microRNAs associated with drought response in the bioenergy crop sugarcane (Saccharum spp.)

Abstract

Sugarcane (Saccharum spp.) is one of the most important crops in the world. Drought stress is a major abiotic stress factor that significantly reduces sugarcane yields. However the gene network that mediates plant responses to water stress remains largely unknown in several crop species. Although several microRNAs that mediate post-transcriptional regulation during water stress have been described in other species, the role of the sugarcane microRNAs during drought stress has not been studied. The objective of this work was to identify sugarcane miRNAs that are differentially expressed under drought stress and to correlate this expression with the behavior of two sugarcane cultivars with different drought tolerances. The sugarcane cultivars RB867515 (higher drought tolerance) and RB855536 (lower drought tolerance) were cultivated in a greenhouse for three months and then subjected to drought for 2, 4, 6 or 8 days. By deep sequencing of small RNAs, we were able to identify 18 miRNA families. Among all of the miRNAs thus identified, seven were differentially expressed during drought. Six of these miRNAs were differentially expressed at two days of stress, and five miRNAs were differentially expressed at four days. The expression levels of five miRNAs (ssp-miR164, ssp-miR394, ssp-miR397, ssp-miR399-seq 1 and miR528) were validated by RT-qPCR (quantitative reverse transcriptase PCR). Six precursors and the targets of the differentially expressed miRNA were predicted using an in silico approach and validated by RT-qPCR; many of these targets may play important roles in drought tolerance. These findings constitute a significant increase in the number of identified miRNAs in sugarcane and contribute to the elucidation of the complex regulatory network that is activated by drought stress.

Figure 1. Real-time PCR of a sugarcane gene encoding a dehydrin (SCQGLR1085F11.g).

RB867515 (higher drought tolerance) and RB855536 (lower drought tolerance) plants were irrigated (black bars) or subjected to drought stress by withholding irrigation (gray bars) for two (A) and four (B) days. Error bars represent the standard error (n = 4). * p<0.07 and ** p<0.05. Statistics was calculated between irrigated and drought treatments in each cultivar using the permutation mean test. The expression in irrigated RB867515 plants was considered as 1.

Figure 2. Size distribution of small RNA (sRNA) redundant sequences in two sugarcane cultivars.

Cultivars RB867515 (A and C) and RB855536 (B and D) were irrigated (gray bars) or drought-stressed (black bars) for two (A and B) and four (C and D) days. RB867515 is the higher drought tolerant genotype, and RB855536 is the lower drought tolerant genotype.

Figure 3. Size distribution of small RNA (sRNA) non-redundant sequences in two sugarcane cultivars.

HTD: RB867515 (higher tolerance cultivar) plants without watering. HTI: RB867515 plants under irrigated conditions. LTD: RB855536 (lower tolerance cultivar) plants without watering. LTI: RB855536 plants under irrigated conditions.

Figure 4. Composition of nucleotides in the first base positions of all 21 nt sequences.

A: Control and stressed plants after two days of drought conditions. B: Control and stressed plants after four days of drought conditions. HTD: RB867515 (higher tolerance cultivar) plants without watering. HTI: RB867515 (higher tolerance cultivar) plants under irrigated conditions. LTD: RB855536 (lower tolerance cultivar) plants without watering. LTI: RB855536 (lower tolerance cultivar) plants under irrigated conditions. The total numbers of 21 nt reads in each library are shown at the tops of the bars.

Figure 5. Primary transcripts containing the predicted stem-loop structures of the precursors of the sugarcane miRNAs.

The mature miRNAs identified in the sugarcane sRNA library are highlighted in black. The sizes of the precursors may be slightly longer than represented. The colors represent the probabilities for sequence alignment. Red is the highest probability of alignment (1), and purple is the lowest probability of alignment (0).

Figure 6. Expression profiles of seven sugarcane miRNAs modulated by drought stress.

The values are expressed as the number of transcripts per million (TPM) for irrigated plants (control, black bars) and drought-stressed plants (gray bars). RB867515 (higher drought tolerance) and RB855536 (lower drought tolerance) plants were evaluated after two and four days of stress, as indicated below each graphic. * p<0.05 and fold change >2.0. Statistics was calculated between irrigated and drought treatments in each cultivar using the Audic-Claverie method .

Figure 7. RT-qPCR expression profiles of five sugarcane miRNAs modulated by drought stress.

The values are expressed as fold changes relative to the irrigated control for each gene. The bars represent the average of the irrigated plants (control, black bars), drought-stressed plants (gray bars) and rehydrated plants (white bars) for RB867515 (higher drought tolerance) after two (A) and four (B) days of stress. Error bars represent the standard error (n = 4). Means followed by different letters are statistically different (p<0.05) using the permutation mean test.