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Plant Physiol. (1998) 118: 399-406
A Fiberless Seed Mutation in Cotton Is Associated with Lack of
Fiber Cell Initiation in Ovule Epidermis and Alterations in Sucrose
Synthase Expression and
Carbon Partitioning in Developing
Seeds1
Yong-Ling Ruan2 and
Prem S. Chourey*
Program in Plant Molecular and Cellular Biology and Department of
Plant Pathology, University of Florida (Y.-L.R., P.S.C.), and
United States Department of Agriculture-Agricultural Research Service
(P.S.C.), Gainesville, Florida 32611-0680
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ABSTRACT |
Fiber
cell initiation in the epidermal cells of cotton (Gossypium
hirsutum L.) ovules represents a unique example of trichome development in higher plants. Little is known about the molecular and
metabolic mechanisms controlling this process. Here we report a
comparative analysis of a fiberless
seed (fls) mutant (lacking fibers) and a
normal (FLS) mutant to better understand the initial cytological events in fiber development and to analyze the metabolic changes that are associated with the loss of a major sink for sucrose
during cellulose biosynthesis in the mutant seeds. On the day of
anthesis (0 DAA), the mutant ovular epidermal cells lacked the typical
bud-like projections that are seen in FLS ovules and are
required for commitment to the fiber development pathway. Cell-specific
gene expression analyses at 0 DAA showed that sucrose synthase (SuSy)
RNA and protein were undetectable in fls ovules but were
in abundant, steady-state levels in initiating fiber cells of the
FLS ovules. Tissue-level analyses of developing seeds 15 to 35 DAA revealed an altered temporal pattern of SuSy expression in
the mutant relative to the normal genotype. Whether the altered programming of SuSy expression is the cause or the result of the mutation is unknown. The developing seeds of the fls
mutant have also shown several correlated changes that represent
altered carbon partitioning in seed coats and cotyledons as compared
with the FLS genotype.
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INTRODUCTION |
Developing cotton (Gossypium hirsutum L.) seeds are an
excellent system with which to study diverse patterns of carbon
partitioning, including cellulose, starch, and oil biosynthesis (Ruan
et al., 1997 ). There has been substantial progress in our understanding of cellulose synthesis in developing cotton fibers (Basra and Malik,
1984; Amor et al., 1995; Delmer and Amor, 1995; Ruan et al., 1997). In
this regard, the cloning of cellulose synthase genes from cotton (Pear
et al., 1996) and Arabidopsis (Arioli et al., 1998) is a remarkable
achievement and opens a new era of research in cellulose biosynthesis
in plants. By comparison, however, little is known about the early
events controlling fiber cell initiation from the outermost layer of
ovule epidermis. It has long been recognized that although all
epidermal cells are potential fibers, only about 30% of these cells
actually differentiate into fibers (Basra and Malik, 1984; Tiwari and
Wilkins, 1995). Thus, an understanding of the mechanisms that determine
which epidermal cells differentiate would provide the knowledge that is
essential for increasing fiber productivity through genetic engineering.
Morphologically, the initiation of each fiber cell is associated with
the spherical expansion and protrusion of one epidermal cell above the
ovular surface during anthesis (Basra and Malik, 1984; Trelease et al.,
1986). The appearance of SuSy (EC 2.4.1.13) marks one of the first
signals of the onset of this process (Nolte et al., 1995), and the
expression of this protein correlates with cellulose biosynthesis (Amor
et al., 1995; Ruan et al., 1997). It should be pointed out, however,
that rapid secondary cell wall cellulose biosynthesis in developing
cotton fibers starts at about 16 DAA (Basra and Malik, 1984; Ruan et
al., 1997). The functional basis for SuSy expression during the fiber
cell initiation phase (0 DAA) remains to be elucidated. One approach to
a better understanding of this problem would be the use of a mutant
with reduced SuSy expression or fiber cell
initiation.
