
wavelengths similarly. ............................................... 109

CHAPTER V.

. Flowering of Viola xwittrockiana ‘Crystal Bowl Yellow’ under photoperiods
consisting of 9-h natural days extended with light from incandescent lamps.

N1 = 9-h photoperiods plus 4-h night interruption. Plants were considered
nonflowering if they did not reach anthesis within 98 to 100 d from seed.

Days to flower with the same letter are not statistically different at P = 0.05. . . . . 133

. Flowering and peduncle length of Viola xwittrockiana ‘Crystal Bowl Yellow’

grown under a neutral (N) filter (Figs. A, C, E, and G) or one that selectively
reduced the transmission of far-red (FR, 700 to 800 nm) light (Figs. B, D, F,

xii

and H). Except for the 9-h photoperiod, a 16-h base photoperiod was provided

by natural photoperiods extended with light from high-pressure sodium lamps

from 0600 to 2200 HR. Light rich in FR was provided under the FR filter for

periods during the day or night, as indicated. Values with the same letter

within measurement are not statistically different at P = 0.05. Letters are not
provided when all treatments are statistically similar. ....................... 135

. Flowering and stem length of Viola xwittrockiana ‘Crystal Bowl Yellow’

grown under a 9-h neutral (N) filter (Figs. A, C, and E) or a filter that

selectively reduced the transmission of far-red (FR, 700 to 800 nm) light

(Figs. B, D, and F). Night interruption (NI) lighting was provided for varying
durations by lamps delivering a low (0.56), moderate (1.28), or high (7.29) red

(R, 600 to 700 nm) to FR ratio (Table 2). In each graph, open or dark symbols
represent means are significantly different (at P = 0.05) from or similar to that
without an NT, respectively. Error bars represent 95 % confidence intervals,

and except for Figs. A and B, are not presented when NI lighting treatments

were statistically similar. ............................................. 137

. Flowering and stem length of Viola xwittrockiana ‘Crystal Bowl Yellow’

grown under a neutral (N) or far-red deficient (FRd) filter with night

interruption (N 1) treatments as described in Fig. 3. Data for plants under the

N and FRd filters were pooled, since the effects of base photoperiod were
insignificant. Error bars represent 95 % confidence intervals, and are not

presented when NI lighting treatments were statistically similar. .............. 138

. Flowering and stem extension of Viola Xwittrockiana ‘Crystal Bowl Yellow’

grown under 26 combinations of filter and night interruption (NI) treatments,

as described in Fig. 3. Linear regression analysis was used to relate the

relative promotion of stem extension with flowering; see text for equations.

m = significant at P 5 0.0001. ......................................... 139

xiii

CHAPTER I
PHYTOCHROME A DOES NOT MEDIATE STEM EXTENSION
IN RELATION TO COOL-TEMPERATURE TREATMENTS

Runkle, E. S. and Pearson, S. 1998. Phytochrome A does not mediate reduced stem
extension from cool day-temperature treatments. Physiol. Plant. 104(4):596-602.

Phytochrome A does not mediate reduced stem extension from cool day-temperature

treatments

Erik S. Runkle

Department of Horticulture, Michigan State Univ., East Lansing, MI 48824-1325 USA

Simon Pearson

Department of Horticulture, The Univ. of Reading, Reading, Berkshire, RG6 6AS UK

Key words — DIF, DROP, fri mutant, internode elongation, Lycopersicon esculentum,
phytochrome, potato, Solanum lycopersicon, Solanum tuberosum, stem extension,

tomato, tri mutant.

Abstract

Stem elongation can be suppressed by a temperature drop at the onset of the photoperiod
(DROP) or with a cooler day than night temperature (DT and NT, respectively),
commonly described as DIF (DT - NT). To test our hypothesis that phytochrome A

(phyA) mediated the reduction of stem elongation caused by —DIF and DROP, we

conducted experiments with photomorphogenic mutants of tomato (Solanum

lycopersicon L.) and transgenic potato (Solanum tuberosum L.). The plants studied were
tomato mutants fri’ (deficient in phyA) and tri’ (deficient in phytochrome Bl [phyB1])
and their isogenic wild-type (WT) cv. Moneymaker, nontransformed potato, and two lines

each of antisense phyA (15-9 and 15-11) and overexpressed phyA (P82 and PS4). Plants

were placed in three temperature regimens with a daily mean of 20°C: a constant 20°C (0
DIF), an 8°C DROP for 3 h, and a -8°C DIF. For all tomato genotypes, —DIF and DROP
reduced internode length by 221% and stem elongation by 30% compared to that of
plants at 0 DIF. Interactions between temperature treatment and genotype were
nonsignificant. For potato, —DIF, but not DROP, significantly reduced internode length
of WT (by 39%) and both antisense lines (by 36 or 48%) but only one of the two lines of
overexpressed phyA plants (by 18%). The —DIF significantly reduced stem length for
only antisense phyA (by 36 or 48%) and WT (by 35%) plants. Thus, at least for tomato
and potato, it appears that phyA does not control stem extension in relation to cool-

temperature treatments.

Introduction

For many species, stem extension is suppressed when the day temperature (DT) is lower
than the night temperature (NT) (Erwin et al. 1989a, b, Erwin and Heins 1995, Myster
and Moe 1995). The inverse of this is also true: a warmer DT than NT promotes stem
elongation. Erwin et al. (1989b) quantified this phenomenon in relation to the sign and
magnitude of the difference between DT and NT (DIF); e.g., a +6 DIF signifies that
plants are grown with a day temperature 6°C warmer than the night temperature. For
many but not all species that respond to DIF, stem elongation also can be suppressed by a
transient (i.e., 2- to 4-h) drop in temperature (DROP), which is often most effective when
timed with the onset of the photoperiod (Erwin et al. 1989a, Langton et al. 1992,

Cockshull et al. 1995). Stem extension is perhaps more accurately related by the absolute

values of the day and night temperatures, not the differences between the two (Langton
and Cockshull 1997).

The physiological mechanism of the suppression of stem extension caused by DIF
or DROP is not understood, but several studies have implicated gibberellin (GA)
involvement. Application of GAl to DIF-sensitive Begonia xhiemalis Fotch grown under
a — 10°C DIF caused internode-length development similar to that of untreated plants
under a constant temperature regimen (Myster et al. 1995). Studies with tomato
(Solanum lycopersicon L. [synz Lycopersicon esculentum Mill.]), pea (Pisum sativum L.),
and campanula (Campanula isophylla Moretti cv. Hvit) suggest that -DIF suppresses
internode elongation by altering rates of gibberellin metabolism (Jensen et al. 1996,
Langton et al. 1997, Grindal et a1. 1998). However, in some instances, the effects of DIF
on stem elongation could not be attributed completely to changes in gibberellin
biosynthesis, metabolism, or sensitivity. In addition, application of GA to two Lilium
spp. only partially overcame the reduced stem extension cause by a - 8°C DIF (Zieslin and
Tsujita 1988).

Light plays a key regulatory role in mediating DIF or DROP responses; cool
temperatures limit stem extension only when delivered with the photoperiod, and
effective DROP temperature regimens are usually most effective when delivered in
coordination with the light-on signal (Langton et al. 1992, Cockshull et a1. 1995). In
addition, DROP treatments during the dark period did not reduce plant height (Bertram
1992, Gertsson 1992). Moe and Heins (1990) postulated that one of the major
photoreceptors found in green plants, phytochrome, could mediate the effect of

contrasting day and night temperatures on stem elongation.

The phytochromes mediate many physiological events, including germination, de-
etiolation, the shade-avoidance response, and flowering. Phytochrome exists in a
photoequilibrium of two interconvertible forms, Pr and P1,, which absorb maximally in the
red (R) (660 nm) and far-red (FR) (730 nm) regions of the spectrum, respectively. Thus,
at any one time, depending on the spectral quality of intercepted light, the proportion of
phytochrome in the Pf, (presumed active) state varies (Smith 1995). This proportion is
referred to as the phytochrome photoequilibrium (Pa/P), and light with a high R : FR
yields a Pfi/P high value. Furthermore, phytochrome mediates stem extension in response
to R and FR light. DIF affects stem elongation only when plants are grown with a high R
: FR ratio, and plants grown with a low R : FR ratio are phenotypically similar to those
grown with a +DIF (Moe and Heins 1990).

There are several members of the phytochrome family: e. g., five genes and their
encoded proteins have been identified in tomato (Hauser et al. 1995). The two most
abundant and best characterized types of phytochrome are phytochrome A (phyA) and
phytochrome B (phyB). PhyA is a light-labile (type I) phytochrome that accumulates in
the dark and is rapidly depleted in light. In contrast, phyB is a light-stable (type II)
phytochrome and is present in roughly equal amounts in the light and dark. The Pf, form
of phyA (PfiA) is considered the active form and in etiolated tissues has a 100-fold higher
turnover rate (half-life zl to 2 h) than the Pr form of phyA (P,A) (half-life =1 week)

(V ierstra 1994, Clough and Vierstra 1997). In green plants, the turnover of phyA as Pf,
probably occurs at a slower rate (J abben 1980). In addition, the rate of Pr, degradation

depends on temperature (Schafer and Schmidt 1974).

Given the accumulation of phyA in darkness, its metabolic instability in light and
the sensitivity of Pf, to temperature, we conducted experiments with photomorphogenic
mutants of tomato and transgenic potato (Solanum tuberosum L.) to determine whether
phyA could be implicated in mediating reduced stem elongation caused by —DIF and
DROP. We postulated that phyA could be involved for the following reasons: (1) in
potatoes, the overexpression of phyA reduces internode length (Heyer et al. 1995); (2)
phyA is degraded by light at the onset of the photoperiod, when DROP treatments are
effective; and (3) phyA degradation appears to be temperature-dependent, and reducing
the temperature in the morning via DROP may reduce the rate of phyA degradation,
thereby leading to reduction in internode elongation.

Tomato was selected as an ideal model plant to test this hypothesis since it
responds to DIF and DROP (Heuvelink 1989, Gertsson 1992, J acobsen et al. 1992), and
phyA and phyBl mutants exist (van Tuinen et al. 1995a, b, Kerckhoffs et al. 1996).
Potato was selected since it responds to +DIF (Bennett et al. 1991) and transgenic plants
that expressed very low (antisense) or high (overexpressed) levels of phyA have been
constructed (Heyer et a1. 1995). Furthermore, PHYA has been implicated in controlling
stem extension in potato in relation to the R : FR ratio (Heyer et al. 1995).

In tomato, PH YA and PH YBI are the first and second most abundant mRNAs,
respectively (Pratt et al. 1997). Tomato phyA mutants are insensitive to far-red light and
hence named fi'i mutants; phyBl mutants are temporarily insensitive to red light and
named tri mutants (van Tuinen et al. 1995a, b). If our hypothesis were true, we would
expect fri mutants and antisense-phyA potato to show negligible or no -DIF or DROP

response and plants with overexpressed phyA to Show an enhanced ~DIF or DROP

response compared to that of the WT. Our data Show that phyA does control stem

extension in light-grown WT plants but has no specific effect in cool-temperature

treatments.

Abbreviations - DIF, day temperature — night temperature; +DIF, day temperature >
night temperature; -DIF, day temperature < night temperature; 0 DIF, day temperature =
night temperature; DROP, morning temperature drop; FR, far-red light; phyA,

phytochrome A; phyBl, phytochrome B1; R, red light; WT, wild type.

Materials and methods

Tomato plant material

Seeds of fi-i’ and tri3 (subsequently referred to as fri and tri) and their isogenic WT cv.
Moneymaker were sown on 6 October 1997 in 3-cm (18-ml volume) plug trays
containing peat and vermiculite compost (SHL Professional Seed Sowing/Modular
Compost, Lincoln, UK). Seedlings were grown in a constant 20°C controlled-
environment room irradiated for 16 h at ca 65 umol m'2 s‘1 PPFD (400 to 700 nm) at
canopy level from cool-white fluorescent lamps (L40W/23; Osram Sylvania, Wembley,
UK), as measured with a Li-Cor quantum (model 03017-7901; Li-Cor, Lincoln, NE,
USA) sensor attached to a DC microvoltmeter (type 1221; Comark Electronics, Ltd,
Littlehampton, UK). Extra seedlings were grown so that plants could be selected for
uniformity at transplanting.

Three-week-old seedlings were transplanted into 9.5-cm (370-ml volume) pots

containing 75% (by volume) peat (SHL Professional Potting Compost) and 25%

medium-grade Perlite. Plants were fertilized at every irrigation with well water and
180N—80P-150K (mg 1") fertilizer (Sangral 111, SHL) applied by top watering with
minimal leaching. Plants were transferred to growth cabinets three days later and grown
at a constant 20°C with 12-h photoperiods. Twenty-five 27-day-old plants (with
approximately three or four leaves >1.5 cm) of each genotype were apportioned randomly

to each temperature treatment.

Potato plant material

Unless otherwise noted, materials and methods are identical to those described above.

A nontransformed control, two lines of antisense phyA (15-9 and 15-11), and two
lines of overexpressed phyA (PS2 and PS4) potato plants (transformed as described by
Heyer et al. [1995]) were received in agar on 31 Oct. 1997 and held at a constant 20°C.
PH YA in overexpressed lines was approximately twice that in WT; antisense constructs
had a 7- or lO-fold reduction (15-9 or 15-11, respectively) in PH YA (Heyer et a1. 1995).
For antisense constructs, there was some evidence for slightly lower PH YB mRNA levels.
Plants were removed from agar 7 (1 later and grown for one week in enclosed propagation
domes containing peat and vermiculite; the lowest three to four nodes were buried below
the soil surface to facilitate adventitious rooting. Plants were then potted into 9.5-cm pots
with peat and Perlite and transferred to growth cabinets set at a constant 20°C with 12-h
photoperiods. Afier 10 d of acclimation, 8 to 10 plants of each line were apportioned
randomly to each temperature treatment. Plants had generally developed 8 to 11 true

leaves above the soil surface at the onset of temperature treatments.

Light and temperature treatments

For each experiment, three temperature regimens were assigned randomly to growth
cabinets, each with a daily mean of 20°C. Cabinet 1 was a constant 20°C; cabinet 2 was
13°C from 0600 to 0900 h and 21°C fi'om 0900 to 0600 h (an 8°C DROP for 3 h); and
cabinet 3 was 16 or 24°C from 0600 to 1800 h or 1800 to 0600 h, respectively (a -8°C
DIF) (Fig. 1). Temperature fluctuations were completed within 10 min.

Air temperatures at canopy level were monitored with 36-gauge (0.127-mm-
diameter) type-E thermocouples connected to a datalogger (Datataker DT5 00; Data
Electronics, Letchworth Garden City, UK). The datalogger collected temperature data
every 15 s and recorded the average every 10 min. Cabinet settings were adjusted
regularly to maintain air temperatures at canopy level to those desired. Actual average
temperatures were calculated and varied to within O.2°C of the desired settings.

Each cabinet was illuminated from 0600 to 1800 h at ca 170 umol m'2 S“1 PPF D at
canopy level from a mixture of cool-white fluorescent lamps (VHOF48T12; Osram
Sylvania, Wembley, UK) and incandescent lamps (18% incandescent calculated by
nominal wattage). Using the model of Hayward (1984), the calculated Pfi/P using a
narrow-band absorption (1 nm) for each wavelength were 0.77, as measured with a
spectroradiometer (Bentham 605 with dual Bentham TM300 monochrometers; Reading,

UK).

Data collection and analysis

At the onset of the tomato experiment, the first proximal internode <1 mm in length was

identified, and the node below it was tagged. After 10 days of temperature treatment, the

following measurements were made: total plant height (from soil level to the apical
meristem), hypocotyl length, length of the designated internode of interest, and stem
length from and including that internode to the apical meristem. Total stem length from
the hypocotyl to the apical meristem was calculated. In all temperature treatments and
tomato genotypes, ca 5% of plants developed opposite or subopposite phyllotaxy at or
near the internode of interest, and in such instances, internode lengths were not included
in the results. The experiment was replicated with more mature plants and similar results
were obtained (data not shown).

For the potato experiment, the most recent partially expanded leaf (>5 mm) was
marked on each plant immediately before the beginning of the temperature treatments.
After 14 days of temperature treatments, stem length from the marked leaf to the apex
was measured and the number of developed nodes was counted. Average internode
length was calculated by dividing the developed stem length by the number of nodes
developed during temperature treatments.

A completely randomized design was used with 22 to 25 or 8 to 10 observations
of tomato or potato, respectively, for each genotype and temperature treatment. Data
were analyzed using SAS (SAS Institute, Cary, NC, USA) analysis of variance

(ANOVA) and general linear models (GLM) procedures.

.10

Results
Tomato

Compared to WT, light-grown photomorphogenic tomato mutants, especially phyBl
mutants, had increased hypocotyl lengths: hypocotyls were 49, 55, or 73 mm for WT, fri,
or tri plants, respectively, significantly different at P <0.001 (data not shown).

Across all temperature treatments, total plant height (not including the
hypocotyls) of fri and tri were ca 1 cm shorter than WT plants (statistically different at P
= 0.009) (data not shown). The interaction between temperature treatment and genotype
was nonsignificant.

For all genotypes, -DIF and DROP reduced the internode length of interest by 30
or 21%, respectively (Fig. 2A). Regardless of temperature treatment, internode length of
fri plants was ca 3 mm shorter than that of the WT (significantly different at P = 0.043).
The interaction between temperature treatment and genotype was statistically
nonsignificant. Similarly, -DIF and DROP reduced stem elongation of WT, fri, and tri
during the temperature treatments by 30%, or 2.5 cm (significantly different at P <0.001)

(Fig. 2B). Developed stem length did not differ among genotypes.

Potato

Combined across all temperature treatments, antisense phyA plants (15-9 and 15-11)
developed stems and intemodes that were 22 or 19% longer, respectively, than that of
WT or overexpressed phyA plants (PS-2 and PS-4) (Fig. 3). Measured parameters of

overexpressed phyA plants were statistically similar to that of WT. However, for all

11

measured parameters, there was a strong interaction between genotype and temperature
treatments (P 50.014), primarily from the response of the PS-4 overexpressed phyA line.

