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Screen of Genes Linked to High-Sugar Content in Stems by Comparative Genomics

Screen of Genes Linked to High-Sugar Content in Stems by Comparative Genomics Rice (2008) 1:166–176 DOI 10.1007/s12284-008-9012-9 Screen of Genes Linked to High-Sugar Content in Stems by Comparative Genomics Martín Calviño & Rémy Bruggmann & Joachim Messing Received: 1 May 2008 /Accepted: 20 August 2008 /Published online: 24 September 2008 Springer Science + Business Media, LLC 2008 Abstract One of the great advantages of the fully sequenced practical aspect of such a concept is of great importance for rice genome is to serve as a reference for other cereal agronomical purposes because a useful trait in one species genomes in particular for identifying genes linked to unique could be transferred to another. A relevant example could traits. A trait of great interest is reduced lignocellulose in the be carbohydrate partitioning and allocation. In cereals such stem of related species in favor of fermentable sugars as a as wheat, sorghum, and rice, the process of grain filling source of biofuels. While sugarcane is one of the most demands carbon from photosynthesis assimilation as well efficient biofuel crops, little is known about the underlying as the remobilization of pre-stored carbohydrates in the gene repertoire involved in it. Here, we take advantage of the stem before and after anthesis [45]. It has been estimated natural variation of sweet and grain sorghum to uncover that about 30% of the total yield in rice depends on the genes that are conserved in rice, sorghum, and sugarcane but carbohydrate content accumulated in the stem before differently expressed in sweet versus grain sorghum by using heading [18]. For these reasons, characterization of genes a microarray platform and the syntenous alignment of rice involved in carbohydrate metabolism and accumulation can and sorghum genomic regions containing these genes. lead to the development of improved cereal crops. Indeed, enzymes involved in carbohydrate accumulation In recent years, there has been an increasing demand on and those that reduce lignocellulose can be identified. biomass for the production of ethanol as a renewable resource for fuel. The biggest producers of ethanol in the . . Keywords Integrative genetics Sugar accumulation world are Brazil and the USA [34]. In Brazil, it is derived . . . Cell wall synthesis Microarray analysis Synteny from sugarcane, while in the USA, ethanol is derived from Biofuel crops the grain of corn. Because of the use of the entire plant as a source for fermentable sugars, carbohydrate accumulation and partitioning has been extensively studied in sugarcane, Introduction probably more than in any other species [30]. However, genes involved in these processes cannot easily be identified Comparisons of genetic maps and sequences of several because of the complex genome of sugarcane, with several grass species have shown that there is global conservation cultivars differing greatly in their ploidy levels from 2n=100 of gene content and order [11]. Therefore, grasses have to 2n=130 chromosomes [9, 13]. Even if one could make been considered as a “single genetic system” [2]. The further improvements to sugarcane, it has the disadvantage of being a crop restricted to tropical growing areas. Electronic supplementary material The online version of this article On the other hand, the use of corn grain for ethanol (doi:10.1007/s12284-008-9012-9) contains supplementary material, production poses a major conflict because of its dual use as which is available to authorized users. food and fuel. Therefore, it has been proposed to use grain : : M. Calviño R. Bruggmann J. Messing (*) solely for food and only the stover as a source for ethanol. Waksman Institute of Microbiology, Rutgers University, A major impediment to this approach is that, in contrast to 190 Frelinghuysen Road, sugarcane, corn stover consists mainly of lignocellulose, Piscataway, NJ 08854-8020, USA which is more costly to process than fermentable sugars [8]. e-mail: messing@waksman.rutgers.edu Rice (2008) 1:166–176 167 167 Therefore, it would be attractive to identify corn varieties sweet sorghum lines (Dale, Della, M81-E, Rio, Top76-6, and with reduced lignocellulose. Interestingly, there is extensive Simon) and one line from grain sorghum (BTx623). As an natural intra-species variation for sugar content in sorghum estimation of the total amount of sugars present in the juice with cultivars that do not accumulate sugars (referred to as of sorghum stems, we measured the Brix degree of each grain sorghums) in contrast to those that accumulate large internode along the main stem at the time of flowering. We amounts of sugars in their stems [16]. Such intra-species found great variation in flowering time as well as in Brix variation can serve as a platform to identify genes linked to degree between the sweet sorghum lines when compared to increased sugar content and reduced lignocellulose [4]. grain sorghum BTx623 (Fig. 1a,b). In general, the Brix Moreover, if these genes are conserved by ancestry in degree was lower in the mature and immature internodes of related species, one could envision the introduction of such the stem, in contrast to maturing internodes. These findings a trait by the import of specific regulatory regions. are in agreement with previous studies [16, 25]. Consistent Conservation of gene order between closely related species with the inability of grain sorghum to accumulate significant permits the alignment of orthologous chromosomal segments. levels of sugars in the stem, the Brix degree in BTx623 was Non-collinear genes would constitute paralogous copies [29]. low and remained fairly constant for all the internodes along To facilitate such alignments, the use of rice with one of the the stem. Among the sweet sorghum cultivars, Rio had the smallest cereal genomes that has been sequenced [17] highest Brix degree and Simon the lowest. Furthermore, the increasingly becomes the anchor genome for other grasses difference in flowering time between BTx623 and Rio was [28]. In this sense, we can use rice as a reference genome for smaller than in the rest of sweet sorghum lines with high Brix biofuel crops such as sugarcane and sorghum. degrees. For this reason, we decided to perform a comparative While rice offers an excellent reference as a compact analysis of transcripts in the stem of the Rio and BTx623 genome from an evolutionary point of view, it is less sorghum lines. suitable as a reference for a phenotype of reduced lignocellulose. Moreover, rice is a bambusoid C3 cereal plant, and sorghum and sugarcane are panicoid C4 cereal plants, which branched out 50 mya [23]. Sorghum and sugarcane belong to the Saccharinae clade and diverged from each other only 8–9mya [14, 20]. Therefore, sugarcane and its reduced lignocellulose can serve as a trait reference for sorghum varieties that differ in the cellulose content of their stems. Consequently, we took advantage of a GeneChip that was created to study gene expression in Btx 623 Dale Della M81-E Rio Simon Top 76-6 sugarcane and its role in the accumulation of sugar in the stem during development [6] for the comparison of grain b and sweet sorghum genes. One would expect that sweet sorghum and sugarcane use similar gene products for enhanced sugar accumulation in their stems. Indeed, we not only identified genes involved in sugar accumulation and lignocellulose synthesis, whose expression levels are correlated with the trait, but also demonstrate their ancestry through the alignment of orthologous regions of the rice and sorghum genomes. Therefore, the same genes could also be used to improve other biofuel crops like switchgrass and Miscanthus, validating a translational genomic approach. Results Sugar accumulation in the stem of grain and sweet Fig. 1 Variation in flowering time and Brix degree. a Comparison of sorghum cultivars flowering time between grain sorghum BTx623 and six sweet sorghum cultivars. Time to flowering was measured as days required reaching 50% anthesis. b Comparison of Brix degree along the main Previous reports have indicated that in sorghum stems, stem between grain sorghum BTx623 and six sweet sorghum sugars start to accumulate at flowering stage [16, 25]. We genotypes. The Brix degree was measured for each internode, and compared the accumulation of sugars in the stem between six the average of a triplicate experiment was plotted. 168 Rice (2008) 1:166–176 Microarray analysis of transcripts from sorghum found that almost 16% of the transcripts that were stem tissues differentially expressed between BTx623 and Rio corre- sponded to transcripts affecting carbohydrate metabolism In order to identify genes expressed in the stem with a (Tables 1 and 2). Based on the link between hypothetical potential role in sugar accumulation and reduced lignocellu- function and the sweet sorghum trait, we selected 37 lose [4], we compared transcript profiles between grain candidate genes, of which differential expression could be (BTx623) and sweet sorghum (Rio). Such a genome-wide considered to be the cause or the consequence of increased analysis became possible because of the recently designed sugar and decreased lignocellulose in sorghum stems. GeneChip of sugarcane [6]. This array was specifically Clearly such a screen would not detect candidate genes developed with sequences that were obtained from several that might play a qualitative rather than a quantitative role. cDNA libraries representing distinct tissue types including For instance, one might expect that the role of sucrose stem from 15 sugarcane varieties. The use of this GeneChip phosphate synthase in carbohydrate metabolism should be permitted us to directly compare gene expression data of two differentially expressed, but it appears to be unchanged different sorghum cultivars with the previously generated under the parameters examined. We therefore can discover data from sugarcane. Three independent plants for each mainly genes that are differentially expressed. Extrinsic BTx623 and Rio were grown until anthesis and RNA was evidence for the link between these candidate genes and extracted from the same maturing internode for all six plants. their potential function is described in more detail in These RNAs were used to prepare biotylinated cRNAs for “Discussion.” hybridization, each sample separately hybridized to one array. Among these, transcripts that were up-regulated include The sugarcane array comprised 8,224 probe sets, of hexokinase 8 and carbohydrate phosphorylase (starch and which more than 70% (5,900) gave a positive signal with sucrose metabolism), nicotinamide adenine dinucleotide sorghum RNA samples. When a twofold cut-off value was phosphate (NADP) malic enzyme (C4 photosynthesis), a applied as criterion to distinguish differentially expressed D-mannose binding lectin (sugar binding), and a lysin motif transcripts between grain and sweet sorghum, a total of 195 (LysM) domain protein possibly involved in cell wall transcripts were identified, with 132 transcripts being degradation. Transcripts that were down-regulated included down-regulated and 63 transcripts up-regulated in Rio, sucrose synthase 2 and fructokinase 2 (starch and sucrose respectively (Electronic Supplementary Material Tables S1 metabolism), alpha-galactosidase and beta-galactosidase and S2). Because some probe sets identify the same gene, (hydrolysis of glycosidic bonds), and cellulose synthase 1, the number of genes that is down-regulated is 103 and up- 7, and 9 together with cellulose synthase catalytic subunit regulated 51, respectively. Based on the annotation of the 12 (cell wall metabolism). In addition, several others sorghum genes, we were able to infer the possible function transcripts with a cell-wall-related role that were down- for most of the differentially expressed transcripts. regulated included cinnamoyl CoA reductase, cinnamyl Among the transcripts that were up-regulated in Rio, a alcohol dehydrogenase, 4-coumarate coenzyme A ligase, saposin-like type B gene displayed the highest differential caffeoyl-CoA O-methyltransferase, xyloglucan endo- expression. Saposins are involved in the degradation of transglycosylase/hydrolase, peroxidase and phenylalanine, sphingolipids [31]. Other transcripts encoding stress-related and histidine ammonia-lyase. proteins such as heat shock protein 70 (HSP70) and HSP90 were up-regulated, consistent with an osmotic stress Validation of microarray data by quantitative reverse imposed