Honghong Hu

Abiotic stresses reduce rice growth and yield, yet the mechanisms underlying multiple stress tolerance are unknown. Here, we show that the E3 ubiquitin ligase WIDTH OF LEAF AND GRAIN (WLG) enhances drought, salt, and heat tolerance by ubiquitinating and degrading DROUGHT AND SALT TOLERANCE (DST). This degradation represses PEROXIDASE 24 PRECURSOR (Prx24) expression, increasing guard-cell H₂O₂ accumulation, reducing stomatal conductance and transpiration rate, thereby improving water retention and limiting sodium uptake under abiotic stress conditions. Elevated H₂O₂ also induces the expression of heat shock proteins, contributing to enhanced tolerance to heat stress. DST, in turn, transcriptionally represses WLG, establishing a negative feedback loop that fine-tunes H₂O₂ homeostasis and stress responses. Moreover, WLG^hap.B promotes stronger DST degradation than WLG^hap.A. Our findings elucidated the WLG-DST-Prx24 module as a central regulatory mechanism governing H₂O₂ dynamics under multiple abiotic stresses, highlighting the potential of WLG^hap.B for developing multiple stress-resistant rice varieties in a changing climate.

Climate change threatens global food security by imposing concurrent abiotic and biotic stresses on major crops such as rice. Genetic resources conferring dual resistance to drought and blast disease are rare, limiting the development of climate-resilient crops. Here, using a genome-wide association study, we identify NLR8-Hap2, a natural allele of the rice resistance gene NLR8 that enhances resistance to drought and blast disease. A single-base deletion in the NLR8-Hap2 coding sequence generates a premature stop codon, resulting in a protein lacking the C-terminal leucine-rich repeat (LRR) domain. The dual protective effects of NLR8-Hap2 arise from the enhanced association with and stabilization of OsbHLH148 and OsBIHD1, which regulate drought tolerance and blast defense, respectively. Introgression of NLR8-Hap2 into elite indica and japonica rice cultivars significantly improves dual-stress resistance without detectable yield penalties under normal field conditions. Together, these findings highlight NLR8-Hap2 as a genetic resource for developing multi-stress-resilient rice.

Chloroplast division is essential for plant growth and photosynthesis, and the assembly of the FtsZ‐ring is tightly regulated by ARC3 (ACCUMULATION AND REPLICATION OF CHLOROPLASTS 3). However, the mechanisms controlling its protein turnover remain poorly understood. This study reveals that ARC3 stability is co‐regulated by both the ubiquitin‐proteasome system (UPS) and chloroplast protease pathways. We demonstrate that the E3 ubiquitin ligase PUB52 mediates cytosolic degradation of ARC3 precursors via the 26S proteasome, whereas the chloroplast chaperone‐protease CLPC1 (CLPC HOMOLOGUE 1) promotes ARC3 degradation within chloroplasts. Conversely, ARC2 stabilizes ARC3 by protecting it from proteolytic degradation, ensuring proper FtsZ‐ring assembly. Either disruption or overexpression of PUB52 , CLPC1 , or ARC2 leads to aberrant chloroplast division phenotypes that resemble those of ARC3 dysregulation mutants. Genetic analyses place these regulators upstream of ARC3 in the chloroplast division pathway. These findings establish a post‐translational regulatory network where PUB52, CLPC1, and ARC2 dynamically control ARC3 levels to fine‐tune chloroplast division. This work provides important insights into the coordination of cytoplasmic and chloroplast protein homeostasis systems in maintaining organellar homeostasis, with broader implications for organelle biogenesis and plant stress adaptation.

The Ca ²⁺ -independent OST1 and Ca ²⁺ -dependent protein kinases CPKs both activate the anion channel SLAC1 during ABA-induced stomatal closure pathway. However, the mechanism by which OST1 regulates SLAC1 activation and its relationship with CPKs remain unclear. Here, we identify that OST1 primarily activates SLAC1 in this process through CPK15. Mutation of CPK15 significantly impairs ABA-induced stomatal closure and increases drought sensitivity. OST1 interacts with CPK15 and phosphorylates it at T103, which is essential for ABA-induced stomatal closure. Moreover, CPK15 can phosphorylate eight sites in the N terminus of SLAC1 to activate its anion currents in oocytes. Expression of SLAC1 ^8D (a phosphomimetic form) in oocytes constitutively activates anion channel activity and effectively restores the impaired ABA-induced stomatal closure of cpk15-1 but not by SLAC1 ^8A (a phospho-dead form). Furthermore, activated CPK15 by OST1 and Ca ²⁺ enhances its activity toward SLAC1, and mutations of both OST1 and CPK15 have additive effect on ABA-induced stomatal closure, suggesting that CPK15 activates SLAC1 through both direct and indirect mechanisms. These findings demonstrate the key role of CPK15 in ABA-induced stomatal closure, revealing a connection between OST1 and CPKs in both Ca ²⁺ -independent and Ca ²⁺ -dependent pathways in ABA-induced stomatal closure.

