De novo DNA methylation, orchestrated by the RNA-directed DNA methylation (RdDM) pathway, is essential for gene regulation and transposon silencing. While CLASSY (CLSY) proteins facilitate RNA POLYMERASE IV (Pol IV) recruitment to initiate the RdDM pathway in plants, their roles in crops are incompletely explored. Here, we report OsCLSY4 as the dominant regulator within the OsCLSY family, driving Pol IV-mediated epigenomic patterns and influencing diverse agricultural traits. Epigenomics analyses reveal that OsCLSY4 controls over 95% of Pol IV-dependent 24-nucleotide small interfering RNA (24-nt siRNA) clusters and more than 70% of Pol IV-dependent hypomethylated CHH differentially methylated regions (DMRs), predominantly at miniature inverted-repeat transposable elements (MITEs). Loss of OsCLSY4 leads to dysregulation of MADS22 and GA20ox1 in a DNA methylation-dependent manner. SunTag-mediated targeted demethylation confirms that reduced DNA methylation in promoter regions leads to MADS22 activation and GA20ox1 repression to influence grain size, linking epigenetic changes to phenotypic outcomes of osclsy4. Moreover, OsCLSY4 governs tissue-specific methylation patterns in panicle and seedling. Mechanistically, OsCLSY4 is the predominantly expressed OsCLSY family member and interacts with Pol IV. Collectively, our findings position OsCLSY4 as a central hub for Pol IV-mediated epigenomic regulation in rice and suggest its potential utility in epigenetic breeding strategies.

Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase, a key acyltransferase in the phenylpropanoid pathway and a canonical member of the BAHD acyltransferase family (BAHD), catalyzes the formation of pivotal intermediates in the biosynthesis of secondary metabolites such as lignin, chlorogenic acid, and flavonoids. These compounds serve indispensable protective functions in terrestrial plants, underpinning their adaptive responses to abiotic stresses such as drought, ultraviolet (UV) radiation, and oxidative damage. Although the role of HCT/HQT in the core phenylpropanoid pathway has been extensively characterized, its precise functional contributions to the flavonoid biosynthetic branch—particularly with respect to substrate selectivity, kinetic regulation, and metabolic channeling—remain incompletely understood. This review systematically analyzes the structural features, spatial conformation, catalytic mechanism, and substrate promiscuity of HCT/HQT to clarify its molecular determinants of activity and specificity. Furthermore, it highlights regulatory factors influencing HCT/HQT gene expression, such as transcription factors (MYB, bHLH, WRKY), phytohormones (GA3, Eth, MeJA, 6-BA, MT), and abiotic/biotic stressors (temperature, blue light, nitric oxide, nano-selenium). Collectively, these insights illuminate how plants dynamically fine-tune phenylpropanoid metabolism in coordination with developmental programs and environmental challenges. This work provides a foundation for further research on HCT/HQT and supports efforts to develop improved crop varieties through targeted regulation of this central metabolic node.

This paper considered bolt end distance, connection thickness, bolt spacing, and number of bolts as influencing factors, through experiments and numerical simulations, the mechanical performance and failure types of steel-bolted connection of laminated-flattened bamboo (LFB) under perpendicular-to-grain load were studied. The study results indicated that when subjected to perpendicular-to-grain load, the failure of the LFB steel-bolted connection was brittle, and the failure type was mainly a mixed type of splitting and shear failure between bamboo fibers. When the bolt end distance was large, the bamboo at the bolt slot could experience bearing failure perpendicular-to-grain; when the connection thickness was large, the bolt no longer remained straight but yielded. The end distance, connection thickness, bolt spacing, and number of bolts all affected the cracking load and yield load of the connection. Thus, this paper evaluated the applicability of existing equations for wood connections in LFB connections. Based on the van der Put model, by introducing the bolt number parameter, bolt spacing parameter, and effective connection thickness parameter, a semi-empirical equation for calculating the load capacity of steel-bolted LFB connection under perpendicular-to-grain load was proposed.

