The HB40-JUB1 transcriptional regulatory network controls gibberellin homeostasisin Arabidopsis

The phytohormones gibberellins (GAs) play fundamental roles in almost every aspect of plant growth and development. Although there is good knowledge about GA biosynthetic and signaling pathways, factors contributing to the mechanisms homeostatically controlling GA levels remain largely unclear. Here, we demonstrate that homeobox transcription factor HB40 of the HD-Zip family in Arabidopsis thaliana regulates GA content at two additive control levels. We show that HB40 expression is induced by GA and in turn reduces the levels of endogenous bioactive GAs by a simultaneous reduction of GA biosynthesis and increased GA deactivation. Hence, HB40 overexpression leads to typical GA-deficiency traits, such as small rosettes, reduced plant height, delayed flowering, and male sterility. In contrast, a loss-of-function hb40 mutation enhances GA-controlled growth. Genome-wide RNA-sequencing combined with molecular-genetic analyses revealed that HB40 directly activates transcription of JUNGBRUNNEN1 (JUB1), a key TF repressing growth by suppressing GA biosynthesis and signaling. HB40 also activates genes encoding GA 2-oxidases (GA2oxs) which are major GA catabolic enzymes. The effect of HB40 is ultimately mediated through induction of nuclear growth-repressing DELLA proteins. Our results thus uncover an important role of the HB40/JUB1/GA2ox/DELLA regulatory network in controlling GA homeostasis during plant growth.Plant science

Calcium-Dependent Protein Kinase CPK1 Controls Cell Death by In Vivo Phosphorylation of Senescence Master Regulator ORE1

Calcium-regulated protein kinases are key components of intracellular signaling in plants that mediate rapid stress-induced responses to changes in the environment. To identify in vivo phosphorylation substrates of CALCIUM-DEPENDENT PROTEIN KINASE1 (CPK1), we analyzed the conditional expression of constitutively active CPK1 in conjunction with in vivo phosphoproteomics. We identified Arabidopsis (Arabidopsis thaliana) ORESARA1 (ORE1), the developmental master regulator of senescence, as a direct CPK1 phosphorylation substrate. CPK1 phosphorylates ORE1 at a hotspot within an intrinsically disordered region. This augments transcriptional activation by ORE1 of its downstream target gene BIFUNCTIONAL NUCLEASE1 (BFN1). Plants that overexpress ORE1, but not an ORE1 variant lacking the CPK1 phosphorylation hotspot, promote early senescence. Furthermore, ORE1 is required for enhanced cell death induced by CPK1 signaling. Our data validate the use of conditional expression of an active enzyme combined with phosphoproteomics to decipher specific kinase target proteins of low abundance, of transient phosphorylation, or in yet-undescribed biological contexts. Here, we have identified that senescence is not just under molecular surveillance manifested by stringent gene regulatory control over ORE1. In addition, the decision to die is superimposed by an additional layer of control toward ORE1 via its posttranslational modification linked to the calcium-regulatory network through CPK1

The plastid metalloprotease FtsH6 and small heat shock protein HSP21 jointly regulate thermomemory in Arabidopsis

Acquired tolerance to heat stress is an increased resistance to elevated temperature following a prior exposure to heat. The maintenance of acquired thermotolerance in the absence of intervening stress is called ‘thermomemory’ but the mechanistic basis for this memory is not well defined. Here we show that Arabidopsis HSP21, a plastidial small heat shock protein that rapidly accumulates after heat stress and remains abundant during the thermomemory phase, is a crucial component of thermomemory. Sustained memory requires that HSP21 levels remain high. Through pharmacological interrogation and transcriptome profiling, we show that the plastid-localized metalloprotease FtsH6 regulates HSP21 abundance. Lack of a functional FtsH6 protein promotes HSP21 accumulation during the later stages of thermomemory and increases thermomemory capacity. Our results thus reveal the presence of a plastidial FtsH6–HSP21 control module for thermomemory in plants.</p

The plastid metalloprotease FtsH6 and small heat shock protein HSP21 jointly regulate thermomemory in Arabidopsis

