BACKGROUND
cereal crops are of critical importance in agriculture.
the top three cereals in global production  are maize, wheat, and rice, with  <dig>   <dig> and  <dig> m tonnes, respectively .
cereal crops constitute our largest primary food source and are also highly used in food and non-food industrial applications.
contributing factors to the importance of cereals are that they can be bred to be very high yielding, that cereal grains lend themselves to long-term storage, and that the grain can accumulate different types of carbohydrates and lipids.
major carbohydrates in cereal caryopses are the starch components amylose and amylopectin, cell wall components, such as different types of arabinoxylan, mixed-linkage β-glucan and cellulose, fructooligosaccharides, fructan, and sucrose
<cit> .
interestingly significant amounts of oil can also be stored in the endosperm, especially in oats
<cit> .
the composition of the cereal grain dictates the end use of the crop.
for example, the cereal endosperm is the most important source of starch worldwide
<cit>  and is therefore of tremendous value for food security.
there is an ongoing search for genotypes with high content of amylose, β-glucan and/or fructan for different applications within the functional food sector
<cit> .
at the other end of the spectrum are efforts to develop cereals that redirect carbon flux from carbohydrates to oils for production of high-density biofuels
<cit> .
a thorough understanding of the mechanisms for the partitioning of photosynthates in cereals is crucial for our ability to boost starch yield, to develop specialty crops for the functional food industry, such as barley with enhanced ß-glucan levels, and to tailor cereal production for the non-food industry.
carbon partitioning in higher plants has been studied at the whole-plant level
<cit> , for certain types of plant tissues
<cit> , and for plant cells
<cit> .
however, many questions remain unanswered.
for example, we need to identify and map the actions of key elements that determine carbon allocation between source and sink tissues and that govern carbon flux along pathways for synthesis of different carbohydrate and oil sinks.
it is also imperative that we gain insight into how environmental factors influence carbon partitioning
<cit> .
several proteins have been implicated as important players in carbon partitioning in plants.
they include proteins involved in sugar transport and metabolism, such as sucrose transporters
<cit> , sucrose invertases
<cit>  and sucrose synthases
<cit> , and in hexose metabolism and transport, such as hexose kinases
<cit>  and monosaccharide transporters
<cit> .
other examples include proteins controlling the flux in polysaccharide biosynthesis, such as adp-glucose pyrophosphorylase
<cit> , and udp-glucose pyrophosphorylase
<cit> , and regulatory proteins, such as sucrose non-fermenting-1-related protein kinase
<cit> , trehalose-6-phosphate synthase
<cit> , and transcription factors
<cit> .
we are interested in identifying molecular switches in cereals that direct carbon flux to different tissues and into the specific end products.
we are particularly concerned with carbon partitioning between amylose, amylopectin, oil, β-glucan and fructan in cereal seeds.
for the present study, we chose a rice waxy mutant, gm <dig>  which is deficient in amylose biosynthesis.
we examined carbon partitioning between amylose, amylopectin, oil, β-glucan, fructan and other dietary fibers in the gm <dig> background, a nearly isogenic waxy line.
we constructed a suppression subtractive hybridization  cdna library between the mutant and the corresponding wild type to identify potential candidates involved in carbon partitioning.
we used qpcr to verify results from the ssh experiments and to study how gene regulation controls carbon allocation in the absence of amylose biosynthesis.
RESULTS
the gm <dig> rice is a waxy mutant
waxy rice has been drawing much attention in rice breeding in china as it has many applications in traditional chinese food and brewing.
this has resulted in a large collection of waxy rice in the chinese rice germplasm repositories and also in a number of breeding programs on the different qualities of waxy rice
<cit> .
we selected one waxy rice cultivar, gm <dig> , mainly based on the following factors: i) it is a stable mutant with a nearly isogenic background; ii) it has a relatively low amylose content  compared to other waxy mutants; iii) with the exception of its waxy grain character, gm <dig> is phenotypically similar to its wild-type counterpart bp <dig> , an elite variety of indica rice   .
when the grains of gm <dig> were cut transversely and stained with an iodine solution, a typical reddish color of waxy starch was revealed in the endosperm
<cit> .
we have further characterized the grain starch of gm <dig> by recording the light absorbance of the starch-iodine complex between  <dig> nm and  <dig> nm with a scanning spectrophotometer.
we included internal standards of starch with known contents of amylose.
as seen in additional file
<dig>  the absorbance value around  <dig> nm for the amylose-iodine complex was reduced proportionally with the amylose content in the starch samples, including those from the wild type  and mutant .
based on the absorbance, the estimated amylose content of bp <dig> and gm <dig> is between standards  <dig>  and  <dig> , and standards  <dig>  and  <dig> , respectively.
the estimations were confirmed with chemical analyses revealing a significant difference  in amylose content of  <dig> % and  <dig> % in kernels of bp <dig> and gmo <dig>  respectively.
the starch content was around 67% in both types of rice grains  .
<cit> .
1no significant difference of total starch content between bp <dig> and gm <dig> .
