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Boron and Sour Cherry (Prunus cerasus)

presented by

Yufei Xu

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BORON AND SOUR CHERRY (PRUNUS CERASUS)
By

Yufei Xu

A THESIS

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

MASTER OF SCIENCE
Department of Horticulture

2005

ABSTRACT
BORON AND SOUR CHERRY
By
Yufei Xu
In Michigan boron (B) deficiencies in sour cherry have resulted in routine use of B sprays
to enhance fruit set and increase fruit yield. However, field observations indicate that
high B levels are associated with premature softening, making fruit unacceptable for
processing. Our fertilization studies show that fruit B levels are higher but B treatments
generally had little or no effect on fruit size, maturity, color, or pull force. However, in
some locations, B applications increased the number of sofi fruit, especially when harvest
was delayed well after the optimum maturity date (as indicated by pull force). The
results suggest that B-induced yield increases can be achieved without inducing excessive
fruit softening by careful monitoring of fruit maturation and prompt harvest.
Determination of leaf and fruit B concentrations show that fruit B, but not leaf B levels

are a good indicator of tree B status.

ACKNOWLEDGMENTS

I would like to express my most sincere thanks to my major professor and
supervisor, Dr. Wayne Loescher: thank you for your patience, guidance, professionalism,
and providing me an opportunity to take my master‘s study in horticulture as well as an

opportunity to explore American culture.

I would also acknowledge great support from Dr. C. Peter Wolk and Dr. Eric

Hanson.

The following individuals provided advice and assistance to my cherry project,
Dr. Randy Beaudry, Steve Berkheimer, Matt Blanchard, Bill Chase, Dr. James Flore, Dr.
Zhifang Gao, Dr. Sastry Jayanty, Dr. Ning Jiang, Tad Johson, Dr. Alexandra
Kravchenko, Dr. Joseph Kuhl, Dr. James Nugent, Dr. Paolo Sabbatini, Dr. Ken Sink, Dr.

Guoqing Song, Dario Stefanelli and Dr. Steve Van Nocker.

Last but not least, I would like to thank my parents for supporting me all the time,

while I could not do much for them.

iii

TABLE OF CONTENT

LIST OF TABLES .................................................................................... v
LIST OF FIGURES .................................................................................. vi
INTRODUCTION .................................................................................... 1
LITERATURE REVIEW ............................................................................ 4
MATERIALS AND METHODS ................................................................. 23
RESULTS AND DISCUSSION .................................................................. 27
Foliar application of B influences fi'uit B concentration .................................. 27
Pit B levels ....................................................................................... 34
Leaf B levels .................................................................................... 38
Effect of the previous year’s treatment ...................................................... 40
Splat test ......................................................................................... 44
LITERATURE CITED ............................................................................ 48

iv

LIST OF TABLES

Table 1. B treatments from 2002 to 2004 ................................................ 24

Table 2. Amount of mesocarp B (pg) per fruit in 2004.

Treatments as described in ‘Materials and methods’. Data shows means : SE of 10
replicates. DAFB= Days After Full Bloom. Means with the same letter are not
significantly different from each other ................................................... 35

Table 3. Amount of pit B (pg) per fruit in 2004.

Treatments as described in ‘Materials and methods’. Data shows means j; SE of 10
replicates. DAFB= Days After Full Bloom. Means with the same letter are not
significantly different from each other .................................................... 35

Table 4. Amount of leaf B (pg) per leaf in 2004.

Treatments as described in ‘Materials and Methods’. Data shows means 3; SE of 10
replicates. DAFB= Days After Full Bloom. Means with the same letter are not
significantly different from each other .................................................... 38

Table 5. Splat test results in 2003. .

Splat tests were performed within 12 h after fruits were sampled from HTRC. Good
fruit were defined as fruit which were not cracked and still firm after the splat test.
................................................................................................. 45

Table 6. Splat test results in 2004.

Splat tests were performed within 12 h after fruits were sampled from HTRC. Good
fruit were defined as fruit which were not cracked and still firm after the splat test
.................................................................................................. 45

LIST OF FIGURES

Figure 1. 2002 fruit quality parameters ........................................................... 28
Figure 2. 2003 fruit quality parameters ........................................................... 29
Figure 3. 2004 fruit quality parameters ........................................................... 30
Figure 4. Amount of mesocarp B (pg) on a per fruit basis, 2003 .............................. 32
Figure 5. Amount ofmesocarp B (pg) on a per gram ofdry weight basis, 2003...... ......32
Figure 6. Amount of mesocarp B (pg) on a per fruit basis, 2004 .............................. 33
Figure 7. Amount of mesocarp B (pg) on a per gram of dry weight basis, 2004. . .33
Figure 8. Amount of pit B (pg) on a per fruit basis, 2003 ..................................... 36
Figure 9. Amount of pit B (pg) on a per gram of dry weight basis, 2003 ................... 36
Figure 10. Amount of pit B (pg) on a per fruit basis, 2004 ..................................... 37
Figure 11. Amount of pit B (pg) on a per gram of dry weight basis, 2004 ................... 37
Figure 12. Amount of B (pg) on a per leaf basis, 2004, after 2004 B spray ................. 39

Figure 13. Amount of B (pg) on a per dry weight (g) basis, 2004, after 2004 B spray. . .39

Figure 14. Amount of mesocarp B (pg) on a per fruit basis, 2004, before 2004 B spray
started ................................................................................................. 40

Figure 15. Amount of mesocarp B (pg) on a per fruit basis, 2004, before 2004 B spray
started ................................................................................................. 41

Figure 16. Amount of B (pg) per leaf, 2004, before 2004 B spray started ................... 41

Figure 17. Amount of B (pg) per gram of dry weight, 2004, before 2004 B spray started.
.......................................................................................................... 42

Figure 18. Amount of bud B (pg) on a per gram of dry weight basis, 04/20/2004, before
2004 B spray started ................................................................................. 42

Figure 19. Amount of bud B (pg) on a per gram of dry weight basis, 04/20/2004, before
2004 B spray started ................................................................................. 43

vi

Figure 20. Amount of flower B (pg) on a per gram of dry weight basis, 04/30/2004,

before 2004 B spray started ....................................................................... 43
Figure 21. Percentage of good fruit of sour cherry afier splat test in 2003 ................. 46
Figure 22. Percentage of good fruit of sour cherry after splat test in 2004 ................. 46

vii

INTRODUCTION

The interest of biologists in B has largely been focused on its role in plants where B was
first established as essential in 1923 (Warington, 1923). Evidence that B has a biological
role in other organisms was later indicated by the establishment of essentiality of B for
diatoms (Smyth and Dugger, 1981) and cyanobacteria (Bonilla et al. 1990; Bonilla et a1.
1995). Recently, B was shown to stimulate growth in yeast (Bennett et al. 1999) and to be
essential for zebrafish (Danio rerio) (Eckhert and Rowe, 1999) and possibly for trout
(Oncorhynchus mykiss) (Eckhert, 1998; Rowe et al. 1998), frogs (Xenopus laevis) (Fort et
al. 1998), and mouse (Lanoue et al. 1998). There is also preliminary evidence to suggest

that B has at least a beneficial role in humans (Nielsen, 2000).

B and sour cherry

Soft fruit has become an increasingly common problem in the sour cherry industry in
Michigan. Affected fruit often rupture during mechanical harvesting or lose their integrity
during pitting and processing. This reduces yields of usable pitted cherries, and may
render entire lots unsuited for processing. Soft fruit may be rejected by processors or
simply not harvested. The economic losses resulting from the soft fruit problem in
Michigan, the state that ranks first in the nation in the production of sour cherry, have
averaged 86.3 M a year, with more severe losses averaging $14.3 M in 1992, 1995 and

1998.