The —DIF significantly (P <0.001) reduced average internode length by 39% for
WT and 36 or 48% for 15-9 or 15-11 antisense phyA plants, respectively, but the
overexpressed lines showed a proportionately reduced response, with only one of the two
(PS-2) showing significantly reduced (27%) internode elongation (Fig. 3A). The DROP
treatment did not reduce average internode length for any genotype. Regardless of PH YA
level, average internode length under -DIF was 9.1 to 11.2 mm. At 0 DIF or DROP,
intemodes of antisense phyA plants were 226% longer than those of overexpressed phyA
plants.

Compared to that of plants at 0 DIF, —DIF significantly reduced stem length of
antisense phyA (by 36 or 48%) and WT (by 35%) plants, but for overexpressed phyA
plants, the 27 or 16% reduction in stem length was not significant at the P = 0.05 level
(Fig. 38). All potato genotypes grown with a —DIF developed similar stem lengths,

ranging from 5.7 to 6.7 cm. Unexpectedly, the DROP treatment did not reduce stem

length for any genotype.

Discussion

We tested the hypothesis that phyA mediated the reduction in stem extension caused by

cool day—temperature treatments. The —DIF treatment effectively reduced internode and
stem extension (by at least 30%) for WT, phyA and phyB] tomato and WT and antisense
phyA potato. The overexpressed phyA potato plants were apparently less responsive to

the temperature regimens than WT. The DROP treatment reduced internode elongation

12

of all genotypes of tomato but did not reduce stem extension of potato, which was
surprising, given their genetic similarity. Thus, at least for tomato and potato, it appears
that phyA does not control stem extension in relation to cool day-temperature treatments.
In addition, phyBl, which is thought to control primarily stem extension in tomato
(Kerckhoffs et al. 1997), can be eliminated as the only mediator of DIF and DROP
responses in tomato. However, phyBl is believed to be partially redundant because of
the other phyB in tomato, phyB2 (Kerckhoffs et a1. 1996, Kendrick et al. 1997), so phyB
cannot be eliminated as mediating temperature-influenced stem extension.

Contrary to our hypothesis, our results suggest that DIF responsiveness in potato
and tomato increases as PH YA expression decreases. Potato stem growth was reduced by
-DIF the least in overexpressed phyA plants (16 or 27%), moderately in WT (35%), and
the most in antisense phyA plants (36 or 48%). Potato intemodes responded similarly.
Comparably, -DIF suppressed stem extension more in phyA tomato mutants (38%) than
in WT (22%) and DROP reduced stem extension by 35% in phyA mutants but only by
16% in WT.

Results with WT tomato are very similar to that of other DIF and DROP studies.
The —8°C DIF temperature regimen suppressed tomato internode elongation by 30%; at a
similar temperature regimen, internode extension was reduced by 26 or 22% for cvs
Moneymaker or Metador, respectively (J acobsen et al. 1992, Langton and Cockshull
1997). A 3-h 4.8°C DROP at the beginning of the light-on signal reduced stem
elongation of tomato cvs Solentos or Elin by 22 or 14%, respectively (Gertsson 1992),

which compares favorably to our 30% reduction in stem extension with a 3-h 8°C DROP.

l3

Although the tomato intemodes of interest were not completely elongated at
measurement, our data provide an adequate measurement of the initial elongation rate.

No previous DROP or —DIF studies with potato were available for comparison,
but the remarkable suppression of stem extension we observed was unexpected.
Compared to that of plants grown with 0 DIF, 90 days with +8°C DIF promoted stem
extension of potato cvs. Norland and Denali by no more than 16% (Bennett et a1. 1991).
In contrast, the —8°C DIF reduced stem elongation of our WT potato by 35% compared to
those at 0 DIF. The difference in the magnitude of these responses is surprising, since
+DIF generally promotes stern extension more than -DIF with the same absolute value
suppresses it (Erwin et al. 1989a). Our data with stem extension in phyA transgenic
potatoes are consistent with that of Heyer et al. (1995): antisense phyA potatoes were
taller than WT, and overexpressed phyA lines were similar to or slightly shorter than WT.
For overexpressed lines, the difference in the -DIF response could reflect different
amounts of phyA in the lines.

Phytochrome A in tomato is very similar to that in potato (Pratt et al. 1997).
Thus, we expected stem extension responses of phyA tomato to be similar to that for
antisense phyA potato, but this did not occur. These differences could reflect differences
in GA status or in control of GA biosynthesis. The contrast between these species is of
considerable interest, given their genetic similarity, and may indicate that the DROP and
DIF responses are completely separate phenomena with different physiological bases.
However, contrary to our original hypothesis, it is clear that DROP responses in potato

are not associated with the action of phyA.

14

Our data show that phyA affects internode elongation; stem extension was
enhanced in tomato or potato plants with little or no phyA and was reduced in potato with
high levels of phyA. Studies with photomorphogenic mutants show that light and GAs
interact to affect stem extension (Chory and Li 1997). Overexpression of oat phyA in
transgenic tobacco (Nicotiana tabacum L.) (Jordan et al. 1995) and hybrid aspen (Olsen
et al. 1997) reduced levels of active GAs and produced dwarfed phenotypes, suggesting
that overexpression of phyA reduces GA biosynthesis. In addition, light (presumably
mediated through phytochrome) affects the accumulation of GA 20-oxidase mRN A,
which catalyzes the conversion of inactive GA”, to the active GAI (Wu et al. 1996).

Thus, compared to that of the WT, the suppression of internode elongation in
overexpressed phyA potato could be attributed to reduced GA biosynthesis, and vice
versa for the phyA mutant tomatoes and antisense phyA potatoes.

In summary, our results indicate that, while phyA affects internode elongation, it
alone does not mediate thermomorphogenic reduction of stem extension. However, other
phytochromes, alone or in synergy, may be the mediators, which in turn could influence
gibberellin biosynthesis or responsiveness and affect stem elongation. The use of
phytochrome double mutants (e. g., the fri, tri mutant; Kendrick et al. 1997) or
chromophore mutants (e.g., the au tomato mutant; Casal and Kendrick 1993) lend
themselves to further thermomorphogenic studies. Alternatively, other photoreceptors

(e.g., cryptochrome) may mediate temperature-regulated stem extension directly.

15

Acknowledgments
We wish to thank B. Thomas for generously providing the potatoes; R. E. Kendrick for
the tomato seed; P. Hadley for his assistance; and R. D. Heins, S. Jackson, and J. A. D.

Zeevaart for critically reviewing the manuscript.

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21

 

 

 

 

 

 

 

 

24
A 22 - ,
53:; 20 :‘:‘::1‘ _ _l,'“::‘:‘:: :f:‘::;
5 ‘8” I l
161 : :
? 14 ’ ‘I___,I _, — Constant
E 12D : 93:: L

 

/i /,
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

 

Fig. 1. Air temperature regimens at plant canopy level. Actual temperatures measured at
plant height were within i0.2°C of the indicated settings. The dark and light periods are

indicated by closed and open bars, respectively.

22

 

5O

20~

10~

Intemode length (mm)

 

 

 

 

 

 

 

 

 

 

100

80~ I

60~ _
4o- ,
20— .
0

WT

fri’ tn‘3

 

 

Developed stem length (mm)

 

 

 

 

 

 

 

1:3 Constant —D|F — DROP

Fig. 2. Intemode length (Fig. 2A) and developed stem length (Fig. 2B) of tomato WT and
fli- and tri-mutant plants after 10 days of temperature treatments. Error bars are 95%

confidence intervals (n = 22 to 25).

23

30

25- I _
20— 11 J
,5_ l l 1 -

 

 

 

Average internode length (mm)

 

 

 

 

 

 

 

 

 

 

 

 

18
16 - -
14 ~ —

12~ l I _

 

Developed stern length (cm)
8
j——-l
]——-a

 

 

 

 

 

 

 

 

 

 

ONACDCD
I
1

 

 

 

WT 15-9 15—11 PS-2 PS-4

[:| Constant —DIF - DROP

Fig. 3. Average internode length (Fig. 3A) and developed stem length (Fig. 3B) of WT (n
= 10) and four lines of transgenic potato: 15-9 and 15-11 are antisense phyA (n = 9); PS2
and PS4 are with overexpressed phyA (n = 8). Measurements were taken after 14 days of

temperature treatments. Error bars are 95% confidence intervals.

24

CHAPTER II
PHOTOCONTROL OF F LOWERING AND STEM EXTENSION OF THE
INTERMEDIATE-DAY PLANT ECHINACEA PURPUREA MOENCH.

Runkle, E.S., R.D. Heins, A.C. Cameron, and W.H. Carlson. Photocontrol of flowering
and stem extension of the intermediate-day plant Echinacea purpurea Moench.
Submitted to Physiol. Plant.

25

Photocontrol of Flowering and Stem Extension of the Intermediate-Day Plant Echinacea

purpurea

Erik S. Runkle, Royal D. Heins, Arthur C. Cameron, and William H. Carlson
Department of Horticulture, Michigan State University, East Lansing, MI 48824-1325

USA

Abstract

Intermediate-day plants (IDP) flower most rapidly and completely under intermediate
photoperiods (e.g., 12 to 14 h of light), but few species have been identified and their
flowering responses are not well understood. We identified Echinacea purpurea Moench
as an IDP and, based on our results, propose a novel mechanism for flowering of IDP.
Two genotypes of Echinacea purpurea (‘Bravado’ and ‘Magnus’) flowered most
completely (2 79%) and rapidly and at the youngest physiological age under intermediate
photoperiods of 13 to 15 h. Few (s 14%) plants flowered under 10- or 24-h
photoperiods, indicating E. purpurea is a strongly quantitative IDP. Plants were also
induced to flower when 15-h dark periods were interrupted with as few as 7.5 min of low-
intensity lighting (night interruption, NI). Flowering was progressively earlier as the NI
increased to 1 h, but was delayed when the NI was extended to 4 h. Stem length
increased by 2 230% as the photoperiod or NI duration increased, until plants received a
saturating duration (at 14 h or 1 h, respectively). Flowering was inhibited when 16-h
photoperiods were deficient in red (R, 600 to 700 nm) light, [creating a low phytochrome

photoequilibrium (P,,/P)] and was promoted when photoperiods were deficient in far-red

26

(F R, 700 to 800 nm) light (creating a high Pfi/P). Because of our results, we propose the
flowering behavior of IDP such as E. purpurea is composed of two mechanisms: a dark-
dependent response Operating through a high Pf/P in which flowering is promoted by a
short night, and a light-dependent response operating through a low Pfi/P in which

flowering is inhibited by a long day.

Abbreviations — B, blue light (400 to 500 nm); Bd, blue-deficient light; DE, day-extension
lighting; FR, far-red light (700 to 800 nm); F Rd, far-red deficient light; F R“, far-red light
(725 to 735 nm); IDP, intermediate-day plant; LD, long-day; LDP, long-day plant; N,
neutral-density filter; NI, night-interruption lighting; R, red light (600 to 700 nm); Rd,
red-deficient light; R1,, red light (655 to 665 nm); SD, short day; SDP, short-day plant;

VB, visible bud.

Introduction
Plants have been classified into flowering response groups based on how photoperiod
influences flowering (Vince-Prue 1975). Day-neutral plants flower irrespective of
photoperiod, and short-day plants (SDP) and long-day plants (LDP) flower, or flower
most rapidly, when photoperiods are less than or greater than some genotype-specific
“critical” photoperiod, respectively. Some species have more specialized photoperiodic
responses, such as intermediate-day plants (IDP) in which flowering is promoted most
under intermediate photoperiods.

Under SD, an interruption of the dark period with light [known as night

interruption (NI) lighting] promotes flowering in LDP and suppresses it in SDP. Night

27

interruption is usually most effective at or near the middle of the night when the dark
duration is long (e.g., 15 hours) (Lane et al. 1965). Short durations (e.g., 30 min) of NI
lighting are often sufficient to keep SDP vegetative, but most LDP require a long (e. g., 2
2 h) duration of NI to promote rapid reproductive development ( Lane et al. 1965, Vince-
Prue 1975).

A distinction has been made between plants in which flowering is controlled
primarily by dark processes (dark-dominant) or light processes (light-dominant; Thomas
and Vince-Prue 1997). Light-dominant plants show a more or less quantitative
relationship between irradiance of the night break and the magnitude of the flowering
response, until a saturation light intensity, duration, or both, are reached. In contrast,
dark-dominant plants are those in which a short lighting duration (e. g., s 30 min) during a
long night regulates plant development. In most instances, SDP and LDP are dark- and
light-dominant plants, respectively, but a few exceptions exist. For example, Fuchsia
thbrida Hort. ex Vilm. ‘Lord Byron’ has been classified as a dark-dominant LDP
(Thomas and Vince-Prue 1997).

Phytochrome is a family of photoreceptors that mediate flowering and stem
extension in many plants. There are several phytochromes found in plants (e.g., at least
five have been identified in tomato and Arabidopsis thaliana Heynh.), each with distinct
functions in some physiological processes and overlapping roles in others (Clack et al.
1994, Hauser et al. 1995). For any one phytochrome, there exists a photoequilibrium of
two interconvertible forms: the red (R, 600 to 700 nm) and far-red (FR, 700 to 800 nm)
absorbing forms, which are known as P, and P,,, respectively. A molecule is synthesized

as P, in darkness but is converted to the active Pf, form in light. P, becomes the

28

predominant form in darkness or upon FR exposure, but intermediate forms exist. Thus,
depending on the R : FR of light, a photoequilibrium (known as P,,/(P, + P,,), or Pf/P) is
established, where a high R : FR creates a high Pf/P, and vice versa.

For flowering in light-dominant LDP, various experiments suggest that R light (or
a high Pfi/P) is required at least during the early part of the photoperiod, and FR light (or a
lower P,,/P) is required toward the end of the photoperiod (Lane et al. 1965, Thomas and
Vince-Prue 1997). The low Pf, requirement is supported by many studies in which NI and
day-extension (DE) lighting containing R and FR more effectively induce flowering (i.e.,
flowering is more complete and rapid) than light deficient in FR. Some exceptions exist:
Whitman et al. (1998) found that lamps with various R : FR (from 0.7 to 8.8) induced
flowering similarly in three species that Runkle et al. (1998) found were light-dominant
LDP. Light quality is not critical for day-extension or NI lighting to be effective in dark-
dominant plants (Thomas and Vince-Prue 1995). In addition, environments with a low R
: FR, thus a low Pf/P, promote stem elongation, while a high R : FR suppresses it.
Therefore, FR causes rapid flowering in LDP but also promotes stem extension.

Brassicaceae are especially sensitive to blue (B, 400 to 500 nm) light. Recently,
two B photoreceptors, cryptochromes, were identified in the Brassicaceae member
Arabidopsis, through which some specific roles of B light have been identified.
Cryptochrome acts throughout the Arabidopsis life cycle, including in the promotion of
flower induction and inhibition of stem extension (Mozley and Thomas 1995, Bagnall et
al. 1996, Lin et al. 1996). Some of the cryptochrome actions are independent of

phytochrome, while others are interactive (Poppe et al. 1998).

29

We performed experiments to determine how light regulates growth and
development of Echinacea purpurea, a herbaceous Asteraceae perennial grown for its
reported medicinal properties and its aesthetic value in the landscape. Our objectives
were to determine (1) the differential sensitivity of plants to R, FR, and B light, (2) the
photoperiodic flowering response, and (3) if flowering was dark- or light-dependent.
Here, we report that E. purpurea is an IDP with an optimum reproductive photoperiod of
13 to 15 h. Short durations of NI (as little as 7.5 min) during 15-h dark periods induced
flowering in most plants, indicating that E. purpurea is a dark-dominant plant. Light
deficient in B or R reduced flowering percentages, suggesting specific, independent roles

of phytochrome and cryptochrome in flower initiation of E. purpurea.

Materials and methods
Plant material

Seed were sown into 128-cell plug trays (IO-ml volume) by a wholesale plug producer
(Rakers Acres, Litchfield, Michigan) and grown at 22.5 i 1.5 °C. Seedlings were initially
grown under photoperiods 2 l4 and _<. 16 h, since under short photoperiods, leaf size is
small and development is slow. Seeding, shipping, and forcing dates are provided in
Table 1. Following shipping, plugs were thinned to one plant per cell and were held at 20
°C. At the onset of experiments, plants were transplanted into 13-cm square plastic
containers (1.1-1 volume) and node counts were recorded (Table 1).

Plant culture, 1994 to 1996

Plants were grown in a commercial soilless medium composed of composted pine bark,

horticultural vermiculite, Canadian Sphagnum peat moss, processed bark ash, and washed

30

sand (MetroMix 510, Scotts-Sierra Horticultural Products Co., Marysville, Ohio). At
every in'igation, plants were fertilized with well water (EC of 0.65 mS cm" and 105, 35,
and 23 (mg L") of Ca, Mg, and S, respectively) acidified (two parts H3PO4 plus one part
H2804, which provided P at =80 mg L") to a titratable alkalinity of 2130 mg CaCO, L".
The nutrient solution (200-155 N-K mg L" from KNO3 and NH4NO3) was applied by top-
watering with minimal leaching. Micronutrients were added with a commercially
available blended chelated material [Compound 111 (1.50 Fe-0.12 Mn— 0.08 Zn—0.11

Cu-0.23 B-0.11 Mo), Scotts, Marysville, Ohio] at a constant 50 mg L".

Plant culture, 1997 to 1999

Plants were grown in a commercial soilless medium composed of composted pine bark,
vermiculite, Canadian sphagnum peat, coarse perlite with a wetting agent, and lime (High
Porosity Mix, Strong-Lite Products, Pine Bluff, AR). Plants were fertilized at every
irrigation with a nutrient solution of well water acidified with HZSO4 to a titratable
alkalinity of 2130 mg CaCO3 L" and water soluble fertilizer [125-12-125 N-P-K mg L"
plus 1.0-0.5-0.5-0.5-0.1-0.1 (Fe, Mn, Zn, Cu, B, Mo) mg L" (MSU Special, Greencare

Fertilizers, Chicago, IL)].

Greenhouse temperature control

All plants were grown in a glass greenhouse at 20 °C. Air temperatures on each bench
were monitored with 36-gauge (0.127-mm-diameter) type B thermocouples connected to
CR10 dataloggers (Campbell Scientific, Logan, Utah). To provide uniform night

temperatures, dataloggers controlled 1500-W electric heaters under each bench, which

31

provided supplemental heat as needed throughout the night. The dataloggers collected
temperature data every 10 s and recorded the hourly averages. For each experiment,
actual average daily air temperatures from the beginning of forcing until the average date

of flowering for every treatment were calculated (Table 1).

