Ethylene-mediated improvement in sucrose accumulation in ripening sugarcane involves increased sink strength

Abstract

Background: Sugarcane is a major crop producing about 80% of sugar globally. Increasing sugar content is a top priority for sugarcane breeding programs worldwide, however, the progress is extremely slow. Owing to its commercial significance, the physiology of sucrose accumulation has been studied extensively but it did not lead to any significant practical outcomes. Recent molecular studies are beginning to recognize genes and gene networks associated with this phenomenon. To further advance our molecular understanding of sucrose accumulation, we altered sucrose content of sugarcane genotypes with inherently large variation for sucrose accumulation using a sugarcane ripener, ethylene, and studied their transcriptomes to identify genes associated with the phenomenon.

Results: Sucrose content variation in the experimental genotypes was substantial, with the top-performing clone producing almost 60% more sucrose than the poorest performer. Ethylene treatment increased stem sucrose content but that occurred only in low-sugar genotype. Transcriptomic analyses have identified about 160,000 unigenes of which 86,000 annotated genes were classified into functional groups associated with carbohydrate metabolism, signaling, localization, transport, hydrolysis, growth, catalytic activity, membrane and storage, suggesting the structural and functional specification, including sucrose accumulation, occurring in maturing internodes. About 25,000 genes were differentially expressed between all genotypes and treatments combined. Genotype had a dominant effect on differential gene expression than ethylene treatment. Sucrose and starch metabolism genes were more responsive to ethylene treatment in low-sugar genotype. Ethylene caused differential gene expression of many stress-related transcription factors, carbohydrate metabolism, hormone metabolism and epigenetic modification. Ethylene-induced expression of ethylene-responsive transcription factors, cytosolic acid- and cell wall-bound invertases, and ATPase was more pronounced in low- than in high-sugar genotype, suggesting an ethylene-stimulated sink activity and consequent increased sucrose accumulation in low-sugar genotype.

Conclusion: Ethylene-induced sucrose accumulation is more pronounced in low-sugar sugarcane genotype, and this is possibly achieved by the preferential activation of genes such as invertases that increase sink strength in the stem. The relatively high enrichment of differentially expressed genes associated with hormone metabolism and signaling and stress suggests a strong hormonal regulation of source-sink activity, growth and sucrose accumulation in sugarcane.

Keywords: Ethylene; Sucrose accumulation; Sugarcane; Transcriptomics.

Fig. 1

Effect of ethylene on sucrose content in mature stems of three sugarcane genotypes, ROC22, YT71–210 and GT86–887. Stem sugar content was quantified before (0 d) and 3, 5 and 7 days after ethylene treatment. The control treatment received water. The ordinate headline shows the ratio of sugar content to fresh weight (mg/g) ± SD. Data were analysed by ANOVA following a repeated measurements model using Genstat (VSNC, UK). Genotype (LSD 3.4) and treatment (LSD 2.8) effects are highly significant (p < 0.001) for day 5 and day 7

Fig. 2

Statistics of transcripts and unigenes obtained from RNA-seq data. a Transcripts assembled from the RNA-seq data. b Unigenes obtained from the transcripts assembled

Fig. 3

Statistics of DEGs identified in maturing stem tissue in different sugarcane genotypes treated with ethephon. Venn diagram of pairwise comparisons DEGs in three genotypes (a) before ethephon treatment (b) after ethephon treatment and (c) before and after ethephon treatment. (d) Number of up-regulated and down-regulated DEGs before and after ethephon treatment in 3 genotypes. (e) Heatmap of DEGs in 3 genotypes before and after ethephon treatment. Expression values are presented as RPKM normalized log₂ transformed counts. Red and blue colors indicate up- and down- regulated transcripts, respectively. CK- check (water control), T- ethephon treatment, HS: high-sugar variety, MS: medium-sugar variety, LS: low-sugar variety

Fig. 4

Analysis of differential expression of unigenes associated with carbohydrate metabolism. Pairwise comparison of differentially expressed unigenes involved in starch and sucrose metabolism (a). The significance in each comparison pair is indicated using log-transformed P-value (red); the dark gray areas represent missing values. Expression of invertases (b), ATPase (c) and SPS/SUS in HS and LS (d) as affected by ethylene treatment. CK- check (water control), T- ethephon treatment, HS: high-sugar variety, MS: medium-sugar variety, LS: low-sugar variety

Fig. 5

Analysis of differential expression of unigenes associated with transcription. Pairwise comparison of differentially expressed unigenes involved in transcriptional regulation (a), phosphorylation (b) and DNA methylation (c). The significance in each comparison pair is indicated using log-transformed P-value (red); the dark gray areas represent missing values. CK- check (water control), T- ethephon treatment, HS: high-sugar variety, MS: medium-sugar variety, LS: low-sugar variety

Fig. 6

Functional enrichment analysis of differentially expressed genes in low-sugar (LS) sugarcane before and after ethylene treatment. a The up-regulated functions are displayed with respect to their significance (p < 0.01). Different colors correspond to the ratio of genes in each category. It’s noteworthy that the most enriched function is RNA-related transcription (corresponding to ethylene response factor; ERFs) and hormone-related metabolism and signaling (ethylene-related). These enriched modules will guid the search for the functional networks underpinning ethylene-regulated sugar production/translocation in sugarcane. b the expression of ERFs in HS and LS following ethylene treatment. CK- check (water control), T- ethephon treatment, HS: high-sugar variety, MS: medium-sugar variety, LS: low-sugar variety

Fig. 7

Schematic presentation of ethylene-regulated sugar production and translocation in sugarcane. The identified ethylene-regulated are integrated into a functional network of sugar production (source), sugar transportation and sugar aaccumulation (to sink). When leaves were treated with ethylene (Eth), Rubisco activity was enhanced; sucrose synthase (SUS), invertases (INV) and sucrose phosphate synthase (SPS) were up-regulated by translational regulation by ERFs, stimulating sucrose biosynthesis. At this time, sucrose and glucose could be acting as regulatory signals to control photosynthesis. After synthesis, sucrose will be transported from leaf to phloem through plasmodesmata (by sugar transporter, SUT - the symplastic route) and cell wall (by cell wall invertase- the apoplastic route). Then, sucrose was unloaded at the sink mainly through SUT. Sugar translocation from the leaves to phloem creates a sucrose gradient that facilitates the biochemical balance of sugar synthesis at the source