Functional genomics: Transcriptomics of sugarcane-current status and future prospects

Systems biology is the science of relating genes and metabolic pathways across multiple levels, which may range from an individual to an entire crop. Transcriptomics, the global analysis of expression of RNA is an integral component of this and is widely applied in plant biology in both model systems and crop plants, such as sugarcane. This chapter reviews sugarcane transcriptomics, including the tools developed, the use of the transcriptome to improve marker discovery and the relevance of resources developed for related species. The use of the new high-throughput sequencing technologies is discussed as is the possible impact of sugarcane transcriptomics in genomicsassisted breeding. All of these techniques will inform efforts to expedite enhanced breeding of sugarcane either by traditional or precision breeding strategies

Circadian rhythms of sense and antisense transcription in sugarcane, a highly polyploid crop.

Commercial sugarcane (Saccharum hybrid) is a highly polyploid and aneuploid grass that stores large amounts of sucrose in its stem. We have measured circadian rhythms of sense and antisense transcription in a commercial cultivar (RB855453) using a custom oligoarray with 14,521 probes that hybridize to sense transcripts (SS) and 7,380 probes that hybridize to antisense transcripts (AS).We estimated that 32% of SS probes and 22% AS probes were rhythmic. This is a higher proportion of rhythmic probes than the usually found in similar experiments in other plant species. Orthologs and inparalogs of Arabidopsis thaliana, sugarcane, rice, maize and sorghum were grouped in ortholog clusters. When ortholog clusters were used to compare probes among different datasets, sugarcane also showed a higher proportion of rhythmic elements than the other species. Thus, it is possible that a higher proportion of transcripts are regulated by the sugarcane circadian clock. Thirty-six percent of the identified AS/SS pairs had significant correlated time courses and 64% had uncorrelated expression patterns. The clustering of transcripts with similar function, the anticipation of daily environmental changes and the temporal compartmentation of metabolic processes were some properties identified in the circadian sugarcane transcriptome. During the day, there was a dominance of transcripts associated with photosynthesis and carbohydrate metabolism, including sucrose and starch synthesis. During the night, there was dominance of transcripts associated with genetic processing, such as histone regulation and RNA polymerase, ribosome and protein synthesis. Finally, the circadian clock also regulated hormone signalling pathways: a large proportion of auxin and ABA signalling components were regulated by the circadian clock in an unusual biphasic distribution

Sense and antisense transcripts are modulated differently by the circadian clock.

<p>(<b>A</b>) Overlap between SAS that had their probes for sense transcripts (SS) considered circadian and SAS that had their probes for antisense transcripts (AS) considered circadian. (<b>B</b>) Distribution of Spearman's rank correlation coefficient (ρ) for each of all the 428 pairs of SS/AS (light blue) and only the 207 pairs of SS/AS that had at least one probe considered circadian (orange). If ρ >0.56, correlation is positive and significant. If ρ <−0.56, correlation is negative and significant. (<b>C to F</b>) Z-score normalized expression levels of SS (red) and AS (dark blue) for a gene that have (<b>C</b>) both SS and AS in the same phase (RuBisCo activase; SCBGLR1044D06.g,), (<b>D</b>) SS and AS in opposite phases (urease, SCSGLR1045A02.g), (<b>E</b>) AS peaking before SS (glucose 1-phosphate adenylyltransferase, SCVPCL6061A06.g) and (<b>F</b>) SS peaking before AS (violaxanthin de-epoxidase, SCVPHR1095C07.g). White boxes represent periods of subjective day and light grey boxes represent periods of subjective night.</p

Rhythmic probes associated with sugar storage.

<p>Z-score normalized time courses of rhythmic probes for transcripts associated with sugar storage pathways were separated into (<b>A</b>) sucrose synthesis (light green); (<b>B</b>) sucrose degradation (dark green); (<b>C</b>) starch synthesis (dark red); (<b>D</b>) starch degradation (cyan); (<b>E</b>) starch branching (red); and starch debranching (light blue). White boxes represent periods of subjective day and light grey boxes represent periods of subjective night.</p

Proportion of rhythmic probes in different plant species using different algorithms.