General lighting conditions, Expts. l and 2

Opaque black cloth was pulled at 1700 h and opened at 0800 h every day on all benches
so plants received a similar daily light integral within cold treatment or replication. From
0800 to 1700 HR, high-pressure sodium (HPS) lamps provided a supplemental
photosynthetic photon flux (PPF) of :75 umol m'2 s" at plant level when the ambient PPF
outside the greenhouse was < 200 pmol m'2 s" and were shut off when the ambient PPF
was > 400 umol m‘2 s". Incandescent (tungsten-filament) lamps, which delivered 1 to 3

pmol m“2 s" at canopy level, were used in the photoperiodic and NI studies.

Photoperiod experiment (Expt. 1)

To determine the photoperiodic flowering response, noncooled and cooled plants were
grown under one of eight photoperiods. The cold treatments consisted in holding half of
the plugs in a controlled-environment chamber for 10 (‘Magnus’) or 15 (‘Bravado’)
weeks at 5 °C. The chamber was illuminated by cool-white fluorescent lamps
(VHOF96T12; Philips, Bloomfield, NJ.) from 0800 to 1700 h at 20 to 40 umol rn‘2 s" at
canopy level, as measured with a LI-COR quantum sensor (model LI-189; LI-COR, Inc.,
Lincoln, Nebr.). While in the cooler, plants were watered with well water acidified (93%

HZSO4) to a titratable alkalinity of CaCO, at z 100 mg L".

32

Ten noncooled or cooled plugs of each cultivar were planted and placed under one
of eight photoperiods: 10, 12, 13, 14, 15, 16, or 24 h of continual light or 9 h with a 4-h
NI. For the continual photoperiodic treatments, lamps provided DE; they were turned on
at 1700 h and turned off after each photoperiod was completed. The NI was delivered

from 2200 to 0200 HR.

NI duration experiment (Expt. 2)

To further characterize the NI response of E. purpurea, plants were provided with one of
six NI durations: 0, 7 .5, 15, 30, 60, or 240 min. All NT were provided during the middle
of the 15-h dark periods. An additional treatment was a 14-h photoperiod with DE
lighting. Twenty plants of ‘Bravado’ and ‘Magnus’ were placed under each lighting

regimen.

Spectral filter experiment (Expt. 3)

A reciprocal transfer experiment with four different light quality environments was
conducted to determine the specific effects of modified light wave bands from sunlight on
flowering and stem extension of E. purpurea. Cladding materials used were neutral (N)
density or plastics that selectively reduced the transmission of B (B deficient, Ed), R (R
deficient, Rd), or FR (FR deficient, FRd) light. Filter treatments were designed to transmit
a similar PPF. The following filters (one layer each) enclosed greenhouse benches to
provide the light quality treatments: N, OLSSO (Ludvig Svensson, Charlotte, NC) + PLS
Clear (Ludvig Svensson); Bd, Lee filter 101 (Andover, UK) + OLS40 (Ludvig Svensson);

Rd, Lee filter 115; FR, experimental FR filter (van Haeringen, 1998) + PLS Clear.

33

Spectral transmissions from 400 to 800 nm were measured by a spectroradiometer (L1—
1800, LI-COR Inc.) and are shown in Fig. 1. Quantum ratios (R : FR, B : R, and B : FR)
and the estimated Pfi/P (Sager et a1. 1988) were calculated (Table 2).

A 16-h photoperiod was delivered with a combination of sunlight and HPS lamps
positioned above filters. From 0600 to 2200 HR, HPS lamps provided a supplemental
PPF of :35 umol m”2 s" at plant level when the ambient greenhouse PPF was < 200 umol
m'2 s", and was terminated when the ambient PPF was > 400 nmol m‘2 5". Since HPS
lamps emit a high proportion of R light, an additional lamp was placed above the Rd filter
so that the supplemental PPF was similar among treatments. The light quantum ratios
under the filters were calculated when the lamps were the only light source (Table 2).
Under each filter treatment, the average daily light integral was measured at canopy level
with line quantum sensors connected to a CR10 datalogger (Campbell Scientific), each of
which was independently calibrated under the filters by using the spectroradiometer
(Table 2).

Twenty plants were placed under each of the Bd, Rd, and FRd filter treatments, and
40 under the N filter. At VB, ten plants under the Bd, Rd, and FR, environments were
transferred to the N filter, and ten were transferred at VB from the N filter to each
photoselective filter. Filter effects on flowering and stem extension before and after

flower initiation could thus be separated.

Data collection and analysis

Experiments were replicated in time (Table 1) and were arranged in a completely

randomized design. The date the first flower bud was visible (without dissection) and the

34

date the terminal flower reached anthesis (flowering) were recorded for each plant. At
flowering, visible inflorescences and nodes on the main stem were counted, and total
plant height (not including the container) was measured. Plant height at VB was also
measured in Expts. 2 and 3. Plants that did not have visible inflorescences after 15 weeks
(Expts. 1 and 2) or 18 weeks (Expt. 3) of forcing were considered nonflowering and
discarded. Days to VB, days from VB to flower, days to flower, and node-count increase
from the start of forcing were calculated.

Data were analyzed using SAS’s (SAS Institute, Cary, NC.) analysis of variance
(AN OVA), general linear models (GLM) procedures, and a mean separation procedure
for unequal observation numbers (pdiff) with P = 0.05. Regression analysis was
performed by Sigma Plot (SPSS, Inc., Chicago). Data were pooled when cold treatment

(Expt. 1) did not significantly influence the parameters measured.

Results
Photoperiod experiment

Flowering percentage for E. purpurea ‘Bravado’ and ‘Magnus’ was greatest under
photoperiods of 13 to 15 h or a 4-h NI (Fig. 2A). Less than 20% of the plants under 10 or
24 h of light reached VB within 15 weeks of forcing. Cold treatment did not influence
flowering percentage (data not shown). Further data for ‘Bravado’ under lO-h and
‘Magnus’ under 10- and 24—h photoperiods are not presented, since the low number of
flowering observations prevented statistical analysis.

Under photoperiods of 13 to 16 h or a 4-h NI, ‘Bravado’ flowered in 87 to 95

days, and ‘Magnus’ in 95 to 100 days (Fig. 2B). Flowering was significantly delayed (by

35

2 14 days) for both cultivars under 12-h photoperiods and, for ‘Bravado’, under 24-h.
Cold treatment did not significantly affect time to flower. Time from VB to flowering
was not consistently influenced by photoperiod and averaged 29 days for ‘Bravado’ and
27 for ‘Magnus’ (data not shown).

From the start of forcing under N1 or photoperiods of 14 to 16 h, flowering plants
of both cultivars developed 13 to 15 nodes below the inflorescence (Fig. 2C). Plants
under shorter or longer photoperiods developed significantly more nodes before
flowering. The effect of cold treatment on node count before flowering was not
significant at P = 0.05.

‘Bravado’ plant height at flowering increased from 23 to 2 58 cm as the
photoperiod increased from 12 to 2 14 h (Fig. 2D). Similarly, plant height of ‘Magnus’
reached a plateau under photoperiods 214 h. Flowering height under N1 was similar to

that under 2 14-h photoperiods. Cold treatment did not modify plant height.

NI duration experiment

Flowering percentage of ‘Bravado’ and ‘Magnus’ was < 25 without N1 and 2 74 when the
15-h night was interrupted with a 7.5 min or longer NI (Fig. 3A). There was a quadratic
(P < 0.001) effect of NI duration on flowering percentage, and flowering percentage was
highest under 30 or 60 min of NI or a 14-h photoperiod.

The duration of NI lighting had a quadratic (P < 0.001 for ‘Bravado’, P = 0.017
for ‘Magnus’) effect on time to flower and was most rapid under 30 or 60 min of NI (Fig.
3B). Flowering of both cultivars was less uniform (as indicated by the large 95%

confidence intervals) and delayed (by 2 14 days) under the 4-h NI. Flowering of

36

‘Bravado’ was also delayed (by 2 20 days) and was less uniform when the NI was s 15
min. Night interruption duration had no consistent effect on time from VB to flowering
and averaged 25 and 27 days for ‘Bravado’ and ‘Magnus’, respectively (data not shown).

The effect of NI duration on node development before flowering was negatively
correlated with the NI effect on flowering time (Fig. 3C). Night interruption duration had
a quadratic (P < 0.001 for ‘Bravado’, P = 0.010 for ‘Magnus’) effect on the increase in
node number, with the fewest nodes formed under the 60-min NI. Both cultivars under s
15 min of N1 developed 2 3.0 more nodes before flowering compared with plants under
the 60-min NI.

Plant height at VB and flowering was markedly influenced by NI duration.
Inflorescence height at VB was < 5 cm for both cultivars under s 15 min of NI (data not
shown). ‘Bravado’ height at VB increased from 4.0 to 21.6 cm as NI increased from 15
to 60 min and reached a maximum with =90 min of NI, when VB height leveled (:26
cm). Similarly, ‘Magnus’ height at VB increased from 4.3 to 18.3 cm as NI increased
from 15 to 60 min, and longer NI durations did not increase stem elongation. At
flowering, an increase in NI from 15 to 60 min increased ‘Bravado’ and ‘Magnus’

flowering height by 105% and 119%, respectively (Fig. 3D).

Spectral filter experiment

Flowering percentage was highest under the N and F Rd filters (Fig. 4). Significantly
fewer plants flowered under the B, filter (43%), and flowering percentage was lowest
(10%) under the Rd filter. Plants reached VB 14 days (17%) earlier under the F Rd filter

than under the N filter. Plant height at VB and flower, time from VB to flower, and the

37

node count increase of flowering plants were not significantly influenced by filter type or

transfer (data not shown).

Discussion

Both cultivars of E. purpurea had the highest flowering percentage and flowered most
rapidly under intermediate photoperiods of 13 to 15 h, and flowering was strongly
inhibited under short (3 12 h) photoperiods or continual light. In addition, plants
flowered at the youngest physiological age (e.g., node count) under these intermediate
photoperiods. The photoperiodic response was similar in cooled and noncooled plants.
Therefore, E. purpurea can be classified as an IDP. Few IDP have been identified: only
13 of the > 500 species classified into photoperiodic responses by Thomas and Vince-
Prue (1997) were IDP.

Echinacea purpurea is native to the east central United States (lat. 32-42° N), and
flowers naturally in June, when biological photoperiods are 15 to 16 h. Bolting, and
hence flower initiation, occurs 6 to 8 weeks earlier, when photoperiods are approximately
60 to 90 min shorter. Thus, the optimum reproductive photoperiod (:13 to 15 h) in our
studies is similar to the natural photoperiod when Echinacea is induced to flower.

Many of the identified IDP have a weakly quantitative flowering response, where
plants flower under a broad range of photoperiods but flowering occurs slightly earlier
under intermediate day lengths. In contrast, E. purpurea has a strong quantitative
photoperiodic flowering response, where flowering is primarily restricted to intermediate

photoperiods. Flowering of plants under short (10 h) or long (24 h) photoperiods could

be at least partially attributed to exposure to inductive photoperiods at the seedling stage.

38

The most inductive photoperiod for many [DP is similar to that of E. purpurea: 2 13 and
< 16 h (Allard 1938, EA. Clough 1999. Thesis, Michigan State Univ., East Lansing, MI,
USA). For some species, however, the most inductive photoperiod is slightly shorter
(e.g., 12 h in Capsicum frutescens L.; Cochran 1942).

Compared to SDP and LDP, few details are known about flowering of IDP
(Thomas and Vince-Prue 1997). Takeno et a1. (1995) concluded that the quantitative
intermediate-day behavior of Salsola komarovii Iljim was a type of SD response, since all
plants under SD eventually flowered and at the same physiological age (e. g., node count)
as those under the most inductive intermediate photoperiods. Sachs (1956) suggested that
IDP are actually plants that require dual day length (e. g., SD followed by LD) in which
intermediate photoperiods satisfy the requirements of both SD and LD. In our studies,
nearly all plants remained vegetative under short (5 10 h) or long (24 h) photoperiods,
and of those that did become reproductive, flowering occurred at an older physiological
and chronological age. Thus, the flowering behavior of E. purpurea cannot be considered
a type of SD or LD response. In addition, E. purpurea was induced to flower relatively
rapidly when a light break interrupted a long night. Since SD were never provided, E.
purpurea cannot be a plant that requires dual day length. Therefore, we conclude that a
true intermediate photoperiodic flowering response exists.

Short-day plants and LDP are generally dark or light dominant, respectively, but
to our knowledge, the roles of light and dark processes on IDP have not been described.
We reported the sensitivity of E. purpurea ‘Bravado’ to short (30 min) durations of NI
(Runkle et al. 1998). To our knowledge, no NI studies have been performed on any other

IDP. In this study, we found that even shorter (7.5 min) durations of low-intensity

39

lighting induced flowering of both cultivars during 15-h dark periods. Increasing the NI
duration to 30 min increased flowering percentage and decreased time to flower and node
count at flowering. Thus, E. purpurea can be labeled a dark-dominant IDP, but the
response has some similarities to those of SDP and some LDP. A short NI regulates
development of most SDP, but the response is an inhibition, not promotion, of flowering.
In contrast, short NI durations are not sufficiently long to induce a flowering response in
most LDP, since they are light dominant.

As with light-dependent plants, the spectral distribution of light delivered during
the photoperiod had a marked effect on the reproductive status of Echinacea. Flowering
of light-dominant plants is most rapid when long photoperiods contain at least some FR
light, particularly toward the end of the photoperiod (Thomas and Vince-Prue 1995).
When Echinacea was exposed to 16-h photoperiods deficient in FR light (e.g., under the
F R, filter), flowering was more rapid than plants under the N filter. When photoperiods
were extended to 2 16-h with light rich in FR (e. g., day-extension lighting with
incandescent lamps), flowering was inhibited. Thus, our data suggest that a light
dominant response, operating through a low Pfi/P, inhibits flowering in Echinacea.
Furthermore, the 4-h NI was repeatedly less inductive than shorter NT durations, which
also suggests that flowering is inhibited by a light-dependent response. Flowering was
strongly suppressed under the Rd filter (which created a low Pfi/P), as would be expected
with an inhibitory light-dominant response. However, this could also be attributed to a
lack of flowering promotion through the dark-dominant response, which is known to

require P5.

40

We propose that the intermediate day behavior of E. purpurea is the result of two
mechanisms: a dark-dependent response in which flowering is promoted by a short night,
and a light-dependent response in which flowering is inhibited by a long day. Flowering
occurs when conditions allow the dark-dominant mechanism to promote flowering but do
not allow the light-dominant mechanism to inhibit flowering. Thus, flowering occurs
when the duration of darkness is less than the critical night length of 11 h, but is
decreased when the photoperiod is 2 16 h and the light-dominant mechanism inhibits
flowering. A short NI prevents the inhibitory effect of a long night and induces
flowering. However, when the N1 is sufficiently long, the light-dominant mechanism
becomes effective and flowering is depressed.

We have repeatedly observed a high flowering percentage (2 85%) when
Echinacea was forced under natural day lengths extended to 16 h with HPS lamps at 75
pmol ‘2 s" PPF (unpublished data). These lamps create a higher predicted Pfi/P (0.850,
Table 2) than under incandescent lamps (0.645, data not shown) used in our photoperiod
and night interruption studies. In accordance with our theory, the inhibitory light-
dominant mechanism did not operate in plants under the HPS lamps, since a high Pfi/P
was maintained. However, under the incandescent lamps, the Pfi/P was low and the light-
dominant mechanism inhibited flowering.

Flower induction of ‘Bravado’ was also inhibited when photoperiods were
deficient in B light. While the absolute quantity of FR light was greater under the B,
filter compared to the N filter, the R : FR and P,,/P under the B, treatment were nearly
identical to that under the N treatment in which flowering occurred (Table 2). Thus, the

reduced flowering in the absence of B light could be attributed to the lack of positive

41

action of cryptochrome. This response is consistent with delayed flowering of the
cryptochrome mutants of Arabidopsis (Mozley and Thomas 1995, Bagnall et al. 1996,
Lin et al. 1996), and suggests that cryptochrome could be involved in the promotion of
flowering outside Brassicaceae.

Here, we present a newly identified IDP for flower initiation, with a suggested
mechanism, arguments against previous mechanistic proposals in the literature, and
reasons to support our hypothesis. Echinacea purpurea showed a strongly quantitative
flowering response with an optimum reproductive photoperiod of 13 to 15 h of light.
Plants also initiated flowering when 9-h days/ 15-h nights were interrupted with short
durations of light. Stem extension increased with an increase in photoperiod or NI, until
a saturating lighting duration of about 60 min was reached. Thus, our data suggest that a
true intermediate photoperiodic response type exists and has similarities to both SDP and
LDP. We propose that in IDP such as E. purpurea, a dark-dependent response promotes
flowering under a short night and flowering is inhibited by a long day in a light-
dependent response operating through a low Pfi/P. However, further experiments are

warranted to confirm that these mechanisms are indeed involved.

Acknowledgments

We wish to thank the Michigan Agricultural Experiment Station and funding by
greenhouse growers supportive of Michigan State University floricultural research. We
alsoithank Dan Tschirhart and Thomas F. Wallace, Jr. for their greenhouse technical help,
Simon Pearson and British Visqueen for the experimental FR filter, and Daphne Vince-

Prue for her editorial comments.

42

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filter; FRd, far-red (700 to 800 nm) deficient filter. See Table 2 for light wave band

ratios.

48

Fig. 2. Flowering of Echinacea purpurea ‘Bravado’ and ‘Magnus’ under continual
photoperiods consisting in 9-h natural days extended with light from incandescent lamps
(N1 = 4-h night interruption). At first open flower, the number of nodes on the main stem
below the inflorescence was counted and total plant height was measured. Data for
noncooled and cooled plants were pooled, since cold treatment effects were insignificant.
Legend in D applies to all figures. Values with the same letter (‘Bravado’ in uppercase,

‘Magnus’ in lowercase) are not statistically different at P = 0.05.