by high concentration of sugars (Electronic transcription polymerase chain reaction Supplementary Material Tables S1 and S2)[3]. Our results show that in Rio, down-regulated genes outnumber those To validate the data obtained by microarray analysis, we that are up-regulated by a factor of 2. The most reduced randomly sampled 14 of the 37 candidate genes and transcript has a fasciclin domain. This domain has been compared their expression levels in both Rio and BTx623 shown to be involved in cell adhesion (Table 1)[10, 22]. by performing quantitative reverse transcription polymerase chain reaction (qRT-PCR; Fig. 2a). In Rio, the expression Genes with altered expression in carbohydrate metabolism of saposin, carbohydrate phosphorylase, hexokinase-8, and in sweet sorghum NADP malic enzyme is up-regulated in comparison with their expression in BTx623. In contrast, the expression of Based on Gene Ontology (GO) terms (http://www.geneon fasciclin-like protein FLA15, cellulose synthase 1 and 7, tology.org/), the sucrose and starch metabolic pathway from fructokinase-2, 4-coumarate coenzyme A ligase, sucrose the Kyoto Encyclopedia of Genes and Genomes (KEGG; synthase 2, laccase, cinnamoyl CoA reductase, beta-galac- http://www.genome.jp/kegg/), and the Carbohydrate-Active tosidase 3 precursor, and alpha-galactosidase precursor enzymes (CAZy) database (http://www.cazy.org/), we were down-regulated in Rio. Although the levels of gene Rice (2008) 1:166–176 169 169 Table 1 List of “Trait-Specific” Genes that Are Syntenic with Rice a b Gene Rice Sorghum Expression Starch and sucrose metabolism Hexokinase 8 Os01g0190400 Sb03g003190.1 2.3 Hexokinase 8 Os05g0187100 Sb09g005840.1 Carbohydrate phosphorylase Os01g0851700 Sb03g040060.1 1.2 Sucrose synthase 2 Os03g0401300 Sb01g033060.1 −1.3 Sucrose synthase 2 Os07g0616800 Fructokinase-2 Os08g0113100 Sb07g001320.1 −1.7 Sorbitol dehydrogenase Os08g0545200 Sb07g025220.1 1.6 Sugar binding D-mannose binding lectin Os06g0165200 Sb10g022730.1 2 CO assimilation NADP-dependent malic enzyme Os01g0723400 Sb03g033250.1 2 Cell-wall-related LysM domain protein/cell wall catabolism Os03g0110600 Sb01g049890.1 1.2 Cellulose synthase-7 Os03g0837100 Sb01g002050.1 −1 Cellulose synthase-1 Os05g0176100 Sb09g005280.1 −1.1 Cellulose synthase-9 Os07g0208500 Sb02g006290.1 −1.1 Cellulose synthase-9 Os03g0808100 Sb01g004210.1 Cellulose synthase catalytic subunit 12 Os09g0422500 Sb02g025020.1 −4.7 Alpha-galactosidase precursor Os10g0493600 Sb01g018400.1 −1.8 Beta-galactosidase 3 precursor Os01g0875500 Sb03g041450.1 −2.4 Beta-galactosidase 3 precursor Os05g0428100 Sb03g041450.1 Cinnamoyl CoA reductase Os08g0441500 Sb07g021680.1 −2.9 Cinnamoyl CoA reductase Os09g0419200 Sb10g005700.1 Laccase Os01g0842400 Sb03g039520.1 −3.5 4-Coumarate coenzyme A ligase Os02g0177600 Sb04g005210.1 −3.7 4-Coumarate coenzyme A ligase Os06g0656500 Sb10g026130.1 Fasciclin domain Os03g0788600 Sb01g005770.1 −1.75 Fasciclin domain Os07g0160600 Sb02g003410.1 Fasciclin-like protein FLA15 Os05g0563600 Sb09g028490.1 −6.5 Caffeoyl-CoA O-methyltransferase 2 Os06g0165800 Sb10g004540.1 −2.15 Caffeoyl-CoA O-methyltransferase Os08g0498100 Sb07g028530.1 −5.3 Caffeoyl-CoA O-methyltransferase Os09g0481400 Sb02g027930.1 In boldface: sorghum genes that correspond to sugarcane probe set IDs previously reported by [6] Paralogs in italics Mean Log2 Ratio of sweet versus grain sorghum Sorghum gene to which a sugarcane probe set was mapped expression between the microarray and the qRT-PCR flowering and measured the expression of both genes by method differ to some extent, there is no difference in the qRT-PCR. We found that the saposin-type B gene is also classification of up- or down-regulated genes. This 100% highly expressed in Dale and Della when compared to grain correspondence of microarray with qRT-PCR data illus- sorghum and that the opposite is true for the expression of trates that the microarray platform can be used as an fasciclin-like protein FLA15, highly expressed in BTx623 effective method for screening large amounts of genes for a compared to Dale and Della (Fig. 2b). particular trait across closely related species. Before one would embark on any further experimentation, a much Genomic location of differentially expressed genes smaller candidate gene set can then be tested by more labor- intensive methods for gene expression between cultivars of In order to see if genes that were differentially expressed the same species. In order to see if the expression difference between grain and sweet sorghum cluster together in a between BTx623 and Rio for the transcripts encoding a particular region of the sorghum genome, we generated a saposin-type B protein and a fasciclin-like protein FLA15 “transcriptome map” (Fig. 3). We mapped the sequences of also extended to other sweet sorghum lines, we extracted all up- and down-regulated sugarcane probes to the recently RNA from maturing stems of BTx623, Dale, and Della at sequenced sorghum genome (BTx623; http://www.phyto 170 Rice (2008) 1:166–176 Table 2 List of “Trait-Specific” Gene Sorghum Expression Genes that Are Not Syntenic with Rice Cell-wall-related Alcohol dehydrogenase Sb10g006290 1 Cinnamyl alcohol dehydrogenase Sb04g011550 −1.5 Dolichyl-diphospho-oligosaccharide Sb02g006330 −1.4 Xyloglucan endo-transglycosylase/hydrolase Sb06g015880 −1.1 Putative Xylanase inhibitor Sb05g027350 −1.5 Putative Xylanase inhibitor Sb02g004660 −1.5 In boldface: sorghum genes Glycoside hydrolase family 1 Sb02g029640 −1.1 that correspond to sugarcane Phenylalanine and histidine ammonia-lyase Sb04g026520 −2 probe set IDs previously Peroxidase Sb02g037840 −1.5 reported by [6] Mean Log2 Ratio of sweet Similar to Saposin type B protein Sb09g013990 5.7 versus grain sorghum zome.net/cgi-bin/gbrowse/sorghum/) using GenomeThreader Discussion [12]. From a total of 195 probe sets, 176 could be mapped to the sorghum genome based on their alignment with a Translational genomics sorghum gene (“Materials and methods”). In addition, six probe sets could be mapped to the genome but do not The non-renewable nature of fossil oil imposes an increasing correlate with the current sorghum gene annotation, and for pressure to develop alternative energies in order to support another 13 probe sets, we were not able to map them to the and secure social and economic growth in the near future sorghum genome. Genes that were differentially expressed [34]. Currently, there is a worldwide interest to develop between grain and sweet sorghum do not appear to cluster in biofuel crops, the best example being sugarcane, used in any particular region of the genome but rather reflect random Brazil since the 1970s. Besides sugarcane, other grasses distribution (Fig. 3). such as Brachypodium distachyon, Miscanthus, maize, rice, sweet sorghum, and switchgrass are considered as crops for Trait-specific syntenic gene pairs between rice and sorghum biofuel research and production. Recently, the entire gene cluster of ten sorghum kafirin genes contained within a It can be considered that important gene functions have chromosomal segment of 45 kb was intact and stably been conserved by ancestry and that divergence is mainly inserted into the maize genome. Expression analysis then due to changes in regulatory control regions of genes. To has shown that kafirins accumulated in maize endosperm in determine the ancestry of genes, however, requires the a developmental and tissue-specific manner [38]. Such alignment of syntenic regions. Because we know now the transfer of genomic DNA between species that cannot be positions of the sorghum genes in their respective chromo- crossed could then be used in advanced breeding techniques somes, we can align them with the rice genome as a to introduce desirable traits from one species to another. reference [17] and determine whether the aligned regions Here, we integrate the traits of sugar accumulation and are collinear between rice and sorghum. Indeed, we found lignocellulose content with genomic and expression data of that from a total of 154 differentially expressed sorghum the three species, sugarcane, sorghum, and rice. We used the genes, 123 have an orthologous copy in syntenic positions recently developed Affymetrix sugarcane genome array [6] in rice (Electronic Supplementary Material Table S1). This as a tool for the identification of genes differentially collection includes 28 candidate genes for the sweet expressed in maturing stems of grain and sweet sorghum. sorghum trait (Table 1). Interestingly, one of these The intra-species variation for sugar content in sorghum is candidate genes, sucrose synthase 2, is duplicated in rice more pronounced than between sugarcane varieties, making but not in grain sorghum. So the question arose whether the sorghum a more suitable model to study this trait. On the gene is duplicated in sweet sorghum thereby explaining the other hand, because we can map sorghum genes to their difference in gene expression simply by gene duplication. chromosomal positions, we can use rice as a reference Because we have only the sequence of grain sorghum, we genome to identify genes by their ancestry. performed a Southern blot analysis of genomic DNA of sweet sorghum. When genomic DNA from BTx623 and Cross-referencing tissue-specific transcripts Rio are compared, both possess a single copy of sucrose synthase 2 (data not shown). Therefore, it is unlikely that Sorghum and sugarcane belong to the Saccharinae clade gene duplication plays a role in changing the level of gene and diverged from each other only 8 to 9 mya [20], while expression. rice is a more distant relative and separated from this clade Rice (2008) 1:166–176 171 171 Hexokinase 8 NADP Malic enzyme Beta-galactosidase 3 precursor Fasciclin-like protein FLA15 1.2 1.2 1.4 1.2 1.2 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0 0 0 0 Carbohydrate phosphorylate Cellulose synthase 7 Cinnamoyl CoA reductase Saposin type B 1.2 1.4 1.4 1.4 1.2 1.2 1.2 1 1 1 1 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0 0 0 Sucrose synthase 2 Cellulose synthase 1 Laccase 1.2 1.6 1.4 BTx623 1.4 1.2 1 1.2 0.8 Rio 0.8 0.6 0.8 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 Fructokinase 2 Alpha-galactoside precursor 4-Coumarate CoA ligase 1.2 1.4 1.4 1.2 1.2 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 Saposin type B Fasciclin-like protein FLA15 1.6 1.6 1.4 1.4 1.2 1.2 1 1 BTx623 0.8 0.8 Dale Della 0.6 0.6 0.4 0.4 0.2 0.2 0 0 Fig. 2 Validation of microarray data by quantitative RT-PCR. a The abundance for each gene is presented as relative fold expression. b expression of 14 genes selected from Table 1 was analyzed through qRT-PCR comparing the expression of saposin type B and fasciclin- quantitative RT-PCR. The results of three independent experiments for like protein FLA15 in BTx623 and two sweet sorghum lines Della and both BTx623 and Rio are shown. The quantification of the mRNA Dale. 50 mya [23]. Because sorghum and sugarcane belong to the of stem-derived RNAs from sorghum to 5,900 sugarcane same clade, we reasoned that, by hybridizing RNA from probes of a GeneChip comprising 8,224 probe sets in grain and sweet sorghum onto the sugarcane GeneChip, we total is a good indication of such cross-referencing. By could correlate changes in transcript levels with traits from applying a twofold cut-off value as a parameter to filter out sweet sorghum such as sugar content and reduced ligno- differentially expressed transcripts, a total of 195 probe sets cellulose. Given the tissue specificity and the rather small were identified, of which 63 corresponded to transcripts gene set of the sugarcane GeneChip, the positive hybridization that were up-regulated (51 genes) and 132 (103 genes) Relative fold expression Relative fold change 172 Rice (2008) 1:166–176 of the transcripts involved in sucrose and starch metabolism and in cell-wall-related processes were differentially expressed between BTx623 and Rio. This is particularly interesting because a previous study with cDNAs from immature and maturing stem of sugarcane identified only 2.4% of the transcripts related to carbohydrate metabolism [7]. Furthermore, because sorghum stems are fully elongated at the anthesis stage, tissue samples from maturing internodes were also more suitable in profiling changes in gene expression associated with carbohydrate metabolism. The implication is that screening of differentially expressed genes can greatly be enhanced by genetic variability and selection of tissue. Function of genes with elevated expression in sweet sorghum The highest elevated transcript identified