Grain size, which encompasses length, width, and thickness, is a critical agricultural trait that influences both grain yield and quality in rice. Although numerous grain size regulators have been identified, the molecular mechanisms governing grain size and the lignin content remain largely elusive. In this study, we clone and characterize GRAIN LENGTH AND GRAIN WIDTH 10 (GLGW10), a regulator of grain size in rice. Loss-of-function mutations in GLGW10 result in reduced grain size. GLGW10 encodes an evolutionarily conserved protein, the function of which has not been previously characterized in higher plants. Biochemical assays reveal that GLGW10 may interact with the transcription factor OsMYB108, which acts as a negative regulator of the lignin content. Knockout of OsMYB108 leads to longer and slender grain size, accompanied by increased lignin content, indicating that OsMYB108 negatively regulates both grain size and lignin content. Analysis of natural variations and haplotypes in GLGW10 reveals an association with grain size, suggesting an artificial selection on GLGW10 during rice domestication. In summary, our findings identify regulators of grain size and elucidate potential mechanisms linking grain size and lignin metabolism in rice, thereby providing essential insights for improving crop yields.

Stress granules (SGs) are liquid-liquid phase-separated condensates that sequester RNA, proteins, and metabolites to modulate cellular physiology under stress. In Arabidopsis, the RNA-binding protein RBP47b is a canonical SG marker, yet its functional contribution to thermotolerance remains unresolved. Here, we combined mTurboID proximity labeling with multi-omics profiling to define the RBP47b interactome and its physiological impact. mTurboID identified a stress-specific enrichment of 40S ribosomal subunits within RBP47b SGs, implicating these condensates in translational control. Surprisingly, quadruple mutant plants lacking all four RBP47 paralogues ( rbp47abcc′ ) displayed enhanced survival, attenuated growth delay, and faster recovery of photosynthetic efficiency after severe heat stress. Integrated transcriptome, proteome, and metabolome analyses revealed that this gain of thermotolerance is associated with (i) accelerated re-initiation of translation, (ii) constitutively elevated jasmonate and oxylipin pools, and (iii) reduced ROS accumulation during heat and recovery. We conclude that the RBP47 family acts as a negative regulator of heat tolerance by sequestering 40S subunits and limiting translational restart; loss of these SG scaffolds pre-primes jasmonate-dependent detoxification pathways and expedites proteome rebuilding, thereby conferring superior thermotolerance. ### Competing Interest Statement The authors have declared no competing interest.

High-valent iron-oxo species (FeIV═O) is a fascinating enzymatic agent with excellent anti-interference abilities in various oxidation processes. However, selective and high-yield production of FeIV═O remains challenging. Herein, Fe diatomic pairs are rationally fabricated with an assisted S bridge to tune their neighbor distances and increase their loading to 11.8 wt.%. This geometry regulated the d-band center of Fe atoms, favoring their bonding with the terminal and hydroxyl O sites of peroxymonosulfate (PMS) via heterolytic cleavage of O─O, improving the PMS utilization (70%), and selective generation of FeIV═O (>90%) at a high yield (63% of PMS) offers competitive performance against state-of-the-art catalysts. These continuous reactions in a fabricated device and technol-economic assessment further verified the catalyst with impressive long-term activity and scale-up potential for sustainable water treatment. Altogether, this heteroatom-bridge strategy of diatomic pairs constitutes a promising platform for selective and efficient synthesis of high-valent metal-oxo species.

Most of kiwifruit cultivars (e.g. Actinidia chinensis cv. Donghong, "DH") were sensitive to waterlogging, thus, waterlogging resistant rootstocks (e.g. Actinidia valvata Dunn, "Dunn") were widely used for kiwifruit industry. Those different species provided ideal materials to understand the waterlogging responses in kiwifruit. Compared to the weaken growth and root activities in "DH", "Dunn" maintained the relative high root activities under the prolonged waterlogging. Based on comparative analysis, transcript levels of pyruvate decarboxylase (PDCs) and alcohol dehydrogenase (ADHs) showed significantly difference between these two species. Both PDCs and ADHs had been significantly increased by waterlogging in "DH", while they were only limitedly triggered by 2 days stress and subsided during the prolonged waterlogging in "Dunn". Thus, 19 differentially expressed transcript factors (DETFs) had been isolated using weighted gene co-expression network analysis combined with transcriptomics and transcript levels of PDCs and ADHs in waterlogged "DH". Among these DETFs, dual luciferase and electrophoretic mobility shift assays indicated AcMYB68 could bind to and trigger the activity of AcPDC2 promoter. The stable over-expression of AcMYB68 significantly up-regulated the transcript levels of PDCs but inhibited the plant growth, especially the roots. Moreover, the enzyme activities of PDC in 35S::AcMYB68 were significantly enhanced during the waterlogging response than that in wild type plants. Most interestingly, comparative analysis indicated that the expression patterns of AcMYB68 and the previously characterized AcERF74/75 (the direct regulator on ADHs) either showed no responses (AcMYB68 and AcERF74) or very limited response (AcERF75) in "Dunn". Taken together, the restricted responses of AcMYB68 and AcERF74/75 in "Dunn" endow its waterlogging tolerance.