Summary: Stomata are produced through a series of asymmetric cell divisions (ACDs) and one symmetric division. In the Arabidopsis stomatal lineage, the number of ACDs in meristemoids is strictly limited to 1–3 rounds before meristemoids differentiate into guard mother cells. However, the precise regulatory mechanisms that govern this pattern formation remain largely elusive. Here, we identify a unique mechanism of SCREAM (SCRM) in terminating proliferative ACDs of meristemoids by directly suppressing SPEECHLESS (SPCH), a master initiator of stomatal ACDs. The mutation of SCRM, rather than SCRM2, results in a delayed proliferation‐to‐differentiation transition, as well as an excessive phenotype of ACDs. Conversely, overexpression of SCRM promotes the timely termination of proliferative divisions. Expression of stomatal‐linage marker genes is upregulated in scrm and downregulated in SCRM_OE plants. Multiple assays have demonstrated that SCRM, activated by SPCH, directly binds to the promoter of SPCH and represses its expression. Genetic analysis supports that SCRM acts upstream of SPCH, thus forming a loop of SPCH‐SCRM‐SPCH to maintain the balance between the initiation and termination of ACDs. Our findings reveal a distinct role of SCRM in regulating proliferative divisions and provide insight into the mechanism governing the limited rounds of ACDs in meristemoids.

Drought stress seriously threatens plant growth. The improvement of plant water use efficiency (WUE) and drought tolerance through stomatal regulation is an effective strategy for coping with water shortages. Epidermal patterning factor (EPF)/EPF-like (EPFL) family proteins regulate stomatal formation and development in plants and thus contribute to plant stress adaptation. Here, our analysis revealed the presence of 14 PeEPF members in the Populus euphratica genome, which exhibited a relatively conserved gene structure with 1–3 introns. Subcellular localisation prediction revealed that 9 PeEPF members were distributed in the chloroplasts of P. euphratica, and 5 were located extracellularly. Phylogenetic analysis indicated that PeEPFs can be divided into three clades, with genes within the same clade revealing a relatively conserved structure. Furthermore, we observed the evolutionary conservation of PeEPFs and AtEPF/EPFLs in certain domains, which suggests their conserved function. The analysis of cis-acting elements suggested the possible involvement of PeEPFs in plant response to multiple hormones. Transcriptomic analysis revealed considerable changes in the expression level of PeEPFs during treatment with polyethylene glycol and abscisic acid. The overexpression of PeEPF2 resulted in low stomatal density in transgenetic lines. These findings provide a basis for gaining insights into the function of PeEPFs in response to abiotic stress.

Drought stress inhibits plant growth and agricultural production. Improving plant instantaneous water use efficiency (iWUE), which is strictly regulated by stomata, is an effective way to cope with drought stress. However, the mechanisms of iWUE regulation are poorly understood. Through genetic screening for suppressors of mpk12-4, an Arabidopsis (Arabidopsis thaliana) mutant with a major iWUE quantitative trait locus gene MITOGEN-ACTIVATED PROTEIN KINASE12 deleted, we identified HIGH LEAF TEMPERATURE1 (HT1). Genetic interaction and physiological analyses showed that MPK12 controls iWUE through multiple modules in a high CO₂-induced stomatal closing pathway that regulate SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1) activity. HT1 acts downstream of MPK12, whereas OPEN STOMATA1 (OST1) and GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) function downstream of HT1 by activating SLAC1 in iWUE. Photosynthetic-CO₂ response curves and biomass analyses under different water-supply conditions showed that HT1 dysfunction improved iWUE and also increased plant growth capacity, and products of HT1 putative orthologs from Brassica (Brassica napus) and rice (Oryza sativa) exhibited functions similar to that of Arabidopsis HT1 in iWUE and the CO₂-signaling pathway. Our study revealed the mechanism of MPK12-mediated iWUE regulation in Arabidopsis and provided insight into the internal relationship between iWUE and CO₂ signaling in guard cells and a potential target for improving crop iWUE and drought tolerance.

Grain and flag leaf size are two important agronomic traits that influence grain yield in rice (Oryza sativa). Many QTLs and genes that regulate these traits individually have been identified, however, few QTLs and genes that simultaneously control these two traits have been identified. In this study, we conducted a genome-wide association analysis in rice and detected a major locus, WIDTH OF LEAF AND GRAIN (WLG), that associated with both grain and flag leaf width. WLG encodes a RING-domain E3 ubiquitin ligase. WLG^hap.B, which possesses five SNP variations compared to WLG^hap.A, encodes a protein with enhanced ubiquitination activity that confers increased rice leaf width and grain size, whereas mutation of WLG leads to narrower leaves and smaller grains. Both WLG^hap.A and WLG^hap.B interact with LARGE2, a HETC-type E3 ligase, however, WLG^hap.B exhibits stronger interaction with LARGE2, thus higher ubiquitination activity towards LARGE2 compared with WLG^hap.A. Lysine1021 is crucial for the ubiquitination of LARGE2 by WLG. Loss-of-function of LARGE2 in wlg-1 phenocopies large2-c in grain and leaf width, suggesting that WLG acts upstream of LARGE2. These findings reveal the genetic and molecular mechanism by which the WLG–LARGE2 module mediates grain and leaf size in rice, and suggest the potential of WLG^hap.B in improving rice yield.

In the face of climate-induced challenges, understanding the intricate molecular mechanisms underlying drought tolerance in plants has become imperative