49

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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50

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Fig. 3. Flowering of Echinacea purpurea ‘Bravado’ and ‘Magnus’ under various
durations of night interruption lighting or with a 5-h day extension (14-h photoperiod).
Photoperiods consisted in 9-h natural days with light from incandescent lamps during the
middle of the dark period (night interruption) or following the natural photoperiod for 5 h
(14-h photoperiod). Error bars are 95% confidence intervals. Legend in D applies to all
figures. Nonlinear regression analysis was used to describe plant height (cm) as a
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51

 

 

 

 

 

 

 

 

 

 

 

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Fig. 4. Flowering of Echinacea purpurea ‘Bravado’ under a neutral-density filter or
filters that selectively reduced transmission of red (Rd, 600 to 700 nm), blue (Bd, 400 to
500 nm), or far-red (FR, 700 to 800 nm) light. See Fig. l and Table 2 for filter
transmission properties. Photoperiods consisted in day lengths extended with light from
supplemental high-pressure sodium lamps positioned above filters from 0600 to 2200 HR.

Values with the same letter are not statistically different at P = 0.05.

53

CHAPTER III

SPECIFIC FUNCTIONS OF RED, FAR-RED, AND BLUE LIGHT IN
FLOWERING AND STEM EXTENSION OF LONG-DAY PLANTS

Runkle, ES. and RD. Heins. 2001. Specific functions of red, far-red, and blue light in
flowering and stem extension of long-day plants. J. Amer. Soc. Hort. Sci. (In press).

54

Specific Functions of Red, Far-Red, and Blue Light in Flowering and Stem Extension of

Long-Day Plants

Erik S. Runkle and Royal D. Heins'

Department of Horticulture, Michigan State University, East Lansing, MI 48824-1325

Additional index words. Campanula carpatica, Coreopsis xgrandiflora, far-red filter,
height control, Lobelia Xspeciosa, phytochrome, Pisum sativum, spectral filters, Viola

Xwittrockiana

Abstract

For many long-day plants (LDP), adding far-red light (FR, 700 to 800 nm) to red light (R,
600 to 700 nm) to extend the day or interrupt the night promotes extension growth and
flowering. Blue light (B, 400 to 500 nm) independently inhibits extension growth, but its
effect on flowering is not well described. Here, we determined how R-, FR-, or B-

deficient (Rd, FRd, or Bd, respectively) photoperiods influenced stem extension and

flowering in five LDP species: Campanula carpatica J acq., Coreopsis Xgrandiflora
Hogg ex Sweet, Lobelia ><speciosa Sweet, Pisum sativum L., and Viola ><wittrockiana
Gams. Plants were exposed to Rd, FRd, Bd, or normal (control) 16-hour photoperiods,
each of which had a similar photosynthetic (400 to 700 nm) photon flux. Compared with
that of the control, the Rd environment promoted extension grth in C. carpatica (by 65
%), C. Xgrandiflora (by 26 %), P. sativum (by 23 %), and V. Xwittrockiana (by 31 %).

The PR, environment suppressed extension growth in C. Xgrandiflora (by 21 %), P.

55

sativum (by 17 %), and V. Xwittrockiana (by 14 %). Independent of the R : FR ratio, the
B, environment promoted stem extension (by 10 % to 100 %) in all species, but there was
little or no effect on flowering percentage and time to flower. Extension grth was
generally linearly related to the incident wide band (100 nm) R : FR or estimated
phytochrome photoequilibrium except when B light was specifically reduced. A high R :
FR (i.e., under the FR, filter) delayed flower initiation (but not development) in C.
carpatica and C. ><granrliflora and inhibited flower development (but not initiation) in V.
Xwittrockiana. Therefore, B light and the R : FR independently regulate extension
growth by varying magnitudes in LDP, and in some species, an FR, environment can

suppress flower initiation or development.

Introduction
Plants detect light quality by at least three families of photoreceptors: phytochromes,
cryptochromes, and an unidentified UV light receptor. Phytochrome absorbance peaks
are in the red (R, 600 to 700 nm) and far-red (FR, 700 to 800 nm) light, and to a lesser
extent in blue (B, 400 to 500 nm) light. For any one phytochrome, there exists a
photoequilibrium of two interconvertible forms: the R and FR absorbing forms, which are
known as P, and P,,, respectively. P, is considered to elicit physiological responses but P,
could be the active form of phytochrome A (Shinomura et al., 2000). In addition,
intermediate short-lived forms between P, and P, exist.

Depending on light quality, a phytochrome photoequilibrium [P,-,/(P, + P,), or
P,,/P] is established, where a high R : FR creates a high Pfi/P, and vice versa. Models

have been developed (Hayward, 1984; Sager et al., 1988) to estimate the Pfi/P based on

56

the distribution of incident spectral radiation from 300 to 800 mn. These models are
based on cross-section phytochrome A data from etilated oats, but its validity in
estimating Pfi/P in light-grown plants or for other phytochromes is unknown.
Nevertheless, these estimates and R : FR ratios are useful in associating
photomorphogenic responses with light quality (Smith, 1994).

In addition to phytochromes, B light is absorbed by cryptochromes.
Cryptochromes participate in the inhibition of stem extension, particularly those in
Brassicaceae (Kigel and Cosgrove, 1991). The suppression of extension growth by B
light has been well documented, especially in hypocotyls and epicotyls (Casal and Smith,
1989; Laskowski and Briggs, 1989; Liscum et al., 1992; Warpeha and Kaufinan, 1989).
In addition to inhibiting stem extension, B light participates in flower induction in some
LDP, such as Hyoscyamus niger L. (Schneider et al., 1967; Stolwijk and Zeevaart, 1955)
and Arabidopsis thaliana Heynh. (Bagnall et al., 1996; Lin et al., 1996; Mozley and
Thomas 1995). Some of the cryptochrome effects are independent of phytochrome, while
others are conditionally interactive (Casal and Mazzella, 1998; Poppe et al., 1998).
Compared with data on phytochrome, however, relatively little is known about how B
light regulates flowering.

Plants are classified by their photoperiodic flowering response [e.g., short-day
plants (SDP) or LDP]. Alternatively, plants that are controlled primarily by light or dark
processes can be classified as light- or dark-dominant plants, respectively (Thomas and
Vince-Prue, 1997). Most light- and dark-dominant plants are LDP and SDP,
respectively, but some exceptions exist. Light-dominant LDP show two general

characteristics: 1) a quantitative relationship exists between the irradiance of the night

57

break and the magnitude of the flowering response, until a saturation light intensity,
duration, or both, are reached; 2) flowering is often most rapid when photoperiods
contain some minimal amount of FR. In contrast, flowering of dark-dominant plants,
which include most SDP, can be regulated by a short night break (e. g., s 30 min)
containing little or no FR light.

Artificial long days (LD) can be created by lighting at the end of the natural
photoperiod or by interrupting the dark period with a light break. Flowering of LDP is
promoted most when artificial lighting contains R and FR (creating a moderately low
PfilP) compared with light deficient in PR (creating a high Pf/P; Downs and Thomas,
1982; Lane et al., 1965). However, extension growth of a wide range of species shows an
inverse linear relationship with estimated Pfi/P (Smith, 1982, 1994). Therefore, incident
light creating a moderately low Pfi/P simultaneously promotes flowering and stem
extension, and that creating a high Pfi/P is inhibitory to both responses.

Plastic filters that selectively reduce transmission of red light have been used
experimentally to promote extension growth in various herbaceous plants (Kubota et al.,
2000; Murakami et al., 1996; Rajapakse et al., 1999). To limit stem extension, flexible
plastic filters have been developed to reduce transmission of FR radiation (Oyaert et al.,
1999; Rajapakse et al., 1999; van Haeringen et al., 1998). These FR, filters have reduced
extension grth in a variety of species, including vegetables and ornamental plants.
However, an FR, environment can delay flowering in some plants, but in many reports,
this delay has not been addressed.

The primary objective of our experiments was to determine how flowering and

stem extension in LDP were regulated by photoperiods deficient in R, FR, and B. First,

58

specific wave band effects were identified by comparing plant responses under
photoselective filters with those under a neutral (DD filter that transmitted a similar
photosynthetic photon flux (PPF). Two subsequent experiments were performed to
further quantify how light deficient in FR regulated flower initiation and development.
These studies illustrate the variability in species’ responses to light quality and
underscore the complexity of how light regulates flowering and stem extension in LDP.
In addition, while an FR, environment can suppress extension grth in plants, it can

also delay flowering in some LDP.

Materials and Methods
Stem extension and flowering under photoselective filters (Expt. 1)

Plant material. Seed of Campanula carpatica ‘Blue Clips’, Coreopsis
xgrantiiflora ‘Early Sunrise’, and Lobelia Xspeciosa ‘Compliment Scarlet’ were sown
into 128-cell (10-mL) plug trays and that of Viola Xwittrockz'ana ‘Crystal Bowl Yellow’
into 288-cell (6-mL) plug trays by a wholesale plug producer (Rakers Acres, Litchfield,
Mich.) Seedlings were grown under photoperiods s 12 h at 22.0 :t 2 °C. Pisum sativum
‘Utrillo’ seed were sown into 50-cell (85-mL) plug trays. Seeding, shipping, and forcing
(the onset of treatments) dates are provided in Table 1. Plants were thinned to one per

plug and held at 20 °C until plugs were mature. At the onset of experiments, plants were

transplanted into l3-cm (1.1-L) square plastic containers and node counts were recorded
(Table 1).
Plant culture. Plants were grown in a commercial soilless medium composed of

composted pine bark, vermiculite, Canadian sphagnum peat, coarse perlite with a wetting

59

agent, and lime (High Porosity Mix, Strong-Lite Products, Pine Bluff, Ark.). Plants were
fertilized at every irrigation with a nutrient solution of well water acidified with HZSO4 to
a titratable alkalinity of = 130 mg CaCO3-L" and water soluble fertilizer
[125N—12P—125K(mg-L") plus 1.0Fe—0.5Mn—0.SZn—0.5Cu—0.lB—0.1Mo (mg-L"; MSU
Special, Greencare Fertilizers, Chicago, Ill.)].

Lighting and filter treatments. A reciprocal transfer experiment with four different
light quality environments was conducted using a neutral (N) density metalized woven
fabric or plastics that selectively reduced the transmission of B (B deficient, B,,), R (R
deficient, R,), or PR (FR deficient, FR,) light. Filter treatments were designed to transmit
a similar PPF. The following filters (one layer each) enclosed greenhouse benches to
provide the light quality treatments: N, OLS50 (Ludvig Svensson, Charlotte, NO) + PLS
Clear (Ludvig Svensson); B,, Lee filter 101 (Andover, UK) + OLS40 (Ludvig Svensson);
R,, Lee filter 115; FR,, experimental FR filter (van Haeringen et al., 1998) + PLS Clear.
Solar spectra transmissions from 400 to 800 nm were measured under filters by a
spectroradiometer (LI-1800, LI-COR, Inc., Lincoln, Nebr.) and are shown in Fig. 1.
Quantum ratios (R : FR, B : R, and B : FR), the estimated PfilP (Sager et al., 1988), and
the relative quantum efficiency (McCree, 1972) were quantified for each light treatment
(Table 2).

Twenty plants of each species were placed (=8 cm apart) under each of the B,, R,,
and FR, treatments, and 40 under the N filter. When flower buds were visible [visible
bud (VB)], 10 plants under the B,, R,, and FR, environments were transferred to the N

light treatment, and 10 were transferred at VB fi'om the N environment to each other light

60

environments. Filter effects on flowering and stem extension before and alter flower
initiation could thus be separated. The experiment was performed twice.

A 16-h photoperiod was delivered with a combination of sunlight and high-
pressure sodium (HPS) lamps positioned above filters. From 0600 to 2200 HR, I-IPS
lamps provided at plant level a supplemental PPF of =35 pmol-m'2~s" when the ambient
greenhouse PPF was < 200 umol-m'z-s" and were shut off when the ambient PPF was >
400 umol-m‘Z-s". The light quantum ratios under the filters were calculated at night when
the lamps were the only light source (Table 2). Under each filter treatment, the average
photosynthetic daily light integral (DLI) was measured at canopy level with line quantum
sensors that included 18 G2711 photodiodes (Hamamatsu Co., Hamarnatsu, Japan)
connected to a CR10 datalogger (Campbell Scientific, Logan, Utah). Each line quantum
sensor was independently calibrated under the filters by using the spectroradiometer
(Table 1).

Greenhouse temperature control. All plants were grown in a glass greenhouse at
20 °C. Air temperatures under each filter treatment were monitored with 36-gauge
(0.127-mm-diameter) type B thermocouples connected to CR10 dataloggers (Campbell
Scientific). To provide uniform night temperatures, dataloggers were used to control
1500-W electric heaters under each bench, which provided supplemental heat as needed
throughout the night. To improve temperature uniformity under filters during the day,
dataloggers were used to control portable fans to vent each bench as needed. The
dataloggers collected temperature data every 10 s and recorded the hourly averages. For
each experiment, average daily air temperatures from the beginning of treatment until the

average date of VB under each filter were calculated (Table 1).

61

Data collection and analysis. Experiments were replicated in time and were
arranged in a completely randomized design. The date the first flower bud was visible
(without dissection) and the date the first flower reached anthesis (flowering) were
recorded for each plant. At flowering, visible flower buds or inflorescences and nodes on
the main stem were counted. Except for V. xwittrockiana, plant height (fi'om soil level to
the top of inflorescence) at VB was measured. Total plant height at flowering was
measured for all species except P. sativum. Plants were considered nonflowering if
flower buds were not visible after 32 or 63 d of treatments for P. sativum and V.
Xwittrockiana, respectively, and after 15 weeks for C. carpatica, C. Xgrandiflora, and L.
Xspeciosa. Leaves and stems of P. sativum were weighed after 32 d of treatments, and
dry weight was measured following two days at 55 °C. Days to VB, days from VB to
flower, days to flower, and node-count increase from the start of treatments were
calculated. Data were" analyzed by using SAS’s (SAS Institute, Cary, NC.) analysis of
variance (AN OVA), general linear models (GLM) procedures, and a mean separation
procedure for unequal observation numbers (pdifi) with P = 0.05. Unless otherwise

stated, all comparisons made are relative to responses under the N filter.

Viola reciprocal transfer experiment (Expt. 2)

A separate experiment was performed with ‘Crystal Bowl Yellow’ to determine
how plant age influenced the sensitivity of V. Xwittrockiana to the flowering inhibition
under the FR, filter. Plants were placed under the N and FR, filters, then were transferred
to the other filter after 5, 10, 15, 20, 25, 30, 35, or 40 d. The corresponding average node

numbers at transfer times were 1.9, 2.8, 4.0, 5.0, 5.9, 6.9, 7.3, and 8.8. Experimental

62

conditions were as described above unless otherwise stated. Seeding, shipping, and
forcing dates, average temperatures, and DLI are provided in Table 1. The following data
were recorded: date of flowering (anthesis), node count increase to the first VB and first
open flower, and whether first flowering was on the primary or an axillary shoot.
Flowering percentage, days to flower, node-count increase to the first VB and flowering,
undeveloped buds below the first open flower, and axillary flowering percentage were

calculated. The experiment was terminated 21 weeks after initiation.

Coreopsis transfer experiment (Expt. 3)

Only approximately half of the C. xgrandiflora flowered in Expt. 1. We
attributed the low flowering percentage to the relatively low DLI provided to plants.
Therefore, a third experiment was performed to determine if plants could be induced
under naturally high light then transferred to the N or FR, filters until flowering. Plants
were grown under unfiltered, natural photoperiods supplemented from 0600 to 2200 HR
with HPS lamps (as described above) but with a PPF = 100 umol-m'Z-s". After 0, 2, 3, and
4 weeks under high light, 10 plants were transferred to the N and FR, filters until
flowering. Experimental conditions were as described above unless otherwise stated.
Seeding, shipping, and forcing dates and average temperature and DLI from forcing to
flowering are provided in Table 1. The dates of VB and flowering were recorded. At
flowering, visible flower buds and nodes on the main stem were counted and total plant
height was measured. Flowering percentage, days to flower, and node-count increase to

flowering were calculated.

63

Results
Experiment 1

Stem extension. The R, environment increased plant height from forcing to VB by
65 % in C. carpatica and 23 % in P. sativum compared with that in the N environment
(Fig. 2). The B, environment promoted internode elongation in all species studied.
Compared with that under the N filter, the B, filter increased stem length from forcing
until VB by 100 %, 72 %, l7 %, and 16 % in C. carpatica, C. xgrandiflora, L.
Xspeciosa, and P. sativum, respectively.

The R, treatment increased stem and inflorescence elongation from VB to
flowering by 26 % or 30 % in C. Xgrandiflora and V. ><wittrockiana, respectively (Fig.
3). The FR, filter suppressed extension grth in only two species, P. sativum (stem
length by 20 %) and V. Xwittrockiana (stem and peduncle length by 14 % and 33 %,
respectively). Changes in R or FR light did not influence stem extension of L. xspeciosa.
From VB to flowering, the B, environment increased stem and inflorescence elongation
in C. ><grandiflora, L. Xspeciosa, and V. xwittrockiana by 31 %, 11 %, and 10 %,
respectively. In addition, the B, filter increased peduncle length of V. xwittrockiana by
27 %.

Flowering. The FR, environment inhibited flowering in V. Xwittrockiana; 88 %
and 65 % of plants under the FR, filter reached VB and flowering, respectively, whereas
all plants flowered in the other three light environments (data not shown). The B,, R,, and
FR, environments had no significant effect on flowering percentage of the other species
studied. Essentially all C. carpatica, L. Xspeciosa, and P. sativum reached VB and

flowered under all light quality treatments, but irrespective of filter treatment (including

64

that under the N filter), only 50 % and 42 % of C. Xgrandiflora reached VB and anthesis,
respectively (data not shown).

Light deficient in R delayed time to VB by 4 or 1 d in C. carpatica and P. sativum
and hastened it by 4 d in V. xwittrockiana (Figs. 4A and 5A). However, the delay in time
to VB under the R, filter was not accompanied by the formation of more nodes before VB
appearance in any species (Fig. 4B). The R, environment had no effect on time from VB
to flowering for any species (data not shown). However, the R, treatment reduced flower
number by 54 % or 33 % in C. carpatica and L. Xspeciosa, respectively, and reduced dry
matter accumulation by 40 % in P. sativum (Figs. 4C and 4D).