in our study encodes a saposin-like type B domain. Increased expression has also been validated and tested in other sweet sorghum lines by qRT-PCR. We also found a higher expression in Dale and Della compared to that in BTx623 (Fig. 2b). Saposins are water soluble proteins that interact with the lysosomal membrane and are involved in the catabolism of glycosphingolipids in animals [31, 39]. Although it was unexpected that such a function could be related to a role in sugar accumulation, it underscores the value of a microarray- based screen to detect possible new network effects. For instance, we could hypothesize that the removal of sugars from glycosphingolipids in the membrane alters its structure in such a way that it constitutes an early step in carbohydrate partitioning. Additional transcripts that were increased in sweet sorghum included hexokinase 8, sorbitol dehydroge- nase, and carbohydrate phosphorylase (starch phosphorylase). Hexokinase has a role not only in glycolysis but also as a glucose sensor that controls gene expression [19]. Sorbitol dehydrogenase is an enzyme involved in carbohydrate I II III IV V VI VII VIII IX X metabolism that converts the sugar alcohol form of glucose (sorbitol) into fructose [47]. Increased transcript levels of Fig. 3 Localization of differentially expressed genes on the physical map of sorghum. Each sugarcane probe set representing a differen- carbohydrate phosphorylase suggest that enhanced starch tially expressed gene between BTx623 and Rio with a fold change of degradation in Rio may contribute to sugar accumulation. two or higher was mapped to the sorghum genome and plotted on the Another increased transcript encodes a NADP-malic enzyme physical map. Up-regulated genes are in green and down-regulated suggesting that carbon fixation is enhanced in the stems of genes are in red. sweet versus grain sorghum. Indeed, the activity of enzymes involved in photosynthesis and the expression of their corresponded to transcripts that were down-regulated in the transcripts are modulated by sink strength. In sugarcane, sweet sorghum Rio line, respectively. Each differentially the accumulation of sucrose in the maturing and mature expressed sorghum transcript was classified based on the internodes of the stem contribute greatly to sink strength Pfam domains of their encoded proteins and their GO term [27]. Kinetic models have been proposed to explain sucrose (“Materials and methods”). accumulation in sugarcane [35, 41]. These models support Based on the sucrose and starch metabolic pathway from the notion that sucrose accumulates in the vacuole against a the KEGG (http://www.genome.jp/kegg/) and the CAZy concentration gradient. Indeed, we found that a transcript database (http://www.cazy.org/), we found that almost 16% encoding a vacuolar adenosine triphosphate (ATP) synthase Rice (2008) 1:166–176 173 173 catalytic subunit A had an increased expression in sweet hemicellulose polymer in cereals and is degraded by plant sorghum, consistent with the role of this ATP synthase in the endoxylanases [21]. This suggests that, in sweet sorghum, generation of an electrochemical gradient across the vacuolar the degradation of hemicellulose is promoted by suppressing membrane to propel the transport of sucrose. the expression of xylanases inhibitors. The only cell-wall-related transcript that was up-regulat- In other cases, a decrease in the expression of cellulose ed in sweet sorghum encodes a lysine motif containing synthase genes in wheat genotypes with high water-soluble protein. The LysM domain is widespread in bacterial carbohydrate content has also been observed [44]. In proteins that degrade cell walls but is also present in addition, Casu et al. [6] have recently characterized the eukaryotes. They are assumed to have a general role in expression of several cellulose synthase and cellulose peptidoglycan binding [1]. synthase-like genes in sugarcane stem and found that their expression is highly variable depending on internode Mobilization of sugars in the stems of sweet sorghum maturity [6]. Interestingly, genes with reduced transcript levels outpaced Reduced higher-order components in sweet sorghum stems those with increased levels by a 2:1 margin. Down-regulated transcripts involved in the starch and sucrose metabolic In addition to cellulose synthesis, the geometric deposition pathway found in our study included alpha-galactosidase, of cellulose fibrils generally perpendicular to the axis of cell beta-galactosidase, sucrose synthase 2, and fructokinase 2. elongation is a critical step in cell wall formation. There is Alpha and beta-galactosidase enzymes are O-glycosyl evidence that the orientation of cellulose deposition is hydrolases that hydrolyse the glycosidic bond between somehow assisted by microtubules [37]. An example of this two or more carbohydrates or between a carbohydrate and a is the fiber fragile mutant fra1 encoding a kinesin-like non-carbohydrate moiety [15]. Sucrose synthase is involved protein. In this mutant, cellulose deposition displayed an in the reversible conversion of sucrose to uridine diphosphate abnormal orientation [5]. Consistent with these observations, (UDP)-glucose and -fructose [24]. UDP-glucose can then be the expression of two transcripts encoding tubulin alpha-2/ used as a substrate for starch and cell wall synthesis. alpha-4 chain and tubulin folding cofactor A, in conjunction Fructose instead is converted into fructose-6-phosphate by with a transcript encoding a protein with kinesin motor fructokinase and further metabolized through glycolysis [33]. domain, were all down-regulated in sweet sorghum. Our findings are in agreement with previous reports showing Less clear, but also related to cell wall formation, is that the onset of sucrose accumulation in Rio was accom- fasciclin. Interestingly, the most strongly down-regulated panied by a decrease in sucrose synthase activity in stem transcript in sweet sorghum encodes a protein with a fasciclin domain. Fasciclin domains are found in animal tissue [25]. Similarly, Tarpley et al. [40]proposed thata decline in the levels of sucrose synthase may be necessary arabinogalactan proteins that have a role in cell adhesion for sucrose accumulation at stem maturity in sorghum. and communication [22]. These proteins are structural Consistent with our findings, Xue et al. [44]have recently components that mediate the interaction between the plasma reported the down-regulation in the expression of both membrane and the cell wall. However, their specific role in sucrose synthase and fructokinase genes in the stems of plants is still unknown [10]. A loss-of-function mutant in wheat genotypes with high water-soluble carbohydrates. the Arabidopsis gene fasciclin-like arabinogalactan 4 (AtFLA4) displayed thinner cell walls and increased Reduced expression of cellulose and lignocellulose-related sensitivity to salinity [46]. genes in sweet sorghum stems Reduced cross-linking in sweet sorghum stems Several transcripts involved in cell-wall-related processes were identified as down-regulated in sweet sorghum. These Other transcripts that were also down-regulated encode a included cellulose synthase 1, 7, and 9 as well as cellulose peroxidase and a laccase. It has been shown that peroxidases synthase catalytic subunit 12 in cellulose synthesis. In the have an important role in cell wall modification [32]. By case of lignin biosynthesis, we found transcripts such as controlling the abundance of H O in the cell wall, a 2 2 phenylalanine and histidine ammonia-lyase, cinnamoyl necessary step for the cross linking of phenolic compounds, CoA reductase, 4-coumarate coenzyme A ligase, and peroxidases act to inhibit cell elongation and, in conjunction caffeoyl-CoA O-methyltransferases. Interestingly, the ex- with laccases, are assumed to be involved in monolingol unit pression of two transcripts encoding for xylanase inhibitors oxidation, a reaction necessary for lignin assembly. Further- were also down-regulated in sweet sorghum. Xylanase more, it is known that peroxidase activity can be controlled inhibitor proteins belong to the group of protein inhibitors by ascorbate. Indeed, the expression of a transcript encoding of cell wall degrading enzymes. Xylan is the major a protein similar to guanosine diphosphate (GDP)-mannose 174 Rice (2008) 1:166–176 3, 5-epimerase was increased in sweet sorghum. This protein syntenic regions, whereas nine genes appeared to be catalyzes the reversible conversion of GDP-mannose either paralogous copies (Tables 1 and 2). into GDP-L-galactose or a novel intermediate, GDP-gulose, a step necessary for the biosynthesis of vitamin C in plants Outlook [43]. In addition, GDP-mannose is used to incorporate mannose residues into cell wall polymers [26]. For these Given the synteny of these genes between rice and sorghum, reasons, it is considered that GDP-mannose 3,5 epimerase one can assume that they are allelic between different could modulate the carbon flux into the vitamin C pathway sorghum cultivars. Therefore, future genetic mapping experi- as well as the demand for GDP-mannose into the cell wall ments should provide a direct link of allelic variation and the biosynthesis [43]. Indeed, it is known that the stem of high- sweet sorghum trait. Most likely, such allelic variations sucrose-accumulating genotypes of sugarcane are high in extend to the control regions of these genes because of their moisture content and low in fiber, whereas the stem of low- differential expression. Transgenic experiments can then be sucrose-accumulating genotypes are low in moisture content, used to verify such functional aspects for biofuel properties. thin, and fibrous [4]. Moreover, gain of function experiments could be used to import desirable traits such as accumulation of fermentable Compensation of osmotic shock in sweet sorghum stems sugars from sweet sorghum into maize. The generation of “sweet sorghum-like transgenic corn” will alleviate in part the Consistent with the idea that high concentration of sugars increasing pressure of growing corn either for food or for imposes osmotic stress to the cell, we found increased biofuel since it would then be possible to use the grain for transcripts encoding heat shock proteins HSP70 and food and at the same time to extract fermentable sugars from HSP90. Additionally, a transcript encoding a poly the stem to use in ethanol production. adenosine diphosphate (ADP)-ribose polymerase 2 (PARP 2) was significantly down-regulated in sweet sorghum. This is in agreement with a recent report in Materials and methods which Arabidopsis and Brassica napus transgenic plants with reduced levels of PARP 2 displayed resistance to Plant materials and growth conditions various abiotic stresses [42]. Poly ADP-ribosylation involves the tagging of proteins with long-branched poly Seeds from both grain and sweet sorghum (Sorghum ADP-ribose polymers and is mediated by PARP enzymes bicolor (L.) Moench) were sown in pro-mix soil (Premiere [36]. Poly ADP-ribosylation has important roles in the Horticulture Inc., USA) and grown in our greenhouse with a day length of 15 h light: 9 h dark at constant temperature cellular response to genotoxic stress, influence DNA synthesis and repair, and is also involved in chromatin of 23°C. The genotype representing grain sorghum in our structure and transcription. study was BTx623, whereas the genotypes representing sweet sorghum were Dale, Della, M81-E, Rio, Simon, and Mapping genes linked to sugar content and cell wall Top76-6. The seeds from sweet sorghum were kindly metabolism in sorghum and rice provided by Dr. William L. Rooney of Texas A&M, College Station, TX, USA. Although sugarcane has not been sequenced yet, we can use the sequenced genome of sorghum to construct a Measurement of “Brix degree” from sorghum stem’s juice “transcriptome map” with the genes found in our study. Assuming that gene order has been largely conserved The juice from internodes of the main stem in both grain between these closely related species, the “transcriptome and sweet sorghum was harvested at the time of anthesis. A map” of sorghum serves as a valuable reference for section of approximately 6 cm long was dissected from the sugarcane. We could not find any particular clustering of middle of each internode, and 300 μl of juice was extracted these genes but did observe that most of the genes are by pressing each internode with a garlic squeezer. The located towards the telomeres and only a few of them near concentration of total soluble sugars in the juice was the centromeres. We