Tissue-specific transcriptome analysis within the maturing sugarcane stalk reveals spatial regulation in the expression of cellulose synthase and sucrose transporter gene families.

Sugarcane (Saccharum spp. hybrids) accumulates high concentrations of sucrose in its mature stalk and a considerable portion of carbohydrate metabolism is also devoted to cell wall synthesis and fibre production. We examined tissue-specific expression patterns to explore the spatial deployment of pathways responsible for sucrose accumulation and fibre synthesis within the stalk. We performed expression profiling of storage parenchyma, vascular bundles and rind dissected from a maturing stalk internode of sugarcane, identifying ten cellulose synthase subunit genes and examining significant differences in the expression of their corresponding transcripts and those of several sugar transporters. These were correlated with differential expression patterns for transcripts of genes encoding COBRA-like proteins and other cell wall metabolism-related proteins. The sugar transporters genes ShPST2a, ShPST2b and ShSUT4 were significantly up-regulated in storage parenchyma while ShSUT1 was up-regulated in vascular bundles. Two co-ordinately expressed groups of cell wall related transcripts were also identified. One group, associated with primary cell wall synthesis (ShCesA1, ShCesA7, ShCesA9 and Shbk2l3), was up-regulated in parenchyma. The other group, associated with secondary cell wall synthesis (ShCesA10, ShCesA11, ShCesA12 and Shbk-2), was up-regulated in rind. In transformed sugarcane plants, the ShCesA7 promoter conferred stable expression of green fluorescent protein preferentially in the storage parenchyma of the maturing stalk internode. Our results indicate that there is spatial separation for elevated expression of these important targets in both sucrose accumulation and cell wall synthesis, allowing for increased clarity in our understanding of sucrose transport and fibre synthesis in sugarcane.

Enrichment of genomic DNA for polymorphism detection in a non-model highly polyploid crop plant.

Large polyploid genomes of non-model species remain challenging targets for DNA polymorphism discovery despite the increasing throughput and continued reductions in cost of sequencing with new technologies. For these species especially, there remains a requirement to enrich genomic DNA to discover polymorphisms in regions of interest because of large genome size and to provide the sequence depth to enable estimation of copy number. Various methods of enriching DNA have been utilised, but some recent methods enable the efficient sampling of large regions (e.g. the exome). We have utilised one of these methods, solution-based hybridization (Agilent SureSelect), to capture regions of the genome of two sugarcane genotypes (one Saccharum officinarum and one Saccharum hybrid) based mainly on gene sequences from the close relative Sorghum bicolor. The capture probes span approximately 5.8 megabases (Mb). The enrichment over whole-genome shotgun sequencing was 10-11-fold for the two genotypes tested. This level of enrichment has important consequences for detecting single nucleotide polymorphisms (SNPs) from a single lane of Illumina (Genome Analyzer) sequence reads. The detection of polymorphisms was enabled by the depth of sequence at or near probe sites and enabled the detection of 270 000-280 000 SNPs within each genotype from a single lane of sequence using stringent detection parameters. The SNPs were present in 13 000-16 000 targeted genes, which would enable mapping of a large number of these chosen genes. SNP validation from 454 sequencing and between-genotype confirmations gave an 87%-91% validation rate.

Genotypic variation in the accumulation of water soluble carbohydrates in wheat.

Water-soluble carbohydrates (WSC) stored in the stems and leaf sheaths of winter cereals provide an important source of assimilate for remobilisation during grain-filling. Consequently, WSC are a major contributor to wheat grain yield and grain size in all environments but especially where photosynthesis is compromised as occurs where water is limiting. Breeding programs targeting greater WSC should provide improved varieties with greater and more stable yields in stress environments. To facilitate selection for WSC, genetic and genomic approaches are being used to determine the genetic basis of - and define DNA probes for - marker-aided selection for this important drought-adaptive trait. Empirical studies have identified both WSC concentration and content to be under complex genetic control of many genes. Quantitative trait loci (QTL) for WSC have been identified in several wheat populations with individual QTL explaining small amounts of phenotypic variation, typically of less than 20%. Many of these QTL are common across multiple, genetically-unrelated wheat populations. Evaluation of gene expression in high and low WSC wheat progeny lines from a well characterised wheat population has identified significant differences in expression of genes from different gene categories. For example, high WSC progeny lines have higher levels of expression of genes involved in carbohydrate metabolism and lower levels of expression of genes involved in cell wall and amino acid metabolism than low WSC lines. Genetic mapping reveals several candidate genes co-locating with QTL for WSC. In addition, expression QTL (eQTL) for selected candidate genes co-locate with WSC QTL; co-location of the genes and eQTL with WSC QTL make these genes stronger candidate genes for the WSC trait.

