Alternative Splicing of Circadian Clock Genes Correlates With Temperature in Field-Grown Sugarcane

Abstract

Alternative Splicing (AS) is a mechanism that generates different mature transcripts from precursor mRNAs (pre-mRNAs) of the same gene. In plants, a wide range of physiological and metabolic events are related to AS, as well as fast responses to changes in temperature. AS is present in around 60% of intron-containing genes in Arabidopsis, 46% in rice, and 38% in maize and it is widespread among the circadian clock genes. Little is known about how AS influences the circadian clock of C4 plants, like commercial sugarcane, a C4 crop with a complex hybrid genome. This work aims to test if the daily dynamics of AS forms of circadian clock genes are regulated by environmental factors, such as temperature, in the field. A systematic search for AS in five sugarcane clock genes, ScLHY, ScPRR37, ScPRR73, ScPRR95, and ScTOC1 using different organs of sugarcane sampled during winter, with 4 months old plants, and during summer, with 9 months old plants, revealed temperature- and organ-dependent expression of at least one alternatively spliced isoform in all genes. Expression of AS isoforms varied according to the season. Our results suggest that AS events in circadian clock genes are correlated with temperature.

Keywords: alternative splicing; circadian clock; diel rhythms; field experiment; gene expression; sugarcane.

Figure 1

Alternative splicing events identified in the sugarcane circadian clock genes. Alternative splicing (AS) events are shown in the gene structure of ScLHY, ScPRR37, ScPRR73, ScPRR95, and ScTOC1. White boxes show 5′ and 3′ UTR regions; black boxes show exons; black lines represent introns. The main protein domains are in grey. AS events are shown in colored solid lines, colored dotted lines or in dotted white boxes and the splicing events are illustrated below each event. Different colors were chosen to mark the AS events investigated later, whereas events that were not investigated further are in dark red. IR, intron retention; ES, exon skipping; Alt 5′ ss, alternative 5′ splice sites; Alt 3′ ss, alternative 3′ splice sites; Alt Ex, alternative exon.

Figure 2

Diel expression profile of fully spliced and alternative transcript isoforms in different seasons. Biological replicates (circles and triangles) and their LOESS curve (continuous lines ± SE) of fully spliced (FS, black) and alternative transcript forms (AS, colored) for the winter samples (4-month-old plants, left) the summer samples (9-month-old plants, right). (A, B) ScLHY gene expression shows levels of I1R (orange) and (C, D) I5R events (blue). (E, F) ScPRR37 gene expression shows levels of I6R (green), and (G, H) I6R (yellow); (I, J) ScPRR73 gene expression shows levels of I2R (purple). Inverted triangles show the time of the maximum value of the LOESS curve. The light-gray boxes represent the night period. Statistical significance was analyzed by paired Student’s t-test, ^*p < 0.05.

Figure 3

Diel expression profile of fully spliced and alternative spliced transcript isoforms in internodes. Biological replicates (circles and triangles) and their LOESS curve (continuous lines ± SE) of fully spliced (FS, black) and alternative spliced (AS, colored) transcript forms of sugarcane circadian clock genes during the summer harvest in internode 1 and 2 (left) and internode 5 (right). (A, B) ScLHY gene expression shows levels of I1R (orange) and (C, D) I5R (blue); (E, F) ScPRR37 gene expression shows levels of I6R (green) and (G, H) I7R (yellow). Inverted triangles show the time of the maximum value of the LOESS curve. The light-gray boxes represent the night period. Statistical significance was analyzed by paired Student’s t-test, ^*p < 0.05.

Figure 4

Alternative splicing is rhythmic in sugarcane in field conditions. (A–D) The logarithm of the ratio of the expression levels of an AS isoform to its FS isoform, annotated as the log(AS/FS), was plotted against the normalized time of the day (ZT). (A) ScLHY I1R (orange) and I5R (blue); ScPRR37 I3R (dark blue), (B) I6R (green) and I7R (light yellow); (C) ScPRR73 I2R (gold) and I6R (purple); and (D) ScPRR37 E3S (red). (E–F) Normalized expression levels of rhythmic splicing-related transcripts taken from oligo array data (Dantas et al., 2019) in (E) leaves +1 (L1, green), and internodes 1 and 2 (I1, red) and (F) internode 5 (I5, yellow). Individual expression profiles were drawn in gray. LOESS regression was used to draw the trends in the data in all panels (continuous line ± SE). The light-gray boxes represent the night period.

Figure 5

The proportion of alternative spliced and fully spliced forms has a negative correlation with ambient temperature. The logarithm of the ratio of the expression levels of AS and FS isoforms, annotated as the log(AS/FS), was plotted against the ambient temperature for ScLHY I1R (orange) and I5R (blue). Regression lines were added for each group of ratios. R² and P-value were calculated for each regression. Negative correlations were significant in leaf +1 (L1) in the (A) winter (4-month-old plants), and (B) summer, (C) internode 1 and 2 (I1), and (D) internode 5 (I5), both in the summer (9-month-old plants).