also could not find any of these genes measured with a pocket refractometer (Atago Inc., Japan). in the telomeric region on the long arm of chromosome six. Comparing this map with the rice genome demonstrated Isolation of total RNA from stem tissue that, out of 154 differentially expressed genes, 123 were in syntenic positions. With respect to the subset of genes Both grain sorghum BTx623 and sweet sorghum Rio were involved in the accumulation of fermentable sugars and grown until anthesis and total RNA from internode 8 for reduced lignocellulose, 21 genes were also found in each genotype (internodes were numbered from the base Rice (2008) 1:166–176 175 175 towards the apex of the stem) was extracted using the and is available at http://genlisea-rs1.waksman.rutgers. RNeasy Plant Mini Kit (QIAGEN Inc., USA). edu/cgi-bin/gbrowse/sbic/. GeneChip sugarcane genome array hybridization Physical location of differentially expressed transcripts in the sorghum genome Sorghum RNA from internode 8 was hybridized to the Affymetrix GeneChip Sugarcane Genome Array (Affyme- The sugarcane probe sets that were up- and down-regulated trix Inc., USA). Probe set information can be found at in Sorghum, respectively, were mapped to the genome by NetAffx Analysis Center’s web page (http://www.affyme using GenomeThreader [12]. Spliced alignments were only trix.com/analysis/index.affx). The One-Cycle Eukaryotic considered if 75% (score >0.75) or more bases could be Target Labeling Assay protocol was used. The labeling, aligned between the genomic sequence and a probe set. If a hybridization, and data collection were done at the probe could be mapped to the genome and if it also Transcription Profiling Facility, Cancer Institute of New overlapped with a sorghum gene, we assigned the annotation Jersey, Department of Pediatrics, Robert Wood Johnson of the sorghum gene to the probe. Medical School. Acknowledgments We thank William L. Rooney of Texas A&M, College Station, TX, USA, for providing the sweet sorghum seeds Data analysis used in this study. We also thank Mike Peterzack and Marc Probasco for their technical assistance with greenhouse work, Drs. Todd Probe sets that were absent in all chips were eliminated. Michael and Randall Kerstetter for the use of their MyiQ Real-Time About 5,900 out of the original 8,300 probe sets passed this PCR Detection System and the NanoDrop 1000 spectrophotometer, respectively. This work was supported in part by the sponsorship from test. Next, a t test was applied to BTx623 and Rio groups the International Institute of Education (IIE) and the Fulbright (three replicates for each) with an alpha value of 0.001, and Commission in Uruguay to MC. The research described in this the Benjamini–Hochberg multiple-testing correction was manuscript was supported by a grant from the DOE (# DE-FG05- applied. From the probe sets that passed the criteria, only 95ER20194) to JM. those with a fold change of at least two were considered. Validation of microarray data through quantitative RT-PCR References cDNA synthesis and PCR amplification was performed 1. Bateman A, Bycroft M. The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD). J in the same tube from 50 ng of total RNA using the Mol Biol. 2000;299:1113–9. iScript One-Step RT-PCR Kit with SYBR Green (BIO- 2. Bennetzen JL, Freeling M. Grasses as a single genetic system: RAD Laboratories, Inc.). The reaction condition used genome composition, collinearity and compatibility. Trends was as specified in the kit, with an annealing temperature Genet. 1993;9:259–61. 3. Buchanan CD, Lim S, Salzman RA, Kagiampakis I, Morishige DT, of 55°C and 45 cycles for the data collection step. The Weers BD, et al. Sorghum bicolors transcriptome response to qRT-PCR reaction was done using the MyiQ Real-Time dehydration, high salinity and ABA. Plant Mol Biol. 2005;58:699– PCR Detection System (BIO-RAD Laboratories, Inc.). Total RNA was accurately measured for each sample 4. Bull T, Glasziou K. The evolutionary significance of sugar accumulation in Saccarhum. Aust J Biol Sci. 1963;16:737–42. with the extremely sensitive NanoDrop 1000 spectropho- 5. Burk DH, Ye ZH. Alteration of oriented deposition of cellulose tometer (Thermo Scientific, Inc.). A relative quantifica- microfibrils by mutation of a katanin-like microtubule-severing tion normalized against unit mass (50 ng of total RNA) protein. Plant Cell. 2002;14:2145–60. was used to analyze the expression data with the 6. Casu RE, Jarmey JM, Bonnett GD, Manners JM. Identification of ΔCT transcripts associated with cell wall metabolism and development equation: RatioðÞ test=calibrator¼ 2 , as suggested in in the stem of sugarcane by Affymetrix GeneChip Sugarcane Real-Time Applications Guide from Bio-Rad. The primers Genome Array expression profiling. Funct Integr Genomics. for each gene were designed based on the region of 2007;7:153–67. homology (usually in the last exon or 3′untranslated 7. Casu RE, Grof CP, Rae AL, McIntyre CL, Dimmock CM, Manners JM. Identification of a novel sugar transporter homo- region) between the sugarcane probe set sequence and logue strongly expressed in maturing stem vascular tissues of the sorghum gene sequence and are listed in Electronic sugarcane by expressed sequence tag and microarray analysis. Supplementary Material Table S3. The sequence for each Plant Mol Biol. 2003;52:371–86. sugarcane probe set is freely available at the Affymetrix 8. Chapple C, Carpita N. Plant cell walls as targets for biotechnology. Curr Opin Plant Biol. 1998;1:179–85. website: http://www.affymetrix.com/analysis/index.affx. 9. D’Hont A, Grivet L, Feldmann P, Rao S, Berding N, Glaszmann JC. In addition, the genomic location of each sugarcane Characterization of the double genome structure of modern sugar- probe set in sorghum (BTx623) identified in our work cane cultivars (Saccharum spp.) by molecular cytogenetics. Mol has been up-loaded to our Sorghum Genome Browser Gen Genet. 1996;250:405–13. 176 Rice (2008) 1:166–176 10. Faik A, Abouzouhair J, Sarhan F. Putative fasciclin-like arabino- 29. Messing J, Bennetzen J. Grass genome structure and evolution. galactan-proteins (FLA) in wheat (Triticum aestivum) and rice Genome Dynamics. 2008;4:41–56. (Oryza sativa): identification and bioinformatic analyses. Mol 30. Ming R, Liu SC, Moore PH, Irvine JE, Paterson AH. QTL Genet Genomics. 2006;276:478–94. analysis in a complex autopolyploid: genetic control of sugar 11. Gale MD, Devos KM. Comparative genetics in the grasses. Proc content in sugarcane. Genome Res. 2001;11:2075–84. Natl Acad Sci U S A. 1998;95:1971–4. 31. Munford RS, Sheppard PO, O, Hara PJ. Saposin-like proteins 12. Gremme G, Brendel V, Sparks ME, Kurtz S. Engineering a (SAPLIP) carry out diverse functions on a common backbone software tool for gene structure prediction in higher organisms. Inf structure. J Lipid Res. 1995;36:1653–63. Softw Technol. 2005;47:965. 32. Passardi F, Penel C, Dunand C. Performing the paradoxical: how 13. Grivet L, Arruda P. Sugarcane genomics: depicting the complex plant peroxidases modify the cell wall. Trends Plant Sci. genome of an important tropical crop. Curr Opin Plant Biol. 2004;9:534–40. 2002;5:122–7. 33. Pego JV, Smeekens SC. Plant fructokinases: a sweet family get- 14. Guimaraes CT, Sills GR, Sobral BW. Comparative mapping of together. Trends Plant Sci. 2000;5:531–6. andropogoneae: Saccharum L. (sugarcane) and its relation to 34. Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, sorghum and maize. Proc Natl Acad Sci U S A. 1997;94:14261–6. Eckert CA, et al. The path forward for biofuels and biomaterials. 15. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, Davies G. Science 2006;311:484–9. Conserved catalytic machinery and the prediction of a common fold 35. Rohwer JM, Botha FC. Analysis of sucrose accumulation in the for several families of glycosyl hydrolases. Proc Natl Acad Sci U S sugar cane culm on the basis of in vitro kinetic data. Biochem J. A. 1996;93:5674. 2001;358:437–45. 16. Hoffman-Thoma G, Hinkel K, Nicolay P, Willenbrink J. Sucrose 36. Schreiber V, Dantzer F, Ame JC, de Murcia G. Poly(ADP-ribose): accumulation in sweet sorghum stem internodes in relation to novel functions for an old molecule. Nat Rev Mol Cell Biol. growth. Physiol Plant. 1996;97:277–84. 2006;7:517–28. 17. International Rice Genome Sequencing P. The map-based se- 37. Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, quence of the rice genome. Nature 2005;436:793–800. Milne J, et al. Toward a systems approach to understanding plant 18. Ishimaru K, Hirotsu N, Madoka Y, Kashiwagi T. Quantitative trait cell walls. Science 2004;306:2206–11. loci for sucrose, starch, and hexose accumulation before heading 38. Song R, Segal G, Messing J. Expression of the sorghum 10- in rice. Plant Physiol Biochem. 2007;45:799–804. member kafirin gene cluster in maize endosperm. Nucleic Acids 19. Jang JC, Leon P, Zhou L, Sheen J. Hexokinase as a sugar sensor Res. 2004;32:e189. in higher plants. Plant Cell. 1997;9:5–19. 39. Stokeley D, Bemporad D, Gavaghan D, Sansom MS. Conforma- 20. Jannoo N, Grivet L, Chantret N, Garsmeur O, Glaszmann JC, tional dynamics of a lipid-interacting protein: MD simulations of Arruda P, et al. Orthologous comparison in a gene-rich region saposin B. Biochemistry 2007;46:13573–80. among grasses reveals stability in the sugarcane polyploid 40. Tarpley L, Lingle S, Vietor DM, Andrews D, Miller F. Enzymatic genome. Plant J. 2007;50:574–85. control of nonstructural carbohydrate concentrations in stems and 21. Juge N, Nohr J, Le Gal-Coeffet MF, Kramhoft B, Furniss CS, panicles of sorghum. Crop Science. 1994;34:446–52. Planchot V, et al. The activity of barley alpha-amylase on starch 41. Uys L, Botha FC, Hofmeyr JH, Rohwer JM. Kinetic model of granules is enhanced by fusion of a starch binding domain from sucrose accumulation in maturing sugarcane culm tissue. Phyto- Aspergillus niger glucoamylase. Biochim Biophys Acta. chemistry 2007;68:2375–92. 2006;1764:275–84. 42. Vanderauwera S, De Block M, Van de Steene N, van de Cotte B, 22. Kawamoto T, Noshiro M, Shen M, Nakamasu K, Hashimoto K, Metzlaff M, Van Breusegem F. Silencing of poly(ADP-ribose) Kawashima-Ohya Y, et al. Structural and phylogenetic analyses of polymerase in plants alters abiotic stress signal transduction. Proc RGD-CAP/beta ig-h3, a fasciclin-like adhesion protein expressed Natl Acad Sci U S A. 2007;104:15150–5. in chick chondrocytes. Biochim Biophys Acta. 1998;1395:288–92. 43. Wolucka BA, Van Montagu M. GDP-mannose 3′,5′-epimerase forms 23. Kellogg EA. Evolutionary history of the grasses. Plant Physiol. GDP-L-gulose, a putative intermediate for the de novo biosynthesis 2001;125:1198–205. of vitamin C in plants. J Biol Chem. 2003;278:47483–90. 24. Koch K. Sucrose metabolism: regulatory mechanisms and pivotal 44. Xue GP, McIntyre CL, Jenkins CL, Glassop D, van Herwaarden AF, roles in sugar sensing and plant development. Curr Opin Plant Shorter R. Molecular dissection of variation in carbohydrate Biol. 2004;7:235–46. metabolism related to water-soluble carbohydrate accumulation in 25. Lingle S. Sucrose metabolism in the primary culm of sweet stems of wheat. Plant Physiol. 2008;146:441–54. sorghum during development. Crop Science. 1987;27:1214–9. 45. Yang J, Zhang J. Grain filling of cereals under soil drying. New 26. Lukowitz W, Nickle TC, Meinke DW, Last RL, Conklin PL, Phytol. 2006;169:223–36. Somerville CR. Arabidopsis cyt1 mutants are deficient in a 46. Yang J, Sardar HS, McGovern KR, Zhang Y, Showalter AM. A mannose-1-phosphate guanylyltransferase and point to a require- lysine-rich arabinogalactan protein in Arabidopsis is essential for ment of N-linked glycosylation for cellulose biosynthesis. Proc plant growth and development, including cell division and Natl Acad Sci U S A. 2001;98:2262–7. expansion. Plant J. 2007;49:629–40. 27. McCormick AJ, Cramer MD, Watt DA. Sink strength regulates 47. Zhou R, Cheng L, Dandekar AM. Down-regulation of sorbitol photosynthesis in sugarcane. New Phytol. 2006;171:759–70. dehydrogenase and up-regulation of sucrose synthase in shoot tips 28. Messing J, Llaca V. Importance of anchor genomes for any plant of the transgenic apple trees with decreased sorbitol synthesis. J genome project. Proc Natl Acad Sci U S A. 1998;95:2017–20. Exp Bot. 2006;57:3647–57. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Rice Springer Journals

Screen of Genes Linked to High-Sugar Content in Stems by Comparative Genomics

Rice , Volume 1 (2) – Dec 1, 2008

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Springer Journals
Copyright