B has been considered as the cause of the soft fruit problem. Guyer (unpublished)

observed a positive correlation between soft fruit and leaf B concentrations. Flore

(unpublished) showed that foliar B sprays increased splitting, one symptom of soft fruit.
All this suggested high plant B levels may directly or indirectly contribute to the soft
cherry problem. Nonetheless, B use in Michigan cherry orchards has increased over the
past decade and B application on sour cherry is now common in industry because
growers believe that B application increases fruit set. Studies on sour cherry showed that
fruit set and production of sour cherry trees containing leaf B levels of 19 to 25 pg‘ g 7'
dry weight can ofien be increased by B applications, although the mechanism by which B
influences fruit set is unknown (Hanson, 1991a). However, the increase in fruit set and
production is not always observed. In Hanson‘s experiments (1991b), an increase as
much as 100% was found in one trial while no increases were reported in several other
trials. Current Michigan recommendations call for B applications when cherry leaf tissue
contains less than 30 ppm of B. However, leaf B level may not be a good standard to

monitor tree B level. In addition, cherries and most other tree fruit crops are considered

sensitive to high soil B levels (Anonymous, 2000).

Besides sour cherry, B applications have had variable effects on fruit quality in many
species, including a reduction in the firmness of prunes (Prunus domestica) (Wojcik,
1999), lowbush blueberries (Vaccinium angustifolium) (Chen et a1. 1998), and an increase
in the tendency of apples to develop internal breakdown and watercore (Bramlage et al.

1962; Martin et al. 1976).

These observations are difficult to reconcile with the recent evidence for borate in cross-

linking cell wall constituents (Matoh et a1. 1993). It is hypothesized that these covalently

cross-linked borate ester linkages affect the assembly or maintain the structure of cell
walls (Fleischer et al. 1999). Although this theory is consistent with the gross anatomical
changes in the walls of B-deficient cells (Spurr, 1957), whether B esters are essential to

the structure integrity of all plant cell walls is unknown.

Another confounding factor is that the mobility of B in the phloem varies greatly among
species. Species exhibiting high mobility of B, including sour cherry (Hanson, 1991a),
utilize sorbitol as a primary transport carbohydrate. Not only sorbitol, but other sugar
alcohols such as mannitol (Loescher and Everard, 2000) were proved to form complexes
with B, suggesting a way to facilitate transport of B in the phloem (Bellaloui et al. 1999,
Brown and Hu, 1996). When B supplies are abundant, polyol translocating species with
consequent high B mobility may accumulate high levels of B in sinks such as fruits
(Bellaloui et al. 1999). Alternatively, high B levels may facilitate transport of polyols,
contributing to fruit osmotic potential (and internal water potential) and thus a

susceptibility to splitting, a problem related to the soft fruit.

In this project, we use sour cherry fruit as a model system to study the involvement of B
in fruit quality and maturation. Our hypothesis was that B levels affected soft fruit. We

tested the hypothesis by measuring several fruit quality parameters.

LITERATURE REVIEW

B and the Plant Cell Wall

The role of B in plant cell walls has recently been reviewed extensively by O’Neill et a1.
(2004). Part of this section is a synopsis of that review. B‘s role has long been believed
to be related to the plant cell wall. An early symptom of B deficiency in flowering plants
is the formation of primary walls that have abnormal morphology and mechanical
properties (Dell et al. 1997). Further evidence comes from a study of species variability
in B requirement, when B content was shown to be positively correlated with cell wall
pectin (Hu et al. 1996). For example, in the Poaceae, whose primary walls contain
quantitatively small amounts of pectin, B requirements are much lower than the
dicotyledons and nongraminaceous monocotyledons. All this has suggested a relationship

between B and primary wall pectic polysaccharides (Hu et al. 1994; Matoh et al. 1996).

The chemical structure of primary cell wall pectins

Pectins are a family of complex polysaccharides that contain 1,4-linked a-D-
galactosyluronic acid (GalpA) residues. The major pectic polysaccharides isolated and
structurally characterized from the primary cell walls of gymnosperrns and angiospenns
are homogalacturonan (HG), rhamnogalacturonan I (RG-I), and the substituted
galacturonan (SG) including rhamnogalacturonan II (RG-II) (Ridley et al. 2001 ).
Homogalacturonan (HG) is mostly a linear chain of 1,4-linked a-D-
galactopyranosyluronic acid (GalpA) residues in which some of the carboxyl groups are
methyl esterified, and a few are partially O-acetylated at C-3 or C-2.

Rhamnogalacturonan-I (RG-I) is a family of pectic polysaccharides that contain a

backbone of the repeating disaccharide [—+4)-a-D-GalpA-(1-—+2)-a-L-Rhap-(1—->].
Substituted galacturonans (SG) are a diverse group of polysaccharides that contain a
backbone of linear 1,4-linked a-D-GalpA residues (O‘Neill et al. 1990).
Rhamnogalacturonan II (RG-II) belongs to SG and it is found in all higher plant primary
walls analyzed to date (O‘Neill et al. 1990). More detail will be provided in the section

entitled ‘RG-II’.

These three polysaccharides are covalently linked to one another to form a pectic
macromolecule. A covalent link between RG-II and HG is highly likely because they
both have backbones composed of 1,4-linked u—D-GalA resides. Additional evidence that
RG-II is covalently linked to HG to form a high-molecular—weight (>100kDa) complex
was obtained by characterizing the material that aqueous buffers solubilized from sugar
beet (Beta vulgaris L.) (Ishii et al. 2001), Chenopodium album (Fleischer et al. 1999),
and Arabidopsis cell walls (Reuhs et al. 2003). Further covalent and non-covalent cross-
linking of some glycosyl residues in this macromolecule forms a three-dimensional pectic
network. For example, Ca2+ forms ionic cross-links between some of the carboxylates of
the GalpA residues in HG. A recently discovered covalent HG cross-link involves B. The
first B-polysaccharide complex was isolated and characterized from radish roots by
Matoh and his colleagues in 1993. It was proved to be a B-rhamnogalacturonan-II
complex (Matoh et al. 1993). In 1996, the same complex was also found in sugar beet
pulp by the same research group (Ishii and Matoh, 1996). Moreover, its chemical
structure was characterized to be a cross-linked borate-diol ester (Kobayashi et a1. 1996).

This covalent cross-linking of RG-II and the Ca2+-dependent ionic cross-linking of HG

combine to form a stable three-dimensional pectic network in muro. There are in primary
cell walls two other networks, the load-bearing cellulose microfibrils and the structural
glycoproteins. The interactions within and between these networks give the wall its

mechanical strength.

RG-II

RG-II was first identified in 1978 as a structurally complex yet quantitatively minor
polysaccharide that is solubilized by endopolygalacturonase (EPG) treatment of
suspension-cultured sycamore cell walls (Darvill et al. 1978). RG-II belongs to a group of
pectic polysaccharides referred to as substituted galacturonans. A common feature of this
group is that these polysaccharides all have a backbone composed of linear 1,4-linked a—

D- GalpA residues.

A localization study showed that RG-II is distributed throughout the primary wall and
that regions of the wall that are close to the plasma membrane may be somewhat enriched
with RG-II, while little if any RG-II is detected in the middle lamella (Matoh et al. 1998).
RG-II is ubiquitously found in the cell walls of all gymnosperms and angiosperrns. It has
been isolated from cell walls of a variety of plants including suspension-cultured
sycamore cells (O‘Neill et al. 1996), etiolated pea stems (O‘Neill et al. 1996), sugar beet
(Beta vulgaris) pulp (Ishii et al. 1996), apple fruit (Doco et al. 1997), carrot tuber (Doco
et al. 1997), tomato fruit (Doco et al. 1997), bamboo (Phyllostachys edulis) shoot
(Kaneko et a1. 1997), ginseng (Panax ginseng) leaf (Shin et al. 1997), radish and rice

roots (Matoh et al. 1998), cultured tobacco cells (Matoh et a1. 1998), red clover root

nodules (Matoh et al. 1998), red pine (Pinus densiflora) (Shimokawa et al. 1999),
suspension-cultured Chenopodium album cells (Fleischer et al. 1999), grape berry (Vidal
et al. 2001), pumpkin leaf (Ishii et al. 2001), red beet (Beta vulgaris L var conditiva)

(Strasser et a1. 2001), and lily pollen (Holdaway-Clarke et al. 2003).

RG-II accounts for between 1% and 4% of the pectin-rich primary walls of dicots, non-
graminaceous monocots, and gymnosperms, but less than 0.1% of the pectin-poor
primary walls of the Poaceae (Matoh et al. 1996). This is consistent with the observation

that Poaceae crops require relatively low B.

