Transcript and Metabolite Changes during the Early Phase of ABA-mediated Induction of CAM in Talinum triangulare

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 (ABA). RNA-sequencing and metabolite measurements were employed to analyse the changes underlying CAM induction and identify potential CAM regulators. Non-negative matrix factorisation 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 ABA 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. 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. Talinum triangulare seeds were germinated in multiplication substrate (Floraton 3, Floragard) and four-week-old seedlings were transferred to pots with D 400 soil and Cocopor (Stender). Two weeks before the treatment, the plants were placed to controlled environment plant chamber (MobyLux GroBanks, CLF Plant Climatics) with the following growth conditions: 25 °C/23 °C, 12 h light/12 h dark. The light intensity at the leaf level was 150-200 µmoles s-1 m-2. To avoid unintended CAM induction by drought, all experimental plants were watered as needed and with the same amount of water per plant. At the age of two months, two mature and fully illuminated leaves of each plant were treated either with (+/-) ABA (Sigma-Aldrich; 200 µM solution in 0.095 % (v/v) methanol) or mock solution (0.095 % (v/v) methanol). Both solutions contained 0.02 % (v/v) Tween-20 (PanReac AppliChem). The first treatment took place after four hours in the light, the second treatment followed four hours later. Treated leaves were harvested and shock-frozen in liquid nitrogen 40, 80, 160, 320, 640 and 1280 min after the first treatment. Two biological replicates originating from two independent plants were harvested per time point. The treated leaves from the same plant were pooled prior to all analyses. Please note that the sample information (conditions and replicates) has been corrected on Aug 6, 2020.