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This is to certify that the
thesis entitled
MORPHOLOGICAL AND GENETIC
FACTORS AFFECTING CHLOROPLAST NUMBER
IN DIPLOID AND TETRAPLOID ALFALFA

presented by

PETER WEBB CALLOW

has been accepted towards fulfillment
of the requirements for

Master oLSniennLdegree in We

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Major professor

Date 9-7-93

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MORPHOLOGICAL AND GENETIC FACTORS AFFECTING
CHLOROPLAST NUMBER IN DIPLOID AND
TETRAPLOID ALFALEA

BY

Peter Webb Callow

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1993

ABSTRACT

MORPHOLOGICAL AND GENETIC FACTORS AFFECTING CHLOROPLAST
NUMBER IN DIPLOID AND TETRAPLOID ALFALFA

By

Peter Webb Callow

Two theories have been proposed concerning the control of chloroplast
number per cell. One suggests that cell size is the primary regulating
factor, the other proposes that genome size plays the predominant role.
Chloroplast and cell face area were analyzed in cells of diploid and
tetraploid genotypes and a chimeric plant.

There was significant variation in plastid density at both ploidy
levels, and there was overlap in the mean number of plastids in the
diploid and the tetraploid genotypes. Plastid density values in
reciprocal populations appeared to be maternally influenced, although
plastid numbers in the chimeric tissues were not significantly different.

Chloroplast number may be partially controlled by nuclear genes
independent to those regulating cell size, and genome size does not
appear to be as important a regulating factor as progenitor genotype.
Chloroplast density is largely controlled by the nucleus, but cytoplasmic

factors separate from the plastid may be involved.

DEDICATION

To Mollie, Sam and Marty and

to the memory of my grandfather, Pete McMullen.

iii

ACKNOWLEDGMENTS

I would like to give my heartfelt thanks to my major professor, Jim
Hancock. Jim’s wealth of enthusiasm, good ideas and sense of humor were
a tremendous help to me and kept me going through the many times I felt I
would never finish this project.

I would also like to thank my committee members, Amy Iezzoni, Jim
Flore and Barb Sears for their patience and valuable suggestions.

Thanks are also due to Dr. Joanne Whalon and Dr. Ken Sink for the use
of their microscopes. Dr. Rebecca Grumet was very generous with her
computer. Gloria Blake helped me through the bureaucratic maze of
extension and re—admission. Julie Heck did a wonderful job on my tables
and was a tremendous help getting this thesis in final form. Roger May
stayed late after work with me to help me put my graphs together and also
helped. me with. much advice on. photography. Colleen. Mulinix 'very
graciously supplied.me with plants left over from'her MJS. that I used for
my controlled crosses experiment and helped me numerous times with my
computer problems. Carol Schumann not only helped me get my project
started but also has had a major impact on my development as a scientist.
My lab mates, Stan, Karen, Jean, Muso and Lu Ping always supported me and
helped me keep the faith. Finally, I want to thank Mollie for her love

and support.

iv

TABLE OF CONTENTS

Page

List of Tables ........................................... vi
List of Figures .......................................... vii
Introduction ............................................. 1
Materials and Methods .................................... 7
Results .................................................. ll
Genotype screens ..................................... ll
Reciprocal crosses ................................... 20
Discussion ............................................... 23
Factors affecting plastid numbers .................... 23
Polyploidy and plastid numbers ....................... 25
Bibliography ............................................. 27

Table

Table

Table

Table

Table

Table

Table

LIST OF TABLES

Page

Mean number of chloroplasts per cell and guard cell
length in different accessions of diploid and tetraploid
alfalfa grown in the greenhouse ......................... 8

Mean cell face area and chloroplast number in spongy
mesophyll cells of different genotypes of alfalfa grown
in the greenhouse ....................................... 15

Mean cell face area and chloroplast number in spongy
mesophyll cells of different genotypes of alfalfa grown
in the growth chamber ................................... 16

Comparison of diploid and tetraploid genotypes at low light
levels (LL), 400 pmoles/sec/cm?‘photosynthetic photon flux
(PPF) and high light levels (HL), 900 pmoles/sec/cm? PPF.
Chloroplast number - CPT No., cell face area - CFA,
chloroplast face area - CPFA, chloroplast density

(CPA/CPT No.) - CPD and total plastid area per cell area -
TPA/CFA ................................................. 18

Mean cell face area (CFA), chloroplast number (CPT No.),
chloroplast face area (CPFA), chloroplast density (CPD) and
total plastid area per cell area (TPA/CFA) in spongy
mesophyll cells of different genotypes of alfalfa grown
under artifical light. Values are averages of replicates
grown at 400 and 900 pmoles/sec/cm? PPF ................. 19

Mean cell face area (CFA), chloroplast number (CPT No.),
chloroplast face area (CPFA) and chloroplast density (CPD) in
spongy mesophyll cells of the accessions 299049-1 (S),
W7l-42-2 (F) and their self and reciprocal

progeny ................................................. 21

Mean chloroplast number (CPT No.), cell face area (CFA),
chloroplast face area (CPFA) and chloroplast density (CPD)
in chimeric sectors of cross SxF 11. SxF 11d has S plastome
SxF lla has F plastome .................................. 22

vi

Figure 1.

Figure 2.

Figure 3.

LIST OF FIGURES

A plot of guard cell length (p) and number of chloroplasts per
cell for seven diploid and twelve tetraploid alfalfa
genotypes. Each point represents the mean of thirty cells.
Diploids, r-0.470, not significant; tetraploids, r-0.20, not
significant.

A plot of mesophyll cell face area (pmz) and number of
chloroplasts per cell for five diploid and ten tetraploid
alfalfa genotypes grown in a greenhouse. Each point
represents the mean of fifty cells. Diploids, r-0.093, not
significant; tetraploids, r-0.501, not significant.