Copyright © Springer Science + Business Media, LLC 2008
Subject
Life Sciences; Plant Sciences; Plant Genetics & Genomics; Plant Breeding/Biotechnology; Agriculture; Plant Ecology
ISSN
1939-8425
eISSN
1939-8433
DOI
10.1007/s12284-008-9012-9
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Abstract

Rice (2008) 1:166–176 DOI 10.1007/s12284-008-9012-9 Screen of Genes Linked to High-Sugar Content in Stems by Comparative Genomics Martín Calviño & Rémy Bruggmann & Joachim Messing Received: 1 May 2008 /Accepted: 20 August 2008 /Published online: 24 September 2008 Springer Science + Business Media, LLC 2008 Abstract One of the great advantages of the fully sequenced practical aspect of such a concept is of great importance for rice genome is to serve as a reference for other cereal agronomical purposes because a useful trait in one species genomes in particular for identifying genes linked to unique could be transferred to another. A relevant example could traits. A trait of great interest is reduced lignocellulose in the be carbohydrate partitioning and allocation. In cereals such stem of related species in favor of fermentable sugars as a as wheat, sorghum, and rice, the process of grain filling source of biofuels. While sugarcane is one of the most demands carbon from photosynthesis assimilation as well efficient biofuel crops, little is known about the underlying as the remobilization of pre-stored carbohydrates in the gene repertoire involved in it. Here, we take advantage of the stem before and after anthesis [45]. It has been estimated natural variation of sweet and grain sorghum to uncover that about 30% of the total yield in rice depends on the genes that are conserved in rice, sorghum, and sugarcane but carbohydrate content accumulated in the stem before differently expressed in sweet versus grain sorghum by using heading [18]. For these reasons, characterization of genes a microarray platform and the syntenous alignment of rice involved in carbohydrate metabolism and accumulation can and sorghum genomic regions containing these genes. lead to the development of improved cereal crops. Indeed, enzymes involved in carbohydrate accumulation In recent years, there has been an increasing demand on and those that reduce lignocellulose can be identified. biomass for the production of ethanol as a renewable resource for fuel. The biggest producers of ethanol in the . . Keywords Integrative genetics Sugar accumulation world are Brazil and the USA [34]. In Brazil, it is derived . . . Cell wall synthesis Microarray analysis Synteny from sugarcane, while in the USA, ethanol is derived from Biofuel crops the grain of corn. Because of the use of the entire plant as a source for fermentable sugars, carbohydrate accumulation and partitioning has been extensively studied in sugarcane, Introduction probably more than in any other species [30]. However, genes involved in these processes cannot easily be identified Comparisons of genetic maps and sequences of several because of the complex genome of sugarcane, with several grass species have shown that there is global conservation cultivars differing greatly in their ploidy levels from 2n=100 of gene content and order [11]. Therefore, grasses have to 2n=130 chromosomes [9, 13]. Even if one could make been considered as a “single genetic system” [2]. The further improvements to sugarcane, it has the disadvantage of being a crop restricted to tropical growing areas. Electronic supplementary material The online version of this article On the other hand, the use of corn grain for ethanol (doi:10.1007/s12284-008-9012-9) contains supplementary material, production poses a major conflict because of its dual use as which is available to authorized users. food and fuel. Therefore, it has been proposed to use grain : : M. Calviño R. Bruggmann J. Messing (*) solely for food and only the stover as a source for ethanol. Waksman Institute of Microbiology, Rutgers University, A major impediment to this approach is that, in contrast to 190 Frelinghuysen Road, sugarcane, corn stover consists mainly of lignocellulose, Piscataway, NJ 08854-8020, USA which is more costly to process than fermentable sugars [8]. e-mail: messing@waksman.rutgers.edu Rice (2008) 1:166–176 167 167 Therefore, it would be attractive to identify corn varieties sweet sorghum lines (Dale, Della, M81-E, Rio, Top76-6, and with reduced lignocellulose. Interestingly, there is extensive Simon) and one line from grain sorghum (BTx623). As an natural intra-species variation for sugar content in sorghum estimation of the total amount of sugars present in the juice with cultivars that do not accumulate sugars (referred to as of sorghum stems, we measured the Brix degree of each grain sorghums) in contrast to those that accumulate large internode along the main stem at the time of flowering. We amounts of sugars in their stems [16]. Such intra-species found great variation in flowering time as well as in Brix variation can serve as a platform to identify genes linked to degree between the sweet sorghum lines when compared to increased sugar content and reduced lignocellulose [4]. grain sorghum BTx623 (Fig. 1a,b). In general, the Brix Moreover, if these genes are conserved by ancestry in degree was lower in the mature and immature internodes of related species, one could envision the introduction of such the stem, in contrast to maturing internodes. These findings a trait by the import of specific regulatory regions. are in agreement with previous studies [16, 25]. Consistent Conservation of gene order between closely related species with the inability of grain sorghum to accumulate significant permits the alignment of orthologous chromosomal segments. levels of sugars in the stem, the Brix degree in BTx623 was Non-collinear genes would constitute paralogous copies [29]. low and remained fairly constant for all the internodes along To facilitate such alignments, the use of rice with one of the the stem. Among the sweet sorghum cultivars, Rio had the smallest cereal genomes that has been sequenced [17] highest Brix degree and Simon the lowest. Furthermore, the increasingly becomes the anchor genome for other grasses difference in flowering time between BTx623 and Rio was [28]. In this sense, we can use rice as a reference genome for smaller than in the rest of sweet sorghum lines with high Brix biofuel crops such as sugarcane and sorghum. degrees. For this reason, we decided to perform a comparative While rice offers an excellent reference as a compact analysis of transcripts in the stem of the Rio and BTx623 genome from an evolutionary point of view, it is less sorghum lines. suitable as a reference for a phenotype of reduced lignocellulose. Moreover, rice is a bambusoid C3 cereal plant, and sorghum and sugarcane are panicoid C4 cereal plants, which branched out 50 mya [23]. Sorghum and sugarcane belong to the Saccharinae clade and diverged from each other only 8–9mya [14, 20]. Therefore, sugarcane and its reduced lignocellulose can serve as a trait reference for sorghum varieties that differ in the cellulose content of their stems. Consequently, we took advantage of a GeneChip that was created to study gene expression in Btx 623 Dale Della M81-E Rio Simon Top 76-6 sugarcane and its role in the accumulation of sugar in the stem during development [6] for the comparison of grain b and sweet sorghum genes. One would expect that sweet sorghum and sugarcane use similar gene products for enhanced sugar accumulation in their stems. Indeed, we not only identified genes involved in sugar accumulation and lignocellulose synthesis, whose expression levels are correlated with the trait, but also demonstrate their ancestry through the alignment of orthologous regions of the rice and sorghum genomes. Therefore, the same genes could also be used to improve other biofuel crops like switchgrass and Miscanthus, validating a translational genomic approach. Results Sugar accumulation in the stem of grain and sweet Fig. 1 Variation in flowering time and Brix degree. a Comparison of sorghum cultivars flowering time between grain sorghum BTx623 and six sweet sorghum cultivars. Time to flowering was measured as days required reaching 50% anthesis. b Comparison of Brix degree along the main Previous reports have indicated that in sorghum stems, stem between grain sorghum BTx623 and six sweet sorghum sugars start to accumulate at flowering stage [16, 25]. We genotypes. The Brix degree was measured for each internode, and compared the accumulation of sugars in the stem between six the average of a triplicate experiment was plotted. 168 Rice (2008) 1:166–176 Microarray analysis of transcripts from sorghum found that almost 16% of the transcripts that were stem tissues differentially expressed between BTx623 and Rio corre- sponded to transcripts affecting carbohydrate metabolism In order to identify genes expressed in the stem with a (Tables 1 and 2). Based on the link between hypothetical potential role in sugar accumulation and reduced lignocellu- function and the sweet sorghum trait, we selected 37 lose [4], we compared transcript profiles between grain candidate genes, of which differential expression could be (BTx623) and sweet sorghum (Rio). Such a genome-wide considered to be the cause or the consequence of increased analysis became possible because of the recently designed sugar and decreased lignocellulose in sorghum stems. GeneChip of sugarcane [6]. This array was specifically Clearly such a screen would not detect candidate genes developed with sequences that were obtained from several that might play a qualitative rather than a quantitative role. cDNA libraries representing distinct tissue types including For instance, one might expect that the role of sucrose stem from 15 sugarcane varieties. The use of this GeneChip phosphate synthase in carbohydrate metabolism should be permitted us to directly compare gene expression data of two differentially expressed, but it appears to be unchanged different sorghum cultivars with the previously generated under the parameters examined. We therefore can discover data from sugarcane. Three independent plants for each mainly genes that are differentially expressed. Extrinsic BTx623 and Rio were grown until anthesis and RNA was evidence for the link between these candidate genes and extracted from the same maturing internode for all six plants. their potential function is described in more detail in These RNAs were used to prepare biotylinated cRNAs for “Discussion.” hybridization, each sample separately hybridized to one array. Among these, transcripts that were up-regulated include The sugarcane array comprised 8,224 probe sets, of hexokinase 8 and carbohydrate phosphorylase (starch and which more than 70% (5,900) gave a positive signal with sucrose metabolism), nicotinamide adenine dinucleotide sorghum RNA samples. When a twofold cut-off value was phosphate (NADP) malic enzyme (C4 photosynthesis), a applied as criterion to distinguish differentially expressed D-mannose binding lectin (sugar binding), and a lysin motif transcripts between grain and sweet sorghum, a total of 195 (LysM) domain protein possibly involved in cell wall transcripts were identified, with 132 transcripts being degradation. Transcripts that were down-regulated included down-regulated and 63 transcripts up-regulated in Rio, sucrose synthase 2 and fructokinase 2 (starch and sucrose respectively (Electronic Supplementary Material Tables S1 metabolism), alpha-galactosidase and beta-galactosidase and S2). Because some probe sets identify the same gene, (hydrolysis of glycosidic bonds), and cellulose synthase 1, the number of genes that is down-regulated is 103 and up- 7, and 9 together with cellulose synthase catalytic subunit regulated 51, respectively. Based on the annotation of the 12 (cell wall metabolism). In addition, several others sorghum genes, we were able to infer the possible function transcripts with a cell-wall-related role that were down- for most of the differentially expressed transcripts. regulated included cinnamoyl CoA reductase, cinnamyl Among the transcripts that were up-regulated in Rio, a alcohol dehydrogenase, 4-coumarate coenzyme A ligase, saposin-like type B gene displayed the highest differential caffeoyl-CoA O-methyltransferase, xyloglucan endo- expression. Saposins are involved in the degradation of transglycosylase/hydrolase, peroxidase and phenylalanine, sphingolipids [31]. Other transcripts encoding stress-related and histidine ammonia-lyase. proteins such as heat shock protein 70 (HSP70) and HSP90 were up-regulated, consistent with an osmotic