Here we present the results of our studies to characterize a
fiberless seed (fls)
mutant, using a cDNA (SS3) encoding cotton SuSy and a polyclonal
antibody raised against this protein (Ruan et al., 1997). There was a
dramatic reduction in the expression of both SuSy mRNA and protein in
the ovule epidermis of the fls mutant, which correlates with
the lack of fiber cell initiation from the ovules. In the wild-type
ovules (FLS), only those epidermal cells that show high
levels of SuSy expression protrude as fibers. Together, these
observations provide strong evidence that SuSy may play a critical role
in fiber cell initiation. Finally, the impact of the absence of fibers,
a strong sink, on carbon partitioning in the fls seed was
analyzed. The results are discussed in the context of feedback
regulation of Suc unloading and allocation in developing cotton seed.
Overall, characterization of the fls mutation is important
for a better understanding of the nature of the controls over the
process of fiber cell initiation, an important economic trait, and to
elucidate certain basic aspects of a plant developmental process.
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MATERIALS AND METHODS |
Plant Material
Two lines of cotton (Gossypium hirsutum L.), DH 59-64
as the wild type (FLS) and SL-171 as the mutant
(fls), were grown under controlled conditions as previously
described (Ruan et al., 1997). Seeds of the mutant stock SL-171 were a
gift from Dr. Jim Heitholt (USDA-ARS Cotton Physiology and Genetic
Research, Stoneville, MS). Cotton fruit age was determined by tagging
the petioles of a flower when it was fully opened. All samples, unless
otherwise specified, were frozen in liquid nitrogen and then stored at
 80°C until analysis. Frozen, developing seeds were separated into
fibers, seed coats, and cotyledons on dry ice before analysis.
In Situ Hybridization
In situ hybridization using paraffin-embedded sections was carried
out according to the procedure described by Marrison and Leech (1994).
Cotton ovules were collected from fully opened flowers and immediately
fixed in formalin-acetic acid. The fixed ovules were dehydrated through
a tertiary butyl alcohol series and embedded in paraffin. To eliminate
possible RNase contamination, all solutions used prior to and during
the hybridization were incubated in 0.1% (v/v) diethylpyrocarbonate
overnight at 37°C before autoclaving. Cross-sections (12 µm) were
placed on dampened, precharged slides (Probe On Plus, Fisher
Scientific) in a water bath at 40°C to 42°C and left to dry on a
hot plate overnight at 40°C. For a better comparison, sections from
fls and FLS were arranged on the same slides with
each genotype section divided into two parts and treated with sense and
antisense SuSy RNA probes, respectively.
SS3 cDNA was linearized with ApaI or EcoRI for
sense and antisense RNA preparation, respectively. Digoxigenin-labeled
sense and antisense RNA probes were synthesized by in vitro
transcription reactions using 1 µg of linearized SuSy DNA template,
digoxigenin-11-UTP and T3, or T7 RNA polymerases, according to the
protocol of the digoxigenin RNA-labeling kit (Boehringer Mannheim). The
RNA probes were hydrolyzed to an average size of 150 nucleotides to
allow better tissue penetration (Cox and Goldberg, 1988). The
hybridized SuSy mRNA was immunologically detected by using
anti-digoxigenin antibody conjugated with alkaline phosphatase as
described in the digoxigenin nucleic acid detection kit, with
modifications as indicated by Marrison and Leech (1994).
Immunolocalization
Immunogold silver staining was conducted according to the
procedure recommended by the Histogold kit (Zymed Laboratories, Inc.,
San Francisco, CA). Paraffin-embedded cross-sections of cotton ovules
were cut (12 µm), affixed to slides, deparaffined, rehydrated, and
washed with PBS. Thereafter, slides were incubated with serum-blocking
solution for 10 min and incubated with 1:1500 diluted SuSy polyclonal
antibodies or preimmune serum in a humid environment for 1.5 h.
After the slides were washed with PBS, they were incubated for 30 min
in a solution of secondary antibody (goat anti-rabbit IgG linked to
colloidal gold). Slides were then washed thoroughly with PBS (four
times, 3 min each), incubated for 5 min with freshly prepared
silver-enhancement reagents, and washed with excess distilled water.
Slides were dehydrated in an ethanol series and permanently mounted in
Permount (Fisher Scientific) for microscopic examination. Pairs
of pre- and immunostained sections from both fls and
FLS were treated on the same slide for a better comparison.
RNA and Protein Gel Blots
Total RNA was isolated from developing cotyledons of cotton seeds
as previously described (Wadsworth et al., 1988; Ruan et al., 1997).