2significant difference of total amylose content between bp <dig> and gm <dig> .
qpcr analysis of expression levels for gbssi and gbssii.
dw , gbss .
the statistical difference between bp <dig> and gm <dig> is presented as “significantly decreased”  and “increased” , respectively.
it is generally accepted that amylose synthesis is carried out by granule-bound starch synthases .
cereals have two forms of gbss, gbssi and gbssii
<cit> .
gbssi is responsible for amylose synthesis in storage tissues, such as endosperm, whereas gbssii is present in green tissues, including the pericarp of seeds.
we used qpcr to analyze gene expression for both gbss genes in rice seeds with the ubiquitin gene, ubq <dig>  as an internal standard.
the qpcr results showed that, in gm <dig> seeds, gene expression of gbssi was significantly reduced  and the expression of ubq <dig> is about the same as in the control bp <dig>  expression of gbssii was significantly increased in gm <dig> as compared with bp <dig>
we have shown that the gm <dig> rice is a waxy mutant caused by down-regulation of gbssi.
yield, kernel weight and starch content were similar between the waxy mutant and the corresponding wild type .to gain insight into the redistribution of carbon in the gm <dig> seed, we subjected the mutant and wild-type lines to carbohydrate and oil analyses.
the major carbon from amylose is redistributed to amylopectin in the waxy mutant
carbohydrate analyses revealed that both gm <dig> and the parental bp <dig> lines contained about 3% dietary fiber with similar compositions .
arabinoxylan and cellulose were major dietary fiber components  while mixed-linkage β-glucan and fructan were minor components.
consequently, no extra carbon was distributed into the cell walls or to the β-glucan or fructan sink in the gmo <dig> mutant.
the starch content was slightly reduced in the waxy mutant  compared to the wild type  .
the amylose content was normal in the wild type  but highly reduced in the waxy mutant  .
thus the amylose content in the seed was reduced from 17% of the caryopsis in the wild type to  <dig> % in the waxy mutant .
the reduced content of amylose was mainly compensated for by an increased content of amylopectin in the waxy caryopsis, 65% in the waxy mutant compared to 53% in the wild type.
the content of sucrose and crude oils were the same in the two rice lines .
the glucose content in the gm <dig> mutant  was somewhat higher than the wild type  , but was low in both lines.
*mean value from three independent analytic experiments using randomly selected caryopses from a pool of six plants .
**sugar residue.
the carbohydrate analysis thus indicated that a major fraction of carbon in the waxy mutant gm <dig> was reallocated from amylose to amylopectin synthesis.
this result prompted us to try to identify the genes in gm <dig> responsible for this reallocation.
to this end, we employed the ssh strategy .
suppression subtractive hybridization identified  <dig> unigenes in the waxy mutant
we used gm <dig> as the tester and bp <dig> as the driver to construct a cdna library after pcr amplification and ssh of cdnas from total rna isolated from plants at  <dig> days after flowering .
the resulting ssh library of “gm <dig> vs bp034” contained  <dig> clones with an average length of around  <dig> bp.
all positive clones were applied to sequencing, which returned the identification of  <dig> unigenes.
these  <dig> unigenes were used for the clusters of orthologous groups  functional annotation analysis
<cit>  after blastx and tblastx against the ncbi protein databases.
among the  <dig> unigenes,  <dig> exhibited high similarity  to known protein sequences, and  <dig> showed no similarity to any reported sequence.
within the  <dig> protein sequences,  <dig> lacked functional annotation.
the rest of sequences were categorized in four functional groups: “information storage and processing”, “cellular processes and signaling”, “metabolism”, and “poorly characterized” .
these four functional groups have  <dig>   <dig>   <dig> and  <dig> unigenes, corresponding to  <dig> %,  <dig> %,  <dig> % and  <dig> % of the total unigenes, respectively .
the details of the unigenes and their putative functions are shown in table
<dig>  interestingly, two unigenes , similar to the genes for adp-glucose pyrophosphorylase small subunit  and isoamylase , respectively, were found in the carbohydrate transport and metabolism group.
notably, one clone  in the group of no related cogs showed a high similarity to wrky transcription factor  <dig> .
further sequence analysis of the genes for isoamylase and wrky transcription factor  <dig> revealed that they are rice orthologs to barley isa <dig> and susiba <dig>  respectively, previously described by sun et al.
<cit> .
<cit> .
the number of unigenes in each group is indicated and their percentage in the total number of unigenes is denoted.
validation of the shh results by semi-quantitative pcr
to verify the conclusions from the shh experiment, we selected two housekeeping genes, the gene for the eukaryotic elongation factor- <dig> α subunit  and ubq <dig>  to follow the ssh experiment by semi-quantitative pcr.
when we used the same batch of rna as in the shh experiment, or rna isolated from other stages of seed development, or from other tissues, we found expression levels of the two housekeeping genes to be more or less the same in gm <dig> and bp <dig>  furthermore, expression levels were constant throughout seed development and in different tissues of mutant and wild-type rice .
importantly, we observed that the cdna for eef- <dig> α could be detected in the tester  and driver  samples prior to ssh, but not in the sample after subtraction hybridization , lending support to the validity of the ssh approach.
we also chose some additional genes, related to starch biosynthesis and carbon portioning  to further verify the reliability of the shh experiment and to obtain detailed quantitative data on gene expression in the two rice lines.