A plot of mesophyll cell face area (pmz) and number of
chloroplasts per cell for six diploid and nine tetraploid
alfalfa genotypes grown in the growth chamber. Each point
represents the mean of fifty cells. Diploids, r-0.829
(P<0.05); tetraploids, r-0.949, (P<0.05).

vii

INTRODUCTION

Many scientists believe that plastids evolved from phototrophic
prokaryotic cells. An endocytotic event between a phototrophic prokaryote
and a primitive eukaryote may have resulted in a symbiotic relationship
between the two cell types. The offspring of this organism inherited DNA
not only from the eukaryotic cell but from the bacterial endo-symbiont as
well. Margulis (1971) has characterized endo-symbiosis as "swallowing
without digesting". This type of relationship exists in present day
organisms. For example, Paramecium bursaria has a symbiotic relationship
with the green algae Chlogella (Margulis, 1971, Karakashian et a1. 1968).
However, this is not an obligatory endosymbiosis, as both organisms can
survive alone. When the W and W are separated and then
reconstituted, the alga will multiply until a certain threshold is reached
and thereafter any W that are ingested are digested with no
apparent harm done to the existing algal cells (Margulis, 1971).

Several lines of evidence support an endosymbiotic origin for
plastids. The chloroplast genome is circular like that of bacteria
(Sears, 1983). The chloroplast ribosomal RNA is sensitive to inhibitors
of prokaryotic translation such as chloramphenical, streptomycin, and
tetracycline (Bottomley and Bohnert 1982, Alberts et al. 1983 and Von
Wettstein, 1981). Ribosomal RNA of the chloroplast has sedimentation
A coefficients (163, 23s and 53) that are similar to prokaryotes (Hoober,

1984) . Protein synthesis in chloroplasts begins with n-formylmethionine,

2
as in bacteria, and not with methionine as in the cytosol of eukaryotic
cells (Alberts et a1. 1983, Von Wettstein, 1981). Chloroplast ribosomes
and bacterial tRNAs can be used together in protein synthesis, and
chloroplast mRNAs can be translated by a protein synthesizing extract from
W (Alberts et a1. 1983, Von Wettstein, 1981). There is also a 70-751
homology at the nucleotide level between cyanobacteria and higher plants
with respect to the gene for the large subunit of ribulose bisphosphate

carboxylase/oxygenase (RUBPC/O) (Hoober, 1984).

PLASTID AUTONOMY. Plastid development and physiology may have initially
been autonomous, but chloroplast functions are now dominated largely by
the nucleus (Ellis, 1984). This is most strongly supported by the
Mendelian segregation of most mutations influencing chloroplast
development (Gillham, 1978). Also, the plastid genome is too small to
carry all the genes associated with its metabolism. Genes in the
chloroplast encode components of plastid transcription, translation and
proteins involved in photosynthesis (Taylor, 1989). However,
photosynthesis and the other plastid functions require the products of
several hundred genes of which only about 120 are present in the
approximately 150 kb chloroplast genome (Gruissem, 1989).

When Scott and Timmis (1984) used restriction enzymes to produce
spinach plastid DNA fragments that were subsequently made into
hybridization probes, they found that every cloned fragment of plastid DNA
showed homologies to the spinach nuclear genome. Many of these homologies
occurred in regions of the nuclear DNA that were highly methylated. They
concluded that essentially all of the plastome has homologies with the

nuclear DNA, and that potentially, the nucleus possesses all the genes

3
required to make functional chloroplasts, although they may be in an
interrupted, highly methylated and perhaps inactive form.

While the majority of the plastid constituents are nuclear-encoded,
there are reports of'a possible feedback.mechanism from the chloroplast to
the nucleus that regulates levels of cytosolic mRNA for some chloroplast
proteins. In Sinapgig 51p; L., it has been hypothesized that a signal
from the plastid is required to allow the phytochrome mediated appearance
of translatable mRNA for the small sub-unit (SSU) gene of RUBPC/O and the
light harvesting chlorophyll a/b binding protein (LHCP) of photosystem II
(Oelmuller and Mohr, 1986). The authors observed that phytochrome-
mediated expression of both nuclear genes (or gene families) is only
possible if the plastids are intact. If the plastids are severely
damaged, expression of the genes for SSU and LHCP are almost completely
inhibited even though nuclear genes not related to the plastid are not
adversely affected. Similar results have been observed in.maize, mustard
and tomato with respect to LHCP, glutamine synthase, nitrate reductase and
NADP-glyceraldehyde-3-phosphate dehydrogenase (Taylor, 1989, Edwards and
Coruzzi, 1989, Deane-Drummond and Johnson, 1980, Feierabend and Schubert,

1978 and Reiss et a1. 1983).

PLASTID DIVISION AND GENOME REPLICATION. Many unicellular plants contain
only one or two chloroplasts (Possingham.and.Lawrence, 1983), while higher
plant cells contain many (Hoober, 1984). Chloroplast division occurs
immediately before cytokinesis in most unicellular organisms (Barlow and
Cattolico, 1981, Slankis and Gibbs, 1972 and Cattolico et a1. 1976). In
the mono-plastidic, primitive vascular plant lgggggg, plastid division

occurs during a number of different stages of the cell cycle (Whatley,

4
1974). In higher plants, chloroplast replication occurs at the time of
new cell formation and continues for two to three cycles after cell
division has stopped (Rose et a1. 1975, Whatley, 1980 and Boffey et al.
1979).

Scott and Possingham (1980, 1982) have identified three phases of
plastid development and division in intact leaves of spinach. The first
phase occurs in young leaves that are growing primarily by cell division;
here plastid division and plastome replication keep pace with cell
division. The second phase occurs when growth changes from cell division
to cell expansion. As the cell expands, plastids continue to divide but
there is a twofold increase in plastome numbers per plastid. Chloroplast
DNA synthesis continues until plastome copy number per cell increases from
approximately 1500 to 5000. The third phase occurs when cell division
ceases. Chloroplast division continues for a few more cycles, but
chloroplast DNA synthesis stops. This overall pattern of plastid division
and chloroplast DNA replication has been observed in pea (Possingham,
1980), beet (Possingham, 1980) and wheat (Lamppa et a1. 1980, Boffey and
Leech, 1982).

Several hypotheses have been presented concerning the control of
chloroplast division. Many feel that division is linked to cell expansion
and plastids simply multiply to cover a constant proportion of the cell
surface area (Pyke and Leech, 1987). This conclusion has come from
numerous studies where chloroplast number was significantly correlated
with cell size (Asahi and Toyama, 1982, Chaly et al. 1980, Ellis and
Leech, 1985, Pyke and Leech, 1987). However, there may also be nuclear
genes that directly regulate plastid division. Frandsen (1968) found

several genotypes of m hybrida that had significantly different

5
chloroplast number per cell but a similar cell size. DeMaggio and Stetler
(1971) described lines in lodge barbara where chloroplast number was not
significantly related to cell size or ploidy level.