RG-II accounts for between 0.2% and 2% of the walls of pteridophytes and lycophytes,
which is of a similar order of magnitude to the amounts of RG-II present in the primary
walls of angiosperrns and gymnosperms. The amounts of borate cross-linked RG-II
present in the sporophyte primary walls of members of the most primitive extant vascular
plant groups (Lycopsida, F ilicopsida, Equisetopsida, and Psilopsida) are comparable with
the amounts of RG-II in the primary walls of angiosperrns. By contrast, the gametophyte
generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and
Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of
the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide.
There are data indicating that the amount of RG-II incorporated into the walls of plants
increased during the evolution of vascular plants from their bryophyte-like ancestors
(Matsunaga et al., 2004). Thus, the acquisition of a B-dependent growth habit, may be

correlated with the ability of vascular plants to maintain upright growth and to form

lignified secondary walls. The glycosyl sequence of RG-II remains essentially unchanged
in all spore- and seed-bearing tracheophytes that have been examined to date. The
conserved structures of pteridophyte, lycophyte, and angiosperrn RG-IIs suggest that the
genes and proteins responsible for the biosynthesis of this polysaccharide appeared early
in land plant evolution and that RG-II has a fundamental role in wall structure

(Matsunaga et al., 2004).

So far, nothing is known about RG-II metabolism during fruit ripening. However, the
primary cell walls of the suspension-cultured cells from kiwifi'uit (Actinidia deliciosa)
contained twice the amount of RG-II found in the cell walls of the same intact fruit, while
the composition of RG-II glycosyl residues were very similar in both cultured cells and

the intact kiwifruit (Fischer, 1996).

Borate cross-linking of RG-II

B has the ability to form monoesters and diesters with compounds containing cis-
hydroxyl groups, resulting in enhanced acidity and a negatively charged complex (Lewis,
1980; Loomis and Durst, 1991). A polysaccharide such as rhamnogalacturonan II has
such propertites. IlB-NMR spectroscopic analysis of a B-polysaccharide complex
extracted and purified from radish (cv. Aokubi-daikon) roots demonstrated that most of
the wall-bound B is present as a tetravalent 1:2 borate-diol ester (Matoh et al. 1993).
Later, Kobayashi‘s group showed that the removal of B from the complex reduced the
molecular weight by one-half without causing a significant increase in the number of

reducing end groups (Kobayashi et al. 1996). Their results indicated that B, as boric acid,

links two rhamnogalacturonan II chains together to form the B-polysaccharide complex.

This provides the structural basis for the relationship between B and primary wall pectins.
Subsequent studies confirmed that borate cross-links two chains of RG-II to form a dimer
in the primary walls of angiosperms, gymnosperms (Shimokawa et al. 1999), lycophytes,

and pteridophytes (Matsunaga et al. 2004).

Borate can cross-link molecules because it contains two pairs of hydroxyl moieties that
can form reversible diester bonds with molecules containing cis-diols in a favorable
conformation. Borate esters are believed to form with apiosyl residues of RG-II since
apiose is the only component of RG-II which has the B-D-erythrofuranose configuration
ready to form an ester. One common belief is that two molecules of RG-II are cross-
linked by a single borate diester. One alternative model is that RG-II dimer contains two
B atoms. If such a dimer exists, it would contain one borate diester cross-linking the
apiosyl residue of each side chain A of RG-II and a second that cross-links the apiosyl
residue of each side chain B of RG-II. However, the existence of such a model is only
partially proven by 13C NMR spectroscopic analyses and still awaits further proof (Ishii
and Ono, 1999). In that model, the borate diol diesters of methyl beta-D-apiofuranoside

are present as two diastereomers in approximately equal molar ratios.

Studies with mutant plants further confirmed the relationship between B and RG-II.
Experiments on Arabidopsis murI mutant and tobacco nolac-H18 mutant (Iwai et al.
2002) showed that a seemingly small change in the structure of RG-II could dramatically

reduced its ability to form a borate cross-linked dimer and that these structural changes

adversely affected plant growth and development. Arabidopsis plants carrying the murI
mutation are dwarfed and have brittle stems. This results from the fact that about 50% of
the RG-II in the rosette leaves of murl plants is cross-linked by borate, while at least
95% of the RG-II is cross-linked in wild-type plants. This suggested that the dwarf
phenotype and brittle tissue of mur] plants was a consequence of altered RG-II structure
and therefore its reduced cross-linking (O‘Neill et al. 2001). In addition, the tobacco
nolac-H18 mutant (nonorganogenic callus with loosely attached cells), artificially
generated by T-DNA transformation, has defects in the glucuronic acid of
rhamnogalacturonan II of pectin, suggesting that the mutation drastically reduced the

formation of borate cross-linking of rhamnogalacturonan II ( Iwai et al. 2002).

Experiments with a borate transporter (BORl) also confirmed the relationship between B
and RG-II. In the wild type, about 90% of RG-II was present as the dimeric form (dRG-
II-B), both at low and sufficient B supply. In the bar] -1 mutant, about 60% of RG-II was
in its monomeric form (mRG-II) at low B supply, whereas more than 85% of it was
present as dRG-II-B at sufficient B supply. However, similar to the wild type, mRG-II
derived from the borI-I mutant was able to form dRG-II-B in vitro in the presence of
borate and lead. Sugar composition of cell wall fractions was similar in both genotypes.
This suggests that the polysaccharide composition in the cell wall was not strongly
affected by the bar] -1 mutation. The observed difference in dimerization of RG-II at low
B supply is most likely due to a reduced B concentration in the shoots of the bar] -1

mutant (Noguchi et al. 2003).

10

Borate-dependent molecules are not limited to RG-II. There are B-polyhydric alcohol
complexes identified from phloem extracts (Hu et al. 1997), a bacterial signaling
molecule and its sensor protein (Chen et al. 2002), as well as several antibiotics (Hunt,

2003)

B MOBILITY

B deficiency in crops is more widespread than deficiency of any other micronutrient.
Nutritional disorders in vegetables include brown heart in rutabaga, turnip and radish
roots, and hollow stem in cauliflower and broccoli (Shelp et al. 1995). The occurrence of
these disorders even when B is in ample supply suggests that they are physiological in
nature and related to the mobility of B in the plant (Shelp et al. 1995). The relative
mobility of an element within a plant has important physiological and agricultural
implications. It is because the ability of a plant species to survive or to yield optimally
during a period of nutrients stress is a consequence of both its ability to obtain nutrients
from the soil under limiting conditions and the extent to which the nutrients can be

supplied through redistribution from other tissues within the plant.

B is generally considered to be phloem immobile. It has been observed for years that
plants grown with an adequate B supply have B concentrations that decrease from old
leaves to young leaves. B deficiency symptoms typically occur in meristematic tissues,
while B toxicity symptoms occur first in margins of old leaves. All this indicates that B is
an immobile element in some species. For example, there is considerable experimental

evidence that in some species B is almost immobile. In squash and tomato, B deficiency

ll

symptoms developed rapidly upon transfer of plants from B-replete to B-deficient growth
conditions. The leaf B content established prior to transfer to B-deficient conditions did
not decrease, while the growth of apical tissues was completely inhibited (Oertli, 1993;
Hu and Brown, 1994). B cannot readily be redistributed in most species, therefore even a
brief disruption in soil B supply results in growth depression and yield loss. The extent of
the damage depends upon the duration of the deficiency and the stage of plant growth at
which it occurs (Dell and Huang, 1997). However, this does not occur in plants which
produce sugar alcohols such as sorbitol (Brown and Hu, 1996). Such plants mainly

include species within the genera Pyrus, Malus and Prunus.

Although in some plants B has been proved to be immobile, it does not exclude the
possibility of B transport in the phloem. In broccoli (Brassica oleracea var. italica),
Shelp (1988) found B concentrations in the phloem sap higher than in the xylem sap.
Shelp and coworkers (Shelp et al. 1987; Shelp and Shattuck, 1987; Shelp, 1988) also
reported that the ratios of B concentrations in developing sinks to those in old leaves
were higher with a continuous supply of growth-limiting B than with adequate B.
Campbell et al. (1975) also concluded that transport of B to developing peanut fruit
occurs in the phloem. Therefore, immobility of B in some plants may be the result of
formation of stable and immobile B complexes within the cell (Loomis and Durst, 1992;
Brown and Hu, 1994). However, the extent of B mobility found in plants in a particular
experiment is affected by the ability of the leaves to absorb boric acid, the size and
photosynthetic activity of their source leaves, and the strength of different sinks. Genetic

variability can also be considerable (Stangoulis et al. 2000).