results from those analyses are presented below.
gene expression profiling in the waxy mutant
to further validate the results from the shh experiment and to quantify expression of genes involved in starch biosynthesis and/or carbon portioning, we chose  <dig> genes as representatives for gene expression analysis by qpcr, including two reference genes, eef- <dig> α and ubq <dig> .
according to the results obtained by qpcr, we divided the genes into five groups .
the classification was based on qpcr quantification of the differential gene expression in gm077; “significantly decreased” , “not changed” , “increased” , “significantly increased”  and “not detected”.
intriguingly, among the four significantly increased genes, agps, sbei, isa <dig> and susiba2-like, all except sbei were found in the shh library.
we noted that the expression level for the upregulated genes in gm <dig> correlated well with the expression level for the susiba2-like transcription factor gene .
gene expression correlation of susiba2-like and isa <dig> in the mutant and wild type
sun et al.
<cit>  have demonstrated that isa <dig> and sbeiib in barley were upregulated by the activity of the susiba <dig> transcription factor and a good correlation in gene expression levels has been demonstrated between susiba <dig> and its target genes, such as isa <dig> and sbeiib <cit> .
to learn if this correlation holds true in rice also, and in an effort to find susiba2-like-controlled genes in rice, we selected rice isa <dig> as a representative to study the correlation in expression between susiba2-like and its target genes in rice.
for this study, we chose different tissues and different time points in both the mutant gm <dig> and the wild type bp <dig>  as displayed in figure
5a and b, there was an excellent correlation between expression levels for the two genes in the analyzed samples.
the statistical analysis  indicated that the relative levels of the spatial and temporal expression for the two genes in both rice lines shared a pearson correlation coefficient  of  <dig>  .
discussion
although waxy mutants of higher plants and the responsible gene  have been studied to a large extent, and the high content of amylopectin in the mutant is known
<cit> , little information about carbon partitioning to other carbohydrates and oil fractions in waxy mutants has been reported.
moreover, gene regulation of carbon reallocation to amylopectin in the mutant is poorly understood.
we are interested in the partitioning of photosynthates between starch and other storage compounds in cereal seeds.
in this study, we selected one of the rice waxy mutants to follow carbon partitioning between starch and other carbohydrates when amylose biosynthesis is impeded.
our carbohydrate analysis indicated that when the amylose content is reduced, the vast majority of the assimilated carbon is reallocated to amylopectin, rather than to other carbohydrates or lipids.
interestingly, such a reallocation did not change seed weight but, rather, shifted carbon from one compound  to another  within the starch biosynthesis machinery.
to understand the molecular mechanisms controlling the increase in amylopectin biosynthesis, we set out to identify genes that were upregulated in the waxy mutant.
from the shh experiments we found three candidates that have previously been shown to be directly involved in starch synthesis and/or its regulation, agp <cit> , isa <dig> <cit> , and susiba2-like <cit> .
the functions and regulation of agpase and isoamylase have been reviewed and well documented previously
<cit> .
in cereal endosperm cells, there are two forms of agpase, one cytosolic and one plastidic.
the major fraction of adp-glucose in cereal endosperm is believed to be produced in the cytosol and then transported to the amyloplast for subsequent starch biosynthesis.
isoamylase is suggested to play an important role in amylopectin biosynthesis and starch granule formation
<cit> .
both agpase  and isa <dig> have been demonstrated as important players in amylopectin synthesis and starch granule formation in rice
<cit> .
our qpcr results indicate that agpase s
<cit>  and isa <dig> are instrumental for the accumulation of additional amylopectin in the amylose–reduced mutant gm <dig>  in the shh experiment, we could not confirm that the identified agps corresponded to the cytosolic enzyme as the unigene sequence did not cover the transit peptide sequence region.
however, our qpcr analysis of both cytosolic  and plastidic  forms according to ohdan et al.
<cit>  indicated that clone id no.
<dig> should be the cytosolic form.
the mechanism behind the elevated expression of agp and isa <dig> in the rice mutant remains unclear.
one possibility for the enhanced agp activity could be that the total amount of agpase needs to be increased to provide ample supply of adp-glucose when more plastidic agpase is being recruited to multienzyme complexes for the regulation of carbon partitioning
<cit> .
another possibility is that the extra agpase is required in gm <dig> to convert glc1-p to adp-glucose in the cytosol .
since isa <dig> is generally accepted as an important player in amylopectin synthesis and granule formation
<cit> , it is not surprising that the isa <dig> expression in gm <dig> significantly increased when extra amylopectin was produced in the endosperm.
we also observed that the genes for sucrose synthase  <dig> and udpase  <dig> were upregulated in gm <dig>  since accumulation of other carbohydrates synthesized from the udp-glucose precursor, such as cellulose and β-glucan, were unaffected in the mutant, we suggest that the increased expression of the genes for sucrose synthase  <dig> and udpase  <dig> may be also associated with amylopectin synthesis.
sucrose synthase  <dig> is suggested to be cytosolic
<cit>  and may produce udp-glucose, which is converted by udpase  <dig> to the hexose-phosphate used for amylopectin synthesis
<cit> .
indeed, sucrose synthases  <dig> and  <dig> in arabidopsis, which belong to the same group as rice sucrose synthase 4
<cit> , have been recently reported to direct carbon to starch synthesis
<cit> .
in our experiment, the elevated expression of cytosolic agp supports that notion.
enhanced levels of agpase may be needed to convert the glc 1-p produced by sucrose synthase  <dig> and udpase  <dig> to adp-glucose for additional amylopectin synthesis.
for other forms of sucrose synthases and for udpase  <dig>  we did not find any significant shifts in gene expression between gm <dig> and bp <dig>
this study is centered on carbon partitioning and gene regulation in seeds of a waxy rice mutant.
our results provide no information about how carbon partitioning is regulated at the level of enzyme activity.
lü et al.