Differences in chloroplast number per cell between diploid and
tetraploid.plants should.be expected" Ploidy level has a strong effect on
nuclear size which influences cell size (Pyke and Leech, 1987). Nucleus
and cell size are positively correlateduwith.DNA.content (Ramachandran.and
Narayan, 1985). Over time, the polyploid plant may undergo 'dosage
compensation', where some of these effects are diminished.due to selective
disadvantage, but at least some difference in cell size is usually
maintained (Hancock, 1992).

When Bingham (1968) determined chloroplast number in guard cells of
diploid, triploid, tetraploid and hexaploid alfalfa, he concluded that
ploidy level has a greater influence on chloroplast number than does
genome source, and suggested that chloroplast number per cell could be
used as a method to determine ploidy level. Butterfass (1973, 1979, 1980,
1983 and 1991) suggested that nuclear DNA. amount itself regulates
chloroplast number, basing his argument on the observation that polyploidy
usually results in increases in plastid number per cell. He also
suggested a similar system may operate within genotypes due to
endopolyploidy.

While it is likely that ploidy level influences plastid numbers via
its effects on cell size, some specific genic effects have been noted.
Molin et a1 (1982) found that there were twice as many chloroplasts per
cell in isogenic lines of tetraploid alfalfa as compared to the diploids.
However, there was also considerable variation. among two different

tetraploid genotypes in chloroplast number per cell, indicating a genic

6
effect. Standring et a1. (1990) found no consistent relationship between
ploidy level and chloroplast number in m m (pepino) and
W basses (tamarillo).

Ellis and Leech (1985) found that chloroplast number was inversely
proportional to chloroplast size in W W and W
m indicating that plastid size may play a role in regulating
plastid numbers. Pyke and Leech (1991) found mutants in W that
had aberrant numbers of chloroplasts per cell plane area. One mutant with
a significantly higher number of chloroplasts per cell had unusually small
chloroplasts, and two mutants with significantly lower numbers of
chloroplasts per cell had unusually large chloroplasts. Also, a mutant of
Arabidgpsis with a deficiency in an n-3 desaturase had unusually small
chloroplasts but significantly more chloroplast numbers per cell than the
wild type (McCourt et a1. 1987).

In this study, a diverse array of alfalfa genotypes was evaluated to
determine if nuclear genes exist which influence plastid division
independent of those affecting cell expansion. Chloroplast numbers and
cell face areas were measured in a broad range of diploid and tetraploid
genotypes of alfalfa maintained in common environments. Reciprocal
crosses of high and low chloroplast lines were also examined to determine

if there were cytoplasmic factors controlling chloroplast number per cell.

MATERIALS AND METHODS

Plastid numbers and cell size were measured in guard and mesophyll
cells of both greenhouse and growth chamber grown plants. Seeds were
obtained from the U.S.D.A. North Central Regional Plant Introduction
Station, Ames, Iowa, or from Dr. E. T. Bingham, University of Wisconsin,
Madison (Table 1). We will refer to individual genotypes by the
accession numbers of their donor population. Accessions with the same
number, but with a hyphenated suffix number were different genotypes from
the same seed population. The same genotypes from each population were
not always used in each experiment. Chromosome counts were verified by
Feulgen and acetocarmine squashes of root tips (Schumann, 1988). All
plants were initially grown for three years in a 1:1:1 (soil, peat and
sand) mixture in 10 cm3 plastic pots in a completely randomized design in
a single greenhouse at Michigan State University, E. Lansing, Michigan.
Seasonal conditions ranged from day temperatures of l8-40° C, night
temperatures of 18-27" C and a photosynthetic photon flux (PPF) of 85-225
Irmoles/sec/m2 in winter to 750-1320 pmoles/sec/m2 in summer.

Counts of guard cell chloroplasts were made in September from seven
diploid and twelve tetraploid genotypes held in the greenhouse. The source
material was fully expanded leaves from the third to sixth node. A
section of the lower (abaxial) epidermis was peeled with forceps and

placed in a saturated potassium iodide—iodine (IZKI, which causes starch

 

 

Table 1. Mean number of chloroplasts per cell and guard cell length
in different accessions of diploid and tetraploid alfalfa
grown in the greenhouse.

Ploidy Accession Origin Source Mean number Mean guard

chloroplasts/ cell length
cell (p)
Diploid 172989 Turkey USDA 3.5a' 12.0a
251689 USSR USDA 3.6a 12.0a
262532 Israel USDA 3.8ab 15.5ab
W70-22 Mixed Bingham (Wisc) 3.9ab 16.5ab
251830 Austria USDA 4.0ab 18.5b
DDC 2X Mixed Bingham (Wisc) 4.1b 19.3b
235021 Germany USDA 4.2b l2.a
Tetraploid 299049 USSR USDA 4.2a 20.0b
172983 Turkey USDA 4.4ab 20.4b
239953 Algeria USDA 4.8ab 20.5b
299051-2 USSR USDA 4.9ab 20.0b
299051-1 USSR USDA 3.8ab 12.a
299048 USSR USDA 5.1ab 19.0b
W71-42-2 Mixed Bingham (Wisc) 5.4b 20.0b
DDC 4X Mixed Bingham (Wisc) 5.7bc 19.3b
Vernal Cultivar USDA 5.9bc 19.5b
W71-42-1 Mixed Bingham (Wisc) 5.9bc 20.9b
253443 Yugoslavia USDA 6.7c 19.5b
251205 Yugoslavia USDA 6.7c 21.0b

 

‘Means within columns sharing the same letter are not significantly

different at the 51 level using the Duncan's Multiple Range Test. Mean

comparisons are within ploidy.

9
grains in the chloroplast to turn red) solution for five minutes.
Chloroplasts were counted and cell length measured using a Zeiss
micrometer, in ten randomly selected guard cells per three trifoliate
leaves. Chloroplast counts were made using a Zeiss microscope with phase
optics.

