Principal Investigator
Researcher
Email:fqguo@cemps.ac.cn
Personal Web: http://people.ucas.edu.cn/~fangqingguo

National Key Laboratory of Plant Molecular Genetics

(1) Discovering a new retrograde-regulatory pathway for cellular heat stress Responses in plants  

We has discovered a new regulatory pathway for plants to survive under heat stress and demonstrated that chloroplast is the signaling source for retrograde-activating nuclear heat-responsive gene expression. By proteomic screening of heat-responsive proteins in Arabidopsis, we have identified chloroplast ribosomal protein S1 (RPS1) as a heat-responsive protein. We have demonstrated that RPS1 functions in biosynthesis of thylakoid membranes encoded by chloroplast DNA, and determines the stability of thylakoid membranes at a RPS1 expression level-dependent manner. Importantly, consistent with the notion that the inhibited activation of HsfA2 in response to heat stress in rps1 mutant causes heat-susceptibility, we further demonstrated that constitutive expression of HsfA2 with a viral promoter in the rps1 mutant is sufficient to reestablish lost heat tolerance and recovers heat-susceptible thylakoid stability to wild type levels. Our discoveries suggest chloroplasts as sensors in activating cellular responses, which is especially exciting for the prospects of breeding crops to tolerate heat stress by modulating plastid translation capacity (Yu et al., PLoS Genetics, 2012). 

(2) Identification of core subunits of photosystem II as action sites of HSP21 that is activated by the GUN5-mediated retrograde pathway in Arabidopsis  

In previous studies, we found that an unrecognized retrograde signaling pathway regulates the heat-responsive activation of the key heat shock transcription factor HsfA2 and its downstream target gene HSP21 in Arabidopsis (Yu et al., 2012). Here, we have provided genetic and biochemical evidence that HSP21 is activated by the GUN5-dependent retrograde signaling pathway and stabilizes PSII by directly binding to its core subunits such as D1 and D2 proteins under heat stress. We further demonstrate that the constitutive expression of HSP21 sufficiently rescues the thermo-sensitive stability of PSII and survival defects of the gun5 mutant with dramatically improving granal stacking under heat stress, indicating that HSP21 is a master chaperone protein in maintaining the integrity of thylakoid membrane system under heat stress. In line with our interpretation based on several lines of in vitro and in vivo protein-interaction evidence that HSP21 interacts with core subunits of PSII, the kinetics of HSP21 binding to the D1 and D2 proteins was determined by performing analysis of microscale thermophoresis. Thus, HSP21 plays a major role in protecting the core subunits of PSII from thermal damages and its heat-responsive activation via the heat shock transcription factor HsfA2 is critical for the survival of plants under heat stress. Considering that the main detrimental effects of heat stress on PSII are the destruction of the manganese cluster of the water-oxidizing complex, as well as damage of the D1 (and to a lesser extent the D2) subunit(s), which form the protein core of the PSII reaction center (Berry and Bjorkman, 1980; Sharkey, 2005; Allakhverdiev et al., 2008), identification of D1 and D2 as the protected target proteins of HSP21 under heat stress is of important significance. Our findings reveal an auto-adaptation loop pathway that plant cells optimize particular needs of chloroplasts in stabilizing photosynthetic complexes by relaying the GUN5-dependent plastid signal(s) to activate the heat-responsive expression of HSP21 in the nucleus during adaptation to heat stress in plants (Chen et al., Plant Journal, 2017). 