The FR, environment delayed appearance of VB in C. carpatica by 2 d and C.
Xgrandzflora by 14 d (Fig. 4A) and delayed flowering of V. Xwittrockiana by 21 d (Fig.
5B). Viola Xwittrockiana developed an average of 9.1 or 12.4 nodes before flowering
under the N or FR, treatments, respectively (significantly different at P < 0.05; data not
shown). In addition, C. carpatica and C. ><grandiflora developed more nodes before
flowering under the FR, environment than those under the N filter (Fig. 4B). The FR,
environment increased flower number of L. Xspeciosa by 44 % but not for any other
species (Fig. 4C).

A deficiency in B light hastened time to VB in P. sativum (by 1 d) and V.
><wittrockiana (by 4 d) but did not influence node number (Figs. 4A, 4B, and 5C). The
B, environment had no effect on VB timing of the other three species. In addition, the B,
environment had no effect on time from VB to flowering for any species (data not

shown). However, flower number of C. carpatica and L. Xspeciosa and dry weight of P.

65

sativum were significantly greater under the B, filter (by 15 %, 19 %, and 37 %,
respectively, Figs. 4C and 4D).

Extension growth of a variety of species shows an inverse linear relationship with
estimated Pfi/P (Smith, 1982, 1994). Using data in our studies, we compared how
extension growth was related to R : FR ratios and the estimated P,-,/P under the filter
treatments (Table 2; Fig. 6). Stem or internode length under the N, R,, and B,
environments was compared with that under the FR, filter, in which stems were shortest.
Using results under the R,, N, or FR, environments, stem extension was not linearly
related to narrow band (10 nm) R : FR ratios (Fig. 6A). However, stem extension was
linearly related to a wide band (100 nm) R : FR ratio (Fig. 6B) or estimated PfilP (Fig.
6C). The promotion of stem extension in the B, environment did not fit any of the

relationships.

Experiment 2

In the reciprocal transfer experiment, all V. Xwittrockiana flowered under the N
filter, in an average of 83 (1. Under the F R, environment only 81 % flowered, in an
average of 108 (1 (Table 3). Plants initiated flowers at approximately the seventh node
under both filter treatments, but under the N filter, the first flower developed to anthesis
at least two nodes sooner than under the FR, filter. Thus, the number of initiated but
undeveloped buds below the first open flower was significantly higher under the FR,

filter.

66

Experiment 3

During the first two weeks of the experiment, the average DLI was 11.8 mol-m‘z-d‘
‘ under the unfiltered, high light environment and 4.3 mol-m‘Z-d" under the N filter.
Coreopsis xgrandiflora that received 22 weeks of LD with a high DLI developed about
two fewer nodes and flowered 11 or 12 d earlier than plants held under the same
photoperiod but under the N or PR, filters (Table 4). The FR, filter reduced plant height
by 21 % regardless of the duration of high light exposure but did not influence any other

measured characteristic.

Discussion
Here, we show the variability in LD species’ responses to environments deficient in R,
FR, and B light. Relative to the five LDP studied, the sensitivity of extension growth to
R : FR was small in L. xspeciosa, moderate in P. sativum and V. xwittrockiana, and
large in C. carpatica and C. Xgrandiflora. Independently, stem extension was promoted
in all species under the B, environment, but the lack of B light had little or no effect on
flowering percentage and time to flower. Although the R : FR had no effect on flowering
time of L. xspeciosa and P. sativum, a high R : FR delayed flower initiation in C.
carpatica and C. Xgrandiflora and inhibited flower development in V. Xwittrockiana.
Although the average DLI was relatively low (Table 1), time to flower of the
same cultivars of C. carpatica, C. Xgrandiflora, L. Xspeciosa, and V. xwittrockiana was
similar to that in published research using similar temperatures and identical photoperiods
(Runkle and Heins, 1998; Runkle et al., 1998; Runkle et al., 1999; Whitman et al., 1997).

In addition, a high daily light integral ( > 6 mol-m'Z-d") was required for complete

67

flowering in C. xgrandiflora, since flowering was promoted when plants were provided
with 22 weeks of high light.

Stem extension in C. carpatica, C. ><grandiflora, P. sativum, and V.
Xwittrockiana showed an inverse linear relationship with a wide band R : FR or
estimated PfilP, similar with that reported in other species (Ritchie, 1997; Smith, 1982,
1994). Narrow band R : FR ratios were not linearly related to stem extension of these
species. However, the promotion of stem extension in the B deficient environment did
not fit any of the relationships, including the estimated P,,/P that accounts for
phytochrome absorption in the B region. Thus, our data indicate that relating stern
extension to R : FR ratios or estimated Pfi/P is invalid when B light levels differ
significantly from that in the natural environment.

Blue light inhibits extension growth in a variety of plants [e.g., pea, pepper
(Capsicum annuum L.), and mustard (Sinapis alba)] and tissues (hypocotyls, epicotyls,
and stems; Brown et al., 1995; Casal and Smith, 1989; Laskowski and Briggs, 1989;
Liscum et al., 1992). Our studies suggest that B light plays a role equal to or greater than
that of R : FR in mediating stem extension in LDP; internode extension in all five LDP
species studied was promoted under the B, filter as well as or better than that under the R,
filter. The stem extension responses appear to be independent of phytochrome, since the
R : FR and Pfi/P under the B, treatment were nearly identical to that under the N
treatment (Table 2). In Arabidopsis and Hyoscyamus, the absorption of B light
accelerates flowering (Bagnall et al., 1996; Guo et al., 1998; Lin et al., 1996; Mozley and
Thomas, 1995; Stolwijk and Zeevaart, 1955), but in the five LDP presented here, B light

had little or no effect on flowering percentage or timing. Under the B, filter, transmission

68

of B light was < 10 % of that under the N filter. However, it is possible that enough B
light penetrated the B, filter to saturate any B-mediated effect on flowering without
saturating a B-mediated effect on extension growth.

In LDP, various experiments suggest that flowering is promoted most when R
light (or light creating a high P,/P) is delivered at least during the early part of the
photoperiod and FR light (or light creating a lower Pfi/P) toward the end (Evans, 1976;
Lane et al., 1965; Thomas and Vince-Prue, 1997). In our studies, R, photoperiods had
little or no effect on flowering percentage and timing and node count increase, which
suggests that a high Pfi/P during the first half of the photoperiod may not necessarily be
promotive to flowering in some light-dominant LDP. However, flowering was inhibited
in three of the five species studied when the Pfi/P was high, and was specific to initiation
in C. carpatica and C. Xgrandiflora and development in V. Xwittrockiana. Flower
initiation was delayed in an FR, environment in the LDP snapdragon (Antirrhinum majus
L.; van Haeringen et al., 1998). The requirement for FR light for rapid flower
development, but not initiation, was reported in the Hyoscyamus (Downs and Thomas,
1982). These findings support the hypothesis proposed by Thomas and Vince-Prue
(1997) that an FR response, especially toward the end of the photoperiod, is specific to
postinductive flower development in some LDP rather than an effect on induction. In
other LDP, an FR response is specific to flower initiation. Interestingly, flower
development of the SDP Chrysanthemum [Dendranthema xgranrliflorum (Rarnat)
Kitamura] was delayed under weakly inductive N photoperiods compared to the same

photoperiod deficient in FR, which suggests that, depending on the timing, FR light can

69

have opposite effects on flower development in SDP and LDP (McMahon, 1999;
Rajapakse and Kelly, 1995).

Within the photosynthetically active wave band (400 to 700 nm), photons are not
equally effective at producing photosynthesis. Red light, especially around 600 nm, is
the most efficient wave band, whereas B light is =80 % as efficient (McCree, 1972; Sager
et al., 1988). A model has been developed to quantify the relative quantum efficiency
(RQE), which is useful in predicting the photosynthetic capacity of a given light quality
(Sager et al., 1988). The RQE was lower (by 13 %) under the R, filter and higher (by 5
%) under the B, filter compared with that of the N filter (Table 2). Flower number of C.
carpatica and L. xspeciosa and dry weight of P. sativum were reduced by 33 % to 54 %
under the R, filter and increased by 15 % to 37 % under the B, filter. Similarly, dry
weight of chrysanthemurn was reduced under R, films compared to N filters transmitting
a similar PPF (Oyaert et al., 1999). Therefore, differences in flower number and dry
weight among lighting treatments could be at least partially attributed to the different
RQE and suggest that relatively small changes in RQE can have large effects on growth.

Plants absorb most visible light (400 to 700 nm) but reflect or transmit most FR
light, and thus a low R : FR is created under a canopy. In response to such an
environment, extension grth and flowering are promoted in shade-avoiding species
(Smith, 1994). The ecological strategy of the shade-avoidance syndrome is to promote
and direct extension growth in an attempt to better harvest available sunlight. In contrast,
shade-tolerant species respond to a low R : FR without a significant change in extension
growth. Not surprisingly, in our studies the species most adaptive to shade in the natural

environment (L. Xspeciosa) was the least responsive to R : FR, while the shade-avoiding

7O

species C. carpatica and C. xgrandiflora were sensitive to R : FR (Armitage, 1989).
Light under a canopy is also deficient in B, and since a B, environment promoted
extension grth in all species we studied, an alternative shade-avoiding ecological
strategy could be mediated by B light. These potentially redundant shade-avoiding
strategies are consistent with studies demonstrating independent, interactive, and
redundant actions of the B-absorbing and R- and FR- absorbing photoreceptors
(cryptochrome and phytochrome, respectively) in Arabidopsis (Casal and Mazzella,
1998; Poppe et al., 1998).

The species in which flowering and extension growth was influenced the most by
R : FR was V. Xwittrockiana. Plants under the R, filter (e.g., low R : FR) were strongly
apically dominant; stem extension and flowering were promoted, and in nearly all
instances, first flowering occurred on the primary stem. In contrast, branching was
promoted and flowering was inhibited under the FR, filter (e.g., high R : FR). Of those
that flowered under the FR, filter, a significant proportion (25 % in Expt. 2) of plants first
flowered on an axillary stem (Table 3).

In summary, our data illustrate the variability in how light quality influences
flowering in LDP and demonstrate that although an FR, environment can suppress stem
extension, it can also delay flowering in some species. A deficiency in FR light can
specifically delay flower initiation in some species (e. g., C. carpatica and C.
Xgrandiflora) and flower development in others (e. g., V. xwittrockiana). In the former
(but not latter) species, plants can be induced to flower under N photoperiods then
transferred to an FR, environment to facilitate rapid flowering with reduced stem

extension. The filters used reduced, but did not completely eliminate, the transmission of

71

certain wavebands of light. However, our results suggest that extension growth can be
attributed to a wide (but not narrow) band R : FR or P,/P, except when the B component
is significantly altered. Therefore, blanket statements are inappropriate when discussing
flowering and stem extension responses of LDP to light quality. Currently, we are
performing additional studies to determine if various lighting strategies can suppress stem
extension in sensitive LDP without an inhibition in flowering. Further research is also
merited to determine how B light interacts with the R : FR to regulate extension growth

in plants outside the Brassicaceae.

Acknowledgments

We gratefully acknowledge the support of the Michigan Agricultural Experiment Station
and funding by project GREEEN and greenhouse growers supportive of Michigan State
University floricultural research. We also thank Dan Tschirhart for his greenhouse

assistance and Simon Pearson and British Visqueen for the experimental FR filter.

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80

Table 3. Flowering characteristics of Viola Xwittrockiana under the neutral (N) or far-red
deficient (FRd) filter. Plants were transferred from the N to the F Rd filter, or vice versa,
following 5, 10, 15, 20, 25, 30, 35, or 40 (1. Data were pooled by filter type because
transfer time and filter >< transfer time interaction had no significant effect on any

measured characteristics.

 

Final environment

 

 

Characteristic N FRd Significance
Flowering (%) 100 31 "an
Axillary flowering (%) 4 25 am:
Days to flower 83 108 ***
Node of first visible bud 7.2 6.8 NS
Node of open flower 11.7 14.1 ***
Undeveloped flower buds2 4.6 7.4 ***

 

’Below the first open flower.

”3' ... Nonsignificant or significant at Ps 0.001, respectively.

81

Table 4. Flowering responses of Coreopsis Xgrandz'flora transferred from an unfiltered
16-h photoperiod with supplemental high pressure sodium lamps [delivering a high daily

light integral (DLI)] to a neutral (N) or far-red deficient (FRd) filter.

 

Flowering Days to Increase in Flower Height

 

 

 

(%) flower node no. no. (cm)

Weeks at high DLI

0 90 65.5 8.4 9.9 25.4

2 95 53.5 6.3 8.2 26.5

3 95 54.4 5.9 11.9 26.4

4 100 53.2 6.8 14.7 26.4
Final filter environment

N 98 56.9 6.8 10.6 29.2

FRd 93 56.4 6.8 11.7 23.1
Significance

Weeks high DLI (WHDLI) *** *** *** NS

Final filter (FF) NS NS Ns ***
__ WHDLI >< FF NS NS NS NS
NS, ...

Nonsignificant or significant at Ps 0.001, respectively.

82

 

Light transmission
relatIve to sunlIght

 

 

Light transmission
relative to neutral filter

 

 

 

400 500 i 600 700 800
Wavelength (nm)
Fig. 1. Spectral transmissions of photoselective filters relative to sunlight (A) or relative
to that under the neutral-density filter treatment with an equal photosynthetic photon flux
(B). R,, red (600 to 700 nm) deficient filter; Bd, blue (400 to 500 nm) deficient filter;

FRd, far—red (700 to 800 nm) deficient filter. See Table 2 for light wave band ratios.

83

 

Neutral
— Rd 3
10 " 1:1 FRd
Ba

      

Plant height at visible bud (cm)
0)
Plant height at visible bud (cm)

.W

 

 

 

 

Campanula Coreopsis Lobelia Pisum
carpatica xgrandiflora xspeciosa sativum

Fig. 2. Plant height at visible bud of Campanula carpatica, Coreopsis Xgrandiflora,
Lobelia Xspeciosa, and Pisum sativum under a neutral filter or a light environment
deficient in red (Rd, 600 to 700 nm), far red (FRd, 700 to 800 nm), or blue (Bd, 400 to 500
nm). A 16—h photoperiod was delivered with a combination of sunlight and high-pressure
sodium lamps positioned above filters. Values with the same letter within species are not

statistically different at P = 0.05.

84

 

40

N

a —Rd .

a a .// [:1 FRd
>/,

’ Bd ‘

35 ..

Stern and inflorescence elongation
from visible bud to flowering (cm)

 

 

 

Coreopsis Lobelia Viola Viola

xgrandiflora xspeciosa xwittrockiana xwittrockiana
stem peduncle

Fig. 3. Stern and inflorescence elongation from visible bud to flowering of Coreopsis

X grandiflora, Lobelia Xspeciosa, and Viola Xwittrockiana under a neutral filter or a light
environment deficient in red (Rd, 600 to 700 nm), far red (FRd, 700 to 800 nm), or blue
(Bd, 400 to 500 nm). A l6-h photoperiod was delivered with a combination of sunlight
and high—pressure sodium lamps positioned above filters. Values with the same letter

within species and measurement are not statistically different at P = 0.05.

85

Fig. 4. Days to visible bud (A), node count increase to first open flower (B), and flower
number (C) or dry weight (D) of Campanula carpatica, C 0re0psis ><grandiflora, Lobelia
Xspeciosa, and Pisum sativum under a neutral filter or a light environment deficient in
red (Rd, 600 to 700 nm), far red (FRd, 700 to 800 nm), or blue (Bd, 400 to 500 nm). A 16-
h photoperiod was delivered with a combination of sunlight and high-pressure sodium
lamps positioned above filters. Values with the same letter within species are not

statistically different at P = 0.05. Legend in A applies to all figures. NS = not significant.

86

BO 290; an.

 

 

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Pisum
sativum

Lobe/Ia
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CoreopSIs
xgrandiflora

Campanula
carpatica

87

Fig. 5. Flowering percentage of Viola Xwittrockiana under a neutral filter or a light
environment deficient in red [Rd, 600 to 700 nm, (A)], far red [FRd, 700 to 800 nm, (B)],
or blue [Bd, 400 to 500 11111, (C)] light. Plants were held under the light treatments until
visible bud (until VB), continually, or after visible bud (after VB). Plants were under the
neutral filter at all other times. A 16-h photoperiod was delivered with a combination of
sunlight and high-pressure sodium lamps positioned above filters. Legend in A applies to

all figures.

88

Flowering percentage

100

 

 

80 T

60 4*

     

 

 

 

0 Neutral filter
20 u D Until VB
0 Continual
a After ve Rd
0 1 + . t —‘r i t +
100 r + ‘ . r
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40 l J I
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15 20 25 30 35 40 45 50 55 60 65
Days to flower

 

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E 0 2 4 6 8 0.0 0.4 0.8 1.2 1.6 0.4 0.5 0.6 0.7 0.8
Narrow band R : FR Wide band R : FR Estimated Pfr/P

Fig. 6. Stem extension of Campanula carpatica, Coreopsis Xgrandiflora, Pisum sativum,
and Viola xwittrockiana relative to that under the far-red (FR) deficient filter. Stem
length was related to red (R) : FR ratios and the estimated phytochrome photoequilibria
(Pf/P) under the filter treatments. The R : FR ratios were determined using narrow [10
nm (A)] and wide [100 mm (B)] band widths. See Fig. 1 and Table 2 for spectral data.
Open symbols represent plants exposed to a neutral filter or light deficient in R or FR.
Closed symbols represent plants exposed to light deficient in blue (400 to 500 nm).

Legend in A applies to all figures.

90

CHAPTER IV

STEM EXTENSION AND SUBSEQUENT FLOWERING
OF SEEDLINGS GROWN UNDER A FILM
CREATING A F AR-RED DEF ICIENT ENVIRONMENT

Runkle, ES. and RD. Heins. Stem extension and subsequent flowering of seedlings
grown under a film creating a far-red deficient environment.
To be submitted to HortScience.

