Transcript and metabolite changes during the early phase of abscisic acid-mediated induction of crassulacean acid metabolism in Talinum triangulare

Abstract

Crassulacean acid metabolism (CAM) has evolved as a water-saving strategy, and its engineering into crops offers an opportunity to improve their water use efficiency. This requires a comprehensive understanding of the regulation of the CAM pathway. Here, we use the facultative CAM species Talinum triangulare as a model in which CAM can be induced rapidly by exogenous abscisic acid. RNA sequencing and metabolite measurements were employed to analyse the changes underlying CAM induction and identify potential CAM regulators. Non-negative matrix factorization followed by k-means clustering identified an early CAM-specific cluster and a late one, which was specific for the early light phase. Enrichment analysis revealed abscisic acid metabolism, WRKY-regulated transcription, sugar and nutrient transport, and protein degradation in these clusters. Activation of the CAM pathway was supported by up-regulation of phosphoenolpyruvate carboxylase, cytosolic and chloroplastic malic enzymes, and several transport proteins, as well as by increased end-of-night titratable acidity and malate accumulation. The transcription factors HSFA2, NF-YA9, and JMJ27 were identified as candidate regulators of CAM induction. With this study we promote the model species T. triangulare, in which CAM can be induced in a controlled way, enabling further deciphering of CAM regulation.

Keywords: Talinum triangulare; Abscisic acid; crassulacean acid metabolism; metabolome; time course; transcriptome.

Fig. 1.

Early response of Talinum triangulare to exogenous abscisic acid. (A) Experimental design. Prior to the treatments, T. triangulare plants were adapted to a 12 h/12 h light/dark, 25 °C/23 °C cycle. At 2 months of age, two mature leaves per plant were sprayed with 200 µM ABA or mock solution [0.095% (v/v) methanol]. The first treatment was applied 4 hours into the light period and was followed by a second treatment 4 hours later. Treated leaves were harvested and snap-frozen 40, 80, 160, 320, 640, and 1280 min after the first treatment. (B) Number of significantly (q≤0.01) down- and up-regulated genes (differentially expressed genes; DEGs) in ABA-treated leaves compared with mock-treated leaves at each time point.

Fig. 2.

Exogenous ABA altered the transcript levels of core CAM genes and genes of related pathways, correlating with altered levels of selected metabolites in Talinum triangulare. Transcript abundances and metabolite amounts are expressed as log₂-fold changes of ABA-treated compared with mock-treated leaves on blue–red and yellow–green scales, respectively (expression, n=2; metabolites, n=4). Only genes with significantly different transcript abundances (DESeq with Benjamini–Hochberg correction, q-value ≤0.01) and significantly altered temporal patterns (maSigPro with Benjamini–Hochberg correction, q-value ≤0.01 and R²>0.85) are shown. The significance level for metabolites was 0.05 after Benjamini–Hochberg correction. Substrate conversions are depicted with solid lines, post-translation regulations with dashed lines, and transport processes are shown in grey. Genes whose involvement in the depicted pathways is expected are also included. Protein subcellular localization is based on prediction from Beta vulgaris (TargetP). 1,3-BPG, 1,3-bisphosphoglycerate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate; CBBC, Calvin–Benson–Bassham cycle; DHAP, dihydroxyacetone phosphate; Fru-6-P, fructose-6-phosphate; Fru-1,6-bisP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; Glc-1-P, glucose-1-phosphate; Glc-6-P, glucose-6-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PGlyM, phosphoglycerate mutase.

Fig. 3.

Transcript levels of genes involved in ABA signalling and biosynthesis were influenced by exogenous ABA in Talinum triangulare. Transcript abundances are expressed as log₂-fold changes of ABA-treated compared with mock-treated leaves (n=2). Only genes with significantly different transcript abundances (DESeq with Benjamini–Hochberg correction, q-value≤0.01) and significantly altered temporal patterns (maSigPro with Benjamini–Hochberg correction, q-value≤0.01 and R²>0.85) are shown. The core signalling pathway comprises PYR/PYL/RCAR receptors, clade A phosphatases PP2C, and SnRK2 kinases. In the absence of ABA, PP2C phosphatases bind SnRK2s and prevent them from phosphorylating downstream targets. Upon binding of ABA to the receptors, they capture PP2Cs, releasing phosphorylated SnRK2s. Targets of SnRK2s include transcription factors in particular, many of which regulate gene expression through binding to ABA-responsive element (ABRE) motifs in promoter sequences of target genes. OST1 is involved in the control of stomatal movement. P, phosphorylation.

Fig. 4.

Non-negative matrix factorization and k-means clustering of the Talinum triangulare transcriptome. (A) Five factors were identified based on time-dependent expression of individual genes (for details, see Materials and Methods). (B) Based on the transcriptional contribution of each factor, mapped genes were assigned to nine clusters (for details, see Materials and Methods). Enrichment of MapMan categories in each cluster is shown in green (over-represented categories) and red (under-represented categories). deg, Degradation; FA, fatty acids; GA, gibberellic acid; JA, jasmonic acid; met, metabolism; min. CHO, minor carbohydrate; misc, miscellaneous; PPR, pentatricopeptide repeat; PS, photosynthesis; sec. met., secondary metabolism.