stress Validation of microarray data by quantitative reverse imposed by high concentration of sugars (Electronic transcription polymerase chain reaction Supplementary Material Tables S1 and S2)[3]. Our results show that in Rio, down-regulated genes outnumber those To validate the data obtained by microarray analysis, we that are up-regulated by a factor of 2. The most reduced randomly sampled 14 of the 37 candidate genes and transcript has a fasciclin domain. This domain has been compared their expression levels in both Rio and BTx623 shown to be involved in cell adhesion (Table 1)[10, 22]. by performing quantitative reverse transcription polymerase chain reaction (qRT-PCR; Fig. 2a). In Rio, the expression Genes with altered expression in carbohydrate metabolism of saposin, carbohydrate phosphorylase, hexokinase-8, and in sweet sorghum NADP malic enzyme is up-regulated in comparison with their expression in BTx623. In contrast, the expression of Based on Gene Ontology (GO) terms (http://www.geneon fasciclin-like protein FLA15, cellulose synthase 1 and 7, tology.org/), the sucrose and starch metabolic pathway from fructokinase-2, 4-coumarate coenzyme A ligase, sucrose the Kyoto Encyclopedia of Genes and Genomes (KEGG; synthase 2, laccase, cinnamoyl CoA reductase, beta-galac- http://www.genome.jp/kegg/), and the Carbohydrate-Active tosidase 3 precursor, and alpha-galactosidase precursor enzymes (CAZy) database (http://www.cazy.org/), we were down-regulated in Rio. Although the levels of gene Rice (2008) 1:166–176 169 169 Table 1 List of “Trait-Specific” Genes that Are Syntenic with Rice a b Gene Rice Sorghum Expression Starch and sucrose metabolism Hexokinase 8 Os01g0190400 Sb03g003190.1 2.3 Hexokinase 8 Os05g0187100 Sb09g005840.1 Carbohydrate phosphorylase Os01g0851700 Sb03g040060.1 1.2 Sucrose synthase 2 Os03g0401300 Sb01g033060.1 −1.3 Sucrose synthase 2 Os07g0616800 Fructokinase-2 Os08g0113100 Sb07g001320.1 −1.7 Sorbitol dehydrogenase Os08g0545200 Sb07g025220.1 1.6 Sugar binding D-mannose binding lectin Os06g0165200 Sb10g022730.1 2 CO assimilation NADP-dependent malic enzyme Os01g0723400 Sb03g033250.1 2 Cell-wall-related LysM domain protein/cell wall catabolism Os03g0110600 Sb01g049890.1 1.2 Cellulose synthase-7 Os03g0837100 Sb01g002050.1 −1 Cellulose synthase-1 Os05g0176100 Sb09g005280.1 −1.1 Cellulose synthase-9 Os07g0208500 Sb02g006290.1 −1.1 Cellulose synthase-9 Os03g0808100 Sb01g004210.1 Cellulose synthase catalytic subunit 12 Os09g0422500 Sb02g025020.1 −4.7 Alpha-galactosidase precursor Os10g0493600 Sb01g018400.1 −1.8 Beta-galactosidase 3 precursor Os01g0875500 Sb03g041450.1 −2.4 Beta-galactosidase 3 precursor Os05g0428100 Sb03g041450.1 Cinnamoyl CoA reductase Os08g0441500 Sb07g021680.1 −2.9 Cinnamoyl CoA reductase Os09g0419200 Sb10g005700.1 Laccase Os01g0842400 Sb03g039520.1 −3.5 4-Coumarate coenzyme A ligase Os02g0177600 Sb04g005210.1 −3.7 4-Coumarate coenzyme A ligase Os06g0656500 Sb10g026130.1 Fasciclin domain Os03g0788600 Sb01g005770.1 −1.75 Fasciclin domain Os07g0160600 Sb02g003410.1 Fasciclin-like protein FLA15 Os05g0563600 Sb09g028490.1 −6.5 Caffeoyl-CoA O-methyltransferase 2 Os06g0165800 Sb10g004540.1 −2.15 Caffeoyl-CoA O-methyltransferase Os08g0498100 Sb07g028530.1 −5.3 Caffeoyl-CoA O-methyltransferase Os09g0481400 Sb02g027930.1 In boldface: sorghum genes that correspond to sugarcane probe set IDs previously reported by [6] Paralogs in italics Mean Log2 Ratio of sweet versus grain sorghum Sorghum gene to which a sugarcane probe set was mapped expression between the microarray and the qRT-PCR flowering and measured the expression of both genes by method differ to some extent, there is no difference in the qRT-PCR. We found that the saposin-type B gene is also classification of up- or down-regulated genes. This 100% highly expressed in Dale and Della when compared to grain correspondence of microarray with qRT-PCR data illus- sorghum and that the opposite is true for the expression of trates that the microarray platform can be used as an fasciclin-like protein FLA15, highly expressed in BTx623 effective method for screening large amounts of genes for a compared to Dale and Della (Fig. 2b). particular trait across closely related species. Before one would embark on any further experimentation, a much Genomic location of differentially expressed genes smaller candidate gene set can then be tested by more labor- intensive methods for gene expression between cultivars of In order to see if genes that were differentially expressed the same species. In order to see if the expression difference between grain and sweet sorghum cluster together in a between BTx623 and Rio for the transcripts encoding a particular region of the sorghum genome, we generated a saposin-type B protein and a fasciclin-like protein FLA15 “transcriptome map” (Fig. 3). We mapped the sequences of also extended to other sweet sorghum lines, we extracted all up- and down-regulated sugarcane probes to the recently RNA from maturing stems of BTx623, Dale, and Della at sequenced sorghum genome (BTx623; http://www.phyto 170 Rice (2008) 1:166–176 Table 2 List of “Trait-Specific” Gene Sorghum Expression Genes that Are Not Syntenic with Rice Cell-wall-related Alcohol dehydrogenase Sb10g006290 1 Cinnamyl alcohol dehydrogenase Sb04g011550 −1.5 Dolichyl-diphospho-oligosaccharide Sb02g006330 −1.4 Xyloglucan endo-transglycosylase/hydrolase Sb06g015880 −1.1 Putative Xylanase inhibitor Sb05g027350 −1.5 Putative Xylanase inhibitor Sb02g004660 −1.5 In boldface: sorghum genes Glycoside hydrolase family 1 Sb02g029640 −1.1 that correspond to sugarcane Phenylalanine and histidine ammonia-lyase Sb04g026520 −2 probe set IDs previously Peroxidase Sb02g037840 −1.5 reported by [6] Mean Log2 Ratio of sweet Similar to Saposin type B protein Sb09g013990 5.7 versus grain sorghum zome.net/cgi-bin/gbrowse/sorghum/) using GenomeThreader Discussion [12]. From a total of 195 probe sets, 176 could be mapped to the sorghum genome based on their alignment with a Translational genomics sorghum gene (“Materials and methods”). In addition, six probe sets could be mapped to the genome but do not The non-renewable nature of fossil oil imposes an increasing correlate with the current sorghum gene annotation, and for pressure to develop alternative energies in order to support another 13 probe sets, we were not able to map them to the and secure social and economic growth in the near future sorghum genome. Genes that were differentially expressed [34]. Currently, there is a worldwide interest to develop between grain and sweet sorghum do not appear to cluster in biofuel crops, the best example being sugarcane, used in any particular region of the genome but rather reflect random Brazil since the 1970s. Besides sugarcane, other grasses distribution (Fig. 3). such as Brachypodium distachyon, Miscanthus, maize, rice, sweet sorghum, and switchgrass are considered as crops for Trait-specific syntenic gene pairs between rice and sorghum biofuel research and production. Recently, the entire gene cluster of ten sorghum kafirin genes contained within a It can be considered that important gene functions have chromosomal segment of 45 kb was intact and stably been conserved by ancestry and that divergence is mainly inserted into the maize genome. Expression analysis then due to changes in regulatory control regions of genes. To has shown that kafirins accumulated in maize endosperm in determine the ancestry of genes, however, requires the a developmental and tissue-specific manner [38]. Such alignment of syntenic regions. Because we know now the transfer of genomic DNA between species that cannot be positions of the sorghum genes in their respective chromo- crossed could then be used in advanced breeding techniques somes, we can align them with the rice genome as a to introduce desirable traits from one species to another. reference [17] and determine whether the aligned regions Here, we integrate the traits of sugar accumulation and are collinear between rice and sorghum. Indeed, we found lignocellulose content with genomic and expression data of that from a total of 154 differentially expressed sorghum the three species, sugarcane, sorghum, and rice. We used the genes, 123 have an orthologous copy in syntenic positions recently developed Affymetrix sugarcane genome array [6] in rice (Electronic Supplementary Material Table S1). This as a tool for the identification of genes differentially collection includes 28 candidate genes for the sweet expressed in maturing stems of grain and sweet sorghum. sorghum trait (Table 1). Interestingly, one of these The intra-species variation for sugar content in sorghum is candidate genes, sucrose synthase 2, is duplicated in rice more pronounced than between sugarcane varieties, making but not in grain sorghum. So the question arose whether the sorghum a more suitable model to study this trait. On the gene is duplicated in sweet sorghum thereby explaining the other hand, because we can map sorghum genes to their difference in gene expression simply by gene duplication. chromosomal positions, we can use rice as a reference Because we have only the sequence of grain sorghum, we genome to identify genes by their ancestry. performed a Southern blot analysis of genomic DNA of sweet sorghum. When genomic DNA from BTx623 and Cross-referencing tissue-specific transcripts Rio are compared, both possess a single copy of sucrose synthase 2 (data not shown). Therefore, it is unlikely that Sorghum and sugarcane belong to the Saccharinae clade gene duplication plays a role in changing the level of gene and diverged from each other only 8 to 9 mya [20], while expression. rice is a more distant relative and separated from this clade Rice (2008) 1:166–176 171 171 Hexokinase 8 NADP Malic enzyme Beta-galactosidase 3 precursor Fasciclin-like protein FLA15 1.2 1.2 1.4 1.2 1.2 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0 0 0 0 Carbohydrate phosphorylate Cellulose synthase 7 Cinnamoyl CoA reductase Saposin type B 1.2 1.4 1.4 1.4 1.2 1.2 1.2 1 1 1 1 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0 0 0 Sucrose synthase 2 Cellulose synthase 1 Laccase 1.2 1.6 1.4 BTx623 1.4 1.2 1 1.2 0.8 Rio 0.8 0.6 0.8 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 Fructokinase 2 Alpha-galactoside precursor 4-Coumarate CoA ligase 1.2 1.4 1.4 1.2 1.2 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 Saposin type B Fasciclin-like protein FLA15 1.6 1.6 1.4 1.4 1.2 1.2 1 1 BTx623 0.8 0.8 Dale Della 0.6 0.6 0.4 0.4 0.2 0.2 0 0 Fig. 2 Validation of microarray data by quantitative RT-PCR. a The abundance for each gene is presented as relative fold expression. b expression of 14 genes selected from Table 1 was analyzed through qRT-PCR comparing the expression of saposin type B and fasciclin- quantitative RT-PCR. The results of three independent experiments for like protein FLA15 in BTx623 and two sweet sorghum lines Della and both BTx623 and Rio are shown. The quantification of the mRNA Dale. 50 mya [23]. Because sorghum and sugarcane belong to the of stem-derived RNAs from sorghum to 5,900 sugarcane same clade, we reasoned that, by hybridizing RNA from probes of a GeneChip comprising 8,224 probe sets in grain and sweet sorghum onto the sugarcane GeneChip, we total is a good indication of such cross-referencing. By could correlate changes in transcript levels with traits from applying a twofold cut-off value as a parameter to filter out sweet sorghum such as sugar content and reduced ligno- differentially expressed transcripts, a total of 195 probe sets cellulose. Given the tissue specificity and the rather small were identified, of which 63 corresponded to transcripts gene set of the sugarcane GeneChip, the positive hybridization that were up-regulated (51 genes) and 132 (103 genes) Relative fold expression Relative fold change 172 Rice (2008) 1:166–176 of the transcripts involved in sucrose and starch metabolism and in cell-wall-related processes were differentially expressed between BTx623 and Rio. This is particularly interesting because a previous study with cDNAs from immature and maturing stem of sugarcane identified only 2.4% of the transcripts related to carbohydrate metabolism [7]. Furthermore, because sorghum stems are fully elongated at the anthesis stage, tissue samples from maturing internodes were also more suitable in profiling changes in gene expression associated with carbohydrate metabolism. The implication is that screening of differentially expressed genes can greatly be enhanced by genetic variability and selection of tissue. Function of genes with elevated