Counts of spongey mesophyll cell chloroplasts were also made from
five diploid and ten tetraploid genotypes held in the greenhouse and six
diploid and nine tetraploid genotypes grown in a growth chamber for three
months. The growth chamber plants were maintained at 25:1: 2" C at PPF
between 600 and 700 pmoles/sec/mz. All plants were cut back to crown level
three weeks prior to analysis. Fully expanded leaves from the fifth node
of each plant were used to determine chloroplast number. The lower
epidermis of forty to fifty leaves were peeled with forceps or rubbed with
carborundum (320 grit). The leaf tissue was then floated for one to two
hours on an enzyme solution with 51 pectinase (Sigma Chemical Co., St.
Louis, Mo.), 22 cellulysin (Calbiochem, La Jolla, 0a.), 22 driselase
(Kogyo co., Tokyo, Japan), 91 mannitol (Lesney et a1. 1986) and cell
protoplast wash solution (Frearson et a1. 1973). Chloroplasts were
counted in 50 randomly selected cells using a Zeiss microscope with phase
optics, and the length and width of cells were measured. The enzyme
solution yielded a high number of cells with intact walls. Only cells
with walls were measured to get an accurate representation of cell size in
113m.

To test if differing light intensities had an effect on chloroplast
number per cell, three diploid and tetraploid genotypes with high
(2Nz235021, 4Nz251205), intermediate (2N:172989, 4N:172983) and low

(2Nz262532, 4Nz239953) chloroplast densities in the previous analysis were

10

grown for several weeks in a growth room at 26°C, 16 hour photoperiod and
under PPF of 400 or 900 pmoles/sec/mg. 'Three weeks after being cut back
to crown level the fifth trifoliate leaf was removed from several stems
and immediately placed in a solution of 1.5 to 2.5% glutaraldehyde in a
0.1M potassium phosphate buffer for one hour. The leaves were then placed
in a 0.1M solution of NaEDTA ([ethylene dinitrilo] tetra acetic acid-
disodium salt) at 60°C for three to eight hours (Pyke and Leech, 1987).
The plant tissue was macerated on a slide and viewed with a Zeiss
universal microscope using Nomarski differential interference optics.
Plastids in twenty five randomly selected cells were counted and plastid
and cell sizes were determined. Total plastid area per unit cell face
area (TPA/CFA) was also determined.

To determine if there was a cytoplasmic effect on chloroplast
division, chloroplast numbers per cell, cell face area and plastid face
area were measured in twelve self and reciprocal progeny of two parent
lines previously shown to have distinct plastid numbers (299049-l, or S
cytoplasm and W7l-42-2, or F cytoplasm, Schumann and Hancock, 1989).
Rooted shoots from a chimeric individual (SF-ll) were also examined that
were previously determined to contain different plastid types (SxF-lld, S
cytoplasm and SxF-lla, F cytoplasm, Schumann and Hancock, 1990). All
these plants were grown in the greenhouse at Michigan State University
under the previously described conditions. Ten cells were evaluted from
five leaves taken from the fifth node from the apex of each plant using

the methods described above.

RESULTS

GENOTYPE SCREENS

Mean number of chloroplasts per guard cell ranged from 3,510.8 to
4.2i0.5 among,diploid.and.4.li0.8 to 6.7i1.4 among tetraploid lines (Table
1). There was a positive correlation between chloroplast number and cell
length among ‘both diploid (r-0.470, df-5) and tetraploid. genotypes
(r-0.20, df-lO), but neither was significant at the P<0.05 level. The
tetraploids averaged more chloroplasts per guard cell than the diploids
(5.3 vs. 3.9), although tetraploid line 299051-1 fell within the range of
the diploids (Figure l). The diploid guard cells appeared to reach a
threshold in chloroplast number per cell which was not strongly associated
with cell length, whereas the tetraploid genotypes seemed to reach a
threshold in cell length but not chloroplast numbers (Figure 1).

Spongey mesophyll cells of tetraploids generally had larger face
areas and more chloroplasts per cell than diploids (Tables 2 and 3;
Figures 2 and 3). There was not a significant correlation between spongey
mesophyll cell size and chloroplast number among the greenhouse grown
genotypes (2N: r-0.093, 4N: r-0.501). However, cell sizes and plastid
numbers were significantly correlated among plants grown in the growth
chamber (2N: r-0.829, 4N: r-0.949). Chloroplast numbers and cell face
areas within individual genotypes of spongey mesophyll cells of both

greenhouse

ll

Figure 1. A plot of guard cell length (p) and number of chloroplasts per
cell for seven diploid and twelve tetraploid alfalfa genotypes. Each
point represents the mean of thirty cells. Diploids, r-0.470, not

significant; tetraploids, r-0.20, not significant.

12

Chloroplasts/cell

13

 

 

 

 

 

 

' 2n " 4n
I
I
I
I I
n+299051 - '
10 12 14 16 1a 20

Cell length (pm)

22

14

Figure 2. A plot of mesophyll cell face area (umz) and number of
chloroplasts per cell for five diploid and ten tetraploid alfalfa
genotypes grown in a greenhouse (see text for details). Each point
represents the mean of fifty cells. Diploids, r-0.093, not significant;

tetraploids, r-0.501, not significant.

Chloroplasts/cell

26

24

22

20

18

16

14

12

10

260

15

 

2n'4n

 

 

 

 

300 340 380 420 460

Cell face area (pmz)

 

16

Figure 3. A plot of mesophyll cell face area (pmz) and number of
chloroplasts per cell for six diploid and nine tetraploid alfalfa
genotypes grown in the growth chamber (see text for details). Each point
represents the mean of fifty cells. Diploids, r-0.829; tetraploids,

r-0.949 , (P<0.05) .

Chloroplasts/cell

l7

 

25

Zn '4n

 

 

20

15

10

 

 

 

220

260 300 340 380

Cell face area (pmz)

420

460

18

Table 2. Mean cell face area and chloroplast number in spongy mesophyll
cells of different genotypes of alfalfa grown in a greenhouse

(see text for details).

 

 

Chloroplasts
Ploidy Genotype Face area (#2) No. per cell Density’
2N 235021 330abz 15.6c 21.1a
DDCZx-S 266a 12.2a 21.8a
172989 300ab 13.lb 22.9a
262532 362b 12.6ab 28.7b
W70-22-5 380b 12.7ab 29.9b
4N 299055 350a 19.4ab 18.0a
239953 340a 17.8ab 19.1a
W7l-42-2 488bc 23.7b 20.6ab
172983 390a 17.2a 22.7b
299049 473bc 20.3b 23.3b
DDC4x-l 512c 21.8b 23.5b
299051-2 402ab 16.3a 24.7bc
251205 522C 18.9ab 27.6c
Vernal 428ab 15.4a 27.8c
253443 512c 17.7ab 28.9c

 

’Density is cell face area divided by chloroplast number.