<cit>  used transgenic rice with antisense inhibition of gbssi to examine the activities of major starch synthesis enzymes.
some of the phenotypic traits observed by lü et al.
in the gmo rice were similar to what we found for the gm <dig> mutant, such as no changes in seed weight and only small changes in total starch content.
in accordance with our gene expression analysis, they also noticed an increase in isoamylase activities.
however, they did not observe any changes in activities for agpase or sbes, which seems to disagree with our results at the gene activity level.
we do not yet know the reason for this disparity between gene expression and enzyme activity but it should be noted that the levels of transcripts and proteins in a cell are determined by several factors, like the rate of transcription initiation, mrna stability, efficiency of translation, and protein stability and modifications.
our knowledge about gene regulation and the involvement of putative transcription factors in carbon partitioning is poor.
sun et al.
<cit>  reported that the barley susiba <dig> transcription factor participates in sugar signaling in barley and that it upregulates target genes by binding to the sure-element  within the promoter region
<cit> .
they suggested that the sure-element in promoter regions of sugar-inducible genes may play an important role in susiba2-controlled gene expression.
interestingly, when we searched the promoter region of the nine upregulated genes in gm <dig>  including the rice susiba2-like, we found a number of putative sure elements in all of the genes .
a very good correlation at the gene expression level was found for susiba2-like and isa <dig>  we suggest that upregulation of isa <dig> and other genes in the gm <dig> mutant is mediated by the susiba2-like transcription factor.
this notion is further corroborated by recent transgenic studies in rice .
interestingly, when we performed a bioinformatic analysis on the gene expression patterns of the three selected genes  from the ssh experiment in this study using the publicly available rice and arabidopsis microarray data, we found some correlations between susiba2-like and the other genes .
however, isa <dig> is expressed in arabidopsis leaves but not in rice leaves and the expression level of susiba2-like is generally low in both species for reasons we do not know.
since susiba2-like is a transcription factor, its gene expression level should be low.
what caused the differential expression of isa <dig> in the two species is unclear.
in vitro and in vivo protein-dna interaction studies are under way to further determine the involvement of susiba2-like and sure elements in the regulation of starch biosynthesis in the rice endosperm.
in addition to their high value as starch crops, there is an increasing interest in using cereals for the production of non-starch compounds, such as β-glucan and fructan for functional foods, and oil for biofuel applications
<cit> .
our experimental data implicate three genes of importance for amylopectin synthesis in the rice endosperm, agp, isa <dig>  and susiba2-like.
since agps and susiba2-like likely control the entire metabolic pathway for starch synthesis in cereals, we believe they are good targets for redirecting carbon flux from starch biosynthesis to alternative products.
in fact, approaches to downregulate agps in arabidopsis to enhance oil production at the expense of starch biosynthesis met with success
<cit> .
it will be interesting to explore the potential for modulating susiba <dig> activity as a strategy for rerouting photosynthate from starch biosynthesis to other anabolic pathways in cereal seeds.
CONCLUSIONS
understanding of carbon allocation in cereal seeds is of great importance in plant biology.
in this study we used a rice waxy mutant to gain molecular insights into how amylose deficiency affects carbon allocation in cereal seeds.
analysis of carbohydrate and oil fractions in the waxy mutant showed that when amylose is deficient, carbon is mainly allocated to amylopectin rather than to other carbon end products, such as β-glucan or oil.
gene expression profiling identified several candidate genes implicated in the carbon reallocation response.
these genes included agp and susiba2-like.
we suggest that these two genes are promising targets in efforts to redirect carbon flux in cereal seeds from starch biosynthesis to alternative carbon end products.
to our knowledge, this study is the first comparative analysis of carbon fractions and gene expression profiling on a global scale in a waxy mutant.