(3) SPL6 represses transcriptional activation of the ER-stress sensor IRE1 for determining cell fates during panicle development in rice 

The SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) gene family has been shown to regulate numerous fundamental aspects of plant growth and development. Based on RNA-seq analysis, SPL6 is highly expressed during panicle development in rice. In order to get a better insight into the role of SPL6, the corresponding T-DNA insertion mutant lines were exploited to characterize its functions. The spl6-1 mutant exhibited a severe panicle apical abortion phenotype. Notably, under normal field-growth conditions, the degenerated apical spikelets of the spl6-1 mutant panicles were marked as whitish glumes, easily distinguished from the normal spikelets of wild-type panicles. Given that the spl6-1 apical spikelets underwent programmed cell death (PCD) marked as whitish glumes under normal growth conditions, we reasoned that loss-of-function of SPL6 might alter an intracellular signaling pathway(s) to disrupt maintenance of cell homeostasis, resulting in cell death. Extensive studies in the past two decades revealed that ER stress occurs in response to the accumulation of unfolded proteins in the ER, causing cells into PCD when prolonged/unresolved (Masferrer et al., 2002; koenigshofer et al., 2008; Hetz, 2012). Our results have provided several lines of evidence that the hyperactivation of the ER-stress sensor IRE1 (Inositol-requiring enzyme 1) contributes to the cell death-induced spikelet degeneration in the spl6-1 mutant and SPL6 acts as a transcriptional repressor of IRE1. 

Protein folding is very sensitive to adverse environmental stresses, causing unfolded or misfolded proteins to accumulate in the endoplasmic reticulum (ER). The accumulation of newly synthesized unfolded proteins is identified by transmembrane sensors that signal activation of an elaborate adaptive process known as the unfolded protein response (UPR) (Masferrer et al., 2002). In mammals, chronic activation of UPR signaling promotes oxidative stress, autophagy, and eventually induces an apoptotic (programmed cell death) responses (Hetz, 2012). Despite IRE1 as an administrator/executor of cell fate determination under ER stress, it remains to be elucidated how the activation of IRE1 is regulated in control of cell fate decisions. Thus, as a fundamental mechanism, the IRE1-dependent adaptation process is tightly modulated to reach homeostasis and normal ER functions. Our findings demonstrate that SPL6 functions as a transcriptional repressor of IRE1 and plays as an essential survival factor for suppression of persistent or intense ER stress conditions that ultimately lead to cell death. Although the UPR has long been linked to cell death in plants (Zuppini et al., 2004; Iwata and Koizumi, 2005; Sangster et al., 2008; Qiang et al., 2012), the underlying mechanisms by which plant cells modulate the amplitude and duration of the IRE1-mediated ER stress signaling outputs to determine cell fate remain to be explored. Data reported here provide direct evidence that loss-of-function of SPL6 stimulates the hyperactivation of the ER stress sensor IRE1, which controls ER-stress signaling outputs that hinge on a balance between adaptive and death signals for determining cell fates during ER stress. Collectively, our findings uncover a mechanistic axis directly linking the ER-stress sensing, UPR signaling outputs, cell fate decision under physiological growth conditions. Our findings were recently published as a cover story article in Nature Plants (Wang et al., Nature Plants, 2018).  

(4) Identification of SBPase as a vulnerable enzyme to carbonyl modifications under stress conditions and in dark-induced leaf senescence in Arabidopsis  

Sedoheptulose-1,7-bisphosphatase (SBPase) is a Calvin cycle enzyme and functions in photosynthetic carbon fixation. We found that SBPase was rapidly carbonylated in response to stress conditions or in dark-induced leaf senescence in Arabidopsis. In vitro activity analysis of the purified recombinant SBPase showed that SBPase was carbonylated by hydroxyl radicals, which led to enzyme inactivation in a H₂O₂ dose-dependent manner. To determine the conformity with carbonylation-caused loss in enzymatic activity in response to stresses, we isolated a loss-of-function mutant sbp, which is deficient in SBPase-dependent carbon assimilation and starch biosynthesis. sbp mutant exhibited a severe growth retardation phenotype, especially for the developmental defects in leaves and flowers where SBPASE is highly expressed. The mutation of SBPASE caused growth retardation mainly through inhibition of cell division and expansion, which can be partially rescued by exogenous application of sucrose. Our findings demonstrate that ROS-induced oxidative damage to SBPase affects growth, development and chloroplast biogenesis in Arabidopsis through inhibiting carbon assimilation efficiency (Liu et al., Molecular Plant, 2012).