RNA electrophoresis, blotting, and hybridization conditions were as
described previously (Ruan et al., 1997). For soluble protein
extraction, cotyledons (approximately 0.5 g each) were ground to a
fine powder in liquid nitrogen. The grinding
continued for 5 min in cold extraction buffer (3:1, v/v)
containing 25 mM Hepes/KOH (pH 7.3), 5 mM EDTA,
1 mM DTT, 0.1% soluble PVP (Mr 40,000), 1 mM PMSF, and 0.01 mM leupeptin. The
homogenate was centrifuged at 10,000g for 5 min at 4°C.
The supernatants were collected and denatured by SDS and boiling
treatments. The protein concentrations were determined using the
Bio-Rad DC protein assay kit with BSA as a standard.
Electrophoresis, blotting, and SuSy antigen detection were carried out
as previously described (Ruan et al., 1997). In each case, three
independent experiments were conducted and the same results were
obtained.
Sugar and Starch Analysis
Soluble sugars and starch were extracted and enzymatically
determined as previously described (Ruan et al., 1997).
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RESULTS |
The fls Trait Is Seed Specific and under Maternal
Control
Although the fls mutant SL-171 and the wild-type
genotypes are not lineage related, plants of the two genotypes have
shown an overall similar pattern in growth rate, development, and
timing of flowering. The only exception was the higher levels of red pigmentation in all plant parts of the mutant due to an unknown genetic
constitution of its anthocyanin genes. The most striking difference
between the two genotypes was seen during seed development. Whereas the
developing FLS seeds were covered with young fibers, the
fls seeds showed no detectable fiber growth, except for a few rudimentary fibers detectable only under a dissecting microscope. The size and shape of the seeds and fruits were similar in both genotypes. These observations were in agreement with the previous report of this fls mutant (Turley and Ferguson, 1996).
Maternal origin of fiber cells from the ovule epidermis, as early as
16 h preanthesis, is well documented cytologically (Ramsey and
Berlin, 1976; Nolte et al., 1995). The fls mutant provided an opportunity to examine this aspect genetically. We emasculated flowers of both genotypes 1 d before anthesis and allowed seed development. Unfertilized seeds at 15 DAA showed massive amounts of
fiber in FLS but none in the fls mutant,
confirming the maternal control of both traits. It is interesting that
seeds at 15 DAA of both genotypes from unpollinated flowers were
indistinguishable from the corresponding self-pollinated controls. The
only detectable difference was a smaller embryo size in the
longitudinal sections of the unpollinated flowers (Fig.
1). The same pattern was also seen in the
mutant seed (data not shown). Obviously, normal development of the
embryo was dependent on fertilization, but the maternal tissues,
including integuments and fiber development, were independent of the
pollen parent and pollination.
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| Figure 1.
Longitudinal sections of FLS cotton
seed at 15 DAA from emasculated (A) and pollinated (B) flowers. Note
the much reduced size of the "embryo" in A. Bars = 625 µm.
isc, Inner seed coat; ct, cotyledon. Arrow indicates location of
hypocotyl.
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SuSy Expression in the fls Ovule Epidermal Cells Is
Reduced
In situ hybridization of ovule sections harvested on the day of
anthesis (0 DAA) was done to examine the cellular localization of SuSy
mRNA using antisense and sense RNA probes generated from SS3 cDNA. As
shown in Figure 2, the appearance of
bubble-like structures among the epidermal cells (indicative of the
initiation of fiber cells) in the wild type (Fig. 2, A-C) was
completely missing in the fls mutant (Fig. 2, D-F). The
FLS ovule sections also showed a strong signal for the SuSy
mRNA in the initiating cells, whereas a slightly weak signal was seen
in the two to three cell layers between the inner and outer integuments
inside of the seed (Fig. 2A). Under high magnification the large,
spherical "bubbles" showed the strongest signal for the SuSy mRNA
(Fig. 2B), whereas the smaller, initiating cells showed weaker signals, and the nondifferentiating cells and those underlying the epidermis did
not show any SuSy mRNA (Fig. 2B). In contrast, the mutant fls ovules at 0 DAA showed no signal for SuSy mRNA in the
epidermal cells, nor was there any fiber cell initiation (i.e. no
bubble formation, Fig. 2, D and E). Significantly, the cell layers
between the inner and outer integuments of the fls mutant
ovules also showed no SuSy RNA. No signal was seen in the sections of
either genotype when treated with the sense control probe (Fig. 2, C and F).