High-throughput assessment of transgene copy number in sugarcane using real-time quantitative PCR.

Accurate and timely detection of transgene copy number in sugarcane is currently hampered by the requirement to use Southern blotting, needing relatively large amounts of genomic DNA and, therefore, the continued growth and maintenance of bulky plants in containment glasshouses. In addition, the sugarcane genome is both polyploid and aneuploid, complicating the identification of appropriate genes for use as references in the development of a high-throughput method. Using bioinformatic techniques followed by in vitro testing, two genes that appear to occur once per base genome of sugarcane were identified. Using these genes as reference genes, a high-throughput assay employing RT-qPCR was developed and tested using a group of sugarcane plants that contained unknown numbers of copies of the nptII gene encoding kanamycin resistance. Using this assay, transgene copy numbers from 3 to more than 50 were identified. In comparison, Southern blotting accurately identified the number of transgene copies for one line and by inference for another, but was not able to provide an accurate estimation for transgenic lines containing numerous copies of the nptII gene. Using the reference genes identified in this study, a high-throughput assay for the determination of transgene copy number was developed and tested for sugarcane. This method requires much less input DNA, can be performed much earlier in the production of transgenic sugarcane plants and allows much more efficient assessment of numerous potentially transgenic lines than Southern blotting.

Linked gene networks involved in nitrogen and carbon metabolism and levels of water-soluble carbohydrate accumulation in wheat stems.

High levels of water-soluble carbohydrates (WSC) provide an important source of stored assimilate for grain filling in wheat. To better understand the interaction between carbohydrate metabolism and other metabolic processes associated with the WSC trait, a genome-wide expression analysis was performed using eight field-grown lines from the high and low phenotypic tails of a wheat population segregating for WSC and the Affymetrix wheat genome array. The 259 differentially expressed probe sets could be assigned to 26 functional category bins, as defined using MapMan software. There were major differences in the categories to which the differentially expressed probe sets were assigned; for example, probe sets upregulated in high relative to low WSC lines were assigned to category bins such as amino acid metabolism, protein degradation and transport and to be involved in starch synthesis-related processes (carbohydrate metabolism bin), whereas downregulated probe sets were assigned to cell wall-related bins, amino acid synthesis and stress and were involved in sucrose breakdown. Using the set of differentially expressed genes as input, chemical-protein network analyses demonstrated a linkage between starch and N metabolism via pyridoxal phosphate. Twelve C and N metabolism-related genes were selected for analysis of their expression response to varying N and water treatments in the field in the four high and four low WSC progeny lines; the two nitrogen/amino acid metabolism genes demonstrated a consistent negative association between their level of expression and level of WSC. Our results suggest that the assimilation of nitrogen into amino acids is an important factor that influences the levels of WSC in the stems of field-grown wheat.

Identification of drought-response genes and a study of their expression during sucrose accumulation and water deficit in sugarcane culms.

