Expansion of the circadian transcriptome inBrassica rapaand genome-wide diversification of paralog expression patterns

Abstract

An important challenge of crop improvement strategies is assigning function to paralogs in polyploid crops. Gene expression is one method for determining the activity of paralogs; however, the majority of transcript abundance data represents a static point that does not consider the spatial and temporal dynamics of the transcriptome. Studies in Arabidopsis have estimated up to 90% of the transcriptome to be under diel or circadian control depending on the condition. As a result, time of day effects on the transcriptome have major implications on how we characterize gene activity. In this study, we aimed to resolve the circadian transcriptome in the polyploid crop Brassica rapa and explore the fate of multicopy orthologs of Arabidopsis circadian regulated genes. We performed a high-resolution time course study with 2 h sampling density to capture the genes under circadian control. Strikingly, more than two-thirds of expressed genes exhibited rhythmicity indicative of circadian regulation. To compare the expression patterns of paralogous genes, we developed a program in R called DiPALM (Differential Pattern Analysis by Linear Models) that analyzes time course data to identify transcripts with significant pattern differences. Using DiPALM, we identified genome-wide divergence of expression patterns among retained paralogs. Cross-comparison with a previously generated diel drought experiment in B. rapa revealed evidence for differential drought response for these diverging paralog pairs. Using gene regulatory network models we compared transcription factor targets between B. rapa and Arabidopsis circadian networks to reveal additional evidence for divergence in expression between B. rapa paralogs that may be driven in part by variation in conserved non coding sequences. These findings provide new insight into the rapid expansion and divergence of the transcriptional network in a polyploid crop and offer a new method for assessing paralog activity at the transcript level.SignificanceThe circadian regulation of the transcriptome leads to time of day changes in gene expression that coordinates environmental conditions with physiological responses. Brassica rapa, a morphologically diverse crop species, has undergone whole genome triplication since diverging from Arabidopsis resulting in an expansion of gene copy number. To examine how this expansion has influenced the circadian transcriptome we developed a new method for comparing gene expression patterns. This method facilitated the discovery of genome-wide expansion of expression patterns for genes present in multiple copies and divergence in temporal abiotic stress response. We find support for conserved sequences outside the gene body contributing to these expression pattern differences and ultimately generating new connections in the gene regulatory network.

Closing the protein gap in plant chronobiology

“…Our understanding of plant chronobiology and diel plant cell regulation has been significantly enhanced through gene expression studies, driven by the evolution of technologies from single‐gene quantitative PCR (qPCR) to systems‐level tools such as microarrays and RNA sequencing (RNA‐seq) (Maric and Mas, 2020). Time‐course transcriptomics experimentation has provided, and continues to provide, us with a wealth of knowledge of diel gene regulation across a range of organisms, including Arabidopsis (Kamioka et al ., 2016), soybean ( Glycine max ) (Li et al ., 2019), lettuce ( Lactuca sativa ) (Higashi et al ., 2016) and Brassica rapa (Greenham et al ., 2020). Recently, extensive transcriptomic characterisation of a suite of circadian clock mutants in Arabidopsis has revealed that individual clock components appear to regulate distinct portfolios of genes (Graf et al ., 2017).…”

Abiotic stress through time

“…The pan-genomes of Brassica napus, B. rapa, and B. oleracea (references within Bayer et al, 2020), have genes with PAV that associate with stress response. In B. rapa, the expansion of the circadian transcriptome resulted in divergence in phasing among retained paralogs, as well as differential drought responses (Greenham et al, 2020). A QTL study in B. rapa uncovered GI contributing to natural variation in circadian period, and salt and cold tolerance (Xie et al, 2015), which aligns with a conserved role for GI in abiotic stress response in Arabidopsis (Seo & Mas, 2015).…”

“…Widespread whole genome duplications gave rise to an expanded circadian clock system in angiosperms (Lou et al ., 2012; Greenham et al ., 2020). The RVE/LHY/CCA1 family, with three copies in the basal angiosperm Amborella trichopoda ( LHY , RVE1 and RVE6 ), has expanded to seven copies in rice (Fig.…”

“…In B . rapa , the expansion of the circadian transcriptome resulted in divergence in phasing among retained paralogs, as well as differential drought responses (Greenham et al ., 2020). A QTL study in B .…”