12

Experiments with broccoli and lupin explored whether B retranslocation depends on
plant-B status and extemal-B supply. B acquired during inflorescence development was
an important source of B for reproductive structures, but the relative importance of B
acquired before and after inflorescence emergence appeared to be species dependent. The
occurrence of B retranslocation was not dependent upon the induction of B deficiency

(Marentes et al. 1997).

B and sugar alcohols

B mobility is closely related to the presence of polyols. The pattern of B distribution
within shoot organs and the translocation of foliar-applied, isotopically-enriched 10B was
studied using six tree species including almond (Prunus amygdalus 8.), apple (Malus
domestica B.), nectarine (Prunus persica L. B), fig (Ficus carica L.), pistachio (Pistacia
vera L.) and walnut (Juglans regia L.). In species in which sorbitol is a major sugar
(sorbitol-rich) such as almond, apple and nectarine, B is freely mobile. But, in sorbitol-
poor species, those that produce little or no sorbitol such as fig, pistachio and walnut, B is
largely immobile. Together with the evidence that B forms a stable complex with
sorbitol in sorbitol-rich species, it is suggested that B mobility is mediated by the

formation and transport of B-sorbitol complexes (Brown et al. 1996).

Introduction utilizing molecular techniques of the gene for sorbitol synthesis into a
species can enhance the within-plant nutrient mobility of B (Brown et a1. 1999).
Enhancing sorbitol synthesis by transforming plants with sorbitol-6-phosphate

dehydrogenase gene, a key enzyme for sorbitol production, can facilitate phloem B

13

transport in both tobacco and rice (Bellaloui et al., 1999; Bellaloui et al., 2002). An
increase in B uptake and mobility may contribute to an overall improvement in tolerating
low-B soils and B deficiency (Brown et al.1999). Moreover, in B-immobile plants such
as tobacco, the transgenic enhancement of within-plant nutrient mobility could be a

viable approach to improve plant tolerance of nutrient stress.

A variety of B-polyol complexes have been isolated and characterized from higher plants.
The first successful isolation and characterization of soluble B complexes from higher
plants were accomplished in 1997 (Hu et al. 1997), from the phloem sap of celery (Apium
graveolens L.) and the extra-floral nectar of peach (Prunus persica L.). In celery phloem
sap, B was present as the mannitol-B-mannitol complex. Molecular modeling further
predicted that this complex is present in the 3,4 3’,4’ bis-mannitol configuration. In the
extrafloral nectar of peach, B was present as a mixture of sorbitol-B-sorbitol, fructose-B-
fructose, or sorbitol-B-fructose. These findings provided a mechanistic explanation for

the observed phloem B mobility in these species (Hu et al, 1997).

However, recent studies are somewhat contradictory. A study of deciduous forest trees
proved that B mobility does not require the presence of polyols as expected, and it
appeared that to some degree remobilization occurs in many plant species ( Lehto et al.
2004). Extensive B mobility was found in Sorbus aucuparia, Prunus padus and Ulmus
glabra. The first two species contain high levels of sorbitol, while in Ulmus glabra, only
trace amounts of B-complexing polyols were detected. A medium level of B mobility was

observed in growing leaves in Alnus incana, Fraxinus excelsior, Betula pubescens and

14

Larix sibirica after 10B isotope labeling of mature leaves of seedlings. Mannitol in

F raxinus and pinitol in Larix may also complex with B to facilitate remobilization.
Another finding suggesting that B mobility is not closely related to polyol presence is in
Alnus glutinosa which has almost identical concentration of polyols as A. incana, a
closely related species. Yet A. glutinosa did not remobilize B. One explanation is that in
plants with limited B mobility, the small amounts of polyols are not necessarily loaded to
the phloem in mature leaves and unloaded in new leaves, which would be the prerequisite
for B mobility. Sorbitol is closely related to other plant monosaccharides, including other
polyols, and the very small amounts detected may in some cases be transitional phases in

metabolic reaction chains (Lehto et al. 2004).

A more detailed study was conducted by the same group on Scots pine and Norway
spruce (Lehto et al. 2000; Lehto et al. 2004) with no controversial results. loB —enriched
boric acid was applied onto the needles of both species. Small but significant increases in
the loB isotope were found in the new stem and needles of both species, after a dormancy
period and 9 weeks of growth. The increases were given credit to the possible presence of

B-polyol complexes in these polyol-rich species.

Other mechamisms of B phloem mobility

Other mechanisms for B phloem mobility may be involved in plants which do not
produce polyols. Other soluble B complexes may be biologically important (Brown et al.,
2002), such as B-fructose (Hu et al., 1997) and B-malic acid (Dembitsky et al., 2002).

Complex formation with other compounds and their translocation might explain the B

15

mobility in species that do not contain polyols. In addition, multiple mechanisms of B
efficiency were observed even within one species. A study on three cultivars of Canola
(Brassica napus L.) showed that applications with 10B labeled boric acid retranslocated
from older leaves to younger leaves in one cultivar while the sink remained unknown for

the other two cultivars (Stangoulis et al. 2001).

Interestingly, fungi are involved in B mobility in some plants. Experiments using l0B /1 lB
isotope on silver birch showed that B was up by the mycorrhizal mycelia and transported

to the host plant in this species combination (Lehto et al. 2004).

B TRANSPORTER

B uptake

The subject of B uptake was controversial long before the discovery of a B transporter.
There was evidence supporting both active and passive uptake of B in higher plants. The
major form of B exists in living cells as boric acid, a weak acid with pKa’s of 9. 1 4, 12.74
and 13.8. However, at normal cytosol pH, boric acid exists mostly as an uncharged
H3BO3, which should make it easy to permeate cell membranes and thus making active
pumping unnecessary. However, a study on sunflower root B pools suggested a even
more complicated mechanism (Dannel et al. 2000). Control plants precultured with high
B supply (100 pM) showed a linear response of the '0B concentrations in the root cell sap
and in the xylem exudate to the differential short-term 10B supply, and this was not
affected by metabolic inhibitor treatments. In the control precultured with low B supply

(1 pM), the response of the 10B concentrations in the root cell sap and xylem exudate to

16

the differential short-term 10B supply appeared to be a combination of a saturable and a
linear component. This suggested that B uptake into the root symplasm, as well as xylem
loading, are preformed by two transport mechanisms, with the linear components

representing B transport by passive diffusion.

Hu and Brown (1997) proposed that B uptake, under conditions of adequate or excessive
B supply, is the result of passive assimilation of undissociated boric acid (B[OH]3). This
conclusion was based largely on the theoretical predictions of membrane permeability

proposed by Raven (1980); however, accumulation against concentration gradient exists

(Brown et al. 2002).

bar]

A B transporter was first found as a result of study of the Arabidopsis thaliana mutant
borI-I (N oguchi et al. 1997). The mutant was discovered by a defect in root-shoot
translocation of B. Compartmental analysis of B in wild type and bar] -1 mutant plants of
Arabidopsis thaliana proved that the reduced B content in shoots of the mutant plants at
low B supply only were mainly the B contents in the water soluble fractions (cell sap),
but not the B in the water insoluble residue (WIR). The results suggested that the borI-I
mutation has little or no effect on the binding of B in the cell wall, since B in WIRs
mainly represents cell wall bound B (N oguchi et al. 2000). Uptake experiments with 10B-
enriched tracer B demonstrated that B taken up through roots was preferentially
transported to young leaves compared to old leaves in the wild-type plants under a low B

supply. Such a preferential transport to young leaves was not evident in the mutant plants,

17

suggesting that in Arabidopsis thaliana plants B is preferentially transported to young
organs under a low B supply and that this transport process is controlled at least in part
by the BORI gene (Takano et al. 2001). Further analysis using loB showed that roots of
the mutants contain adequate levels of B, while the plants still suffer from reduced B
delivery to shoots due to the impaired xylem loading. The patterns of B increases in root
cell saps in both wild type and the mutant plants are the same, suggesting that uptake into
roots occurs mainly by passive transport. The concentration of tracer B in xylem exudates
of the borI-I plants also followed a linear concentration dependence, whereas in the
wild-type plants a combination of saturable and linear concentration dependence was

observed, suggesting a B transporter in the wild type (Takano et al. 2002).