 cereal crops are of critical importance in agriculture. the top three cereals in global production are maize, wheat, and rice, with and m tonnes, respectively. cereal crops constitute our largest primary food source and are also highly used in food and non-food industrial applications. contributing factors to the importance of cereals are that they can be bred to be very high yielding, that cereal grains lend themselves to long-term storage, and that the grain can accumulate different types of carbohydrates and lipids. major carbohydrates in cereal caryopses are the starch components amylose and amylopectin, cell wall components, such as different types of arabinoxylan, mixed-linkage β-glucan and cellulose, fructooligosaccharides, fructan, and sucrose. interestingly significant amounts of oil can also be stored in the endosperm, especially in oats. the composition of the cereal grain dictates the end use of the crop. for example, the cereal endosperm is the most important source of starch worldwide and is therefore of tremendous value for food security. there is an ongoing search for genotypes with high content of amylose, β-glucan and/or fructan for different applications within the functional food sector. at the other end of the spectrum are efforts to develop cereals that redirect carbon flux from carbohydrates to oils for production of high-density biofuels. a thorough understanding of the mechanisms for the partitioning of photosynthates in cereals is crucial for our ability to boost starch yield, to develop specialty crops for the functional food industry, such as barley with enhanced ß-glucan levels, and to tailor cereal production for the non-food industry. carbon partitioning in higher plants has been studied at the whole-plant level, for certain types of plant tissues, and for plant cells. however, many questions remain unanswered. for example, we need to identify and map the actions of key elements that determine carbon allocation between source and sink tissues and that govern carbon flux along pathways for synthesis of different carbohydrate and oil sinks. it is also imperative that we gain insight into how environmental factors influence carbon partitioning. several proteins have been implicated as important players in carbon partitioning in plants. they include proteins involved in sugar transport and metabolism, such as sucrose transporters, sucrose invertases and sucrose synthases, and in hexose metabolism and transport, such as hexose kinases and monosaccharide transporters. other examples include proteins controlling the flux in polysaccharide biosynthesis, such as adp-glucose pyrophosphorylase, and udp-glucose pyrophosphorylase, and regulatory proteins, such as sucrose non-fermenting-1-related protein kinase, trehalose-6-phosphate synthase, and transcription factors. we are interested in identifying molecular switches in cereals that direct carbon flux to different tissues and into the specific end products. we are particularly concerned with carbon partitioning between amylose, amylopectin, oil, β-glucan and fructan in cereal seeds. for the present study, we chose a rice waxy mutant, gm which is deficient in amylose biosynthesis. we examined carbon partitioning between amylose, amylopectin, oil, β-glucan, fructan and other dietary fibers in the gm background, a nearly isogenic waxy line. we constructed a suppression subtractive hybridization cdna library between the mutant and the corresponding wild type to identify potential candidates involved in carbon partitioning. we used qpcr to verify results from the ssh experiments and to study how gene regulation controls carbon allocation in the absence of amylose biosynthesis.  the gm rice is a waxy mutant waxy rice has been drawing much attention in rice breeding in china as it has many applications in traditional chinese food and brewing. this has resulted in a large collection of waxy rice in the chinese rice germplasm repositories and also in a number of breeding programs on the different qualities of waxy rice. we selected one waxy rice cultivar, gm, mainly based on the following factors: i) it is a stable mutant with a nearly isogenic background; ii) it has a relatively low amylose content compared to other waxy mutants; iii) with the exception of its waxy grain character, gm is phenotypically similar to its wild-type counterpart bp, an elite variety of indica rice. when the grains of gm were cut transversely and stained with an iodine solution, a typical reddish color of waxy starch was revealed in the endosperm. we have further characterized the grain starch of gm by recording the light absorbance of the starch-iodine complex between nm and nm with a scanning spectrophotometer. we included internal standards of starch with known contents of amylose. as seen in additional file the absorbance value around nm for the amylose-iodine complex was reduced proportionally with the amylose content in the starch samples, including those from the wild type and mutant. based on the absorbance, the estimated amylose content of bp and gm is between standards and, and standards and, respectively. the estimations were confirmed with chemical analyses revealing a significant difference in amylose content of % and % in kernels of bp and gmo respectively. the starch content was around 67% in both types of rice grains.. 1no significant difference of total starch content between bp and gm. 2significant difference of total amylose content between bp and gm. qpcr analysis of expression levels for gbssi and gbssii. dw, gbss. the statistical difference between bp and gm is presented as “significantly decreased” and “increased”, respectively. it is generally accepted that amylose synthesis is carried out by granule-bound starch synthases. cereals have two forms of gbss, gbssi and gbssii. gbssi is responsible for amylose synthesis in storage tissues, such as endosperm, whereas gbssii is present in green tissues, including the pericarp of seeds. we used qpcr to analyze gene expression for both gbss genes in rice seeds with the ubiquitin gene, ubq as an internal standard. the qpcr results showed that, in gm seeds, gene expression of gbssi was significantly reduced and the expression of ubq is about the same as in the control bp expression of gbssii was significantly increased in gm as compared with bp we have shown that the gm rice is a waxy mutant caused by down-regulation of gbssi. yield, kernel weight and starch content were similar between the waxy mutant and the corresponding wild type.to gain insight into the redistribution of carbon in the gm seed, we subjected the mutant and wild-type lines to carbohydrate and oil analyses. the major carbon from amylose is redistributed to amylopectin in the waxy mutant carbohydrate analyses revealed that both gm and the parental bp lines contained about 3% dietary fiber with similar compositions. arabinoxylan and cellulose were major dietary fiber components while mixed-linkage β-glucan and fructan were minor components. consequently, no extra carbon was distributed into the cell walls or to the β-glucan or fructan sink in the gmo mutant. the starch content was slightly reduced in the waxy mutant compared to the wild type. the amylose content was normal in the wild type but highly reduced in the