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| Figure 2.
In situ hybridization of SuSy in cross-sections of
FLS (A-C) and fls (D-F) ovules. The
purple signals represent SuSy mRNA. A, B, D, and E,
Cross-sections were hybridized with an antisense RNA probe
generated from SS3 cDNA. Note in B the very strong SuSy mRNA signals in
the large and spherically shaped initiating fiber cells (arrow), the
weak signals in the small fiber cells (triangle), and the undetectable
signals in the nondifferentiating epidermal cells. Also note in E that
SuSy mRNA was undetectable in the nondifferentiating epidermis of the
fls mutant. C and F, Cross-sections were
hybridized with sense RNA probe. Bars in A and B are 50 and 22 µm,
respectively (magnifications of A and D, B and C, and E and F). f,
Fiber cell; oi, outer integument; ii, inner integument; n, nucellus.
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Ovule sections of the two genotypes were also examined at 0 DAA for the
SuSy protein by immunolocalization. Figure
3B shows large amounts of SuSy protein in
the FLS epidermal cells, but a much-reduced signal was seen
in the fls mutant (Fig. 3E). Cell layers inside the seeds of
both genotypes were not as sharply contrasted in the levels of SuSy
protein as they were for the RNA signal. Under high magnification,
however, the FLS epidermal cells showed a strong signal for
the protein that was lacking in the mutant cells (Fig. 3, C and F).
Preimmune controls for both genotypes are shown in Figure 3, A and D,
and, as expected, showed no immunogold reactivity. Similar
immunohistological analyses of the serial sections of the same ovules
with carrot and maize cell wall invertase antibodies did not detect any
signal in either genotype (data not shown).
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| Figure 3.
Immunogold localization of SuSy protein in
cross-sections of FLS (A-C) and fls
(D-F) ovules. A and D, Cross-sections were treated with
preimmune serum. B, C, E, and F, Cross-sections were treated
with polyclonal antibody against SuSy. Note in C the very strong SuSy
protein signals in initiating fiber cells (arrows) and weak signals in
the nondifferentiating epidermal cells (see the cell between the two
initiating fiber cells indicated by arrows). Also note in F that very
little SuSy protein can be detected in the ovule epidermis of the
fls mutant. Bars in A and C are 77 and 19 µm,
respectively (magnifications of A and B, D and E, and C and F). f,
Fiber cells; oi, outer integument; ii, inner integument; n, nucellus.
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Previously, it was demonstrated that cotyledons but not seed coat cells
are enriched in SuSy mRNA and protein in developing cotton seed (Ruan
et al., 1997). Thus, SuSy expression patterns were also analyzed by
northern- and western-blot analyses in developing cotyledons at 15 DAA
and beyond, a stage corresponding to the elongation, secondary cell
wall biosynthesis, and maturation phases of fiber development.
Steady-state levels of SuSy transcripts in FLS seeds at 35 DAA were markedly reduced to undetectable levels compared with the
levels seen at 15 DAA (Fig. 4A). The
fls seeds, however, showed much greater abundance of SuSy
transcripts at 35 than at 15 DAA. The reversed pattern of temporal
expression between the two genotypes was also manifested in the soluble
fraction of SuSy protein, as observed by western-blot analyses (Fig.
4C). The highest levels of SuSy polypeptides in the FLS
seeds were seen at 15 DAA, followed by a gradual decline, and the least
amounts were seen at 35 DAA. In contrast, the fls seeds
showed a reverse temporal pattern for the SuSy protein, a gradual
increase starting at 15 DAA, and the highest levels at 35 DAA.
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| Figure 4.
A reversed temporal expression of the SuSy gene in
developing cotyledons of the fls mutant compared with
that of developing FLS seeds. Numbers indicate DAA. A,
RNA gel blot with 16 µg of total RNA in each lane. B, The same blot
sequentially hybridized with a maize rRNA probe. C, Protein immunoblot
with 50 µg of protein in each lane from crude extracts.