'Means within columns sharing the same letter are not significantly

different at the 51 level using the Duncan's Multiple Range Test.

comparisons are within ploidy.

Mean

 

19

Table 3. Mean cell face area and chloroplast number in spongy mesophyll

cells of different genotypes of alfalfa grown in the growth

 

 

 

 

chamber.
Chloroplasts
Ploidy Genotype Face area (p2) No. per cell Density’
2N 235021 263a' 13.3a 19.8a
172989 232a 11.4abc 20.3ab
251689 237a ll.lbc 21.3ab
DDC2x-5 255a 11.5abc 21.4ab
W70-22-5 238a 10.8bc 22.0ab
262532 231a 9.7c 23.8b
4N 299051-1 262c 13.7c 19.2a
DDC4x-l 436a 21.2a 20.4a
239953 288bc 13.7c 21.4ab
W71-42-1 321abc 14.9bc 21.8ab
253443 415ab 18.9a 22.0ab
251205 452a 20.4a 22.1ab
299048 332abc 14.2bc 23.3b
299049 452a 19.2a 23.5b
172983 425a 17.8ab 23.8b

 

yDensity is cell face area divided by chloroplast number.
zMeans within columns sharing the same letter are not significantly
different at the 52 level using the Duncan's Multiple Range Test. Mean

comparisons are within ploidy.

20
and growth chamber grown plants were all significantly correlated (P<
0.05).

In. general, the spongey' mesophyll cells of the diploids and
tetraploids had distinct chloroplast numbers, but there were some
overlaps. In the greenhouse grown plants, diploid genotype 235021 had
15.6 chloroplasts per cell while two tetraploid genotypes, ‘Vernal‘ and
299051-2 had 15.4 and 16.3 respectively. In the growth chamber grown
plants, diploid genotype 235021 had 13.3 chloroplasts per cell, while
tetraploid genotypes 299051-1 and 239953 both had 13.7 chloroplasts per
cell (Table 3).

There was significant variation among genotypes in plastid density
and in many cases, genotypes with similar sized cells had very different
plastid numbers (Tables 2 and 3). For example, in the greenhouse grown
diploid genotypes 235021 and 262532, chloroplast number per cell was 15.6
and 12.6 even though their cell face areas were very similar (330 pm? and
362 um?). The tetraploid genotypes 253443 and DDC 4x-1 both had a cell
face area of 512 pm?, yet their chloroplast numbers per cell were 17.7 and
21.8 respectively. Among the growth chamber grown tetraploids, genotype
299051-1 had a chloroplast density (19.2) that was significantly different
from.genotypes 172983 (23.8), 299048 (23.3) and 299049 (23.5). Chloroplast
numbers per cell also varied significantly among growth chamber grown
diploids even though their cell sizes were generally similar. For
example, genotype 235021 had 13.3 chloroplasts per cell and 262532 had
9.7.

The plastid density of individual genotypes was not differentially
affected by light levels (Table 4), however, the genotypes did show

significant variations in their means (Table 5). The same relative

21

 

 

Table 4. Comparison of diploid and tetraploid genotypes at low light
levels (LL), 400 pmoles/sec/cmz, photosynthetic photon.f1ux
(PPF), and high light levels (HL), 900 pmoles/sec/cm? PPF.
Chloroplast number - CPT No., cell face area - CFA,
chloroplast face area - CPFA, chloroplast density (CFA/CP No.)
- CPD and total plastid area per cell area - TPA/CFA.
2N Genotypes CPT No. CFA CPFA CPD TPA/CFA

262532 (LL) 13.7 383.7 21.4 28.1 0.76
(HL) 13.9 405.6 20.7 29.9 0.71

235021 (LL) 17.0 376.7 15.0 22.6 0.68
(HL) 21.0*' 454.4* 15.3 22.2 0.71

172989 (LL) 13.4 350.2 18.7 26.0 0.71
(HL) 15.6* 405.8 20.2 26.9 0.77

4N Genotypes

239953 (LL) 16.0 350.8 20.0 22.4 0.91
(HL) 18.3* 457.5* 21.8 25.2* 0.87

251205 (LL) 19.8 530.8 20.9 27.3 0.78
(HL) 22.7* 591.6 22.8 26.7 0.87

172983 (LL) 14.9 389.1 19.5 25.8 0.75
(HL) 15.0 382.4 20.5 26.0 0.80

 

'Significant at P<0.05, n-25.

22

Table 5. Mean cell face area (CFA), chloroplast number (CPT No.),
chloroplast face area (CPFA), chloroplast density (CPD) and
total plastid area per cell area (TPA/CFA) in spongy
mesophyll cells of different genotypes of alfalfa grown under
artificial light. Values are averages of replicates grown at

400 and 900 pmoles/sec/cm? PPF.

 

 

Ploidy Genotype CFA (p2) CPT No. CPFA D TPA/CFA
2N 235021 415a‘ 19.0b 15.1a 21.8a 0.69s
172989 378a 14.5a 19.41) 26.0b 0.743
262532 396a 13.8a 21.1b 28.7b 0.73a
4N 239953 404a 17.1ab 20.8ab 23.6a 0.88b
172983 385a 14.9a 20.0a 25.8b 0.77s
251205 561b 21.2b 21.8b 26.5b 0.82ab

 

zMeans within columns sharing the same letter are not significantly

different at the 52 level using the Duncan's Multiple Range Test.

 

23
rankings were displayed under the varying light conditions that were
previously observed in.the greenhouse and growth chamber experiments. The
diploid 235021 had a significantly lower plastid density than 172989 and
262532, and thetetraploid 239953 had a significantly lower plastid
density than 172983 or 251205. Plastid face area was significantly
correlated with chloroplast number in the tetraploid population (r-0.883) ,
although it was negatively correlated in the diploid population (r- -
0.871). The tetraploid genotypes had a greater total plastid area per
unit cell face area (TPA/CFA) than the diploids (4N-0.82, 2N-0.72). There
was significant variation among the tetraploid genotypes with respect to

TPA/CFA but not among the diploid genotypes (Table 5).