waxy mutant. thus the amylose content in the seed was reduced from 17% of the caryopsis in the wild type to % in the waxy mutant. the reduced content of amylose was mainly compensated for by an increased content of amylopectin in the waxy caryopsis, 65% in the waxy mutant compared to 53% in the wild type. the content of sucrose and crude oils were the same in the two rice lines. the glucose content in the gm mutant was somewhat higher than the wild type, but was low in both lines. *mean value from three independent analytic experiments using randomly selected caryopses from a pool of six plants. **sugar residue. the carbohydrate analysis thus indicated that a major fraction of carbon in the waxy mutant gm was reallocated from amylose to amylopectin synthesis. this result prompted us to try to identify the genes in gm responsible for this reallocation. to this end, we employed the ssh strategy. suppression subtractive hybridization identified unigenes in the waxy mutant we used gm as the tester and bp as the driver to construct a cdna library after pcr amplification and ssh of cdnas from total rna isolated from plants at days after flowering. the resulting ssh library of “gm vs bp034” contained clones with an average length of around bp. all positive clones were applied to sequencing, which returned the identification of unigenes. these unigenes were used for the clusters of orthologous groups functional annotation analysis after blastx and tblastx against the ncbi protein databases. among the unigenes, exhibited high similarity to known protein sequences, and showed no similarity to any reported sequence. within the protein sequences, lacked functional annotation. the rest of sequences were categorized in four functional groups: “information storage and processing”, “cellular processes and signaling”, “metabolism”, and “poorly characterized”. these four functional groups have and unigenes, corresponding to %, %, % and % of the total unigenes, respectively. the details of the unigenes and their putative functions are shown in table interestingly, two unigenes, similar to the genes for adp-glucose pyrophosphorylase small subunit and isoamylase, respectively, were found in the carbohydrate transport and metabolism group. notably, one clone in the group of no related cogs showed a high similarity to wrky transcription factor. further sequence analysis of the genes for isoamylase and wrky transcription factor revealed that they are rice orthologs to barley isa and susiba respectively, previously described by sun et al... the number of unigenes in each group is indicated and their percentage in the total number of unigenes is denoted. validation of the shh results by semi-quantitative pcr to verify the conclusions from the shh experiment, we selected two housekeeping genes, the gene for the eukaryotic elongation factor- α subunit and ubq to follow the ssh experiment by semi-quantitative pcr. when we used the same batch of rna as in the shh experiment, or rna isolated from other stages of seed development, or from other tissues, we found expression levels of the two housekeeping genes to be more or less the same in gm and bp furthermore, expression levels were constant throughout seed development and in different tissues of mutant and wild-type rice. importantly, we observed that the cdna for eef- α could be detected in the tester and driver samples prior to ssh, but not in the sample after subtraction hybridization, lending support to the validity of the ssh approach. we also chose some additional genes, related to starch biosynthesis and carbon portioning to further verify the reliability of the shh experiment and to obtain detailed quantitative data on gene expression in the two rice lines. results from those analyses are presented below. gene expression profiling in the waxy mutant to further validate the results from the shh experiment and to quantify expression of genes involved in starch biosynthesis and/or carbon portioning, we chose genes as representatives for gene expression analysis by qpcr, including two reference genes, eef- α and ubq. according to the results obtained by qpcr, we divided the genes into five groups. the classification was based on qpcr quantification of the differential gene expression in gm077; “significantly decreased”, “not changed”, “increased”, “significantly increased” and “not detected”. intriguingly, among the four significantly increased genes, agps, sbei, isa and susiba2-like, all except sbei were found in the shh library. we noted that the expression level for the upregulated genes in gm correlated well with the expression level for the susiba2-like transcription factor gene. gene expression correlation of susiba2-like and isa in the mutant and wild type sun et al. have demonstrated that isa and sbeiib in barley were upregulated by the activity of the susiba transcription factor and a good correlation in gene expression levels has been demonstrated between susiba and its target genes, such as isa and sbeiib. to learn if this correlation holds true in rice also, and in an effort to find susiba2-like-controlled genes in rice, we selected rice isa as a representative to study the correlation in expression between susiba2-like and its target genes in rice. for this study, we chose different tissues and different time points in both the mutant gm and the wild type bp as displayed in figure 5a and b, there was an excellent correlation between expression levels for the two genes in the analyzed samples. the statistical analysis indicated that the relative levels of the spatial and temporal expression for the two genes in both rice lines shared a pearson correlation coefficient of. discussion although waxy mutants of higher plants and the responsible gene have been studied to a large extent, and the high content of amylopectin in the mutant is known, little information about carbon partitioning to other carbohydrates and oil fractions in waxy mutants has been reported. moreover, gene regulation of carbon reallocation to amylopectin in the mutant is poorly understood. we are interested in the partitioning of photosynthates between starch and other storage compounds in cereal seeds. in this study, we selected one of the rice waxy mutants to follow carbon partitioning between starch and other carbohydrates when amylose biosynthesis is impeded. our carbohydrate analysis indicated that when the amylose content is reduced, the vast majority of the assimilated carbon is reallocated to amylopectin, rather than to other carbohydrates or lipids. interestingly, such a reallocation did not change seed weight but, rather, shifted carbon from one compound to another within the starch biosynthesis machinery. to understand the molecular mechanisms controlling the increase in amylopectin biosynthesis, we set out to identify genes that were upregulated in the waxy mutant. from the shh experiments we found three candidates that have previously been shown to be directly involved in starch synthesis and/or its regulation, agp, isa, and susiba2-like. the functions and regulation of agpase and isoamylase have been reviewed and well documented previously. in cereal endosperm cells, there are two forms of agpase, one cytosolic and one plastidic. the major fraction of adp-glucose in