Many heat stress transcription factors (Hsfs) and heat shock proteins (Hsps) have been identified to play important roles in the heat tolerance of plants. However, many of the key factors mediating the heat response pathways remain unknown. Here, we report that two genes, which are targets of TAS1 (trans-acting siRNA precursor 1)–derived small interfering RNAs that we named HEAT-INDUCED TAS1 TARGET1 (HTT1) and HTT2, are involved in thermotolerance. Microarray analysis revealed that the HTT1 and HTT2 genes were highly upregulated in Arabidopsis thaliana seedlings in response to heat shock. Overexpression of TAS1a, whose trans-acting small interfering RNAs target the HTT genes, elevated accumulation of TAS1-siRNAs and reduced expression levels of the HTT genes, causing weaker thermotolerance. By contrast, overexpression of HTT1 and HTT2 upregulated several Hsf genes, leading to stronger thermotolerance. In heat-tolerant plants overexpressing HsfA1a, the HTT genes were upregulated, especially at high temperatures. Meanwhile, HsfA1a directly activated HTT1 and HTT2 through binding to their promoters. HTT1 interacted with the heat shock proteins Hsp70-14 and Hsp40 and NUCLEAR FACTOR Y, SUBUNIT C2. Taken together, these results suggest that HTT1 mediates thermotolerance pathways because it is targeted by TAS1a, mainly activated by HsfA1a, and acts as cofactor of Hsp70-14 complexes.

Cold and drought stresses seriously affect cassava (Manihot esculenta) plant growth and yield. Recently, long noncoding RNAs (lncRNAs) have emerged as key regulators of diverse cellular processes in mammals and plants. To date, no systematic screening of lncRNAs under abiotic stress and their regulatory roles in cassava has been reported. In this study, we present the first reference catalog of 682 high-confidence lncRNAs based on analysis of strand-specific RNA-seq data from cassava shoot apices and young leaves under cold, drought stress and control conditions. Among them, 16 lncRNAs were identified as putative target mimics of cassava known miRNAs. Additionally, by comparing with small RNA-seq data, we found 42 lncNATs and sense gene pairs can generate nat-siRNAs. We identified 318 lncRNAs responsive to cold and/or drought stress, which were typically co-expressed concordantly or discordantly with their neighboring genes. Trans-regulatory network analysis suggested that many lncRNAs were associated with hormone signal transduction, secondary metabolites biosynthesis, and sucrose metabolism pathway. The study provides an opportunity for future computational and experimental studies to uncover the functions of lncRNAs in cassava.

Alternative splicing is an essential post-transcriptional regulatory mechanism that can impact mRNA stability and protein diversity of eukaryotic genomes. Although numerous forms of stress-responsive alternative splicing have been identified in model plants, a large-scale study of alternative splicing dynamics under abiotic stress conditions in cassava has not been conducted. Here, we report the parallel employment of isoform-Seq, ssRNA-Seq, and Degradome-Seq to investigate the diversity, abundance, and fate of alternatively spliced isoforms in response to cold and drought stress. We identified 38 164 alternative splicing events, among which 3292 and 1025 events were significantly regulated by cold and drought stress, respectively. Intron retention was the most abundant subtype of alternative splicing. Global analysis of splicing regulators revealed that the number of their alternatively spliced isoforms and the corresponding abundance were specifically modulated by cold stress. We found that 58.5% of cold-regulated alternative splicing events introduced a premature termination codon into the transcripts, and 77.6% of differential alternative splicing events were detected by Degradome-Seq. Our data reveal that cold intensely affects both quantitative and qualitative aspects of gene expression via alternative splicing pathways, and advances our understanding of the high complexity and specificity of gene regulation in response to abiotic stresses. Alternative splicing is responsible for reprogramming of the transcriptome and the sensitivity of cassava plants to cold.

HYL1 is an important regulator of microRNA (miRNA) biogenesis. A loss-of-function mutation of HYL1 causes the reduced accumulation of some miRNAs but fails to display the miRNA-deficient phenotypes of these miRNAs. In Arabidopsis, miR156 mediates phase transition through repression of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) genes. However, it remains unknown whether, and if so how, HYL1 enables phase transition through miR156. This study showed that a loss-of-function mutation of the HYL1 gene caused defects in the timing of the juvenile phase. In the primary leaves of hyl1-2 mutants, abaxial trichomes were generated prematurely, the leaf blades elongated, and the blade base angles enlarged, as is observed for adult leaves. In hyl1-2 p35S::miR156a and hyl1-2 spl9-4 spl15-1 plants, increased accumulation of miR156a and repressed expression of the SPL genes were concomitant with a complete or partial rescue of the hyl1-2 phenotype in phase defects. In contrast, overexpression of the SPL9 gene in hyl1-2 mutants led to total disappearance of the juvenile phase. Moreover, HYL1 prevented the premature accumulation of adult-related transcripts in the primary leaves. Taken together, these results suggest that HYL1 controls the expression levels of miR156-targeted SPL genes and enables plants to undergo the juvenile phase, an important and critical step during plant development to ensure maximum growth and productivity.

Abiotic stress negatively impacts cassava (Manihot esculenta) growth and yield. Several molecular mechanisms of plant response to cold and drought have been identified and described in the literature, however, little is known about the crosstalk of the responses of cassava to these two stresses. To elucidate this question, transcriptome analysis of cassava seedlings under cold or PEG-simulated drought stress treatment was performed. Our results showed that 6103 and 7462 transcripts were significantly regulated by cold and drought stress, respectively. Gene Ontology (GO) annotation revealed that the abscisic (ABA) and jasmonic acid (JA) signaling pathways shared between the two stresses responses. We further identified 2434 common differentially expressed genes (DEGs), including 1130 up-regulated and 841 down-regulated DEGs by the two stresses. These co-induced or co-suppressed genes are grouped as stress signal perception and transduction, transcription factors (TFs), metabolism as well as transport facilitation according to the function annotation. Furthermore, a large proportion of well characterized protein kinases, TF families and ubiquitin proteasome system related genes, such as RLKs, MAPKs, AP2/ERFBPs, WRKYs, MYBs, E2 enzymes and E3 ligases, including three complexes of interacting proteins were shown as key points of crosstalk between cold and drought stress signaling transduction pathways in a hierarchical manner. Our research provides valuable information and new insights for genetically improving the tolerance of crops to multiple abiotic stresses.