Acquired tolerance to heat stress is an increased resistance to elevated temperature following a prior exposure to heat. The maintenance of acquired thermotolerance in the absence of intervening stress is called ‘thermomemory' but the mechanistic basis for this memory is not well defined. Here we show that Arabidopsis HSP21, a plastidial small heat shock protein that rapidly accumulates after heat stress and remains abundant during the thermomemory phase, is a crucial component of thermomemory. Sustained memory requires that HSP21 levels remain high. Through pharmacological interrogation and transcriptome profiling, we show that the plastid-localized metalloprotease FtsH6 regulates HSP21 abundance. Lack of a functional FtsH6 protein promotes HSP21 accumulation during the later stages of thermomemory and increases thermomemory capacity. Our results thus reveal the presence of a plastidial FtsH6–HSP21 control module for thermomemory in plants

Heat shock factor HSFA2 fine-tunes resetting of thermomemory via plastidic metalloprotease FtsH6

Plants ´memorize´ stressful events and protect themselves from future, often more severe, stresses. To maximize growth after stress, plants ´reset´, or ´forget´, memories of stressful situations which requires an intricate balance between stress memory formation and the degree of forgetfulness. HEAT SHOCK PROTEIN 21 (HSP21) encodes a small HSP in plastids of Arabidopsis thaliana. Previous research found that HSP21 functions as a key component of thermomemory, which requires a sustained elevated level of HSP21 during the recovery from heat stress (HS). A heat-induced metalloprotease, filamentation temperature sensitive H6 (FtsH6), degrades HSP21 to establish pre-stress level, thereby resetting memory during the recovery phase. The transcription factor heat shock factor A2 (HSFA2) activates downstream genes essential for mounting thermomemory thereby acting as a positive regulator in the process. Here, by employing a yeast one-hybrid screen we additionally identify HSFA2 as an upstream transactivator of the resetting element FtsH6. Constitutive and inducible overexpression of HSFA2 increases expression of FtsH6 while it is drastically reduced in the hsfa2 knockout mutant. Chromatin immunoprecipitation reveals in planta binding of HSFA2 to the FtsH6 promoter. Importantly, overexpression of HSFA2 improves thermomemory more profoundly in ftsh6 than wild-type plants. Thus, by activating both memory-supporting and -resetting genes HSFA2 acts as a cellular homeostasis factor during thermomemory

Autophagy complements metalloprotease FtsH6 in degrading plastid heat shock protein HSP21 during heat stress recovery

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

Calcium-Dependent Protein Kinase CPK1 Controls Cell Death by In Vivo Phosphorylation of Senescence Master Regulator ORE1

Calcium-regulated protein kinases are key components of intracellular signaling in plants that mediate rapid stress-induced responses to changes in the environment. To identify in vivo phosphorylation substrates of CALCIUM-DEPENDENT PROTEIN KINASE1 (CPK1), we analyzed the conditional expression of constitutively active CPK1 in conjunction with in vivo phosphoproteomics. We identified Arabidopsis (Arabidopsis thaliana) ORESARA1 (ORE1), the developmental master regulator of senescence, as a direct CPK1 phosphorylation substrate. CPK1 phosphorylates ORE1 at a hotspot within an intrinsically disordered region. This augments transcriptional activation by ORE1 of its downstream target gene BIFUNCTIONAL NUCLEASE1 (BFN1). Plants that overexpress ORE1, but not an ORE1 variant lacking the CPK1 phosphorylation hotspot, promote early senescence. Furthermore, ORE1 is required for enhanced cell death induced by CPK1 signaling. Our data validate the use of conditional expression of an active enzyme combined with phosphoproteomics to decipher specific kinase target proteins of low abundance, of transient phosphorylation, or in yet-undescribed biological contexts. Here, we have identified that senescence is not just under molecular surveillance manifested by stringent gene regulatory control over ORE1. In addition, the decision to die is superimposed by an additional layer of control toward ORE1 via its posttranslational modification linked to the calcium-regulatory network through CPK1