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fls Mutant Seeds Display an Altered Mode of Carbon
Partitioning
Carbohydrate partitioning, in particular the levels of soluble
sugars and starch in developing seeds of the two genotypes, was
analyzed to test for possible feedback regulation due to the loss of a
major sink site for Suc utilization in the mutant seeds. Figure
5 depicts the results of such a
comparative analysis of seed coats and cotyledons at 10 DAA (fiber
elongation) through 35 DAA (fiber maturation). Suc levels in the
fls seed coat remained relatively low throughout the entire
development period, whereas the FLS seed-coat extracts
showed a gradual but significant increase in Suc concentration. At 35 DAA, the FLS seed coats displayed levels of Suc that were 8 times higher than the fls genotype (Fig. 5A). The hexose
levels in the fls seed coat were lower at 10 DAA, but
thereafter, the two genotypes were similar (Fig. 5B). Starch levels in
the fls seed coat were reduced to less than one-half that of
the wild type during 10 to 25 DAA (Fig. 5C). However, the overall
pattern of the developmental changes in starch between the
fls and FLS seed coats was similar, with an
increase during 10 to 25 DAA and a sharp decrease thereafter (Fig. 5C).
These data for the normal genotype are in agreement with those of
Hendrix (1990).
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| Figure 5.
Comparisons of Suc, hexose, and starch levels in
developing cotton seed between FLS (hatched bars) and
fls (open bars) plants. Each value is the mean of three
replicates with SE < 11%. FW, Fresh weight.
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In the developing cotyledons Suc levels in the mutant were lower than
that of the FLS up to 15 DAA but increased thereafter and
surpassed the FLS at 25 and 35 DAA (Fig. 5 D). The hexose contents in the fls cotyledons were quite low at 10 to 25 DAA, but the overall pattern of changes in the two genotypes was
similar for the later development stages (Fig. 5E). Cotyledonary starch levels in both genotypes at 35 DAA were the highest (Fig. 5F); however,
during the early stages, 10 and 15 DAA, the FLS cotyledons showed levels of starch higher than that of the mutant.
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DISCUSSION |
One of the most important observations of this study is the
cytological demonstration that, unlike the FLS genotype,
seed epidermal cells of the fls mutant failed to exhibit
detectable protrusions or bubbles at 0 DAA. Such changes in the
epidermis must precede the elongation phase, leading ultimately to the
fiber pathway. We infer from these data that the fls
epidermal cells lack the competence to adapt to a specialized mode of
growth and development and thus remain fiberless at maturity. To the
best of our knowledge, this is the first such description of the
fls mutation in cotton. Fiber development in cotton is
partitioned into four developmental phases. The first two stages
correspond to the initiation and elongation of the epidermal cell, and
the last two represent secondary growth and maturation of the fiber marked by massive levels of cellulose biosynthesis.
Developmentally, the fiber growth is in parallel with seed development,
which spans a period of about 45 DAA. The fiber cell initiation is
analogous to several other systems of tip growth in a single cell, such
as trichomes on a leaf or the development of root/shoot hairs.
Insertional mutants showing impaired tip-growth functions in
Arabidopsis have in general led to genes that encode transcription
factors (for review, see Schiefelbein, 1998). The only known exception
is the trichome mutation Zwichel (zwi) in Arabidopsis, in which the cloned ZWI gene is shown to encode
a member of the kinesin-like family of microtubule proteins
(Oppenheimer et al., 1997). In this regard, nothing is known concerning
the molecular basis of the initial events that lead seed epidermal cells to the fiber cell pathway in cotton. In a previous study, Turley
and Ferguson (1996) made a comparative analysis of the same
fls mutant, SL-171, against an unrelated FLS
inbred using two-dimensional PAGE analysis of the total ovular
proteins. Of the numerous Coomassie blue-stained spots only
five polypeptides are unique to the mutant. However, it is difficult to
assign any physiological significance to these differences because
these proteins are of an unknown nature.
Our studies at the cellular level have also shown undetectable levels
of SuSy RNA and protein at 0 DAA in the fls epidermal cells.