cereal endosperm is believed to be produced in the cytosol and then transported to the amyloplast for subsequent starch biosynthesis. isoamylase is suggested to play an important role in amylopectin biosynthesis and starch granule formation. both agpase and isa have been demonstrated as important players in amylopectin synthesis and starch granule formation in rice. our qpcr results indicate that agpase s and isa are instrumental for the accumulation of additional amylopectin in the amylose–reduced mutant gm in the shh experiment, we could not confirm that the identified agps corresponded to the cytosolic enzyme as the unigene sequence did not cover the transit peptide sequence region. however, our qpcr analysis of both cytosolic and plastidic forms according to ohdan et al. indicated that clone id no. should be the cytosolic form. the mechanism behind the elevated expression of agp and isa in the rice mutant remains unclear. one possibility for the enhanced agp activity could be that the total amount of agpase needs to be increased to provide ample supply of adp-glucose when more plastidic agpase is being recruited to multienzyme complexes for the regulation of carbon partitioning. another possibility is that the extra agpase is required in gm to convert glc1-p to adp-glucose in the cytosol. since isa is generally accepted as an important player in amylopectin synthesis and granule formation, it is not surprising that the isa expression in gm significantly increased when extra amylopectin was produced in the endosperm. we also observed that the genes for sucrose synthase and udpase were upregulated in gm since accumulation of other carbohydrates synthesized from the udp-glucose precursor, such as cellulose and β-glucan, were unaffected in the mutant, we suggest that the increased expression of the genes for sucrose synthase and udpase may be also associated with amylopectin synthesis. sucrose synthase is suggested to be cytosolic and may produce udp-glucose, which is converted by udpase to the hexose-phosphate used for amylopectin synthesis. indeed, sucrose synthases and in arabidopsis, which belong to the same group as rice sucrose synthase 4, have been recently reported to direct carbon to starch synthesis. in our experiment, the elevated expression of cytosolic agp supports that notion. enhanced levels of agpase may be needed to convert the glc 1-p produced by sucrose synthase and udpase to adp-glucose for additional amylopectin synthesis. for other forms of sucrose synthases and for udpase we did not find any significant shifts in gene expression between gm and bp this study is centered on carbon partitioning and gene regulation in seeds of a waxy rice mutant. our results provide no information about how carbon partitioning is regulated at the level of enzyme activity. lü et al. used transgenic rice with antisense inhibition of gbssi to examine the activities of major starch synthesis enzymes. some of the phenotypic traits observed by lü et al. in the gmo rice were similar to what we found for the gm mutant, such as no changes in seed weight and only small changes in total starch content. in accordance with our gene expression analysis, they also noticed an increase in isoamylase activities. however, they did not observe any changes in activities for agpase or sbes, which seems to disagree with our results at the gene activity level. we do not yet know the reason for this disparity between gene expression and enzyme activity but it should be noted that the levels of transcripts and proteins in a cell are determined by several factors, like the rate of transcription initiation, mrna stability, efficiency of translation, and protein stability and modifications. our knowledge about gene regulation and the involvement of putative transcription factors in carbon partitioning is poor. sun et al. reported that the barley susiba transcription factor participates in sugar signaling in barley and that it upregulates target genes by binding to the sure-element within the promoter region. they suggested that the sure-element in promoter regions of sugar-inducible genes may play an important role in susiba2-controlled gene expression. interestingly, when we searched the promoter region of the nine upregulated genes in gm including the rice susiba2-like, we found a number of putative sure elements in all of the genes. a very good correlation at the gene expression level was found for susiba2-like and isa we suggest that upregulation of isa and other genes in the gm mutant is mediated by the susiba2-like transcription factor. this notion is further corroborated by recent transgenic studies in rice. interestingly, when we performed a bioinformatic analysis on the gene expression patterns of the three selected genes from the ssh experiment in this study using the publicly available rice and arabidopsis microarray data, we found some correlations between susiba2-like and the other genes. however, isa is expressed in arabidopsis leaves but not in rice leaves and the expression level of susiba2-like is generally low in both species for reasons we do not know. since susiba2-like is a transcription factor, its gene expression level should be low. what caused the differential expression of isa in the two species is unclear. in vitro and in vivo protein-dna interaction studies are under way to further determine the involvement of susiba2-like and sure elements in the regulation of starch biosynthesis in the rice endosperm. in addition to their high value as starch crops, there is an increasing interest in using cereals for the production of non-starch compounds, such as β-glucan and fructan for functional foods, and oil for biofuel applications. our experimental data implicate three genes of importance for amylopectin synthesis in the rice endosperm, agp, isa and susiba2-like. since agps and susiba2-like likely control the entire metabolic pathway for starch synthesis in cereals, we believe they are good targets for redirecting carbon flux from starch biosynthesis to alternative products. in fact, approaches to downregulate agps in arabidopsis to enhance oil production at the expense of starch biosynthesis met with success. it will be interesting to explore the potential for modulating susiba activity as a strategy for rerouting photosynthate from starch biosynthesis to other anabolic pathways in cereal seeds.  understanding of carbon allocation in cereal seeds is of great importance in plant biology. in this study we used a rice waxy mutant to gain molecular insights into how amylose deficiency affects carbon allocation in cereal seeds. analysis of carbohydrate and oil fractions in the waxy mutant showed that when amylose is deficient, carbon is mainly allocated to amylopectin rather than to other carbon end products, such as β-glucan or oil. gene expression profiling identified several candidate genes implicated in the carbon reallocation response. these genes included agp and susiba2-like. we suggest that these two genes are promising targets in efforts to redirect carbon flux in cereal seeds from starch biosynthesis to alternative carbon end products. to our knowledge, this study is the first comparative analysis of carbon fractions and gene expression profiling on a global scale in a waxy mutant. 