91

Stem Extension and Subsequent Flowering of Seedlings Grown under a Film Creating a

Far-Red Deficient Environment

Erik S. Runkle and Royal D. Heins'

Department of Horticulture, Michigan State University, East Lansing, MI 48824-1325

Additional index words. Antirrhinum majus, far-red light, height control, Impatiens
walleriana, Petunia thbrida, Solanum lycopersicon, spectral filters, Viola

Xwittrockiana

Abstract

Photoselective plastic filters that reduce the transmission of far-red light (FR, 700 to 800
nm) have been developed to potentially reduce stem extension and thus plant height.
However, an FR-deficient (FRd) environment can also delay flowering, particularly in
long-day plants. Our objective was to determine if seedlings could be grown under an
FRd filter to reduce extension growth without subsequently delaying flowering when
grown under natural long photoperiods. Pansy (Viola ><wittrockiana Gams), petunia
(Petunia thbrida Vilm.-Andr.), impatiens (Impatiens walleriana Hook), snapdragon
(Antirrhinum majus L.), and tomato [Solanum lycopersicon L. (syn.: Lycopersicon
esculentum)] were placed under the F Rd filter or a neutral-density filter (N) that
transmitted a similar photosynthetic daily light integral. Since a dense plant canopy
promotes extension growth, a third treatment consisted in transfen'ing plants from the N

to the FR, filter when leaves of each species began to touch (afier 18 or 24 (1 under the N

92

filter). After 25 to 35 treatment days with a 16-hour photoperiod at 20 °C, seedlings were
measured then grown under natural photoperiods (2 14.5 hours) at 20 °C until flowering.
Compared with that of seedlings continually under the N filter, stem or petiole length
under the FR, filter was significantly reduced in impatiens (by 10 %), pansy (by 18 %),
petunia (by 34 %), snapdragon (by 5 %), and tomato (by 24 %). Extension growth in
plants held continually under the FRd filter was similar to that of plants transferred from
the N to FRd filter when leaves first touched. Flowering of plants from plugs under the
FRd filter was delayed by two to three days in snapdragon, petunia, and pansy. At
flowering, flower number and plant height of all species were similar among treatments.
Therefore, the FRd filter presents an attractive method of controlling seedling height with

minimal impact on flowering when plants are subsequently grown under unfiltered light.

Introduction
Plant height is a commonly manipulated component of plant architecture, especially in
the commercial production of floriculture crops. Promotion of stem extension is useful
for production of cut flowers, but more often plant height is suppressed in a variety of
bedding and potted plants, container omamentals, and vegetables. Effectively controlling
plant height is important, since purchasers are demanding strict plant morphological
specifications, namely, narrow windows of acceptable plant heights.

A common method of reducing extension growth is to apply plant growth-
retarding chemicals (e. g., ancymidol, chlormequat, daminozide, paclobutrazol, and
uniconazole), which inhibit various steps in the gibberellin biosynthetic pathway

(Rademacher, 2000). Although these chemicals can be a convenient method of retarding

93

stem extension, efficacy varies by chemical and species, some chemicals add a
considerable production expense, and use has become increasingly restricted.

A variety of cultural and environmental manipulations have been used to reduce
plant height, including temperature (Erwin et al., 1989; Erwin and Heins, 1995),
fertilization (Melton and Dufault, 1991), watering (Liptay et al., 1997), and mechanical
stimulation (Johjima et al., 1992) regimens. Although these strategies are sometimes
effective, each technique has limitations. For example, warm outdoor temperatures can
prevent implementation of cool-temperature treatments. Therefore, alternative strategies
to control extension grth are needed.

Plants absorb most visible light (400 to 700 nm) but reflect or transmit most far-
red (FR, 700 to 800 nm) light. Therefore, a low red (R, 600 to 700 nm) : FR is created
under a canopy or among closely spaced plants. In response to such an environment,
extension growth in shade-avoiding plants is promoted as plants adapt to better harvest
available sunlight (Smith, 1994). In contrast, shade-tolerant species respond to a low R :
FR without a significant change in extension growth.

To limit stem extension, various materials have been used experimentally to
reduce transmission of FR radiation. The recent development of flexible plastic FR filters
[creating an FR-deficient (FRd) environment] offers an alternative method of controlling
plant height (Oyaert et al., 1999; Rajapakse et al., 1999; van Haeringen et al., 1998).
These FRd filters have reduced extension growth in a variety of species, including
vegetables and ornamental plants (Raj apakse et al., 1999). An FRd environment can
delay flowering in some plants (Runkle and Heins, 2001), but in many reports, this delay

has not been addressed.

94

 

 

In this study, we determined how an F R, filter influenced seedling growth of five
common bedding plant species and then quantified what residual effect, if any, the

seedling environment had on subsequent flowering.

Materials and Methods
Seedling stage

Plant material. Seed of Antirrhinum majus ‘Liberty Scarlet’, Impatiens
walleriana ‘Accent Rose’, Petunia thbrida ‘Carpet Pink’, Solanum lycopersicon
‘Beefmaster’ (syn: Lycopersicon esculentum Mill), and Viola xwittrockiana ‘Crystal
Bowl Yellow’ were sown into 288-cell plug trays (6-mL volume) by a wholesale plug
producer (Rakers Acres, Litchfield, Mich.) on 26 March 1999 and 5 May 1999 for Rep I
and II, respectively. Plants were shipped and received six or eight days later, then were
thinned to one per plug.

Lighting and filter treatments. Seedlings were grown on greenhouse benches
enclosed by a neutral [N; PLS Clear (Ludvig Svensson, Charlotte, N.C.)] or F R,, (van
Haeringen et al., 1998) filter, which transmitted similar photosynthetic photon fluxes
(PPF). Since extension grth is promoted most when a canopy becomes closed, a third
treatment consisted in transferring plants from the N filter to the FR, filter when leaves
began to touch (afier 18 d for tomato and 24 d for the other species). For each
replication, three plug trays of each species were cut in half to yield two blocks for each
of the three filter treatments. Solar spectra transmissions were measured under filters by a
spectroradiometer (LI-1800, LI-COR, Inc., Lincoln, Nebr.; Fig. 1). The R : FR and

estimated phytochrome photoequilibrium (Pf/P; Sager et al., 1988) were 1.07 and 0.715

95

under the N filter and 1.74 and 0.798 under the F Rd filter, respectively. The narrow
waveband R : FR [(655 to 665 nm) : (725 to 735 nm )] was 1.06 and 8.22 under the N and
FR.I filters, respectively.

A 16-h photoperiod was delivered with a combination of sunlight and high-
pressure sodium (HPS) lamps positioned above filters. During Rep I and H, the average
natural photoperiod (lat. 43 °N) during the plug stage was 214 and 15.5 h, respectively.
From 0600 to 2200 HR, HPS lamps provided at plant level a supplemental PPF of =40
utmol-m'Z-s‘1 when the ambient greenhouse PPF was < 200 pmol'm‘z-s‘l and were shut off
when the ambient PPF was > 400 pmol-m'Z-s". The average photosynthetic daily light
integral, measured at canopy level with quantum sensors (LI-COR) connected to a CR10
datalogger (Campbell Scientific, Logan, Utah), was 9.4 and 7.3 mol-m'z-d'l under the N
filter and 9.1 and 7.2 mol~m‘21d‘l under the FRd filter during Rep I and II, respectively.

Plant culture. Plants were fertilized at every irrigation with a nutrient solution of
well water acidified with H280, to a titratable alkalinity of z 130 mg CaCO3-L‘l and water
soluble fertilizer [125N—12P—125K(mg~L") plus
1.0Fe—0.5Mn—0.SZn—O.5Cu—0.lB—O.1Mo (mg'L"; MSU Special, Greencare Fertilizers,
Chicago, Ill.)].

Greenhouse temperature control. All plants were grown in a glass greenhouse at
20 °C. Air temperatures on each bench were monitored with 36-gauge (0.127-mm-
diameter) type B thermocouples connected to CR10 dataloggers (Carnpbell Scientific).
To provide more uniform temperatures, dataloggers were used to control 1500-W electric
heaters under each bench during the night and portable fans during the day to heat or vent

each bench as needed. The dataloggers collected temperature data every 10 s and

96

 

recorded the hourly averages. The average temperatures under the N and F R, filters were
20.0 and 19.6 °C during Rep I and 20.8 and 20.9 °C during Rep 11, respectively.

Data collection and analysis. Seedlings of each species were deemed ready for
transplant after 26, 31, 32, 35, and 35 d for tomato, impatiens, snapdragon, petunia, and
pansy, respectively. Typically, seedlings in the outer rows of a plug tray are shorter than
those toward the inner rows. Therefore, node number and shoot (hypocotyl plus stem) or
longest petiole (for pansy only) length were recorded from each block and treatment from
10 plants in the outer two rows (outside), the next inner two rows (middle), and the
innermost rows (inside). Data were analyzed by using SAS’s (SAS Institute, Cary, NC.)
analysis of variance (AN OVA), general linear models (GLM) procedures, and a mean

separation procedure (pdift) with P = 0.05.

Forcing stage

For each species, block, and repetition, 10 seedlings were removed from the
middle two rows of each half-tray and transplanted into lO-cm round (470-mL vol.) pots
containing a commercial soilless medium composed of composted pine bark, vermiculite,
Canadian sphagnum peat, coarse perlite with a wetting agent, and lime (High Porosity
Mix, Strong-Lite Products, Pine Bluff, Ark). Plants were grown under unfiltered natural
photoperiods (2 14.5 h) in a glass greenhouse at 20 0C.

Data collection. Experiments were replicated in time and were arranged in a
completely randomized design. The date the first flower bud was visible (without
dissection) and the date the first flower reached anthesis (flowering) were recorded for

each plant. At flowering, total plant height (not including the container) was measured,

97

 

 

and visible flower buds (VB) or inflorescences and nodes on the main stem were counted.
Days to VB, days from VB to flower, days to flower, and node-count increase from the
start of forcing were calculated. All other experimental conditions were as described

during the seedling stage.

Results
Seedling stage. In snapdragon, stem length of plants held continually under or transferred
to the FRd filter was similar to or shorter than that of plants under the N filter (Fig. 2). In
impatiens, plants on the outer rows of the plug tray were similar under all treatments, but
plants in the middle and inner rows were z 15 % shorter when grown under the FR, filter
continually or upon transfer. Irrespective of position within the plug tray, pansy, petunia,
and tomato were 18 %, 34 %, or 24 % shorter, respectively, than plants held continually
under the N filter. Similarly, stem length of plants transferred from the N to F Rd filter
was 16 %, 29 %, or 23 % shorter in pansy, petunia, and tomato, respectively, than that of
plants under the N filter. Filter treatment had no significant effect on node number,
except for snapdragon, in which plants transferred fi'om the N to PR, filter had an average
of 0.11 fewer nodes than plants held continually under the N filter (data not shown).
Forcing stage. The average time to flower of impatiens and tomato was 49 and 60
(1, respectively, and was not significantly influenced by seedling environment (data not
shown). Under the N filter, pansy, petunia, and snapdragon flowered in 54, 51, and 62 d,
respectively (data not shown). However, when pansy, petunia, and snapdragon seedlings
were grown continually under the FR,l filter, subsequent flowering was delayed by 2, 2,

and 3 (1, respectively, compared to seedlings grown continually under the N filter.

98

 

Flowering of seedlings transferred from the N to PR, filter was delayed (by one (1) only in
snapdragon and pansy. In petunia and snapdragon, the flowering delay in seedlings
raised in the FR, filter was accompanied by the formation of an average of 0.6 more
nodes before flowering (data not shown). No other significant differences in node
number at flowering existed. In addition, seedling treatment had no effect on time from
VB to flowering or plant height and flower number at first flowering for any species

tested (data not shown).

Discussion
The FRd filter used in our study effectively controlled extension growth in all of the
species studied, with little or no delay in flowering or reduction in flower number when
plants were subsequently grown under unfiltered light. In addition, extension grth was
suppressed similarly when seedlings were exposed to the FR, environment continually or
only from the time leaves first began to touch within the plug tray. The species least
sensitive to FR light was snapdragon, in which plants were 5 12 % shorter than plants
under the N filter, depending on the plug tray position. Petunia and tomato were the most
sensitive species studied: the FR, filter suppressed extension grth by 2 23 % in all plug
tray positions. This result suggests that, of the species studied, snapdragon is the most
shade-tolerant plant, and petunia and tomato are the most shade-avoiding species.
Extension growth reduction in our study is similar to that in other FRd filter
studies. In tomato, other FRd filters reduced extension growth in other tomato cultivars,
including ‘Saturn’ (9 % to 17 % reduction) and ‘Mountain Pride’ (25 °/o reduction; Li et

al., 1999; Murakami et al., 1996, 1997; Rajapakse et al., 1999). In snapdragon, Kumai et

99

 

 

al. (1998) did not observe a reduction in stem extension under an F R, filter; in contrast,
van Haeringen et al. (1998) reported a 30 % reduction in internode length. Kumai et al.
(1998) and Kubota et al. (2000) reported a 2:60 % reduction in petunia stem extension,
and Runkle and Heins (2001) reported a 14 % suppression in pansy peduncle length. To

our knowledge, the effects of an F R, filter on extension grth of impatiens, which is the

best selling bedding plant species in the United States (Behe et al., 2000), have not been
published.

The reduction in extension growth by the FRd filter is comparable to that in
studies with other nonchemical height control strategies. For example, extension growth
of tomato was reduced by cool day-temperature regimens (by 22 % to 33 %), decreased N
fertility (by :40 %), and mechanical stimulation (by 20 % to 33 %; Garner et al., 1997;
Gertsson, 1992; Heuvelink, 1989; Johjima et al., 1992; Melton and Dufault, 1991). Each
of these alternatives has limitations: cool temperature treatments cannot be used when
outdoor temperatures are high, decreased N fertility significantly reduced dry matter
accumulation and leaf area, and mechanical stimulation caused some damage to plants.
The primary disadvantage of the FRd filter is its reduction in PPF transmittance (by =25
%), which could limit use under low light conditions (e. g., during the winter).

A distinction has been made between plants in which flowering is controlled
primarily by dark processes (dark-dominant) or light processes (light-dominant; Thomas
and Vince-Prue 1997). Light-dominant plants show a more or less quantitative
relationship between irradiance of the night break and the magnitude of the flowering
response until a saturation light intensity, duration, or both are reached. In addition,

flowering of light-dominant plants is most rapid when light contains some minimal FR

100

 

 

(Downs and Thomas, 1982; Lane et al., 1965; Runkle and Heins, 2001). Most light-
dominant plants are long-day plants (LDP), but a few exceptions exist.

Flowering is delayed in some LDP, such as Coreopsis grandiflora Hogg ex Sweet
‘Early Sunrise’ and pansy, when grown continually in an FRd environment (Runkle and
Heins, 2001). In petunia, an FRd environment delayed flowering in some cultivars but
not in others H.-H. Kim, personal communication; Kubota et al., 2000). In our studies,
the species in which an FRd environment delayed subsequent flowering were the LDP
(pansy, petunia, and snapdragon), which are likely light-dominant; the day-neutral plants
(impatiens and tomato) showed no flowering delay (Adams et al., 1997; Vince-Prue,
1975). Our study indicates that during the early phases of seedling development, light-
dominant plants appear to be relatively insensitive to light quality with respect to
flowering but are responsive with respect to extension growth. When plants develop the
capacity to flower, light quality influences extension growth and flowering
concomitantly. Therefore, if complete and rapid flowering is desired, light-dominant
LDP can be exposed to an FRd environment during the seedling stage for height control,
then can be transferred to an unfiltered environment for normal flowering.

In summary, the F R, filter presents an effective, easy-to-use, alternative method
of controlling plant height. Unlike cool-temperature regimens, the filter could be used
commercially in temperate and tropical locations throughout the year. However, the PPF
is reduced (by =25%), and as with all height control methods, the magnitude of extension
growth suppression varies by species. Use of an F Rd filter can delay flowering in
sensitive species, particularly light dominant LDP, but the delay can be minimized if

seedlings grown in an FRd environment are subsequently grown under natural light.

101

 

 

Although a delay in flowering is generally considered undesirable, the promotion of
vegetative growth under inductive conditions could be useful in some situations, such as

during propagation or seedling establishment.

Acknowledgments

We gratefully acknowledge the support of the Michigan Agricultural Experiment Station
and funding by project GREEEN and the Western Michigan Bedding Plant Association.
We also thank Dan Tschirhart for his greenhouse assistance and British Visqueen and

Simon Pearson for the experimental FR filter.

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and light integral on the time to flowering of pansy cv. Universal Violet (Viola

Xwittrockiana Gams.) Ann. Bot. 80:107—112.

Behe, B., J. Hardy, J. Heilig, and A. Scordalakes. 2000. 1999 Season sales summary.

Ohio Florists’ Assoc. Bull. March21-12.

Downs, RJ. and J .F. Thomas. 1982. Phytochrome regulation of flowering in the long-day

plant, Hyoscyamus niger. Plant Physiol. 70:898—900.

Erwin, J .E., R.D. Heins, R. Berghage, B.J. Kovanda, W.H. Carlson, and J. Biembaum.

1989. Cool mornings can control plant height. GrowerTalks 52:73—74.

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Erwin, J .E. and RD. Heins. 1995. Therrnomorphogenic responses in stem and leaf

development. HortScience 30:940—949.

Garner, L., F.A. Langton, and T. Bjorkman. 1997. Commercial adaptations of mechanical

stimulation for the control of transplant growth. Acta Hort. 435:219—230.

Gertsson, U.E. 1992. Influence of temperature on shoot elongation in young tomato

plants. Acta Hort. 327:71—76.

Heuvelink, E. 1989. Influence of day and night temperatures on the growth of young

tomato plants. Scientia Hort. 38:11—22.

Johjima, T., J .G. Latimer, and H. Wakita. 1992. Brushing influences transplant growth
and subsequent yield of four cultivars of tomato and their hybrid lines. J. Amer. Soc.

Hort. Sci. 117:384—388.

Kubota, S., T. Yamato, T. Hisamatsu, S. Esaki, R. Oi, M. Rob, and M. Koshioka. 2000.
Effects of red- and far-red—rich spectral treatments and diurnal temperature alternation on

the growth and development of Petunia. J. Japan Soc. Hort. Sci. 69:403-409.

Kumai, N., S. Kubota, R. Oi, S. Esaki, and M. Koshioka. 1998. Effect of combined
treatments of diurnal fluctuations between day and night temperatures and red/far-red

intercepting films on the grth of some flowers. Suppl. J. Jpn. Soc. Hort. Sci. 67:275.

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Lane, H.C., H.M. Cathey, and LT. Evans. 1965. The dependence of flowering in several
long-day plants on the spectral composition of light extending the photoperiod. Amer. J.

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Li, 8., NC. Rajapakse, and R. Oi. 1999. Production of compact cucumber, tomato, and
bell pepper transplants by use of photoselective plastic films. HortScience 34:533.