BACKGROUND: The ability of sugarcane to accumulate high concentrations of sucrose in its culm requires adaptation to maintain cellular function under the high solute load. We have investigated the expression of 51 genes implicated in abiotic stress to determine their expression in the context of sucrose accumulation by studying mature and immature culm internodes of a high sucrose accumulating sugarcane cultivar. Using a sub-set of eight genes, expression was examined in mature internode tissues of sugarcane cultivars as well as ancestral and more widely related species with a range of sucrose contents. Expression of these genes was also analysed in internode tissue from a high sucrose cultivar undergoing water deficit stress to compare effects of sucrose accumulation and water deficit. RESULTS: A sub-set of stress-related genes that are potentially associated with sucrose accumulation in sugarcane culms was identified through correlation analysis, and these included genes encoding enzymes involved in amino acid metabolism, a sugar transporter and a transcription factor. Subsequent analysis of the expression of these stress-response genes in sugarcane plants that were under water deficit stress revealed a different transcriptional profile to that which correlated with sucrose accumulation. For example, genes with homology to late embryogenesis abundant-related proteins and dehydrin were strongly induced under water deficit but this did not correlate with sucrose content. The expression of genes encoding proline biosynthesis was associated with both sucrose accumulation and water deficit, but amino acid analysis indicated that proline was negatively correlated with sucrose concentration, and whilst total amino acid concentrations increased about seven-fold under water deficit, the relatively low concentration of proline suggested that it had no osmoprotectant role in sugarcane culms. CONCLUSIONS: The results show that while there was a change in stress-related gene expression associated with sucrose accumulation, different mechanisms are responding to the stress induced by water deficit, because different genes had altered expression under water deficit.

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism.

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (ß-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form.

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing.

Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 x Q165). The sequencing yielded 96,755 (IJ76-514) and 86,241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP--an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent--with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.

Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species.

Sugarcane is a crop of great interest for engineering of sustainable biomaterials and biofuel production. Isolated sugarcane promoters have generally not maintained the expected patterns of reporter transgene expression. This could arise from defective promoters on redundant alleles in the highly polyploid genome, or from efficient transgene silencing. To resolve this question we undertook detailed analysis of a sugarcane gene that combines a simple pattern in genomic Southern hybridization analysis with potentially useful, sink-specific, expression. Sequence analysis indicates that this gene encodes a member of the SHAQYF subfamily of MYB transcription factors. At least eight alleles were revealed by PCR analysis in sugarcane cultivar Q117 and a similar level of heterozygosity was seen in BAC clones from cultivar Q200. Eight distinct promoter sequences were isolated from Q117, of which at least three are associated with expressed alleles. All of the isolated promoter variants were tested for ability to drive reporter gene expression in sugarcane. Most were functional soon after transfer, but none drove reporter activity in mature stems of regenerated plants. These results show that the ineffectiveness of previously tested sugarcane promoters is not simply due to the isolation of non-functional promoter copies from the polyploid genome. If the unpredictable onset of silencing observed in most other plant species is associated with developmental polyploidy, approaches that avoid efficient transgene silencing in polyploid sugarcane are likely to have much wider utility in molecular improvement.

A bioinformatic approach to the identification of a conserved domain in a sugarcane legumain that directs GFP to the lytic vacuole.

Sugarcane is an ideal candidate as a biofactory for the production of alternate higher value products. One way of achieving this is to direct useful proteins into the vacuoles within the sugarcane storage parenchyma tissue. By bioinformatic analysis of gene sequences from putative sugarcane vacuolar proteins a motif has been identified that displays high conservation across plant legumain homologues that are known to function within vacuolar compartments. This five amino acid motif, represented by the sequence IRLPS in sugarcane is shown to direct an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP appeared to be dependent on cell type, suggesting that the vacuolar environment can be hostile to introduced proteins. This targeting motif will be a valuable tool for engineering plants such as sugarcane for production of novel products.

Identification of transcripts associated with cell wall metabolism and development in the stem of sugarcane by Affymetrix GeneChip Sugarcane Genome Array expression profiling.

Sugarcane is an important crop in tropical regions of the world, producing a very large biomass and accumulating large amounts of sucrose in the stem. In this study, we present the first report of transcript profiling using the GeneChip Sugarcane Genome Array. We have identified transcripts that are differentially expressed in the sugarcane stem during development by expression profiling using the array and total RNA derived from three disparate stem tissues (meristem, internodes 1-3, 8, and 20) from four replicates of the sugarcane variety Q117 grown in the field. We have identified 119 transcripts that were highly differentially expressed with development and have characterised members of the cellulose synthase (CesA) and cellulose synthase-like (Csl) gene families, which displayed coordinated expression during stem development. In addition, we determined that many other transcripts involved in cell wall metabolism and lignification were also co-expressed with members of the CesA and Csl gene families, offering additional insights into the dynamics of primary and secondary cell wall synthesis in the developing sugarcane stem.

Characterisation of single nucleotide polymorphisms in sugarcane ESTs.