It was a mutation in the BORI gene that led to symptoms of B deficiency (Takano. 2001).
The BORI locus is located on the lower arm of chromosome 2 (Noguchi et al., 2000). It
is delimited in a 15.1-kilobase (kb) region between newly generated molecular markers at
positions 19,383 kb and 19,399 kb. Nucleotide sequences of this region in the genome of
borI-I and bor1-2 mutants (ethylmethane sulphonate mutants) revealed that each mutant
contains a different single base substitution in the hypothetical open reading frame (ORF)
At2g47160, each causing a different amino-acid substitution in the predicted protein. A
genomic DNA fragment containing wild-type At2g4 7160 was then introduced into the
borl-l mutant and demonstrated to complement the mutation, establishing that

At2g4 7160 corresponds to the BORI gene. Comparison between the cDNA and genomic _
sequences revealed that BORI has 12 exons. On the basis of the nucleotide sequence,

BORI was predicted to encode a polypeptide of 704 amino acids containing 10 putative

18

transmembrane domains. The mutations found in the bar] -1 and bor1-2 alleles were

located within the second transmembrane domain (Takano et al. 2002).

Subcellular localization of the BORI gene product was determined using a construct
containing green fluorescent protein (GFP) under the control of the BORI promoter
region. This suggested that BORl is a plasma-membrane-localized protein, which was
consistent with its putative transporter function. Cell-type specificity expression showed
that BORI localized in the pericycle, located at the outmost layer of the stele and inside

the endoderrnis (Takano et al. 2002).

Phylogenetic analyses showed that BORl is a membrane protein related to the family of
mammalian anion exchangers known as SLC4. Also, this B transporter fell into a clade
with the yeast protein YNL2 75w, human BTRl and six other Arabidopsis proteins:
At1g15460, AtIg74810, At3g06450, At3g622 70, At4g32510 and At5g25430.
Phylogenetically, the yeast transporter seems to be an intermediate between the anion
exchangers and BORI , and so could potentially transport both bicarbonate and borate.
Most importantly, the human BTRl protein, named as being a possible bicarbonate-like
transporter, also falls into the same clade as BORl, suggesting its possible role as a B
transporter (F rommer and Von Wiren, 2002). One other interesting feature of BORl and
its yeast homolog YNL275w shown by the yeast study is that they both have B-efflux

transport activity (Takano et al. 2002).

19

The phylogenetic analysis gave no firm indication of the function of the six other
Arabidopsis proteins. However, some might serve as B transporters that use other
coupling mechanisms and that provide a route, for example, for importing B into cells.
Many years ago, biophysical studies (Lucas, 1975) had indicated that bicarbonate and
borate may use the same transporter. Thus, some of the anion-exchange transporters
similar to YNL275w may transport bicarbonate as well as borate, for example to facilitate
the supply of CO2 for photosynthesis. Study of this transporter family may therefore shed
light not only on the functions of B in metabolism but perhaps also on CO2 movement in
plants. Given the close relationship of bicarbonate and B transporters among anion
exchangers, it could be that relatives of the active bicarbonate transporter from
cyanobacteria may transport borate. Finally, some aquaporins may be permeable to

borate and serve in B transport (Dordas et al. 2001; Ruiz, 2001).

There is another interesting feature of BORl. The surprising similarity of the transport
systems for B and bicarbonate points to a similarity in the binding forms of these two
substrates. In plant cells a high cytoplasmic pH allows the formation of the borate anion,
whereas in kidney cells bicarbonate formation from CO2 is also enzymatically facilitated.
Given the phylogenetic relation between the proteins, the simplest hypothesis is that
BORl also transports anions, and that perhaps borate transport is coupled to the antiport
of a counterion in the same way as bicarbonate. BORl could then function as a
borate/chloride anion exchanger using the chemical gradient established by certain
chloride channels (X-QUAC channels). Alternatively, it could use proton coupling,

instead of chloride coupling, to export borate by a secondary active route. Another

20

possibility is that the negative membrane potential in the pericycle would allow borate
anions to be exported by BORI-mediated uniport (diffusion through a transporter without
coupling to a second ion). Electrophysiological analyses of BORl in various settings

should help to decide this matter (F rommer et al. 2002).

The mammalian homolog of AtBorl was also studied and proved to be a B transporter.
BTRl (Bicarbonate Transporter Related Protein-1) was cloned as a putative bicarbonate
transporter-related protein. BTRl mRNA was reported to be widely expressed in various
tissues, but most strongly in kidney, salivary glands, testis, thyroid and trachea.
Moreover, it may also be responsible for anion transport mechanisms hitherto

unaccounted for in these tissues (Parker et al. 2001).

The mammalian BTRl has unique transport features. In the absence of borate, it conducts
Na+ and OH" (H+). In the presence of borate, BTRl functions as an electrogenic Na+-
coupled borate cotransporter. This is a voltage-regulated, electrogenic transporter with
shallow inward rectification when mediating Na+-B(OH)4‘ influx and with steep outward
rectification when mediating Na+-B(OH)4' efflux. Based on its transport features, Parker
and his colleagues (2001) renamed the transporter as NaBCl. NaBCl (BTRl) plays a
central role in mediating the stimulating and toxic effects of borate on cell growth and

proliferation.

Recently, a novel mutant line 8-21 that requires a high concentration of B for normal

growth has been found in Arabidopsis thaliana (Aoki et al. 2004). Experiments showed

21

that the concentrations of B in the shoot and the root were the same in both wild-type and
the mutant plants, suggesting that the mutant could not utilize B efficiently. Moreover,
Line 8-21 was not allelic to b0r1-1 (Noguchi et al. 1997). A significant portion of F2
plants from the crosses between the wild type and the mutant grew poorly on a low B

media, suggesting segregation of the mutation.

22

MATERIALS AND METHODS

Plant Material for B analysis

Sour cherries (Prunus Cerasus L.cv.Montmorency) trees at the MSU’s Horticulture
Teaching & Research Center (HTRC), Holt, M1 were used. Trees were spaced at 16 feet
(4.88 m) between rows and 12 feet (3.66 m) between trees (227 trees per acre, 561 per
hectare). Trees were planted in May 1988 and grown in Marlette fine sandy loam soil
type. Trees at the HTRC were treated with increasing levels of B from 2002 till 2004 and

sampled following treatments.

B applications

Commercial Solubor DF (greater than 80% sodium pentaborate decahydrate, and less
than 20% boric acid, equivalent to 17.5% elemental B) was used for foliar applications
from 2002 till 2004. Trees were sprayed to the extent that the Solubor DF solution started
to drip. Three levels of B (including the —B control) were applied each year. In 2002, 18
trees on the south end of the cherry tree plot at the HTRC were assigned randomly into
three treatments: 0 ppm (control), 25 ppm, or 50 ppm of B. In 2003, 36 trees on the north
end of the cherry tree plot at the HTRC were assigned randomly into three treatments: 0
ppm (control), 100 ppm, or 200 ppm of B. In 2004, at the HTRC, the 36 trees tested in
2003 were treated with even higher concentrations of B: 0 ppm (control), 400 ppm and
800 ppm. Also, only in 2004, 18 trees at the Clarksville Horticulture Experiment Station
(CHES) were treated with 0 ppm (control), 400 ppm or 800 ppm of B. In 2002, foliar B
sprays were started 29 days after full bloom (AFB) and were continued about every 5

days thereafter unless there was rain predicted on the day of spray. In 2003 and 2004, the

23

first sprays were 26 days and 17 days after the full bloom. The spray intervals of the two
years were 5 days and 7 days, respectively. The sprays began mid May and ended late

June.