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Liptay, A., P. Sikkema, and W. Fonteno. 1997. Transplant production and performance:

Transplant growth control through water stress, p. 51—53. In: C. Vavrina (ed.). Transplant

Proceedings, The Fifth National Symposium on Stand Establishment. Ohio State Univ.,

Columbus.

Melton, RR. and R.J. Dufault. 1991. Nitrogen, phosphorus, and potassium fertility

regimes affect tomato transplant growth. HortScience 26: 141—142.

Murakami, K., H. Cui, M. Kiyota, I. Aiga, and T. Yarnane. 1997. Control of plant grth
by covering materials for greenhouses which alter the spectral distribution of transmitted

light. Acta Hort. 435:123—130.

Murakami, K., H. Cui, M. Kiyota, Y. Takemura, R. Oi, and I. Aiga. 1996. Covering
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Oyaert, E., E. Volckaert, and RC. Debergh. 1999. Growth of Chrysanthemum under
coloured plastic films with different light qualities and quantities. Scientia Hort.

79:195—205.

Rademacher, W. 2000. Growth retardants: effects on gibberellin biosynthesis and other

metabolic pathways. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51:501-531.

Rajapakse, N.C., R.E. Young, M.J. McMahon, and R. Oi. 1999. Plant height control by

photoselective filters: Current status and fiiture prospects. HortTechnology 9:618—624.

 

Runkle, ES. and RD. Heins. 2001. Specific fimctions of red, far-red, and blue light in

flowering and stem extension of long-day plants. J. Amer. Soc. Hort. Sci. (In press.)

Sager, J .C., W.O. Smith, J .L. Edwards, and KL. Cyr. 1988. Use of spectral data to

determine photosynthetic efficiency and phytochrome photoequilibria. Trans. ASAE

31:1882—1889.

Smith, H. 1994. Sensing the light environment: The functions of the phytochrome family,

 

p. 377—416. In: RE. Kendrick and G.H.M. Kronenberg (eds). Photomorphogenesis in

plants. 2nd ed. Kluwer Academic Publishers, Netherlands.

Thomas, B. and D. Vince-Prue. 1997. Photoperiodism in plants. 2nd ed. Academic Press,

London.

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van Haeringen, C.J., J .S. West, F .J . Davis, A. Gilbert, P. Hadley, S. Pearson, A.E.
Wheldon, and R.G.C. Henbest. 1998. The development of solid spectral filters for the

regulation of plant growth. Photochem. Photobiol. 67:407—413.

Vince-Prue, D. 1975. Photoperiodism in plants. McGraw, London.

106

 

 

 

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. l

1:
Q ‘- 1.25,. ... .__ __ -__ ...__ _._ ___ z.__ __ _ __ .__.__....
8 9 ~ .
"’ E 100 -- -__--_. -.- _._ ._..- -.._ a- .A # -.. .--
52 _ 4
gfi 0.75b~- —————----«—- ~—- --—.~ +11 — __ _, —- a
8 .2 t ‘
=2- ‘5 0.50»--—— —»—--—r~—-———~—~-———_—.~— —-—~——- a
“up . .
E 025 -.-_ ..-___. .‘___ 2--.»-.. 2_ an...__-.-___.

0.00 l I l I l I I .

400 500 600 700 800

Wavelength (nm)

Fig. 1. Spectral transmission of sunlight under the far-red (700 to 800 nm) deficient filter
relative to a filter that reduced the transmission of all wavelengths equally. The

photosynthetic daily light integrals under the two filters were similar (see text).

107

 

‘53 .

 

Fig. 2. Percentage reduction in stem length (or longest petiole length for pansy) of
seedlings transferred from the neutral (N) to far-red deficient (FRd) filter when leaves of
each species began to touch within the plug tray ( V7 ), or held continually under the FR,l
filter ( I ), relative to that continually under the N filter (O). Seedlings were under filter
treatments for 26, 31, 32, 35, and 35 d for tomato, impatiens, snapdragon, petunia, and
pansy, respectively. Measurements were taken of seedlings from the outer two rows
(outside), from the next inner two rows (middle), and from the innermost rows (inside)
from each plug tray. Bars represent means with n = 40. Asterisks indicate that height

was significantly (P = 0.05) less than of plants that were continually under the N filter.

108

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6 4 3 1 6 4 3
i
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Middle Inside

Plant position

Outside

109

 

CHAPTER V i

PHOTOCONTROL OF FLOWERING AND EXTENSION GROWTH OF THE
THE LONG-DAY PLANT PANSY

Runkle, ES. and RD. Heins. Photocontrol of flowering
and extension growth of the long-day plant pansy.
To be submitted to J. Amer. Soc. Hort. Sci.

 

110

Photocontrol of Flowering and Extension Growth of the Long-Day Plant Pansy

Erik S. Runkle and Royal D. Heins‘

Department of Horticulture, Michigan State University, East Lansing, MI 48824-1325

Additional index words. Far-red filter, night interruption, photoperiod, phytochrome,

spectral filters, Viola Xwittrockiana

Abstract

Plastics that selectively reduce the transmission of far-red light (FR, 700 to 800 nm) have
been recently developed to reduce extension growth of floricultural crops. However, an
FR deficient (F Rd) environment delays flowering in some long-day plants (LDP),
including ‘Crystal Bowl Yellow’ pansy (Viola Xwittrockiana Gams). Our primary
objective was to determine if some additional amount of FR light could be added to an
otherwise FRd environment to facilitate flowering with minimal extension growth. In one
experiment, plants were grown under a 16-h FRd base photoperiod and F R-rich light was
added during portions of the day or night. For comparison, plants were also grown with a
9-h photoperiod [short day (SD) control] or under a neutral (N) filter with a 16-h
photoperiod (LD control). Apical flowering percentage was 52 or 98 under the SD or LD
controls, respectively, and was reduced to 28 in 16-h FRd photoperiods without
supplemental FR-rich light. Flowering was promoted most (i.g., flowering percentage
increased and time to flower decreased) when F R-rich light was added during the entire

16-h photoperiod, during the last 4 h of the photoperiod, or during the first or second 4 h

111

 

 

of the otherwise dark period. In a separate experiment, pansy was grown under an N or
FRd 9-h base photoperiod with 0, 0.5, l, 2, or 4 h of night interruption (NI) lighting that
delivered a red (R, 600 to 700 nm) to FR ratio of 0.56 (low), 1.28 (moderate), or 7.29

(high). Compared with that under the N filter, the FRd filter reduced flowering

percentage (by 20), delayed time to flowering (by 4 d) and reduced stem length (by 28 %).

Under the N filter, the minimum NI duration that increased flowering percentage was 2 h
with a low or moderate R : FR and 4 h with a high R : FR. Under the FRd filter, 2, 4, or >
4 h of NI lighting with a moderate, low, or high R : FR, respectively, was required to
increase flowering percentage. However, conditions that promoted flowering also

promoted extension growth. Therefore, it appears that in LDP such as pansy, light
duration and quality concomitantly promote extension grth and flowering, and cannot

readily be separated with lighting strategies.

Introduction

Buyers of floricultural crops impose strict morphological specifications, especially plant
height, on crops they purchase. Chemicals that inhibit various steps in the gibberellin
biosynthetic pathway are often used to limit extension growth of herbaceous plants.
However, their use can be expensive, is increasingly restricted, and is perceived by some
as environmentally “unfiiendly”. Recently, flexible plastic filters that absorb far-red light
(FR, 700 to 800 nm) have been developed as an alternative method of controlling plant
height (Rajapakse et al., 1999; van Haeringen et al., 1998). Although an FR-deficient
(FRd) environment effectively retards stem extension in many herbaceous species, it can

delay flower initiation or development in some long-day plants (LDP; Runkle and Heins,

112

.‘l'i- V'. l ‘.}:‘.D -

‘_‘ vl. " "

 

 

2001). Here, we determined if a minimal amount of FR light could be provided to plants
in an FRd environment to promote rapid flowering without promoting stem extension.

Plants have been classified into flowering response groups based on how
photoperiod influences flowering (V ince-Prue 1975). Flowering is promoted when LDP
are exposed to photoperiods longer than some genotype-specific “critical” photoperiod.
Under short days (SD), an interruption of the dark period with light [known as night
interruption (NI) lighting] promotes flowering in LDP and suppresses it in SD plants
(SDP). To promote rapid reproductive development, most LDP require a long (e.g., 2 2
h) duration of night break lighting, which is usually most effective at or near the middle
of a long dark duration (e.g., 15 hours; Lane et al., 1965; Runkle et al., 1998; Vince-Prue,
1 975 ).

While plants are often classified by their photoperiodic flowering response (e. g.,
SDP or LDP), the photo-regulation of flowering can perhaps be more accurately
described by whether flowering is controlled primarily by light or dark processes. Plants
in which flowering is controlled primarily by light processes, which include most LDP,
are known as light-dominant plants (Thomas and Vince-Prue, 1997). Light-dominant
plants show a more or less quantitative relationship between the irradiance of the night
break and the magnitude of the flowering response, until a saturation light intensity,
duration, or both, are reached. In addition, flowering in light-dominant LDP is ofien most
rapid when photoperiods contain some minimal amount of far-red light (FR, 700 to 800
nm; Downs and Thomas, 1982; Lane et al., 1965; Runkle and Heins, 2001). While FR

light promotes flowering in light-dominant plants, it also promotes internode elongation.

113

 

.l‘ “-2

 

Therefore, in light-dominant LDP, a relatively low R : FR simultaneously promotes
flowering and stem extension while a high ratio is inhibitory to both responses.

Red (R, 600 to 700 nm) and FR light are absorbed by the phytochrome family of
photoreceptors, which in many plants regulate growth and development. For any one
phytochrome, in the presence of light there exists a photoequilibrium of two
interconvertible forms: the R and F R absorbing forms, which are known as Pr and PR,
respectively. Depending on light quality, a phytochrome photoequilibrium [known as
Pf/(Pr + Pfi), or Pfi/P] is established, where a high R : FR creates a high Pfi/P, and vice
versa. Models have been developed to estimate the Pfi/P based on the distribution of
incident spectral radiation (Sager et al., 1988). Although these models are based on
cross—section phytochrome A data from oats grown in darkness, these estimates and R :
FR ratios are useful in associating phytochrome—mediated responses with light quality
(Smith, 1994).

We performed lighting and filter experiments with pansy, which is one of the five
best selling bedding plants in the United States (Behe et al., 2000). Pansy ‘Crystal Bowl
Yellow’ was chosen for study because of its sensitivity to light quality with respect to
extension growth and flowering (Runkle and Heins, 2001). Using pansy as a model plant,
the objectives of our experiments were: 1) to quantify the photoperiodic flowering
response, 2) determine if light rich in FR could be added during the day or night in a FRd
light environment to facilitate rapid flowering with minimal extension growth, and 3) to
determine the minimum amount and duration of FR light delivered as an NI for rapid

flowering under a neutral or FRd filter.

114

 

 

Materials and Methods

Plant material and culture. Seed of pansy (V. ><wittrockiana ‘Crystal Bowl Yellow’)
were sown into 288-cell plug trays (6-mL volume) by a wholesale plug producer (Rakers
Acres, Litchfield, Mich.), germinated for z4 din darkness, then grown under
photoperiods 2 14 h at 20 to 24 °C. Plugs were thinned to one plant per cell. Propagation
and forcing dates and node counts at the beginning of experimental treatments are
provided in Table 1. Plants were transplanted into lO-cm (470-mL) round pots in Expts.
l and 2 and into ll-cm (600-mL) round pots in Expt. 3. Media, irrigation, and nutrition
were previously described [Runkle et al., 1998 (Expt. 1), Runkle and Heins, 2001 (Expts.
2 and 3)].

General greenhouse conditions. All plants were grown in glass greenhouses at 20
°C. Air temperatures were controlled and monitored as previously reported (Runkle and
Heins, 2001) and are provided in Table 1. Nine-hour base photoperiods were created by
opening and closing opaque black cloth at 0800 and 1700 HR, respectively. Sixteen-hour
photoperiods were provided by a combination of sunlight and high-pressure sodium
(HPS) lamps positioned above filters. From 0600 to 2200 HR, HPS lamps provided at
plant level a supplemental photosynthetic photon flux (PPF) of 40 to 50 pmol-m‘z-s"
when the ambient greenhouse PPF was < 200 umol-m'z-s'l and were shut off when the
ambient PPF was > 400 pmol'm‘z-s". The average photosynthetic daily light integral
(DLI) was measured at canopy level with LI-COR quantum sensors (model LI-189; LI-
COR, Lincoln, Nebr.) connected to a CR10 datalogger (Campbell Scientific Logan, Utah;

Table 1).

115

---1

 

 

Spectral filters. Spectral filters were used in Expts. 2 and 3 to provide two light
quality environments [neutral (N) or FRd] with similar daily light integrals. Filters were
an N density metalized woven shading fabric [PLS Clear (Ludvig Svensson, Charlotte,
N.C.)] or a plastic that selectively reduced the transmission of FR light (van Haeringen et
al., 1998). Both filters reduced photosynthetic active radiation (PAR) by z25 %. Solar
spectra transmissions through the filters were as previously reported (Runkle and Heins,
2001)

Photoperiod experiment (Expt. 1). To determine the photoperiodic flowering
response of ‘Crystal Bowl Yellow’, eight or ten plants were apportioned to each of seven
photoperiod treatments: 10, 12, l3, 14, 16, or 24 h of continual light or 9 h with a 4-h
(2200 to 0200 HR) NI. Continual photoperiods consisted in 9-h base photoperiods
completed by day-extension lighting; lamps were turned on at 1700 HR and turned off
after each photoperiod was completed. Day-extension and NI lighting were provided by
incandescent (INC) lamps at 1 to 3 pmol-m'z's'l at canopy level. The experiment was
performed twice.

Timing of F R light delivery (Expt. 2). An experiment was performed to determine
if light rich in FR could be added to an otherwise FRd LD to facilitate rapid flowering
with minimal internode extension. Plants were exposed to one of nine filter and lighting
treatments: a 9-h photoperiod, a 16-h photoperiod under the N filter, a 16-h FRd
photoperiod, or a 16-h F Rd photoperiod with lighting from INC lamps (delivering 22.3
and 4.0 p.mol-m"‘-s‘l of R and FR light, respectively) positioned below filters from 0600 to

2200 HR, 0600 to 1000 HR, 1200 to 1600 HR, 1800 to 2200 HR, 2200 to 0200 HR, or 0200

116

 

 

to 0600 HR. The experiment was performed thrice with 20 plants per treatment and
replication.

Night interruption lighting experiment (Expt. 3). An alternative lighting strategy
was performed with ‘Crystal Bowl Yellow’ to determine if NI lighting could be used in
an FRd environment to promote rapid flowering with minimal stem extension. Twenty-
six wooden chambers (55 cm X 75 cm X 64 cm) were constructed with an open top and
southward-facing side. Half of the chambers were covered with the N filter and half with
the F Rd filter. To minimize any temperature increase, chambers were continually
ventilated with exhaust fans (model 4C548; Dayton Electric, Chicago) that provided z5.8

air exchanges-min. The outlet air temperature of each chamber was recorded (Table l).

 

Inside each chamber, one of three light sources provided NI lighting that delivered
a R : FR ratio of 0.56 (low), 1.28 (moderate), or 7.29 (high). The low, high, or moderate
R : FR were provided by an INC lamp, 3 soft-white fluorescent (SWF) lamp, or an INC
and a SWF lamp, respectively. Electrical timers were used to turn lamps on midway
through 15-h dark periods (at 0030 HR) for 0, 0.5, l, 2, or 4 h. All lamps were surrounded
with a filter (Lee filter 101, Andover, UK) to reduce the transmission of blue (400 to 500
nm) light. In addition, an N filter (OLSSO; Ludvig Svensson) surrounded the combined 1

lamps to provide a more similar PPF among light quality treatments. The spectral

fi‘- .

qualities of lighting treatments are provided in Table 2.

 

Data collection and analysis. Experiments were replicated in time and treatments
were arranged in a completely randomized design. Plants were considered nonflowering
if pansy did not reach anthesis within 56 d, 95 d, or 65 d of forcing in Expts. l, 2 and 3,

respectively. The date the first flower bud was visible (without dissection) and the date

117

the first flower reached anthesis on the apical stem [(apical) flowering] or a lateral stem
(lateral flowering) were recorded for each plant. At flowering, visible flower buds above
the first open flower and nodes on the main stem below the first open flower were
counted. In Expts. 2 and 3, total plant height (from soil level) was measured. Node count
increase to the first open flower and days to VB, days from VB to flower, and days to
flower from the start of forcing were calculated. Intemode length of flowering plants was
calculated by dividing stem length by the increase in node count, and for nonflowering
plants, by determining the average internode length of the first 10 nodes from the start of
forcing. Data were analyzed by using analysis of variance (ANOVA), general linear
models (GLM) procedures, and a mean separation procedure for unequal observation
numbers (pdifl) with P = 0.05 (SAS Institute, Cary, NC.) Regression analysis was

performed by Sigma Plot (SPSS, Inc., Chicago)

Results
Expt. 1. Flowering percentage of ‘Crystal Bowl Yellow’ increased from 50 to 100 as the
photoperiod increased from 10 to 16 h (Fig. 1). Time to flower decreased as the
photoperiod increased, and was most rapid under continual (24 h) light or a 4-h NI.
Flowering plants developed 2 10 nodes before flowering under photoperiods s 13 h, and
s 8.4 nodes under longer photoperiods or NI (data not shown).

Expt. 2. Apical flowering percentage was 52 or 98 under natural 9- or 16-h
photoperiods, but was reduced to 28 when 16-h photoperiods were deficient in FR (Figs.
2A and B). Half of the plants flowered on an apical stem when light was added to the 16-

h FRd photoperiod in the morning (0600 to 1000 HR) or mid-day (1200 to 1600 HR).

118

 

 

Apical flowering percentage was further increased (2 72) when light was added before or
after the end of the 16-h base photoperiod, but was not as high as that under natural 16-h
photoperiods. Lateral flowering was generally greatest under conditions that inhibited
apical flowering.

Flowering was most rapid under natural l6-h photoperiods or when INC lighting

was added to F Rd photoperiods after 1800 HR and before 1000 HR (Figs. 2C and D).