RECIPROCAL CROSSES
Mean chloroplast number per cell in 299049-l was 20.9, and in W71-42-
2 was 24.1 (Table 6). Chloroplast density was significantly different
between the two parents (28.0 vs. 22.1, P<0.05) These differences were
mirrored in the selfed crosses (S self-29.7, F self-22.5). Chloroplast
numbers in both reciprocal crosses were not significantly different;
however, chloroplast density was significantly associated with cytoplasmic
source (SxF-25.8, FxS-23.4). In all cases, progenies with S cytoplasms
had higher means than those with F (Table 6).
Chloroplast number, cell face area, chloroplast density, plastid
face area and total plastid area per cell face area were not significantly
different in the shoots from the chimeric plants that contained different

plastid types (SxF 11d and SxF 11a, Table 7).

24

 

 

 

Table 6. Mean cell face area (CFA), chloroplast number (CPT No.),
chloroplast face area (CPFA) and density (CPD) in spongy
mesophyll cells of the accessions 299049-l (S), W71-42-2 (F) and
their self and reciprocal progeny.

Parent or cross CFA (p2) CPT No. CPFA CPD

299049-l (S) 576 20.9 26.8 28.0

W71-42-2 (F) 528 24.1 23.4 22.1*

S self 615 20.9 26.4 29.7

F self 545 24.2*2 21.4 22.5*

SxF 598 23.7 28.2 25.8

FxS 573 24.2 25.0 23.4*

 

' Significant at P-0.05

25

Table 7. Mean chloroplast number (CPT No.), cell face area (CFA),
chloroplast face area (CPFA), and chloroplast density (CPD) of
shoots from chimeric sectors of cross SxF 11. SxF 11d has
chloroplasts containing the S plastome and SxF 11a has

chloroplasts containing the F plastome.

 

 

Sector CPT No. CFA CPFA CPD
plastome
SxF 11d (8) 21.8 567.6 19.7 26.3

SxF 11a (F) 22.7 623.0 21.3 27.3

 

DISCUSSION

ct e atin lastid numbers. Many feel that cell size is the
primary factor that determines chloroplast number per cell. They believe
chloroplasts simply divide until they fill a constant proportion of the
cell surface (Pyke and Leech 1987). Others have proposed that cell size
may set the threshold for a particular number of chloroplasts per cell,
but the tendency to realize that potential is controlled by other factors
(Paolillo and Kass, 1977; Frandsen, 1968; De Maggio and Stetler, 1971).

In this study, we did observe a number of significant, positive
correlations between cell face area and chloroplast number. However,
significant differences were observed in chloroplast densities, and a
number of outlier genotypes were observed with similar cell sizes, but
significantly different plastid densities. This genotypic variation in
plastid density indicates that there may be genes influencing chloroplast
number per cell that are independent of cell size.

In the crosses, F1 hybrids displayed chloroplast number per cell
values intermediate to their parents indicating nuclear control; however,
the individual reciprocal crosses still varied significantly in the
direction of their maternal parent. Normally this would imply that the
plastids themselves exert control over their ultimate densities due to
maternal inheritance of plastids, but Schumann and Hancock (1989)
previously showed that plastids are inherited from the paternal parent in

these populations. It is possible that other cytoplasmic factors are

26

27
regulating plastid densities or our limited population sizes led to
sampling errors. Shoots originating from the distinct sectors of the
chimeric plant displayed no significant differences for any of the
parameters measured. This indicates that control of chloroplast number,
size, density and total chloroplast area must lie within the nucleus or
maternal environment (excluding the chloroplasts).

Ellis and Leech (1985) found that total chloroplast area was
positively correlated with cell size in IIIEIQEE- They suggested that
variation in chloroplast number per cell is due to variation in
chloroplast size and that chloroplast number is inversely regulated by
chloroplast size (Ellis and Leech, 1985). We did find highly significant
(P-0.01) correlations between cell size and chloroplast surface area in
our alfalfa populations, but we only observed a significant inverse
relationship between chloroplast number and size among the diploids.

Light was probably not an important factor influencing plastid
density in this study. When three genotypes (high, medium and low
chloroplast number per cell) of both ploidies were grown under two
different levels of PPF of the greenhouse (900 pmoles/sec/mz) and growth
chamber (400 umoles/sec/mF), the plastid densities of individual genotypes
were significantly different in each of the enviroments, but they
themselves varied little across environments.

While light can induce movement and.inf1uences growth.and.development
of chloroplasts, other work has shown that it has little direct effect on
chloroplast division. Chaly et a1. (1981) found that proplastids and
etioplasts grow and divide in roots and shoot apices where they may
receive little or no light. When spinach leaf discs were precultured in

darkness chloroplasts did divide after exposure to high intensity light,

28

however it was suggested that chloroplast division may depend on high
energy compounds produced from photosynthesis or mitochondrial respiration
rather than direct light (Possingham and Lawrence 1983, Possingham et a1.
1988). In our light experiments, cell sizes were generally larger under
high light, but this had little effect on chlorOplast density.
2glyp1g1gy__§ng__plggtig__gumbg;§. Bingham (1968) found significantly
different numbers of chloroplasts in.diploid.and tetraploid.guard.cells of
alfalfa, and as a result, suggested that chloroplast counts could be used
to determine ploidy level. Based on our results, we do not share Bingham’s
confidence in determining level of ploidy for individual genotypes, since
we found overlap in plastid numbers between the two ploidies. Chloroplast
numbers were more variable among spongy mesophyll cells of diploids and
tetraploids than among guard cells, but there was still considerable
overlap between some 2N and 4N genotypes.

Butterfass (1980) proposed that ploidy level controls chloroplast
number per cell and that a doubling of the ploidy level should result in
a 60-801 increase in chloroplast number per cell. In our comparisons of
cell face area and chloroplast number, there was a smooth transition
between the chloroplast numbers of diploids and tetraploids, rather than
a distinct gap as Butterfass would predict. Such an overlap would not be
observed if nuclear DNA.mass alone regulates chloroplast number per cell.
A similar overlap was seen by by Ellis and Leech (1985) in wheat.
Likewise, Strandring et a1. (1990) found no relationship between genome
size and chloroplast number in tamarillo.

Therefore, the number of chloroplasts found in a polyploid may be
more dependent on the cell size and genotype of the progenitor species

than on ploidy level per se. Molin et a1. (1982) found cell size and

29
chloroplast number to coordinately double in isogenic diploid and
tetraploid lines of alfalfa, but Standring et a1. (1990) found some
trisomics of'pepino to have higher chloroplast numbers than.disomics while
others did not. They concluded that the chromosomes exert varying levels
of control on chloroplast numbers and therefore, some genes have a
stronger effect on chloroplasts per cell than others.