In contrast, the FLS cells showed abundant levels of SuSy, and there was much heterogeneity among the FLS cells. Those
with the highest levels of SuSy appeared to differentiate into the longest cells, whereas cells with the lower or undetectable levels showed smaller or no initiation out of the epidermis (Figs. 3 and 4;
Nolte et al., 1995). The enzyme SuSy catalyzes a reversible conversion
of Suc and UDP to UDP-Glc and Fru and plays a major role in energy
metabolism by mobilizing Suc into diverse pathways, including
metabolic, structural, and storage functions of plant cells. Several
lines of evidence from divergent plant sources (Chourey and Nelson,
1979; Geigenberger and Stitt, 1993; Heim et al., 1993) indicate that
the enzyme preferentially catalyzes Suc degradation to Fru and UDP-Glc,
the immediate precursor for cellulose biosynthesis in cotton fiber
(Amor et al., 1995, and refs. therein). However, it is unclear why such
high levels of SuSy expression were seen as early as 0 DAA, the stage
primarily associated with the initiation phase of the fiber pathway in
the FLS epidermal cells. It is significant that cell wall
invertase was immunohistologically not detectable in serial sections of the same ovules at 0 DAA using either carrot or maize polyclonal antibodies (W. Cheng and P. Chourey, unpublished data). Thus, SuSy is a key enzyme in metabolizing incoming Suc, presumably both for
energy and as a precursor for biosynthetic processes in the initiating
fiber cells.
It is also possible that SuSy may contribute to turgor-related
functions required for initiation and associated protrusion of the
ovular epidermal cells. Such cellular changes are associated with rapid
cell expansion driven by high cell turgor (Cosgrove, 1986). Previous in
vitro studies have shown that the maintenance of turgor potential is
essential for fiber cell expansion and elongation, which is achieved by
increasing osmotically active solutes, mainly soluble sugars,
K+, and malate (Basra and Malik, 1984). The most
recent data of Smart et al. (1998) show high levels of expression of
genes encoding plasma membrane H+-ATPase,
vacuolar H+-ATPase, proton-translocating
pyrophosphatase, PEP carboxylase, and major intrinsic protein, which
are putatively engaged in the control of cell turgor in elongating
cotton fibers. They also observed high levels of these proteins during
the early stages that coincide with fiber initiation. Suc cleavage into
Fru and UDP-Glc by SuSy would double the osmotic contribution of Suc. Thus, the enzyme could play a critical role in establishing and maintaining high turgor potential, driving the fiber cell precursor above the ovule epidermis. The high ratio of hexose to Suc in developing fibers (Ruan et al., 1997) concurs with this viewpoint. In
the fls mutant the loss or the reduced levels of SuSy in the ovule epidermal cells may lead to low turgor and, consequently, no
fiber growth. The role of SuSy in osmotic regulation and maintenance of
cell turgor has also been indicated in guard cells of potato leaves in
response to water stress (Kopka et al., 1997).
Remarkably, developing seeds of the two genotypes were quite different
from each other during later developmental stages, 10 to 35 DAA.
Alterations in the temporal patterns of SuSy expression, based on
northern- and western-blot analyses, are of particular interest.
Overall, the temporal pattern of SuSy expression in the fls
mutant was reversed as compared with the normal genotype. In the
FLS seeds the highest levels of SuSy RNA and protein were seen at 15 DAA and the lowest at 35 DAA; however, in the fls
mutant the highest and the lowest levels were seen at 35 and 15 DAA, respectively. Thus, the mutant seeds have shown a delayed program of
SuSy expression at both the RNA and protein levels, i.e. undetectable levels at 0 DAA, followed by a slow but gradual increase such that the
highest levels of SuSy protein were seen at 35 DAA.
It should be noted that both genotypes were grown in the same
greenhouse and that the samples were harvested at nearly the same time,
thus minimizing possible contributions due to environmental factors. In
addition, although the two genotypes were not lineage related, there
were no obvious developmental differences (such as flowering and
maturity) between them. Thus, it is unlikely that an altered temporal
program of SuSy expression could be due to differences in either the
environment or their inbred backgrounds. Previous reports of
alterations of SuSy expression in maize (Chourey and Nelson, 1976;
Chourey et al., 1988) and potato (Zrenner et al., 1995) show that the
loss or the reduction of tissue-specific gene products are either due
to a mutation in a gene that encodes SuSy or to the antisense
inhibition. Results concerning fls seeds are unique because
they indicate a regulatory type of change, as evidenced by an altered
program of gene expression. Whether such a change in SuSy expression is
a cause or a feedback effect of a mutation in certain upstream
regulatory genes (such as those encoding the transcription factors)
remains to be elucidated.