Kiwifruit plants have a fleshy, shallow root system which is sensitive to waterlogging stress, which results in a decrease in crop yield or even plants death. Although the waterlogging stress responses in kiwifruit have attracted much attention, the underlying molecular mechanism remains unclear. In this study, waterlogging led to drastic inhibition of root growth of 'Donghong' kiwifruit (Actinidia chinensis) plants grown in vitro, which was accompanied by significant elevation of endogenous acetaldehyde and ethanol contents. RNA-seq of roots of plants waterlogged for 0, 1 and 2 days revealed that a total of 149 genes were up- or down-regulated, including seven biosynthetic genes related to the glycolysis/gluconeogenesis pathway and 10 transcription factors. Analyses with real-time PCR, dual-luciferase assays and EMSA demonstrated that AcERF74 and AcERF75, two members of the ERF-VII subfamily, directly upregulated AcADH1 (alcohol dehydrogenase). Moreover, the overexpression of AcERF74/75 in transgenic calli resulted in dramatic increase of endogenous ethanol contents through the triggering of AcADH1 and AcADH2 expression. Although the AcPDC2 (pyruvate decarboxylase) expression was also enhanced in transgenic lines, the endogenous acetaldehyde contents showed no significant changes. These results illustrated that AcERF74/75 are two transcriptional activators on alcoholic fermentation related genes and are responsive to waterlogging stress in kiwifruit.

Tianxingzi Wang Hebei Agricultural University Yue Chen Hebei Agricultural University Zheng Zhu Hebei Agricultural University Yuqing Liu Hebei Agricultural University Gaowei Yan Hebei Agricultural University Shan Xu Hebei Agricultural University Haiqing Xu Hebei Agricultural University Ming Yang Hebei Agricultural University Shijuan Dou Hebei Agricultural University Liyun Li Hebei Agricultural University Guozhen Liu (  gzhliu@hebau.edu.cn ) Hebei Agricultural University https://orcid.org/0000-0002-7072-3357

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activase (RCA) has attracted more and more attention since it is discovered. It becomes one of the most important ifelds of photosynthesis research to study enzymology characteristics, activation of Rubisco both in vivo and in vitro, regulation of photosynthet-ic rate and RCA activity, gene and molecular structure, antisense, and overexpression. In this paper we mainly review the molecular structure, the relationship between RCA and crop photosynthesis and, especially, the role of RCA in resistance of plant to abiotic stresses.

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA) is a nuclear gene that encodes a chloroplast protein that plays an important role in photosynthesis. Some reports have indicated that it may play a role in acclimation to different abiotic stresses. In this paper, we analyzed the stress-responsive elements in the 2.0 kb 5'-upstream regions of the RCA gene promoter and the primary, secondary and tertiary structure of the protein. We identified some cis-elements of multiple stress-related components in the RCA promoter. Amino acid and evolution analyses showed that the RCA protein had conserved regions between different species; however, the size and type varied. The secondary structures, binding sites and tertiary structures of the RCA proteins were also different. This might reflect the differences in the transcription and translation levels of the two RCA isoforms during adaptation to different abiotic stresses. Although both the transcription and translation levels of RCA isoforms in the rice leaves increased under various stresses, the large isoform was increased more significantly in the chloroplast stroma and thylakoid. It can be concluded that RCA, especially RCAL, is also a multiple responder to abiotic stresses in rice, which provides new insights into RCA functions.