Table l. B treatments from 2002 to 2004

 

 

B concentration Spray time Total B a year
(pg/L) (g/tree)
2002 0 From May 22m till July 9‘" 0
South end 25 7 applications 0.25
50 0.50
2003 0 From May 19th till June 27th 0
North end 100 12 applications 1.69
200 3.37
2004 0 From May 24th till June 29‘h 0
North end 400 6 applications . 4.73
800 9.45

 

Two g/ tree a year is the normal amount growers use (Hanson et al. 1987).

Fruit quality assessments

Fruit, 10 per tree, were selected at random on well exposed limbs for determinations of
fresh weight, pulling force, total soluble solids, and drain weight (percentage of pulp
weight over total flesh weight after freezing and thawing; pulp weight = total flesh weight
- juice weight). These parameters were measured at 5-day intervals starting

approximately one month AFB.

Fruit pulling force is the force it takes to remove a fruit from its pedicel. It was measured

with a mechanical force gage (Hunter Spring, Hartfield, Pennsylvania). Total soluble

24

solids were read with a pocket refractometer (Pocket PAL-1, ATAGO). For drain
weights, fruits were pitted prior to freezing at -18°C for 24 h. The frozen fruits were then
thawed in 50 mL Corning tubes containing 4 grams of 3 mm diameter glass beads. The
addition of the beads helped to separate the juice and pulp. Immediately after the fruits
thawed, the tubes were centrifuged at 1,000 g for 5 min. The juice and pulp were
separated and weighed. The drain weight (pulp) was calculated as the percentage of the

pulp weight over total pitted fruit weight.

Colorimetric Analysis of B
Colorimetric analyses of B were performed as described by Lopez (1993) using a

BioSpec-1601 spectrophotometer (Shimadzu) and UVProbe 2.00 software (Shimadzu).

Preparation before conducting colorimetric analysis

Cherry bud, flower, leaf, and fruit were sampled randomly on well exposed limbs, 10 per
tree. Tissues were freeze-dried till constant weight. Samples were ground and an aliquot
of around 0.5 g per sample was weighed, ashed at 550 °C for 6 h and then dissolved in 1

ml of 3 N HNO3,

Splat Test

The splat test is designed to mimic mechanical harvesting. Fruits were dropped two
meters, bouncing twice on 45° inclined wooden boards before they hit the floor. Fruits
were then collected and classified into three categories: good, soft but not cracked, and

cracked.

25

Field plot design and Statistical Analysis
A complete random design (CRD) with subsamples (2 subsamples per plot, 6 plots per

treatment) and repeated measures (sampling days) was used as the field plot design.

Statistical analysis was performed on treatment means using either PROC GLM or PROC

MIXED procedures in SAS version 8.0 (SAS Institute, Cary, NC.) Error bars represent

95% confidence intervals.

26

RESULTS AND DISCUSSION

F oliar application of B influences fruit B concentration

B treatments did not influence fresh weight, pulling force, total soluble solids, and drain
weight three years in a row, from 2002 to 2004 (drain weight data not shown), with
concentrations of B in the sprays ranging from 0 to 800 ppm (Figures 1, 2 and 3).

Fresh weight, pulling force, and total soluble solids data were otherwise typical of normal

development.

Since pulling force is related to abscission, the lack of B treatment effects on pulling
force indicates that B does not influence abscission. Since total soluble solids (TSS) level
represents TSS accumulation or transport of photosynthetic products into the fruit, the
lack of B treatment effects on TSS indicates that B did not influence TSS dynamics in the

fruit.

Fruit B concentrations (Figures 4 to 11) were measured in order to know whether foliar B
applications resulted in transport into the fruit. Mesocarp (flesh) and pit B were
measured separately. Data are presented in two ways: pg of B per fruit and pg of B per
gram of dry weight (Figures 4, 5, 8 and 9). B effects on fruit B concentration in 2002 are
not shown: the highest concentration of B in the 2002 sprays was 50 ppm. The first B
effects were observed in 2003 on 57 days after full bloom (AFB), in fruit mesocarp from

trees treated with 200 ppm B. However, there were no significant differences in mesocarp

27

 

    

 

 

 

 

 

6
E a
:2
E: -O- control
§ 4 _ +25 ppm B
a -o— 50 ppm B
.r:
.E’
Q!
3
a 2 ~
0
I-
u.
0
b
E‘
g 1200 -
0
5
0
9. 80° ‘ -o-control
.2
m +25 ppm B
c
g 400 - +50 ppm B
O.
0
3? C
a 20 _ -o-control
In +25 m B
E + 50 :m B
3 15 -
E
.0
2 10 -
O
In
3 5 .
O
p.
0

 

Days After Full Bloom in 2002

Figure 1. 2002 fruit quality parameters.
a. Fresh weight per fruit. b. Pulling force per fruit. c. Degree of total soluble solids (°Brix)
per fruit. Data shown are the means of 12 replicates.

28

 

    
   
  
  

 

 

  

 

 

 

E
g -0-control
g + 100 ppm B
i 4 ‘ +200 ppm B
u
.r:
.9
on
a 2 -
.r:
m
2
"" a
0
’E
O -0- control
% 1200 ~ +100 ppm B
E + 200 ppm B
at
g 800 -
u.
c»
.E
'5 400 -
o.
b
0
3?
93 20 1 -0- control
‘0'" + 100 ppm B
i; +200 ppm B
m 15 .
.9.
g
I8 10 ‘
in
.73
o 5 *
'- c
0

 

 

 

Days After Full Bloom in 2003

Figure 2. 2003 fruit quality parameters.
a. Fresh weight per fruit. b. Pulling force per fruit. c. Degree of total soluble solids (°Brix)
per fruit. Data shown are the means of 12 replicates.

29

 

 

 

 

 

 

 

 

E
g -o-control
g 4 +400 ppm B
3'; +800 ppm B
a.
H
.:
.9
0
3 2 -
.c
in
2
u_ a
0
E‘
o 1200 _ -o-control
E +400 ppm B
g +800 ppm B
o
2 800 —
.2
a)
.E
3 400 -
n.
0
a
g, 20 ~
..3 c
8 15 -
2
a
2
8 1° ‘ -o—control
% +400 ppm B
I- 5 - +800 ppm B
0

 

 

 

Days After Full Bloom in 2004

Figure 3. 2004 fruit quality parameters.
a. Fresh weight per fruit. b. Pulling force per fruit. c. Degree of total soluble solids (°Brix)
per fruit. Data shown are the means of 12 replicates.

30

B concentrations between the control and 100 ppm treatments, nor between 100 ppm and
200 ppm treatments. On 63 and 68 days AFB 2003, there were significant differences in
mesocarp B among all the treatments (0, 100 and 200 ppm); while on 72 days AFB 2003,

B treatment effects on mesocarp were only observed between the control and 200 ppm B.

Interestingly, in 2004, with higher B applications (0, 400 and 800 ppm), significant
treatment differences in mesocarp B levels were observed starting from 21 days AFB and
continuing until the last day of sampling, 67 days AFB. However, differences between
400 ppm and 800 ppm were only significant on some sampling dates. One possible
reason is that 400 ppm was high enough so that fruit B accumulation (perhaps as cell wall
binding capacity) may have been saturated (Figures 6 and 7). Note that in 2003, foliar
spray of B at 100 ppm was applied 12 times in total, making the total amount of applied
B (1.7 g of B per tree) close to the annual amount growers use (growers apply once a year

1 lb of B over 227 trees per acre, or 2 g of B per year).

Our observations, especially the increase in B fruit levels after sprays stopped, indicated
three things: 1. B is mobile in sour cherry, which is consistent with previous studies
(Hanson, 1991); 2. B from foliar B application is translocated into the fruit; 3. B
accumulation in the fruit is dose-dependent, e.g. with higher B applications, the treatment

effects were evident much earlier in 2004 than in 2003.

31

 

100

75-

50‘

Mesocarp B (pg) per frui

 

 

El control
B 100 ppm B
200 ppm B

 

 

Days After Full Bloom in 2003

Figure 4. Amount of mesocarp B (pg) on a per fruit basis, 2003.
Data shows mean : SE of 10 replicates

 

E 150

120 -

(D
O
1

(JD
0
l

 

 

El control
B 100 ppm B
200 ppm B

 

 

 

Mesocarp B (pg) per g of dry
8

49 54 57 63 68 72
Days After Full Bloom in 2003

Figure 5. Amount of mesocarp B (pg) on a per dry weight (g) basis, 2003.
Data shows mean 1 SE of 10 replicates.