expression in sweet sorghum The highest elevated transcript identified in our study encodes a saposin-like type B domain. Increased expression has also been validated and tested in other sweet sorghum lines by qRT-PCR. We also found a higher expression in Dale and Della compared to that in BTx623 (Fig. 2b). Saposins are water soluble proteins that interact with the lysosomal membrane and are involved in the catabolism of glycosphingolipids in animals [31, 39]. Although it was unexpected that such a function could be related to a role in sugar accumulation, it underscores the value of a microarray- based screen to detect possible new network effects. For instance, we could hypothesize that the removal of sugars from glycosphingolipids in the membrane alters its structure in such a way that it constitutes an early step in carbohydrate partitioning. Additional transcripts that were increased in sweet sorghum included hexokinase 8, sorbitol dehydroge- nase, and carbohydrate phosphorylase (starch phosphorylase). Hexokinase has a role not only in glycolysis but also as a glucose sensor that controls gene expression [19]. Sorbitol dehydrogenase is an enzyme involved in carbohydrate I II III IV V VI VII VIII IX X metabolism that converts the sugar alcohol form of glucose (sorbitol) into fructose [47]. Increased transcript levels of Fig. 3 Localization of differentially expressed genes on the physical map of sorghum. Each sugarcane probe set representing a differen- carbohydrate phosphorylase suggest that enhanced starch tially expressed gene between BTx623 and Rio with a fold change of degradation in Rio may contribute to sugar accumulation. two or higher was mapped to the sorghum genome and plotted on the Another increased transcript encodes a NADP-malic enzyme physical map. Up-regulated genes are in green and down-regulated suggesting that carbon fixation is enhanced in the stems of genes are in red. sweet versus grain sorghum. Indeed, the activity of enzymes involved in photosynthesis and the expression of their corresponded to transcripts that were down-regulated in the transcripts are modulated by sink strength. In sugarcane, sweet sorghum Rio line, respectively. Each differentially the accumulation of sucrose in the maturing and mature expressed sorghum transcript was classified based on the internodes of the stem contribute greatly to sink strength Pfam domains of their encoded proteins and their GO term [27]. Kinetic models have been proposed to explain sucrose (“Materials and methods”). accumulation in sugarcane [35, 41]. These models support Based on the sucrose and starch metabolic pathway from the notion that sucrose accumulates in the vacuole against a the KEGG (http://www.genome.jp/kegg/) and the CAZy concentration gradient. Indeed, we found that a transcript database (http://www.cazy.org/), we found that almost 16% encoding a vacuolar adenosine triphosphate (ATP) synthase Rice (2008) 1:166–176 173 173 catalytic subunit A had an increased expression in sweet hemicellulose polymer in cereals and is degraded by plant sorghum, consistent with the role of this ATP synthase in the endoxylanases [21]. This suggests that, in sweet sorghum, generation of an electrochemical gradient across the vacuolar the degradation of hemicellulose is promoted by suppressing membrane to propel the transport of sucrose. the expression of xylanases inhibitors. The only cell-wall-related transcript that was up-regulat- In other cases, a decrease in the expression of cellulose ed in sweet sorghum encodes a lysine motif containing synthase genes in wheat genotypes with high water-soluble protein. The LysM domain is widespread in bacterial carbohydrate content has also been observed [44]. In proteins that degrade cell walls but is also present in addition, Casu et al. [6] have recently characterized the eukaryotes. They are assumed to have a general role in expression of several cellulose synthase and cellulose peptidoglycan binding [1]. synthase-like genes in sugarcane stem and found that their expression is highly variable depending on internode Mobilization of sugars in the stems of sweet sorghum maturity [6]. Interestingly, genes with reduced transcript levels outpaced Reduced higher-order components in sweet sorghum stems those with increased levels by a 2:1 margin. Down-regulated transcripts involved in the starch and sucrose metabolic In addition to cellulose synthesis, the geometric deposition pathway found in our study included alpha-galactosidase, of cellulose fibrils generally perpendicular to the axis of cell beta-galactosidase, sucrose synthase 2, and fructokinase 2. elongation is a critical step in cell wall formation. There is Alpha and beta-galactosidase enzymes are O-glycosyl evidence that the orientation of cellulose deposition is hydrolases that hydrolyse the glycosidic bond between somehow assisted by microtubules [37]. An example of this two or more carbohydrates or between a carbohydrate and a is the fiber fragile mutant fra1 encoding a kinesin-like non-carbohydrate moiety [15]. Sucrose synthase is involved protein. In this mutant, cellulose deposition displayed an in the reversible conversion of sucrose to uridine diphosphate abnormal orientation [5]. Consistent with these observations, (UDP)-glucose and -fructose [24]. UDP-glucose can then be the expression of two transcripts encoding tubulin alpha-2/ used as a substrate for starch and cell wall synthesis. alpha-4 chain and tubulin folding cofactor A, in conjunction Fructose instead is converted into fructose-6-phosphate by with a transcript encoding a protein with kinesin motor fructokinase and further metabolized through glycolysis [33]. domain, were all down-regulated in sweet sorghum. Our findings are in agreement with previous reports showing Less clear, but also related to cell wall formation, is that the onset of sucrose accumulation in Rio was accom- fasciclin. Interestingly, the most strongly down-regulated panied by a decrease in sucrose synthase activity in stem transcript in sweet sorghum encodes a protein with a fasciclin domain. Fasciclin domains are found in animal tissue [25]. Similarly, Tarpley et al. [40]proposed thata decline in the levels of sucrose synthase may be necessary arabinogalactan proteins that have a role in cell adhesion for sucrose accumulation at stem maturity in sorghum. and communication [22]. These proteins are structural Consistent with our findings, Xue et al. [44]have recently components that mediate the interaction between the plasma reported the down-regulation in the expression of both membrane and the cell wall. However, their specific role in sucrose synthase and fructokinase genes in the stems of plants is still unknown [10]. A loss-of-function mutant in wheat genotypes with high water-soluble carbohydrates. the Arabidopsis gene fasciclin-like arabinogalactan 4 (AtFLA4) displayed thinner cell walls and increased Reduced expression of cellulose and lignocellulose-related sensitivity to salinity [46]. genes in sweet sorghum stems Reduced cross-linking in sweet sorghum stems Several transcripts involved in cell-wall-related processes were identified as down-regulated in sweet sorghum. These Other transcripts that were also down-regulated encode a included cellulose synthase 1, 7, and 9 as well as cellulose peroxidase and a laccase. It has been shown that peroxidases synthase catalytic subunit 12 in cellulose synthesis. In the have an important role in cell wall modification [32]. By case of lignin biosynthesis, we found transcripts such as controlling the abundance of H O in the cell wall, a 2 2 phenylalanine and histidine ammonia-lyase, cinnamoyl necessary step for the cross linking of phenolic compounds, CoA reductase, 4-coumarate coenzyme A ligase, and peroxidases act to inhibit cell elongation and, in conjunction caffeoyl-CoA O-methyltransferases. Interestingly, the ex- with laccases, are assumed to be involved in monolingol unit pression of two transcripts encoding for xylanase inhibitors oxidation, a reaction necessary for lignin assembly. Further- were also down-regulated in sweet sorghum. Xylanase more, it is known that peroxidase activity can be controlled inhibitor proteins belong to the group of protein inhibitors by ascorbate. Indeed, the expression of a transcript encoding of cell wall degrading enzymes. Xylan is the major a protein similar to guanosine diphosphate (GDP)-mannose 174 Rice (2008) 1:166–176 3, 5-epimerase was increased in sweet sorghum. This protein syntenic regions, whereas nine genes appeared to be catalyzes the reversible conversion of GDP-mannose either paralogous copies (Tables 1 and 2). into GDP-L-galactose or a novel intermediate, GDP-gulose, a step necessary for the biosynthesis of vitamin C in plants Outlook [43]. In addition, GDP-mannose is used to incorporate mannose residues into cell wall polymers [26]. For these Given the synteny of these genes between rice and sorghum, reasons, it is considered that GDP-mannose 3,5 epimerase one can assume that they are allelic between different could modulate the carbon flux into the vitamin C pathway sorghum cultivars. Therefore, future genetic mapping experi- as well as the demand for GDP-mannose into the cell wall ments should provide a direct link of allelic variation and the biosynthesis [43]. Indeed, it is known that the stem of high- sweet sorghum trait. Most likely, such allelic variations sucrose-accumulating genotypes of sugarcane are high in extend to the control regions of these genes because of their moisture content and low in fiber, whereas the stem of low- differential expression. Transgenic experiments can then be sucrose-accumulating genotypes are low in moisture content, used to verify such functional aspects for biofuel properties. thin, and fibrous [4]. Moreover, gain of function experiments could be used to import desirable traits such as accumulation of fermentable Compensation of osmotic shock in sweet sorghum stems sugars from sweet sorghum into maize. The generation of “sweet sorghum-like transgenic corn” will alleviate in part the Consistent with the idea that high concentration of sugars increasing pressure of growing corn either for food or for imposes osmotic stress to the cell, we found increased biofuel since it would then be possible to use the grain for transcripts encoding heat shock proteins HSP70 and food and at the same time to extract fermentable sugars from HSP90. Additionally, a transcript encoding a poly the stem to use in ethanol production. adenosine diphosphate (ADP)-ribose polymerase 2 (PARP 2) was significantly down-regulated in sweet sorghum. This is in agreement with a recent report in Materials and methods which Arabidopsis and Brassica napus transgenic plants with reduced levels of PARP 2 displayed resistance to Plant materials and growth conditions various abiotic stresses [42]. Poly ADP-ribosylation involves the tagging of proteins with long-branched poly Seeds from both grain and sweet sorghum (Sorghum ADP-ribose polymers and is mediated by PARP enzymes bicolor (L.) Moench) were sown in pro-mix soil (Premiere [36]. Poly ADP-ribosylation has important roles in the Horticulture Inc., USA) and grown in our greenhouse with a day length of 15 h light: 9 h dark at constant temperature cellular response to genotoxic stress, influence DNA synthesis and repair, and is also involved in chromatin of 23°C. The genotype representing grain sorghum in our structure and transcription. study was BTx623, whereas the genotypes representing sweet sorghum were Dale, Della, M81-E, Rio, Simon, and Mapping genes linked to sugar content and cell wall Top76-6. The seeds from sweet sorghum were kindly metabolism in sorghum and rice provided by Dr. William L. Rooney of Texas A&M, College Station, TX, USA. Although sugarcane has not been sequenced yet, we can use the sequenced genome of sorghum to construct a Measurement of “Brix degree” from sorghum stem’s juice “transcriptome map” with the genes found in our study. Assuming that gene order has been largely conserved The juice from internodes of the main stem in both grain between these closely related species, the “transcriptome and sweet sorghum was harvested at the time of anthesis. A map” of sorghum serves as a valuable reference for section of approximately 6 cm long was dissected from the sugarcane. We could not find any particular clustering of middle of each internode, and 300 μl of juice was extracted these genes but did observe that most of the genes are by pressing each internode with a garlic squeezer. The located towards the telomeres and only a few of them near