1<p>Khan et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Khan1" target="_blank">[18]</a>;</p>2<p>Filichkin et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Filichkin2" target="_blank">[17]</a>;</p>3<p>Covington and Harmer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Covington2" target="_blank">[39]</a>;</p>4<p>Edwards et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Edwards1" target="_blank">[20]</a>;</p>5<p>estimated.</p

Identification of rhythmic sense and antisense transcripts in sugarcane.

<p>(<b>A</b>) Venn diagram showing the number of sense probes that were considered expressed and were considered rhythmic by three algorithms: JTK_CYCLE, COSOPT and Fisher's G-test. Probes identified as rhythmic in at least two algorithms were considered circadian. (<b>B</b>) Phase distribution of rhythmic sense probes. The phase of each rhythmic probe was estimated using JTK_CYCLE and binned in 6 groups, according to the time of their peak. (<b>C</b>) Venn diagram showing the number of antisense probes that were considered expressed and considered rhythmic by three algorithms: JTK_CYCLE, COSOPT and Fisher's G-test. (<b>D</b>) Phase distribution of circadian antisense probes.</p

Temporal coordination of probes associated with sucrose and starch metabolism pathways.

<p>The time of peak of rhythms in transcript levels of genes associated with sucrose and starch metabolism was identified in a schematic metabolic pathway. Each circle corresponds to a specific gene model. Metabolic pathways were colored according to the median of the phase of their constitutive genes. The time of peak of probes associated with starch and sucrose synthesis pathways was between ZT20 and ZT4, while the time of the peak of probes associate to sucrose and starch degradation was between ZT8 to ZT16. Genes that were not circadian (n. c.) were in gray. Rhythmic with a time of peak at ZT0 are in yellow, ZT4 in dark orange, ZT8 in red, ZT12 in blue, ZT16 in dark blue and ZT20 in light blue. Enzymes for sucrose synthesis are: (<b>1</b>) sucrose-phosphate synthase; (<b>2</b>) sucrose phosphatase; (<b>3</b>) sucrose synthase; (<b>4</b>) neutral invertase; (<b>5</b>) hexokinase; (<b>6</b>) fructokinase; (<b>7</b>) glucose-6-phosphate isomerase; (<b>8</b>) phosphoglucomutase; (<b>9</b>) UDP-glucose pyrophosphorylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Rohwer1" target="_blank">[75]</a>. Abbreviations: S6P – sucrose 6-phosphate; UDP-G - UDP-glucose; G1P – glucose 1-phosphate; G6P – glucose 6-phosphate; F6P – fructose 6-phosphate; ADP-G – ADP-glucose; 3P glycerate – 3-phospho glycerate.</p

Time courses of probes associated with the perception of light.

<p>Z-score normalized time courses of rhythmic probes for sugarcane transcripts associated with the perception of light. Expression levels were measured using oligoarrays. Different probes for the same sugarcane genes are represented separately. (<b>A</b>) <i>PHYTOCHROME A</i> (Sc<i>PHYA</i>), (<b>B</b>) <i>ScPHYB</i>, (<b>C</b>) Sc<i>PHYC</i>, (<b>D</b>) <i>CRYPTOCHROME1</i> (Sc<i>CRY1</i>), (<b>E</b>) Sc<i>CRY2</i>, (<b>F</b>) <i>PHOTOTROPIN</i> (<i>ScPHOT</i>) (<b>G</b>) <i>ZEITLUPE</i> (<i>ZTL-1</i>), (<b>H</b>) <i>ZTL</i>-2, (<b>I</b>) Sc<i>UVR8</i>. White boxes represent periods of subjective day and light grey boxes represent periods of subjective night.</p