32

 

 

 

 

 

 

 

V\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\. W %///////////////////E
I
.\\\\\\\\\\\\\\\\\\\\\\\x a M . 7////////////////////é
I 4
m m ll
2 ,
V\\\\\\\\\\\\\\\\\\\\\\\\\ m .m s. s ..7//////////////////////////
I .1 e I
m .m w.
0 h
V\\\\\\\\\\\\\\\\\\\\\. % O ..u P .x////////////////////..
I m cm m I
r. O
I e 1
. 9 I P 7/////////////////////////,
\\\\\\\\\\\\\\\\§ 4 u 3 cm I
I F. n E ll ll
r 0 S
) l
V\\\\\\\\\\\§ M m g +_ .47////////////////////////////
I A mm
B e B B
S .
B B V\\\\\\\\\ :3. y m. m m m ..7///////////////////////
lo m. m. I m c m lo 0. p
0 t
H D. D. 8 w e W P P ,7////////////////,
O 0 0 I f o 0 0
c 4 8 O W C 4 8
U § I . 1 m .m D E I V//////////////////A
\\\fl 2 o s I
M m _ . _ a _
D 0 o 0 o o 0
M n :0. fl 0 6. 5 2 9 6 3
1 e 1 1
2:: con 33 Encomo m E m a m: a
. m s. .w. es :0 ..w A .m .30me
F

63 67

60

Days After Full Bloom in 2004

Figure 7. Amount of mesocarp B (pg) on a per gram of dry weight basis, 2004.
plicates.

35 42 49 56
33

28

21
Data shown are the means + SE of 10 re

Pit B levels

In contrast with mesocarp (around 20 pg of B per fruit in the control and up to 80 pg of B
per fruit with 800 ppm B treatment), there were only trace amounts of B in pits (less than
3 pg of B per fruit for all the treatments; Figures 8 and 9). Considering that the pit could
not be separated from mesocarp until 42 days AFB (2004), at which time the pit already
completed the hardening process, one explanation is that B applications may not get into
pit after pit hardening. However, although the amount measured was small, there was

still a significant treatment effect.

In 2004, from 42 to 67 days AFB, there were always significant treatment effects on pg
of B per pit and pg of B per g of dry weight, but the differences between 400 ppm B and
800 ppm B were insignificant on some sampling dates (Figures 10 and 11). The
differences were, however, always consistent, whether expressed as pg of B per fruit or

as pg of B per g of dry weight.

On some sampling dates, all three B treatments differed on both amount of B per pit and
amount per dry weight basis. While on other dates, there were no significant differences
between 400 ppm and 800 ppm (Tables 1 and 2). One explanation is that pit hardening

started early so that pit deve10pment was complete before B sprays had an effect.

34

Table 2. Amount of mesocarp B (pg) per fruit in 2004.

Treatments as described in ‘Materials and methods’. Data shows means :SE of 10
replicates. DAFB= Days After Full Bloom. Means with the same letter are not
significantly different.

 

 

 

 

 

 

 

 

 

 

 

m
42 49 56 60 63 67
Treatment
800 ppm 1? 50.3 59.5 60.8 71.2 61.2 80.4
@13.4) a @186) a @11.0) " @20.1) " @21.9) a @283) a
400 ppm B 26.8 34.4 38.4 41.4 38.1 55.4
@9.1) b (ill-21’ (:13-3) ’ @12.7) b @125) b @143) b
com-61 11.1 11.6 11.8 12.5 15.1 17.1
@3.7) ° @2.3) ‘ @3.7) ‘ @2.1) b @3.8) ° @4.1) c

 

 

Table 3. Amount of pit B (pg) per fruit in 2004

Treatments as described in ‘Materials and methods’. Data shows means :SE of 10
replicates. DAFB= Days After Full Bloom. Means with the same letter are not

 

 

 

 

 

 

 

 

 

 

 

significantly different.
1321113 42 49 56 60 63 67

Treatment

800 ppm B 2.2 2.0 1.9 2.3 2.1 1.8
@0.6) " @0.7) a @0.7) " @0.5) " @0.5) ‘ @0.4) '

400 ppm T 1.4 1.5 1.2 b 1.8 b 1.8 1.8
@0.3) a @0.3) a @0.3) @0.4) @0.5) " @0.3) "

control 1.0 b 0.8 b 0.7 1.3 1.1 b 1.2 b
@0.3) @0.1) @0.1) ° @0.2) ° @0.2) @0.2)

 

35

 

 

100

 

 

El control

’5 75 _ a 100 ppm B
t
h I 200 ppm B
0
a.
B -
5 50
m
1':
“- 25 -

0 -mMF-n—J

49 54 57 63 68 72

Days After Full Bloom in 2003

Figure 8. Amount of pit B (pg) on a per fruit basis, 2003.
Data shown are the means 1 SE of 10 replicates.

 

100
El control
75 _ E 100 ppm B
I 200 ppm B
50 -

Pit B (pg) per g of dry wt

   

 

 

 

 

 

 

49 54 57 63 68 72

Days After Full Bloom in 2003

Figure 9. Amount of pit B (pg) on a per dry weight (g) basis, 2003.
Data shown are the means 1 SE of 10 replicates.

36

 

100

:- El control
3 1
“I: 75 I400 ppm B
I-
I 800 B
3 ppm
‘3 50 -
a
m
3:
O. 25 _

 

 

0 .W
42 49 56 60 63 67

Days After Full Bloom in 2004

Figure 10. Amount of pit B (pg) on a per fruit basis, 2004.
Data shown are the means : SE of 10 replicates.

 

100
El control
75 - I400 ppm B
I 800 ppm B
50 -

Pit B (pg) per g of dry wt

 

 

 

42 49 .56 60 63 67
Days After Full Bloom in 2004

Figure 11. Amount of pit B (pg) on a per gram of dry weight basis, 2004.
Data shown are the means j; SE of 10 replicates.

37

Leaf B levels

Interestingly, the treatment effect on 2004 leaf B levels (Figures 2.3 a and 2.3 b) was not
as significant as the treatment effect on fruit flesh (mesocarp). On 57 days AFB, there
was no difference in leaf B level between the three treatments. But on 63 and 67 days
AFB, although 400 ppm B and 800 ppm B did not differ significantly, the control was
different from the other two treatments (Table 3; Figures 12 and 13). On 67 days AFB, on
a per leaf basis, there was no difference between the control and the 400 ppm treatment.

It is likely that B is so mobile in sour cherry that it almost immediately relocates to
wherever there is an active sink, namely, fruit from 57 days AFB till 63 days AFB.
Consequently, leaf B may not be a good standard for growers to judge cherry B

deficiency/toxicity levels.

Table 4. Amount of leaf B (pg) per leaf in 2004

Treatments as described in ‘Materials and Methods’. Data shows means 1 SE of 10
replicates. DAFB= Days After Full Bloom. Means with the same letter are not
significantly different.

 

 

 

 

 

DAFB 53 63 67
Treatment
800 ppm B 14.8 (+3.5) " 11.7 (+ 0.8) “ 9.7 (+ 1.7) ‘
400 ppm B 13.7 (+3.3) " 11.2 (+ 2.2) " 10.0 (+ 1.3) "
control 9.7 (+1.3) ‘ 8.1 (+ 1.0) b 13.8 (+ 2.3) " ‘

 

 

 

 

 

38

 

100

 

 

C] control

“5 75 I400 ppm B
7?. I 800 ppm B
m
a.
A 50 -
a:
3
m

25 -

. “MN

       

 

 

 

 

53 63 67
Days After Full Bloom in 2004

Figure 12. Amount of B (pg) on a per leaf basis, 2004, after 2004 B spray.
Data shown are the means 9: SE of 10 replicates.

 

    
   
   

 

 

E 100 Clcontrol
S 2:22:22:
'8‘ 8

E 25 ~ §

.1 0 §

 

 

 

 

 

 

53 63 67
Days After Full Bloom in 2004

Figure 13. Amount of B (pg) on a per dry weight (g) basis, 2004, after 2004 B spray.
Data shown are the means : SE of 10 replicates.

