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Pre-exposing (priming) plants to mild, non-lethal elevated temperature improves their tolerance to a later higher-temperature stress (triggering stimulus), which is of great ecological importance. 'Thermomemory' is maintaining this tolerance for an extended period of time. NAM/ATAF1/2/CUC2 (NAC) proteins are plant-specific transcription factors (TFs) that modulate responses to abiotic stresses, including heat stress (HS). Here, we investigated the potential role of NACs for thermomemory. We determined the expression of 104 Arabidopsis NAC genes after priming and triggering heat stimuli, and found ATAF1 expression is strongly induced right after priming and declines below control levels thereafter during thermorecovery. Knockout mutants of ATAF1 show better thermomemory than wild type, revealing a negative regulatory role. Differential expression analyses of RNA-seq data from ATAF1 overexpressor, ataf1 mutant and wild-type plants after heat priming revealed five genes that might be priming-associated direct targets of ATAF1: AT2G31260 (ATG9), AT2G41640 (GT61), AT3G44990 (XTH31), AT4G27720 and AT3G23540. Based on co-expression analyses applied to the aforementioned RNA-seq profiles, we identified ANAC055 to be transcriptionally co-regulated with ATAF1. Like ataf1, anac055 mutants show improved thermomemory, revealing a potential co-control of both NAC TFs over thermomemory. Our data reveals a core importance of two NAC transcription factors, ATAF1 and ANAC055, for thermomemory.

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Capsicum annuum L. is one of the most cultivated Solanaceae species, and in the open field, water limitation leading to drought stress affects its fruit quality, fruit setting, fruit size and ultimately yield. We identified stage-specific and a common core set of differentially expressed genes, following RNA-seq transcriptome analyses of a breeding line subjected to acute drought stress followed by recovery (rewatering), at three stages of plant development. Among them, two NAC transcription factor (TF) genes, i.e., CaNAC072 and CaNAC104, were always upregulated after drought stress and downregulated after recovery. The two TF proteins were observed to be localized in the nucleus following their transient expression in Nicotiana benthamiana leaves. The expression of the two NACs was also induced by NaCl, polyethylene glycol (PEG) and abscisic acid (ABA) treatments, suggesting that CaNAC072 is an early, while CaNAC104 is a late abiotic stress-responsive gene. Virus-induced gene silencing (VIGS) of CaNAC104 did not affect the pepper plantlet's tolerance to drought stress, while VIGS of CaNAC072 increased drought tolerance. Heterologous expression of CaNAC072 in Arabidopsis thaliana as well as in plants mutated for its homolog ANAC072 did not increase drought stress tolerance. This highlights a different role of the two NAC homologs in the two species. Here, we discuss the complex role of NACs as transcriptional switches in the response to drought stress in bell pepper.

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Moderate and temporary heat stresses (HS) prime plants to tolerate, and survive, a subsequent severe HS. Such acquired thermotolerance can be maintained for several days under normal growth conditions, and create a HS memory. We recently demonstrated that plastid-localized small heat shock protein HSP21 is a key component of HS memory in Arabidopsis thaliana. A sustained high abundance of HSP21 during the HS recovery phase extends HS memory. The level of HSP21 is negatively controlled by plastid-localized metalloprotease FtsH6 during HS recovery. Here, we demonstrate that autophagy, a cellular recycling mechanism, exerts additional control over HSP21 degradation. Genetic and chemical disruption of both, metalloprotease activity and autophagy trigger superior HSP21 accumulation, thereby improving memory. Furthermore, we provide evidence that autophagy cargo receptor ATG8-INTERACTING PROTEIN1 (ATI1) is associated with HS memory. ATI1 bodies colocalize with both autophagosomes and HSP21, and their abundance and transport to the vacuole increase during HS recovery. Together, our results provide new insights into the control module for the regulation of HS memory, in which two distinct protein degradation pathways act in concert to degrade HSP21, thereby enabling cells to recover from the HS effect at the cost of reducing the HS memory.

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As sessile life forms, plants are repeatedly confronted with adverse environmental conditions, which can impair development, growth, and reproduction. During evolution, plants have established mechanisms to orchestrate the delicate balance between growth and stress tolerance, to reset cellular biochemistry once stress vanishes, or to keep a molecular memory, which enables survival of a harsher stress that may arise later. Although there are several examples of memory in diverse plants species, the molecular machinery underlying the formation, duration, and resetting of stress memories is largely unknown so far. We report here that autophagy, a central self-degradative process, assists in resetting cellular memory of heat stress (HS) in Arabidopsis thaliana. Autophagy is induced by thermopriming (moderate HS) and, intriguingly, remains high long after stress termination. We demonstrate that autophagy mediates the specific degradation of heat shock proteins at later stages of the thermorecovery phase leading to the accumulation of protein aggregates after the second HS and a compromised heat tolerance. Autophagy mutants retain heat shock proteins longer than wild type and concomitantly display improved thermomemory. Our findings reveal a novel regulatory mechanism for HS memory in plants.

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Leaf senescence is a key process in plants that culminates in the degradation of cellular constituents and massive reprogramming of metabolism for the recovery of nutrients from aged leaves for their reuse in newly developing sinks. We used molecular-biological and metabolomics approaches to identify NAC transcription factor (TF) RD26 as an important regulator of metabolic reprogramming in Arabidopsis thaliana. RD26 directly activates CHLOROPLAST VESICULATION (CV), encoding a protein crucial for chloroplast protein degradation, concomitant with an enhanced protein loss in RD26 overexpressors during senescence, but a reduced decline of protein in rd26 knockout mutants. RD26 also directly activates LKR/SDH involved in lysine catabolism, and PES1 important for phytol degradation. Metabolic profiling revealed reduced γ-aminobutyric acid (GABA) in RD26 overexpressors, accompanied by the induction of respective catabolic genes. Degradation of lysine, phytol and GABA is instrumental for maintaining mitochondrial respiration in carbon-limiting conditions during senescence. RD26 also supports the degradation of starch and the accumulation of mono- and disaccharides during senescence by directly enhancing the expression of AMY1, SFP1 and SWEET15 involved in carbohydrate metabolism and transport. Collectively, during senescence RD26 acts by controlling the expression of genes across the entire spectrum of the cellular degradation hierarchy.

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Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.

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