among the unigenes, three, agp, isa and susiba2-like, were found to be directly involved in amylopectin synthesis, indicating their possible roles in redirecting carbon flux from amylose to amylopectin. we believe that agp and susiba are two promising targets for classical breeding and/or transgenic plant improvement to control the carbon flux between starch and other components in cereal seeds. in this study, we used a rice mutant, gm with a low content of amylose to gain molecular insight into how a deficiency of amylose affects carbon allocation to other end products and to amylopectin. we used carbohydrate analysis, subtractive cdna libraries, and qpcr to identify candidate genes potentially responsible for the changes in carbon allocation in gm seeds. carbon allocationrice waxy seedssuppression subtractive hybridization quantitative polymerase chain reaction gene expression carbohydrate analysis indicated that the content of amylose in gm seeds was significantly reduced, while that of amylopectin significantly rose as compared to the wild type bp the content of glucose, sucrose, total starch, cell-wall polysaccharides and oil were only slightly affected in the mutant as compared to the wild type. understanding carbon partitioning in cereal seeds is of critical importance to develop cereal crops with enhanced starch yields for food security and for producing specified end-products high in amylose, β-glucan, or fructan, such as functional foods or oils for biofuel applications. analyses of carbohydrate and oil fractions and gene expression profiling on a global scale in the rice waxy mutant gm revealed several candidate genes implicated in the carbon reallocation response to an amylose deficiency, including genes encoding agpase and susiba2-like. waxy mutants of cereals have a high content of amylopectin and have been well characterized. a bioinformatics analysis of the putative susiba2-like binding elements in the promoter regions of the upregulated genes indicated that the susiba2-like transcription factor may be instrumental in promoting the carbon reallocation from amylose to amylopectin. suppression subtractive hybridization experiments generated unigenes in the mutant on the wild-type background. however, the allocation of carbon to other components, such as β-glucan and oils, and the regulation of the altered carbon distribution to amylopectin in a waxy mutant are poorly understood.
 understanding carbon partitioning in cereal seeds is of critical importance to develop cereal crops with enhanced starch yields for food security and for producing specified end-products high in amylose, β-glucan, or fructan, such as functional foods or oils for biofuel applications. waxy mutants of cereals have a high content of amylopectin and have been well characterized. however, the allocation of carbon to other components, such as β-glucan and oils, and the regulation of the altered carbon distribution to amylopectin in a waxy mutant are poorly understood. in this study, we used a rice mutant, gm with a low content of amylose to gain molecular insight into how a deficiency of amylose affects carbon allocation to other end products and to amylopectin. we used carbohydrate analysis, subtractive cdna libraries, and qpcr to identify candidate genes potentially responsible for the changes in carbon allocation in gm seeds.  carbohydrate analysis indicated that the content of amylose in gm seeds was significantly reduced, while that of amylopectin significantly rose as compared to the wild type bp the content of glucose, sucrose, total starch, cell-wall polysaccharides and oil were only slightly affected in the mutant as compared to the wild type. suppression subtractive hybridization experiments generated unigenes in the mutant on the wild-type background. among the unigenes, three, agp, isa and susiba2-like, were found to be directly involved in amylopectin synthesis, indicating their possible roles in redirecting carbon flux from amylose to amylopectin. a bioinformatics analysis of the putative susiba2-like binding elements in the promoter regions of the upregulated genes indicated that the susiba2-like transcription factor may be instrumental in promoting the carbon reallocation from amylose to amylopectin.  analyses of carbohydrate and oil fractions and gene expression profiling on a global scale in the rice waxy mutant gm revealed several candidate genes implicated in the carbon reallocation response to an amylose deficiency, including genes encoding agpase and susiba2-like. we believe that agp and susiba are two promising targets for classical breeding and/or transgenic plant improvement to control the carbon flux between starch and other components in cereal seeds. carbon allocationrice waxy seedssuppression subtractive hybridization quantitative polymerase chain reaction gene expression 