In conclusion, there is often a tight correlation between chloroplast
number per cell and cell face area among diploid and tetraploid genotypes
of alfalfa, but significant differences in plastid density can be found.
This indicates that while the size of the cell wields considerable control
on plastid number, genes still exist which act independently of cell face
area to regulate chloroplast number. Our controlled crosses demonstrated
that Chloroplast number per cell is largely controlled by the nucleus, but

other non-chloroplastic factors appear to play a role.

BIBLIOGRAPHY

BIBLIOGRAPHY

Alberta, 8., D. Bray, J. Lewis, M. Raff, K. Roberts and J. Watson. 1983.
Molecular Biology of the Cell. Garland Pub. Co. pp. 532.

Asahi,Y. and S. Toyama. 1982. Some factors affecting the chloroplast

replication in the moss Elam trichomangg. Protoplasma 112:9-
l6.

Barlow, S.B. and R.A. Cattolico. 1981. Mitosis and cytokinesis in the
Prasinophyceae. I. Mantoniella sguamata (Manton and Parke)
Desikachary. Am. J. Bot. 68:606-615.

Batschauer, A., E. Mosinger, K.Kreuz, I. Dorr and K. Apel. 1986. The
implication of a plastid-derived factor in the transcriptional
control of nuclear genes encoding the light-harvesting chlorophyll
a/b protein. Eur. J. Biochem. 154:625-634.

Bingham, E.T. 1968. Stomatal chloroplasts in alfalfa at four ploidy
levels. Crop Sci. 8:509-510.

Boffey, S.A., J.R. Ellis, G. Sellden and R.M. Leech. 1979. Chloroplast
division and DNA synthesis in light-grown wheat leaves. Plant
Physiol. 64:502-505.

Boffey, S.A. and R.M. Leech. 1982. Chloroplast DNA levels and the
control of chloroplast division in light-grown wheat leaves. Plant
Physiol. 69:1387-1391.

Bottomley, W. and H.J. Bohnert. 1982. The biosynthesis of chloroplast
proteins. Encyclopedia of Plant Phys. 143:532-596.

Butterfass, T. 1973. Control of plastid division by means of nuclear DNA
amount. Protoplasma 76:167-195.

1979. Patterns of chloroplast reproduction: A. developmental
approach to protoplasmic plant anatomy. Cell Biology monographs
Vol. 6, Springer-Verlag, N.Y.

1980. ‘The continuity of plastids and the differentiation.of plastid
populations. Chloroplasts, J. Reinert, ed. Springer-Verlag, N.Y.

1983. A nucleotypic control of chloroplast reproduction.
Protoplasma 118:71-74.

30

31

1991. Cell sizes and chloroplast numbers per cell of hemiploid and
polyploid plants. Cytologia 56:473-478.

Busbice, T.H. and.C.P. Wilsie. 1966. Inbreeding,depression.andfiheterosis
in autotetraploids with applications to Mggigggg.§g§12§. Euphytica
15:52-67.

Cattolico, R.A., J.C. Boothroyd and S.P. Gibbs. 1976. Synchronous growth
and plastid replication in the naturally wall-less alga

Qlisthggiscus lutgug. Plant Physiol. 57:497-503.

Chaly, N. and J.V. Possingham. 1981. Structure of constricted
proplastids in meristematic plant tissues. Biol. Cell 41:203-210.

Chaly, N., J.V. Possingham and W.W. Thomson. 1980. Chloroplast division
in spinach leaves examined by scanning electron microscopy and
freeze etching. J. Cell Sci. 46:87-96.

Deane-Drummond, C.E. and C.B. Johnson. 1980. Absence of nitrate
reductase activity in San 9789 bleached barley seedlings (nggegm
gglgarg cv. Midas). Plant Cell Environ. 3:303-307.

DeMaggio, A.E. and D.A. Stetler. 1971. Polyploidy and gene dosage

effects on chloroplasts of fern gametophytes. Exp. Cell Res.
67:287-294.

Edwards, J.W. and G.M. Coruzzi. 1989. Photorespiration and light act in
concert to regulate the expression of the nuclear gene for
chloroplast glutamine synthetase. The Plant Cell, Vol. 1:241-248.

Ellis, J.R. and RWM. Leech. 1985. Cell size and chloroplast size in
relation to chloroplast replication in light-grown wheat leaves.
Planta 165:120-125.

Ellis, J.R., A.J. Jellings and.R.M. Leech. 1983. Nuclear DNA content and
the control of chloroplast replication in wheat leaves. Planta
157:376-380.

Ellis, J.R., 1984. The nuclear domination of chloroplast development.
Sci. Prog., Oxf. 69:129-142.

Feierabend, J. and B. Schubert. 1978. Comparative investigation of the
action of several chlorosis inducing herbicides on the biogenesis of
chloroplasts and leaf microbodies. Plant Physiol. 61:1017-1022.

Frandsen, N.0. 1968. Die Plastidenzahl als Merkmal bei der Kartoffel.
Theor. Appl. Genetics 38:153-167.

Frearson, E.M., J.B. Power and E.C. Cocking. 1973. The isolation,

culture and regeneration of Petunia leaf protoplasts. Dev. Biol.
33:130-137.

32

Gillham, N.W. 1978. Organelle Heredity, Raven Pr., N.Y. Giuliano, G. and
P.A. Scolnick. 1988. Transcription of two photosynthesis
associated nuclear gene families correlates with the presence of

chloroplasts in leaves of the variegated tomato ghost mutant. Plant
Physiol. 86:7-9.

Gruissem, W. 1989. Chloroplast gene expression: How plants turn their
plastids on. Cell, Vol. 56:161-170.

Hancock, J.F. 1992. Plant evolution and the origin of crop species.
Prentice Hall. N.J. pp. 100.

Harpster, M.H., S.P. Mayfield and.W.C. Taylor. 1984. Effects of pigment
deficient mutants on the accumulation of photosynthetic proteins in
maize. Plant Mol. Biol. 3:59-71.

Hoober, J.R. 1984. Cholroplasts, Plenum Pr., N.Y. and London.