In developing fibers after initiation, although reducing sugar levels
are high (Hendrix, 1990; Ruan et al., 1997), the predominant sugar
nucleotide is UDP-Glc during both primary and secondary cell wall
formation (Carpita and Delmer, 1981), and SuSy is the major enzyme in
degrading Suc. Based on enzyme activity and immunohistological assays,
invertase appears to play a minor role in developing fibers (Hendrix,
1990) or in ovular cells initiating to fibers (Y. Ruan and P. Chourey, unpublished data). Similarly, we have also observed only low to undetectable levels of cell wall invertase and Suc phosphate synthase RNAs in fibers at 15 DAA by northern-blot analyses using carrot and spinach cDNA clones, respectively (however, the same
clone detected invertase RNA in sink leaves and seedlings, and the
spinach clone detected Suc phosphate synthase RNA in source leaves; data not shown).
Seed weights at maturity for FLS (without the fibers) and
fls genotypes were similar (data not shown); however, the
two genotypes showed significant differences in both the relative
distributions and the amounts of soluble sugars and starch in
developing seeds during 10 to 35 DAA (Fig. 5). In developing cotton
seeds, phloem-unloaded Suc is differentially partitioned into three
major sink tissues: fiber, seed coat, and cotyledons (Ruan et al.,
1997). Obviously, the loss of Suc utilization in fiber development in
the fls mutant has led to several secondary alterations in
carbon partitioning. Among these, the most striking changes were in the
reduced levels of Suc and starch in the fls seed coat
throughout the period of 10 to 35 DAA (Fig. 5, A and C). In addition,
the mutant cotyledons had undetectable levels of starch during the very
early (10-15 DAA) phases of development (Fig. 5F). A steady increase
in the levels of Suc in the FLS seed coat would steepen the
gradient of Suc concentration between seed coat and fibers, thus
facilitating symplastic Suc transport to the growing fibers (Ryser,
1992; Ruan et al., 1997). In contrast, the absence of fibers and the
loss of the associated major utilization sink in the mutant may cause a
reduction in the levels of incoming Suc to the seed coat. Reduced levels of starch in the mutant seed coat and cotyledon, particularly during the early developmental phases, were most likely due to the
reduced levels of SuSy protein, as has been previously demonstrated in
SuSy-deficient mutants of maize and potato (Chourey and Nelson, 1976;
Zrenner et al., 1995).
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FOOTNOTES |
1
This work was supported in part by the U.S.
Department of Agriculture (USDA)-Agricultural Research Service (ARS)
and by the United States-Israel Binational Agricultural Research and
Development Fund (grant no. IS-2282-93). It was a cooperative
investigation between the USDA-ARS and the Institute of Food and
Agricultural Sciences, University of Florida. This paper is Florida
Agricultural Experiment Station journal series no. R-06228.
2
Present address: Division of Plant Industry,
Commonwealth Scientific and Industrial Research Organization, GPO Box
1600, Canberra, ACT 2601, Australia.
*
Corresponding author; e-mail psch{at}gnv.ifas.ufl.edu; fax
1-352-392-6532.
Received May 20, 1998;
accepted July 17, 1998.
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ABBREVIATIONS |
Abbreviations:
DAA, days after anthesis.
SuSy, Suc synthase.
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ACKNOWLEDGMENTS |
We thank Drs. Jim Heitholt and Rickey Turley (USDA-ARS,
Stoneville, MS) for many consultations, Deborah P. Delmer (University of California, Davis) and Arnd Sturm (Friedrich Miescher
Institute, Basel, Switzerland) for cotton SuSy and carrot cell
wall invertase cDNA clones and antibodies, Michael Salvucci for the
spinach Suc phosphate synthase cDNA clone, and Earl W. Taliercio and
Susan Carlson for critical reading of the manuscript. We also thank Dr.
W.-H. Cheng for assistance with immunolocalization analyses for
invertases.
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Abstract
Introduction