concentration of total soluble sugars in the juice was the centromeres. We also could not find any of these genes measured with a pocket refractometer (Atago Inc., Japan). in the telomeric region on the long arm of chromosome six. Comparing this map with the rice genome demonstrated Isolation of total RNA from stem tissue that, out of 154 differentially expressed genes, 123 were in syntenic positions. With respect to the subset of genes Both grain sorghum BTx623 and sweet sorghum Rio were involved in the accumulation of fermentable sugars and grown until anthesis and total RNA from internode 8 for reduced lignocellulose, 21 genes were also found in each genotype (internodes were numbered from the base Rice (2008) 1:166–176 175 175 towards the apex of the stem) was extracted using the and is available at http://genlisea-rs1.waksman.rutgers. RNeasy Plant Mini Kit (QIAGEN Inc., USA). edu/cgi-bin/gbrowse/sbic/. GeneChip sugarcane genome array hybridization Physical location of differentially expressed transcripts in the sorghum genome Sorghum RNA from internode 8 was hybridized to the Affymetrix GeneChip Sugarcane Genome Array (Affyme- The sugarcane probe sets that were up- and down-regulated trix Inc., USA). Probe set information can be found at in Sorghum, respectively, were mapped to the genome by NetAffx Analysis Center’s web page (http://www.affyme using GenomeThreader [12]. Spliced alignments were only trix.com/analysis/index.affx). The One-Cycle Eukaryotic considered if 75% (score >0.75) or more bases could be Target Labeling Assay protocol was used. The labeling, aligned between the genomic sequence and a probe set. If a hybridization, and data collection were done at the probe could be mapped to the genome and if it also Transcription Profiling Facility, Cancer Institute of New overlapped with a sorghum gene, we assigned the annotation Jersey, Department of Pediatrics, Robert Wood Johnson of the sorghum gene to the probe. Medical School. Acknowledgments We thank William L. Rooney of Texas A&M, College Station, TX, USA, for providing the sweet sorghum seeds Data analysis used in this study. We also thank Mike Peterzack and Marc Probasco for their technical assistance with greenhouse work, Drs. Todd Probe sets that were absent in all chips were eliminated. Michael and Randall Kerstetter for the use of their MyiQ Real-Time About 5,900 out of the original 8,300 probe sets passed this PCR Detection System and the NanoDrop 1000 spectrophotometer, respectively. This work was supported in part by the sponsorship from test. Next, a t test was applied to BTx623 and Rio groups the International Institute of Education (IIE) and the Fulbright (three replicates for each) with an alpha value of 0.001, and Commission in Uruguay to MC. The research described in this the Benjamini–Hochberg multiple-testing correction was manuscript was supported by a grant from the DOE (# DE-FG05- applied. From the probe sets that passed the criteria, only 95ER20194) to JM. those with a fold change of at least two were considered. Validation of microarray data through quantitative RT-PCR References cDNA synthesis and PCR amplification was performed 1. Bateman A, Bycroft M. The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD). J in the same tube from 50 ng of total RNA using the Mol Biol. 2000;299:1113–9. iScript One-Step RT-PCR Kit with SYBR Green (BIO- 2. Bennetzen JL, Freeling M. Grasses as a single genetic system: RAD Laboratories, Inc.). The reaction condition used genome composition, collinearity and compatibility. Trends was as specified in the kit, with an annealing temperature Genet. 1993;9:259–61. 3. Buchanan CD, Lim S, Salzman RA, Kagiampakis I, Morishige DT, of 55°C and 45 cycles for the data collection step. The Weers BD, et al. Sorghum bicolors transcriptome response to qRT-PCR reaction was done using the MyiQ Real-Time dehydration, high salinity and ABA. Plant Mol Biol. 2005;58:699– PCR Detection System (BIO-RAD Laboratories, Inc.). Total RNA was accurately measured for each sample 4. Bull T, Glasziou K. The evolutionary significance of sugar accumulation in Saccarhum. Aust J Biol Sci. 1963;16:737–42. with the extremely sensitive NanoDrop 1000 spectropho- 5. Burk DH, Ye ZH. Alteration of oriented deposition of cellulose tometer (Thermo Scientific, Inc.). A relative quantifica- microfibrils by mutation of a katanin-like microtubule-severing tion normalized against unit mass (50 ng of total RNA) protein. Plant Cell. 2002;14:2145–60. was used to analyze the expression data with the 6. Casu RE, Jarmey JM, Bonnett GD, Manners JM. Identification of ΔCT transcripts associated with cell wall metabolism and development equation: RatioðÞ test=calibrator¼ 2 , as suggested in in the stem of sugarcane by Affymetrix GeneChip Sugarcane Real-Time Applications Guide from Bio-Rad. The primers Genome Array expression profiling. Funct Integr Genomics. for each gene were designed based on the region of 2007;7:153–67. homology (usually in the last exon or 3′untranslated 7. Casu RE, Grof CP, Rae AL, McIntyre CL, Dimmock CM, Manners JM. Identification of a novel sugar transporter homo- region) between the sugarcane probe set sequence and logue strongly expressed in maturing stem vascular tissues of the sorghum gene sequence and are listed in Electronic sugarcane by expressed sequence tag and microarray analysis. Supplementary Material Table S3. The sequence for each Plant Mol Biol. 2003;52:371–86. sugarcane probe set is freely available at the Affymetrix 8. Chapple C, Carpita N. Plant cell walls as targets for biotechnology. Curr Opin Plant Biol. 1998;1:179–85. website: http://www.affymetrix.com/analysis/index.affx. 9. D’Hont A, Grivet L, Feldmann P, Rao S, Berding N, Glaszmann JC. In addition, the genomic location of each sugarcane Characterization of the double genome structure of modern sugar- probe set in sorghum (BTx623) identified in our work cane cultivars (Saccharum spp.) by molecular cytogenetics. Mol has been up-loaded to our Sorghum Genome Browser Gen Genet. 1996;250:405–13. 176 Rice (2008) 1:166–176 10. Faik A, Abouzouhair J, Sarhan F. Putative fasciclin-like arabino- 29. Messing J, Bennetzen J. Grass genome structure and evolution. galactan-proteins (FLA) in wheat (Triticum aestivum) and rice Genome Dynamics. 2008;4:41–56. (Oryza sativa): identification and bioinformatic analyses. Mol 30. Ming R, Liu SC, Moore PH, Irvine JE, Paterson AH. QTL Genet Genomics. 2006;276:478–94. analysis in a complex autopolyploid: genetic control of sugar 11. Gale MD, Devos KM. Comparative genetics in the grasses. Proc content in sugarcane. Genome Res. 2001;11:2075–84. Natl Acad Sci U S A. 1998;95:1971–4. 31. Munford RS, Sheppard PO, O, Hara PJ. Saposin-like proteins 12. Gremme G, Brendel V, Sparks ME, Kurtz S. Engineering a (SAPLIP) carry out diverse functions on a common backbone software tool for gene structure prediction in higher organisms. Inf structure. J Lipid Res. 1995;36:1653–63. Softw Technol. 2005;47:965. 32. Passardi F, Penel C, Dunand C. Performing the paradoxical: how 13. Grivet L, Arruda P. Sugarcane genomics: depicting the complex plant peroxidases modify the cell wall. Trends Plant Sci. genome of an important tropical crop. Curr Opin Plant Biol. 2004;9:534–40. 2002;5:122–7. 33. Pego JV, Smeekens SC. Plant fructokinases: a sweet family get- 14. Guimaraes CT, Sills GR, Sobral BW. Comparative mapping of together. Trends Plant Sci. 2000;5:531–6. andropogoneae: Saccharum L. (sugarcane) and its relation to 34. Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, sorghum and maize. Proc Natl Acad Sci U S A. 1997;94:14261–6. Eckert CA, et al. The path forward for biofuels and biomaterials. 15. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, Davies G. Science 2006;311:484–9. Conserved catalytic machinery and the prediction of a common fold 35. Rohwer JM, Botha FC. Analysis of sucrose accumulation in the for several families of glycosyl hydrolases. Proc Natl Acad Sci U S sugar cane culm on the basis of in vitro kinetic data. Biochem J. A. 1996;93:5674. 2001;358:437–45. 16. Hoffman-Thoma G, Hinkel K, Nicolay P, Willenbrink J. Sucrose 36. Schreiber V, Dantzer F, Ame JC, de Murcia G. Poly(ADP-ribose): accumulation in sweet sorghum stem internodes in relation to novel functions for an old molecule. Nat Rev Mol Cell Biol. growth. Physiol Plant. 1996;97:277–84. 2006;7:517–28. 17. International Rice Genome Sequencing P. The map-based se- 37. Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, quence of the rice genome. Nature 2005;436:793–800. Milne J, et al. Toward a systems approach to understanding plant 18. Ishimaru K, Hirotsu N, Madoka Y, Kashiwagi T. Quantitative trait cell walls. Science 2004;306:2206–11. loci for sucrose, starch, and hexose accumulation before heading 38. Song R, Segal G, Messing J. Expression of the sorghum 10- in rice. Plant Physiol Biochem. 2007;45:799–804. member kafirin gene cluster in maize endosperm. Nucleic Acids 19. Jang JC, Leon P, Zhou L, Sheen J. Hexokinase as a sugar sensor Res. 2004;32:e189. in higher plants. Plant Cell. 1997;9:5–19. 39. Stokeley D, Bemporad D, Gavaghan D, Sansom MS. Conforma- 20. Jannoo N, Grivet L, Chantret N, Garsmeur O, Glaszmann JC, tional dynamics of a lipid-interacting protein: MD simulations of Arruda P, et al. Orthologous comparison in a gene-rich region saposin B. Biochemistry 2007;46:13573–80. among grasses reveals stability in the sugarcane polyploid 40. Tarpley L, Lingle S, Vietor DM, Andrews D, Miller F. Enzymatic genome. Plant J. 2007;50:574–85. control of nonstructural carbohydrate concentrations in stems and 21. Juge N, Nohr J, Le Gal-Coeffet MF, Kramhoft B, Furniss CS, panicles of sorghum. Crop Science. 1994;34:446–52. Planchot V, et al. The activity of barley alpha-amylase on starch 41. Uys L, Botha FC, Hofmeyr JH, Rohwer JM. Kinetic model of granules is enhanced by fusion of a starch binding domain from sucrose accumulation in maturing sugarcane culm tissue. Phyto- Aspergillus niger glucoamylase. Biochim Biophys Acta. chemistry 2007;68:2375–92. 2006;1764:275–84. 42. Vanderauwera S, De Block M, Van de Steene N, van de Cotte B, 22. Kawamoto T, Noshiro M, Shen M, Nakamasu K, Hashimoto K, Metzlaff M, Van Breusegem F. Silencing of poly(ADP-ribose) Kawashima-Ohya Y, et al. Structural and phylogenetic analyses of polymerase in plants alters abiotic stress signal transduction. Proc RGD-CAP/beta ig-h3, a fasciclin-like adhesion protein expressed Natl Acad Sci U S A. 2007;104:15150–5. in chick chondrocytes. Biochim Biophys Acta. 1998;1395:288–92. 43. Wolucka BA, Van Montagu M. GDP-mannose 3′,5′-epimerase forms 23. Kellogg EA. Evolutionary history of the grasses. Plant Physiol. GDP-L-gulose, a putative intermediate for the de novo biosynthesis 2001;125:1198–205. of vitamin C in plants. J Biol Chem. 2003;278:47483–90. 24. Koch K. Sucrose metabolism: regulatory mechanisms and pivotal 44. Xue GP, McIntyre CL, Jenkins CL, Glassop D, van Herwaarden AF, roles in sugar sensing and plant development. Curr Opin Plant Shorter R. Molecular dissection of variation in carbohydrate Biol. 2004;7:235–46. metabolism related to water-soluble carbohydrate accumulation in 25. Lingle S. Sucrose metabolism in the primary culm of sweet stems of wheat. Plant Physiol. 2008;146:441–54. sorghum during development. Crop Science. 1987;27:1214–9. 45. Yang J, Zhang J. Grain filling of cereals under soil drying. New 26. Lukowitz W, Nickle TC, Meinke DW, Last RL, Conklin PL, Phytol. 2006;169:223–36. Somerville CR. Arabidopsis cyt1 mutants are deficient in a 46. Yang J, Sardar HS, McGovern KR, Zhang Y, Showalter AM. A mannose-1-phosphate guanylyltransferase and point to a require- lysine-rich arabinogalactan protein in Arabidopsis is essential for ment of N-linked glycosylation for cellulose biosynthesis. Proc plant growth and development, including cell division and Natl Acad Sci U S A. 2001;98:2262–7. expansion. Plant J. 2007;49:629–40. 27. McCormick AJ, Cramer MD, Watt DA. Sink strength regulates 47. Zhou R, Cheng L, Dandekar AM. Down-regulation of sorbitol photosynthesis in sugarcane. New Phytol. 2006;171:759–70. dehydrogenase and up-regulation of sucrose synthase in shoot tips 28. Messing J, Llaca V. Importance of anchor genomes for any plant of the transgenic apple trees with decreased sorbitol synthesis. J genome project. Proc Natl Acad Sci U S A. 1998;95:2017–20. Exp Bot. 2006;57:3647–57.

Journal

RiceSpringer Journals

Published: Dec 1, 2008

Keywords: Integrative genetics; Sugar accumulation; Cell wall synthesis; Microarray analysis; Synteny; Biofuel crops

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