Commercial sugarcane cultivars (Saccharum spp. hybrids) are both polyploid and aneuploid with chromosome numbers in excess of 100; these chromosomes can be assigned to 8 homology groups. To determine the utility of single nucleotide polymorphisms (SNPs) as a means of improving our understanding of the complex sugarcane genome, we developed markers to a suite of SNPs identified in a list of sugarcane ESTs. Analysis of 69 EST contigs showed a median of 9 SNPs per EST and an average of 1 SNP per 50 bp of coding sequence. The quantitative presence of each base at 58 SNP loci within 19 contiguous sequence sets was accurately and reliably determined for 9 sugarcane genotypes, including both commercial cultivars and ancestral species, through the use of quantitative light emission technology in pyrophosphate sequencing. Across the 9 genotypes tested, 47 SNP loci were polymorphic and 11 monomorphic. Base frequency at individual SNP loci was found to vary approximately twofold between Australian sugarcane cultivars and more widely between cultivars and wild species. Base quantity was shown to segregate as expected in the IJ76-514 x Q165 sugarcane mapping population, indicating that SNPs that occur on one or two sugarcane chromosomes have the potential to be mapped. The use of SNP base frequencies from five of the developed markers was able to clearly distinguish all genotypes in the population. The use of SNP base frequencies from a further six markers within an EST contig was able to help establish the likely copy number of the locus in two genotypes tested. This is the first instance of a technology that has been able to provide an insight into the copy number of a specific gene locus in hybrid sugarcane. The identification of specific and numerous haplotypes/alleles present in a genotype by pyrophosphate sequencing or alternative techniques ultimately will provide the basis for identifying associations between specific alleles and phenotype and between allele dosage and phenotype in sugarcane.

Identification of differentially expressed transcripts from maturing stem of sugarcane by in silico analysis of stem expressed sequence tags and gene expression profiling.

Sugarcane accumulates high concentrations of sucrose in the mature stem and a number of physiological processes on-going in maturing stem tissue both directly and indirectly allow this process. To identify transcripts that are associated with stem maturation, we compared patterns of gene expression in maturing and immature stem tissue by expression profiling and bioinformatic analysis of sets of stem ESTs. This study complements a previous study of gene expression associated directly with sugar metabolism in sugarcane. A survey of sequences derived from stem tissue identified an abundance of several classes of sequence that are associated with fibre biosynthesis in the maturing stem. A combination of EST analyses and microarray hybridization revealed that genes encoding homologues of the dirigent protein, a protein that assists in the stereospecificity of lignin assembly, were the most abundant and most strongly differentially expressed transcripts in maturing stem tissue. There was also evidence of coordinated expression of other categories of fibre biosynthesis and putative defence- and stress-related transcripts in the maturing stem. This study has demonstrated the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques to highlight the very significant transcriptional investment the maturing stem of sugarcane has placed in fibre biosynthesis and stress tolerance, in addition to its already well-documented role in sugar accumulation.

Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis.

The ability of sugarcane to accumulate sucrose provides an experimental system for the study of gene expression determining carbohydrate partitioning and metabolism. A sequence survey of 7242 ESTs derived from the sucrose-accumulating, maturing stem revealed that transcripts for carbohydrate metabolism gene sequences (CMGs) are relatively rare in this tissue. However, within the CMG group, putative sugar transporter ESTs form one of the most abundant classes observed. A combination of EST analysis and microarray and northern hybridization revealed that one of the putative sugar transporter types, designated PST type 2a, was the most abundant and most strongly differentially expressed CMG in maturing stem tissue. PST type 2a is homologous to members of the major facilitator super-family of transporters, possessing 12 predicted transmembrane domains and a sugar transport conserved domain, interrupted by a large cytoplasmic loop. Its transcript was localized to phloem companion cells and associated parenchyma in maturing stem, suggesting a role in sugar translocation rather than storage. In addition, other categories of CMGs show evidence of coordinated expression, such as enzymes involved in sucrose synthesis and cleavage, and a majority of enzymes involved in glycolysis and the pentose phosphate pathway. This study demonstrates the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques for studies of the developmental regulation of metabolic enzymes and potential transporters in sugarcane.