 

Regardless of photoperiod or lighting treatment, plants initiated flowers at the same node B
(Figs. 2E and F). Conditions that were least favorable for flowering (e.g., 9-h natural or

16-h F Rd photoperiod) developed more nodes before anthesis compared with the most i
inductive treatments. Peduncles of flowering plants were shortest under 9-h days or F Rd *1

photoperiods without or with lighting during the 16-h base photoperiod (Figs. 2G and H).
Except for pansy grown under 9-h days or a 16-h F Rd environment with INC lighting
from 0600 to 1000 HR, total plant height of flowering plants was similar (data not shown).
Expt. 3. Regardless of NI lighting, primary flowering percentage and stem length

of pansy were reduced (by 20 and 28 %, respectively) when the 9-h base photoperiod was
deficient in PR (Table 3). Of the plants that flowered under the FRd base photoperiod,
average time to flower was delayed (by 4 (1) compared to flowering plants under the N
filter. In general, flowering percentage increased as the NI duration increased, but the

response varied by the quality of light provided (Figs. 3A and B). Relative to plants

 

under the N filter without NI lighting, the minimum NI duration that increased flowering
percentage was 2 h when the NI R : FR was low or moderate and 4 h when the ratio was
high (Fig. 3A). Under the FRd filter, 2 or 4 h of NI lighting with a moderate or low R :

FR, respectively, was required to increase flowering percentage compared with plants

119

without an NI (Fig. 3B). A 4-h NI with a high R : FR failed to promote flowering under
an FRd environment. Axillary flowering percentage was greatest under the high R : FR
(13 %) compared with a low (7 %) or moderate (6 %) R : FR and the least when the NI
was for 4 h (2.5 % compared with 2 7.5 % under shorter NI durations; data not shown).
Time to anthesis decreased as the NI duration increased, but the magnitude varied
by the R : FR (Figs. 3C and D). One hour of NI lighting significantly accelerated
flowering under the N filter when delivered with a low or moderate R : FR, but 4 h of r:
lighting with a high R : FR was required to hasten flowering. A 4-h NI with a low or

moderate R : FR hastened flowering by 18 d under the N filter and 14 (1 under the FRd

 

filter. Plants provided with a 4-h NI with a high R : FR flowered 7 or 8 d earlier than
plants without an NI, regardless of filter type.

Light quality during the base photoperiod did not have a significant effect on the
node at flower initiation or anthesis (Table 3), so data within filter treatments were
pooled. Pansy initiated flowers at the same node, regardless of NI duration or quality
(Fig. 4A). However, compared with plants without an NI, pansy developed fewer nodes
before anthesis when the NI was 22 h with a low R : FR or 4 h with a moderate R : FR
ratio (Fig. 4B). An NI with a high R : FR did not reduce the number of nodes developed
before anthesis compared with plants under SD.

An N1 of s 1 h had little or no effect on stem extension, regardless of light quality

 

(Figs. 3E and F). However, compared with plants without an NI, stem length increased
by 2 138 % or 2 103 % when a 4-h NI was delivered with low or moderate R : FR,
respectively, regardless of light quality during the base photoperiod. In contrast, a 4-h NI

with a high R : FR promoted stern extension by s 35 %.

120

Discussion

Half of the Viola ><wittrockiana ‘Crystal Bowl Yellow’ plants under unfiltered 9- or lO-h
photoperiods reached anthesis within the experimental periods. Flowering percentage
increased and time to flower decreased as the photoperiod increased, until =16 h, when
essentially all plants flowered with natural photoperiods extended with INC or HPS

lamps. A 4-h NI with INC lamps promoted flowering similar to that under continual

 

photoperiods 2 16 h. Therefore, ‘Crystal Bowl Yellow’ is a quantitative LDP, which is El"

similar to that reported in another pansy cultivar (Adams et al., 1997). :
In all experiments, ‘Crystal Bowl Yellow’ initiated flowers at approximately the “I

eighth node, regardless of the duration or spectral quality of the photoperiod. However, v

flower bud development was arrested when photoperiods were short or when long
photoperiods were deficient in FR light. When 16-h photoperiods were deficient in FR,
flowering was suppressed greater than that under unfiltered 9-h photoperiods. Therefore,
pansy appears to be day-neutral with respect to flower initiation and a quantitative LDP
with respect to flower development. In addition, pansy can be considered a light-
dominant plant since 1) it required a long period (e.g., 2 2 h) of night break lighting to
maximally promote flowering and 2) the promotion of flowering was dependent upon the
spectral quality of incident radiation, with a low or moderate R : FR more effective than a

high R : FR.

 

Experiments 2 and 3 were performed to determine if pansy could be exposed to a
minimal amount of FR light to promote flower development without promoting stem
extension. Light from INC lamps, which is rich in FR, added during the first 10 h of the

base photoperiod had little or no promotive effect on flowering or stem extension.

121

Flowering was promoted when light from INC lamps was added at the end of or
continually during the base photoperiod, or during the otherwise dark period. Plants
lighted from 2200 to 0200 HR or from 0200 to 0600 HR received a 20—h photoperiod, and
thus at least some of the promotion of flowering could be attributed to the increase in day
length. However, regardless of the addition of FR light, the percentage of plants with a
normal flowering phenotype (with apical flowering) was always lower under the FRd
filter compared with that under the N filter. In addition, when the addition of FR
promoted flowering, it also promoted extension growth. Therefore, exposing plants to a
FR-rich environment for portions of the day or night is not an effective strategy to
promote flowering without promoting extension growth.

The maximum predicted Pfi/P in an environment completely devoid of FR light is
20.89, since there is considerable overlap in the R- and F R-absorbing forms of
phytochrome in the 650 to 700 nm waveband (Sager et al., 1988). The estimated Pfi/P
under the FR filter was 20.78 to 0.80, which is significantly greater than that of unfiltered
sunlight (Pf/P 2 0.71 to 0.72). Although the FR filter reduced the transmission of FR
radiation, particularly around 730 nm, a substantial amount of FR radiation penetrated the
filter when the ambient light intensity was high. However, stem extension and flowering
were suppressed when the Pfr/P was increased even though plants were exposed to some
FR light.

Our studies indicate that a moderately low Pfr/P (e.g., < 0.78) is required toward
the end of the photoperiod for normal flower development in pansy (Expt. 2). A similar
requirement for a high Pf/P, especially toward the end of the photoperiod, has been

reported for rapid initiation in other LDP (Carr-Smith et al., 1989). For example, in the

122

 

 

LDP Lolium temulentum L., FR promoted flowering during the first 26 h of a 16-h night
and R was most promotive toward the end of the night (Evans, 1976; Vince, 1965).
While most studies have described a FR requirement for flower initiation, our results with
pansy are similar to that in Hyoscyamus niger L., where the primary effect of FR light is
on flower development, not initiation (Downs and Thomas, 1982).

A high Pfr/P during the base photoperiod inhibited flowering, regardless of NI

lighting duration or spectral quality (Expt. 3). This suggests that, in addition to the end- ‘7-

. ...—r.

of-day FR requirement, a moderate or low Pfi/P (< 0.80) is required during the base

photoperiod for rapid and complete flowering of pansy. This is supported by Expt. 2, in

 

which flowering percentage under the FRd filter was never as high as that under the N k
filter, regardless of lighting treatment.

Flowering is promoted most in light-dominant LDP when an N1 is long (e.g., 2 2
h) and contains a minimal amount of FR light. In pansy, flowering was promoted most
when the night break was for 4 h and the R : FR was relatively low (e.g., s 1.28). An NI
5 l h had no promotive effect on flowering percentage, regardless of the light quality
provided during the day or night. Except for 1 h of NI with INC lamps, an NI 5 l h did
not significantly promote stem extension in any treatment. The conditions that were most
promotive to flowering (e. g., a 4-h NI with a low or moderate R : FR) were also most

promotive to extension growth. Together, this suggests that flowering and stem extension

 

are promoted similarly by the quality and duration of NI.
To quantify the relationship between stem extension (SE) and flowering

promotion (FP), indices were developed for each of the 26 treatments used in Expt. 3.

123

The F P index (adapted from Lange, 1993) and SE index of each treatment were

determined by:

SE 211‘ 1.1.x" [11

FP = F - FTmm - FT," [2]

where It = treatment average internode length (mm), 1m, = maximum average internode
length (mm), F = flowering (%), FTmin = minimum average flowering time (d), and F Tt =
treatment average flowering time (d). In our studies, plants under the N filter with a 4-h
NI from INC lamps flowered most rapidly and had the greatest internode length, and thus
were the values used for IImam and FTmin (Figs. 3C and E).

Regardless of light quality during the photoperiod or the NI light quality or
duration, the promotion of extension growth was linearly related to the promotion of
reproductive development (Fig. 5). This suggests that in light-dominant LDP such as
pansy, light duration and quality concomitantly promote extension growth and flowering,
and it does not appear that they can readily be separated with lighting strategies.

A variety of phytochrome-mediated processes, including extension grth and
flowering, are mediated by the gibberellin (GA) family of plant hormones. A close
relationship between phytochrome, gibberellins, and extension growth has been
demonstrated by several molecular studies. In transgenic aspen (Populus tremula X
tremuloides), the over-expression of phytochrome A reduced internode length, which was
correlated with low levels of GAs in apical stem and leaf tissue (Olsen et al., 1997).

Similarly, transgenic tobacco and potato with over-expressed phyA induced dwarfism

124

 

 

(Heyer et al., 1995; Jordan et al., 1995). Phytochrome A mutants of Arabidopsis thaliana
Heynh. are relatively insensitive to FR and LD, which suggests that this phytochrome
participates in the regulation of extension growth and flowering (Reed et al., 1994). In
the LDP Nicotiana sylvestris Speg. & Comes, phytochrome B overexpression
concomitantly reduced internode extension and delayed flowering (J. Metzger, personal
communication). Therefore, it appears that regulating the phytochrome status, using
lighting or molecular approaches, is not an effective strategy to suppress extension T
grth without inhibiting flowering. i

In summary, these experiments indicate that while an FR,I environment can limit 3

 

extension growth, it can also delay flower initiation in light-dominant LDP such as pansy. F
Exposure of plants to periods of FR light can partially but not completely overcome a

delay in flowering. However, when light conditions are promotive to flowering, they are

also promotive to extension growth. Therefore, commercial use of an FR filter could be

limited when rapid and complete flowering of LDP is desired.

Acknowledgments
We gratefully acknowledge funding by project GREEEN and greenhouse growers
supportive of Michigan State University floricultural research and the support of the

Michigan Agricultural Experiment Station. We also thank Dan Tschirhart and David

 

J oeright for their greenhouse assistance, British Visqueen for the far-red filter, and

Daphne Vince-Prue for her valuable discussions.

125

Literature Cited
Adams, S.R., S. Pearson, and P. Hadley. 1997. The effects of temperature, photoperiod
and light integral on the time to flowering of pansy cv. Universal Violet (Viola

><wittrockiana Gams.). Ann. Bot. 80: 107—1 12.

Behe, B., J. Hardy, J. Heilig, and A. Scordalakes. 2000. 1999 season sales summary.

Ohio Florists’ Assoc. Bull. Marchzl-12. p”

Carr-Smith, H.D., C.B. Johnson, and B. Thomas. 1989. Action spectrum for the effect of

 

day-extensions on flowering and apex elongation in green, light-grown wheat (T riticum F

aestivum L.). Planta 179:428-432.

Downs, R.J. and J .F. Thomas. 1982. Phytochrome regulation of flowering in the long-day

plant, Hyoscyamus niger. Plant Physiol. 70:898—900.

Evans, LT. 1976. Inflorescence initiation in Lolium temulentum L. XIV. The role of

phytochrome in long day induction. Aust. J. Plant Physiol. 32207-217.

 

Heyer, A. G., D. Mozley, V. Landschiitze, B. Thomas, and C. Gatz. 1995. Function of
phytochrome A in potato plants as revealed through the study of transgenic plants. Plant

Physiol. 109253-61.

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Jordan, E. T., P.M. Hatfield, D. Hondred, M. Talon, J .A.D. Zeevaart, and RD. Vierstra.

1995. Phytochrome A overexpression in transgenic tobacco. Plant Physiol. 107:797-805.

Lane, H.C., H.M. Cathey, and LT. Evans. 1965. The dependence of flowering in several
long-day plants on the spectral composition of light extending the photoperiod. Amer. J.

Bot. 52:1006—1014.

Lange, N. 1993. Modeling flower induction in Lolium longiflorum. MS Thesis, Michigan

State Univ., East Lansing. i

 

Olsen, J. E., O. Junttila, J. Nilsen, M.E. Eriksson, 1. Martinussen, O. Olsson, G.
Sandberg, and T. Moritz. 1997. Ectopic expression of oat phytochrome A in hybrid aspen

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1350.

Rajapakse, N.C., R.E. Young, M.J. McMahon, and R. Oi. 1999. Plant height control by

photoselective filters: Current status and firture prospects. HortTechnology 9:618—624.

Reed, J .W., A. Nagatani, T.D. Elich, M. Fagan, and J. Chory. 1994. Phytochrome A and

 

phytochrome B have overlapping but distinct functions in Arabidopsis development.

Plant Physiol. 104:1139-1149.

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Runkle, ES. and RD. Heins. 2001. Specific functions of red, far-red, and blue light in

flowering and stem extension of long-day plants. J. Amer. Soc. Hort. Sci. (In press.)

Runkle, E.S., R.D. Heins, A.C. Cameron, and W.H. Carlson. 1998. Flowering of
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HortScience 33:672—677.

Sager, J .C., W.O. Smith, J .L. Edwards, and KL. Cyr. 1988. Use of spectral data to
determine photosynthetic efficiency and phytochrome photoequilibria. Trans. ASAE

31:1882—1889.

 

Smith, H. 1994. Sensing the light environment: The functions of the phytochrome family,
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plants. 2nd ed. Kluwer Academic Publishers, Netherlands.

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London.

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Wheldon, and R.G.C. Henbest. 1998. The development of solid spectral filters for the

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Vince, D. 1965. The promoting effect of far-red light on flowering in the long day plant

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Vince-Prue, D. 1975. Photoperiodism in plants. McGraw, London.

129

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Table 2. Spectral radiation and estimated phytochrome photoequilibria (Pf/P; Sager et al.
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10 12 14 16 18 20 22 24 NI 1

Photoperiod (hours)

 

Fig. 1. Flowering of Viola Xwittrockiana ‘Crystal Bowl Yellow’ under photoperiods
consisting of 9-h natural days extended with light from incandescent lamps. N1 = 9—h
photoperiods plus 4-h night interruption. Plants were considered nonflowering if they did
not reach anthesis within 98 to 100 d from seed. Days to flower with the same letter are

not statistically different at P = 0.05.

133

Fig. 2. Flowering and peduncle length of Viola Xwittrockiana ‘Crystal Bowl Yellow’
grown under a neutral (N) filter (Figs. A, C, E, and‘G) or one that selectively reduced the
transmission of far-red (FR, 700 to 800 nm) light (Figs. B, D, F, and H). In Figs. A and
B, - - - represents the total flowering percentage. Except for the 9-h photoperiod, a 16-h
base photoperiod was provided by natural photoperiods extended with light from high-
pressure sodium lamps from 0600 to 2200 HR. Light rich in FR was provided under the
FR filter for periods during the day or night, as indicated. Values with the same letter
within measurement are not statistically different at P = 0.05. Letters are not provided

when all treatments are statistically similar.

134

 

 

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135

Fig. 3. Flowering and stem length of Viola Xwittrockiana ‘Crystal Bowl Yellow’ grown
under a 9-h neutral (N) filter (Figs. A, C, and E) or a filter that selectively reduced the
transmission of far-red (FR, 700 to 800 nm) light (Figs. B, D, and F). Night interruption
(N I) lighting was provided for varying durations by lamps delivering a low (0.56),
moderate (1.28), or high (7.29) red (R, 600 to 700 nm) to FR ratio (Table 2). In each
graph, Open or dark symbols represent means are significantly different (at P = 0.05) from
or similar to that without an NI, respectively. Error bars represent 95 % confidence

intervals, and except for Figs. A and B, are not presented when NI lighting treatments

were statistically similar.

Primary
flowering percentage

Days to anthesis

Ten-node stem length (cm)

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100 “A Neutral filter 1” B FRd filter .0 ‘
// D
// /
80 ‘7 - /// // .
/ /
/’D //

60 - ~~ // // a
. F/ //.’ ///
401 ~~ m” ///” <

o R:FR=O.56 o v7/K/ ,-r’
20 -~ I R:FR=1.28«~ J v’
v R:FR=7.29
0 1 1 1 1 1 1 1 - 1 1 1 1 . 1 e 1 - 1
70 1 1 1 1 1 . 1 1 1 1 1 1 . 1 1 1 f 4
C D
65 1 1 «
60 1"- nh. / \ *
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55 *0 ,_ k. \\ _ ,
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40 L l. .
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0 1 1 1 . 1 1 1 _ 1 1 - 1 - 1 - 1 # 1
0 1 2 3 4 O 1 2 3 4

NI duration (h)

 

 

 

137

 

 

 

 

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8, - R:FR=1.28 ,
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0 1 2 3 4

NI Duration (h)

Fig. 4. Flowering and stem length of Viola xwittrockiana ‘Crystal Bowl Yellow’ grown

under a neutral (N) or far-red deficient (FRd) filter with night interruption (N 1) treatments

as described in Fig. 3. Data for plants under the N and FRd filters were pooled, since the
effects of base photoperiod were insignificant. Error bars represent 95 % confidence

intervals, and are not presented when NI lighting treatments were statistically similar.

138

 

 

 

Stem extension index

 

Y = 0.405X + 0.494

 

 

 

Q R:FR=0.56
I R:FR=1.28

 

 

 

 

0.2 ~-
_. mu ' R I FR = 7.29
(r2 ' 0'78 ) Closed symbol = N filter
Open symbol = FRd filter
0.0 1 1 1 1 1 1
0.0 0.2 0.4 0.6 0.8 1.0

Flower promotion index

Fig. 5. Flowering and stem extension of Viola Xwittrockiana ‘Crystal Bowl Yellow’

grown under 26 combinations of filter and night interruption (NI) treatments, as described

in Fig. 3. Linear regression analysis was used to relate the relative promotion of stem

extension with flowering; see text for equations. ... = significant at P _<. 0.0001.

139

 

 

 

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