Karakashian, S.J., M.W. Karakashian and M. A. Rudzinska. 1968. Electron
microscope observations on the symbiosis of Paramecium Bugggxig and
its intracellular algae. J. Protozool. 15:113-128.

Lamppa, G.K., L.V. Elliot and A.J. Bendach. 1980. Changes in chloroplast
number during pea leaf development. An analysis of a protoplast
population. Planta 148:437-443.

Lesney, M.S., P.W. Callow and K.S. Sink. 1986. A technique for bulk
production of cytoplasts and miniprotoplasts from suspension
culture-derived protoplasts. Plant Cell Rep. 5:115-118.

Lyndon, R.F. and E.S. Robertson. 1976. The quantitative ultrastructure

of the pea shoot apex in relation to leaf initiation. Protoplasma
87:387-402.

Margulis, L. 1971. Symbiosis and evolution. Sci. Amer. 225(2):48-57.

Mayfield, S . P . and W . C . Taylor . 1984 . Carotenoid-deficient maize
seedlings fail to accumulate light-harvesting chlorophyll a/b
bindingprotein (LHCP) mRNA. Eur. J. Biochem. 144:79-84.

McCourt, P., l“ Kunst, J. Browse and C.R. Somerville. 1987. The effects
of reduced amounts of lipid unsaturation on chloroplast
ultrastructure and photosynthesis in a mutant Arabiggpgig. Plant
Phys. 84:353-360.

Molin, W.T., S.P. Meyers, C.R. Baer and L.E. Schrader. 1982. Ploidy
effects in isogenic populations of alfalfa 2: Photosynthesis,
chloroplast number, RUBP, chlorophyll and DNA in protoplasts. Plant
Physiol. 70:1710-1714.

Oelmuller, R. and H. Mohr. 1986. Photo-oxidative destruction of
chloroplasts and its consequences for expression of nuclear genes.
Planta, 167:106-113.

33

Olszewska, M.I., B. Damsz and E. Rabeda. 1983. DNA endoreplication and
increase in number of chloroplasts during leaf differentiation in

five monocotyledonous species with. different 26 DNA. contents.
Protoplasma 116:41-50.

Paolillo, D.J. and J. Kass. 1977. The relation between cell size and
chloroplast number in the spores of a moss, Eglytzighum. J. Exp.
Bot. 28:457-467.

Possingham, J.V. 1980. Plastid replication and development in the life
cycle of higher plants. Ann. Rev. Plant Physiol. 31:113-129.

Possingham, J.V., H. Hashimoto and J. Gross. 1988. Factors that
influence plastid division in higher plants. Division and
segregation of organelles. ed. S. Boffey and A. Lloyd. Society
for experimental biology seminar, series 35. Cambridge pr.

Possingham, J.V. and.M.E. Lawrence. 1983. Controls to plastid division.
International review of cytology. Vol. 84:1-49. Academic pr.

Price, H.J., A.H. Sparrow and A.F. Nauman. 1973. Correlations between
nuclear volume, cell volume and DNA content in meristematic cells of
herbaceous angiosperms. Experientia 29:1028-1029.

Pyke, R.A. and R.M. Leech. 1987. The control of chloroplast number in
wheat mesophyll cells. Planta, 170:416-420.

Ramachandran, D.D., and R.K.J. Narayan.. 1985. Chromosomal variation in
Qucumis. Theor. Appl. Genet. 69:497-502

Reiss, T. R. Bergfeld, G. Link, W. Thien and H. Mdhr. 1983. Photo-
oxidative destruction of chloroplasts and its consequences for
cytosolic enzyme levels and plant development. Planta, 159:518-528.

Rose, R.J. , D.G. Cran and J.V. Possingham. 1975. Changes in DNA
synthesis during cell growth and chloroplast replication in greening
spinach leaf disks. J. Cell Sci. 17:27-41.

Sagar, A.D., B.A. Horwitz, R.C. Elliott, W.F. Thompson and W.R. Briggs.
1988. Light effects on several chloroplast components in
norflurazon-treated pea seedlings. Plant Physiol. 88:340-347.

Schumann, G.M. and.J.F. Hancock. 1989. Paternal inheritance of plastids
in Medigggo sativa. Theor. Appl. Genet. 78:863-866.

Schumann, G.M. 1988. Master of Science thesis. Michigan State Univ. E.
Lansing, Mi.

Scott, N.S. and J.V. Possingham. 1980. Chloroplast DNA in expanding
spinach leaves. J. Exp. Bot. 31:1081-1092.

34

1982. Manipulation and expression of genes in eukaryotes. P.
Nagey, A.W. Linnae, J.W. Peacock and J.A. Pateman, eds. Academic
Pr., N.Y.

Scott, N.S. and J.N. Timmis, 1984. Homologies between nuclear and plastid
DNA in spinach. Theor. app. gen. 67:279-288.

Sears, 3.3., 1983. Genetics and evolution of the chloroplast. Stadler
symp. Vol. 15 (1983), Univ. of Mo., Columbia. p.119-l39.

Simpson, J., M. Van Montague and L. Herrera-Estrella. 1986.
Photosynthesis associated gene families: differences in response to
tissue specific and environmental factors. Sci. 233:34-38.

Slankis, T. and G.P. Gibbs. 1972. The fine structure of mitosis and cell
division in the alga Qchromonag danigg. J. Phycol. 8:243-256.

Standring, L.S., G.J. Pringle and 3.0. Murray. 1990. The control of
chloroplast number in Solanum muricatm Ait. and W We
(Cav.) Sendt. and its value as an indicator of polyploidy.
Euphytica 47:71-77.

Taylor, W.C., 1989. Regulatory interactions between nuclear and plastid
genomes. Annu. Rev. Plant Physiol. Plant Mol. Biol., 40:211-233.

Von Wettstein, D. 1981. Chloroplast and nucleus: Concerted interplay
between genomes of different cell organelles. In International Cell
Biology, 1980-1981. H.G. Schweiger, ed. N.Y., Springer-Verlag.

Whatley, J.N. 1974. The behaviour of chloroplasts during cell division of
Iggeteg laggggris. New Phytol. 73:139-142.

1980. Plastid growth and division in mm Wis. New
Phytol. 86:1-16.

Whatley, J.M. and B.E.S. Gunning. 1981. Chloroplast development in
5:211; roots. New Phytol. 89:129-138.

"Illllllllllllllllllls