Project A5 deals with the fact that plants can be protected from an otherwise deleterious or even lethal heat stress (HS) by a prior moderate (priming) heat treatment. Notably, the capacity of the plant to withstand a damaging HS is maintained over a couple of days after the priming and reflects the presence of a thermomemory. During phase I of the CRC 973 we made two observations that form the basis of the project proposed here for phase II:
(i) The elongation of the hypocotyl is almost solely based on cell expansion, while cell proliferation does not play a significant role. In Arabidopsis thaliana, the model plant used in our studies, hypocotyl elongation after a triggering heat stress (at 44°C) only occurs when seedlings have been thermoprimed before. Most relevant for the current proposal is the existence of a thermomemory enabling the plant to ´remember´ the previous priming HS stress even after two or three days. Furthermore, in phase I of project A5 we discovered biological diversity of the thermomemory trait in different natural accessions of Arabidopsis. The molecular basis underlying and controlling the hypocotyl (cell) elongation thermomemory is virtually unknown at present.
(ii) The shoot apical meristem (SAM) of plants gives rise to above-ground organs including leaves and flowers and is therefore of critical importance for shoot formation. A young seedling typically has only a single apical meristem, while older plants have additional aboveground meristems particularly in their leaf axils. Protecting the SAM from severe damage is therefore of prime importance for seedling survival under conditions of abiotic stress. In phase I of project A5 we discovered that a priming HS entrains the SAM of young Arabidopsis seedlings to survive a subsequent triggering HS stimulus, and that this process has a strong thermomemory component. However, molecular determinants of this entrainment are unknown at present.
Considering the big knowledge gaps that exists with respect to the thermomemory in relation to growth-related processes in Arabidopsis (or plants in general), phase II of project A5 aims at unravelling some of the molecular elements and regulatory networks constituting the growth-related thermomemory. We propose the following research: First, we will characterise the transcriptome of the hypocotyl (from dark-grown seedlings) after a thermopriming stimulus using RNA-seq in two natural Arabidopsis accessions that strongly differ in their thermomemory capacity, namely N13, which has a strong memory, and Col-0, which has a weak memory of thermostress. This will allow us to identify transcription factors (TFs) and gene clusters affected by the thermopriming. Based on the data obtained we will then select individual TFs for functional analysis including the identification of downstream target genes and gene regulatory networks they control. As a second inroad to TFs coordinating transcriptional reprogramming during thermopriming, we propose to identify TFs regulating the expression of cell wall extension-associated genes encoding expansins (EXPs) and xyloglucan endotransglucosylase/hydrolases (XTHs) that are functionally relevant for thermoprimed hypocotyl growth. We will identify thermomemory-relevant EXP/XTH genes by knocking out single or multiple EXP/XTH genes in parallel (in different combinations), using the CRISPR-Cas9 technology. With respect to the shoot apical meristem, we will use light-grown Arabidopsis seedlings of the N13 and Col-0 accessions, subject them to a thermopriming stimulus and microscopically analyse potential effects on the cellular (re-)organisation of their SAMs. Next, we will determine the global transcriptome of shoot apices throughout the memory phase by RNA-seq. Finally, we will study the expression of genes robustly responding to the thermopriming stimulus at spatial resolution within the SAM, using INTACT and RNA in situ hybridisation methods.
Collectively, project A5 will reveal key elements of plant growth affected by thermopriming.