39

Effect of the previous year’s treatment

There was no significant treatment effect (carry-over effect) from 2003 on 2004 fruits
(Figures 14 and 15). The total amount of B per fruit increased over time while the
concentration (marked as B per unit dry weight) slightly decreased, probably because
fruits grow fast at this early development stage. This increase in B per fruit also indicated
a B stock or reserve in the plant. Fruit B levels increased as with time even without B
spray (Figure 14), indicating a pool of B in sour cherry was available to new growth.
Similar results (no significant treatment effects) were also observed in bud, flower and

leaf tissues in 2004 (Figures 16 and 17).

 

    

 

 

 

 

 

 

6
E
': El control
8. 4 5 E 100 ppm B
’5 I 200 ppm B
5 :-
°°
.«.-_
a 2 ‘
2
O
E
0 1 22523252323
0 7 14

Days After Full Bloom in 2004

Figure 14. Amount of mesocarp B (pg) on a per fruit basis, 2004, before 2004 B spray
started. Data shown are the means j; SE of 10 replicates.

40

 

 

 

 

 

E 150
>
6
“5 120 _ Elcontrol
0') E100 ppm B
I- [200 ppm B
a so —
’5
5‘: 60 _
CD
:2
3
q': 30 -
2
O
s . .
0 7 14

Days After Full Bloom in 2004

Figure 15. Amount of mesocarp B (pg) on a per dry weight basis, 2004, before 2004 B
spray started. Data shown are the means : SE of 10 replicates.

 

6

El control
E 100 ppm B
I 200 ppm B

    

B (pg) per leaf

 

 

 

3 14 17
Days After Full Bloom in 2004

Figure 16. Amount of B (pg) per leaf, 2004, before 2004 B spray started.
Data shown are the means : SE of 10 replicates.

41

 

100

El control
80 -
E 100 ppm B
I 200 ppm B
60 —

Leaf B (pg) per g of dry wt

 

 

 

3 14 17
Days After Full Bloom in 2004

Figurel7. Amount of leaf B (pg) per gram of dry weight (g), 2004, before 2004 B
spray started. Data shown are the means 1 SE of 10 replicates.

 

 

 

 

400
Bud B (pg) per g of dry weight,
04I20I2004
300 ~
9 B (119/9)
200 -
100 -
o _
control 100 ppm B 200 ppm B
Treatment

Figure 18. Amount of bud B (pg) on a per gram of dry weight (g) basis, 04/20/2004,
before 2004 B spray started. Data shown are the means t SE of 10 replicates.

42

100

 

Bud B (pg) per g of dry weight,

11l10l2004
75 -

B B (Hg/9)

 

 

 

control 400 ppm 800 ppm
Treatment

Figure 19. Amount of bud B (pg) on a per gram of dry weight basis, 11/10/2004, before
2004 B spray started. Data shown are the means 1 SE of 10 replicates.

 

100
Flower B (pg) per g of dry wt

O4I30I2004
75 -

B lug/g)

 

 

 

control 100 ppm B 200 ppm B

Treatment

Figure 20. Amount of flower B (pg) on a per gram of dry weight (g) basis, 04/30/2004,
before 2004 B spray started. Data shown are the means i SE of 10 replicates.

43

Splat test

In 2003, splat tests were carried out on day 68 and 77 AFB. There was no significant
difference between 100 ppm, 200 ppm, and control treated fruit (Table 4, Figure 21). In
2004, splat tests were carried out on day 63, 67 and 75 AFB. On day 63 AFB, there was a
difference between control and 800 ppm treatments. The 800 ppm treatment had the
lowest percentage of good fruit while the control had the highest. On 67 days AFB, there
were significant differences between all the three treatments, with 800 ppm treatments
having the lowest percentage of good fruit while the control had the highest. On 75 days
AFB, there were no significant differences between the three treatments (Figure 22;

Table 5); however, all fruits were about to abscise.

Fruits appeared to mature faster in 2004 than in 2003. As shown in Table 1, on 77 days
AFB in 2003, the good fruit percentage ranged from 77% to 83%; while in 2004, on 75
days AFB, the good fruit percentage ranged from 8% to 15%. A week after, on 82 days
AFB, all the fruits were gone (abscised). Therefore a time adjustment may be needed
before we can compare 2004 splat test results with 2003 splat test results. Since 75 days
AFB is very late in the harvest season and all fruits become soft eventually, it is within
expectations to see low percentages of good fruits and no significant difference between

the three treatments.

One reason why there was no significant treatment effect on percent of good fruit

observed in 2003 may be due to the low concentration of the B applications. Growers

now use in a single application the equivalent of one 1b (454 g) of elemental B per acre a

44

Table 5. Splat test results in 2003

Splat tests were performed within 12 h after fruits were sampled from HTRC. Good fruit
were defined as fruit which was not cracked and still firm after the splat test. 01 = 0.05
was used as significance level. Means with the same letter are not significantly different.

 

 

 

 

 

 

Treatment Percentage of good fruit Percentage of good fruit
on Day 68 AFB ( :1: SE) on Day 77 AFB ( : SE)

100 ppm B 94 %( i 3%) ' 77 %( i 6%) ‘

200 ppm B 95 %( i 3%) ' 78 %( i 10%) "

control 95 %( i 2%) ' 83 %( i 4%) '

 

 

 

Table 6. Splat test results in 2004
Experimental details are described in Table 2. 01 = 0.05 is used as significance level.
Means with the same letter are not significantly different.

 

 

 

 

 

 

 

 

Treatment Percentage of good Percentage of good Percentage of good
fruit on Day 63 fruit on Day 67 fruit on Day 75
AFB ( :1; SE) AFB ( 1 SE) AFB ( : SE)
400 ppm B 61 %( j 20%) “b 78 %( i 10%) ' 8 %( i 13%) “
800 ppm B 52 %( i 16%) " 65 %( i 8%) b 15 %( i 11%) "
control 72 %( i 16%) b 92 %( j; 7%) ‘ 8 %( i 10%) "

 

45

 

 

 

   

 

 

 

 

m 100%

O

O

N Elcontrol

.E 75% -

‘H E 100 ppm B

E l 200 ppm B

'8 50% —

O

U)

u—

3 25% —

I:

(D

2

g 0% .
63 68 77

Days After Full Bloom in 2003

Figure 21. Percentage of good fruit of sour cherry after splat test in 2003.
Cherries were treated with three levels of B foliar spray (control, 100 ppm B
and 200 ppm B). Data shown are the means 35 SE of 10 replicates.

 

   
   
  

100%
V
a
8 [I control
5 75% - I400 ppm B
'§ I 800 ppm B
:; .4
g 50% —
c»
*
O
E
a) 25% ‘
o
h
a:
O.

0%

 

 

 

 

 

63 67 75
Days After Full Bloom in 2004

Figure 22. Percentage of good fruit of sour cherry after splat test in 2004.

Cherries were treated with three levels of B foliar spray (control, 400 ppm B
and 800 ppm B). Data shown are the means t SE of 10 replicates.

46

year (227 trees per acre, 561 per hectare), which is close to our 100 ppm B application.
As shown in Table 2, early harvest could prevent B treatment effects to some extent (63
days AFB), but a later harvest showed a B treatment effect (67 days AFB). To further
understand whether early harvest (marked by a high percentage of good fruit) would
necessarily avoid a B treatment effect, more frequent splat tests throughout fruit

development with 400/800 ppm B applications are required.

In conclusion, although the correlation between B applications to increase fruit set and
percentage of good fruit is not yet clear, evidence here indicates that early or timely
harvests can avoid the soft fruit problem. Another option is that a 400 ppm treatment, the
equivalent of what growers routinely apply, may also be the threshold of B effects on soft
fruit. Since B foliar applications may increase fruit set and a significant B effect will not
be observed if B application does not exceed 2 g per tree a year, it would be beneficial for
sour cherry industry to use B foliar applications only if fruit B levels are carefully
monitored, i.e. less than 50 pg mesocarp B on a per gram of dry weight basis. If a
significant B treatment effect on fruit is observed, then B applications or concentrations
should be adjusted or simply stopped in order to avoid the possibility of consequent soft
fruit problems. However, based on these results, leaf B level is not recommended as a

way to monitor fruit B dynamics.

47

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366