Cadmium (Cd) toxicity was investigated in cotton cultivar (ZMS-49) using physiological, ultrastructural, and biochemical parameters. Biomass-based tolerance index decreased, and water contents increased at 500 μM Cd. Photosynthetic efficiency determined by chlorophyll fluorescence and photosynthetic pigments declined under Cd stress. Cd contents were more in roots than shoots. A significant decrease in nutrient levels was found in roots and stem. A significant decrease in nutrient levels was found in roots and stems. In response to Cd stress, more MDA and ROS contents were produced in leaves than in other parts of the seedlings. Total soluble proteins were reduced in all parts except in roots at 500 μM Cd. Oxidative metabolism was higher in leaves than aerial parts of the plant. There were insignificant alterations in roots and leaves ultrastructures such as a little increase in nucleoli, vacuoles, starch granules, and plastoglobuli in Cd-imposed stressful conditions. Scanning micrographs at 500 μM Cd showed a reduced number of stomata as well as near absence of closed stomata. Cd depositions were located in cell wall, vacuoles, and intracellular spaces using TEM-EDX technology. Upregulation of oxidative metabolism, less ultrastructural modification, and Cd deposition in dead parts of cells show that ZMS-49 has genetic potential to resist Cd stress, which need to be explored.

Salt stress often induces declination of net photosynthetic rate (Pn), partially resulted from Rubisco degradation. The chloroplast protrusions (CPs) is one of the pathways of Rubisco exclusion from chloroplasts. To explore the relationship between the Rubisco contents and CPs under salt stress, Pn, maximum photochemical efficiency of PSII (F(v)/F(m)), carboxylation efficiency (CE) and concentration of Rubisco, number of CPs and Rubisco-containing Body (RCBs) were investigated with two differently salt-responding varieties in this experiment. We observed that 150 mM salt treatment resulted in not only significant decrease in Pn, CE and Rubisco content, but also obvious increase in the number of CPs and RCBs in salt-sensitive variety. Under salt stress formation of CPs resulted in production of much more RCBs, which could immigrate into and combine with vacuole. It may be a kind of important mechanism for rapid degradation of Rubisco under salt stress. Our conclusion provides a new sight for how Rubisco can be fast degraded under salt stress.

Phosphorus (P) is crucial nutrient element for crop growth and development. However, the network pathway regulating homeostasis of phosphate (Pi) in crops has many molecular breeding unknowns. Here, we report that an auxin response factor, OsARF12, functions in Pi homeostasis. Measurement of element content, quantitative reverse transcription polymerase chain reaction analysis and acid phosphatases (APases) activity assay showed that the osarf12 mutant and osarf12/25 double mutant with P-intoxicated phenotypes had higher P concentrations, up-regulation of the Pi transporter encoding genes and increased APase activity under Pi-sufficient/-deficient (+Pi/-Pi, 0.32/0 mM NaH2 PO4) conditions. Transcript analysis revealed that Pi-responsive genes--Phosphate starvation (OsIPS)1 and OsIPS2, SYG1/Pho81/XPR1(OsSPX1), Sulfoquinovosyldiacylglycerol 2 (OsSQD2), R2R3 MYB transcription factor (OsMYB2P-1) and Transport Inhibitor Response1 (OsTIR1)--were more abundant in the osarf12 and osarf12/25 mutants under +Pi/-Pi conditions. Knockout of OsARF12 also influenced the transcript abundances of the OsPHR2 gene and its downstream components, such as OsMiR399j, OsPHO2, OsMiR827, OsSPX-MFS1 and OsSPX-MFS2. Results from -Pi/1-naphthylphthalamic acid (NPA) treatments, and auxin reporter DR5::GUS staining suggest that root system alteration and Pi-induced auxin response were at least partially controlled by OsARF12. These findings enrich our understanding of the biological functions of OsARF12, which also acts in regulating Pi homeostasis.

• Auxin has an important role in maintaining optimal root system architecture (RSA) that can cope with growth reductions of crops caused by water or nutrient shortages. However, the mechanism of controlling RSA remains largely unclear. Here, we found a limiting factor of RSA--OsARF12--an auxin response factor whose knockout led to decreased primary root length in rice (Oryza sativa). • OsARF12 as a transcription activator can facilitate the expression of the auxin response element DR5::GFP, and OsARF12 was inhibited by osa-miRNA167d by transient expression in tobacco and rice callus. • The root elongation zones of osarf12 and osarf12/25, which had lower auxin concentrations, were distinctly shorter than for the wild-type, possibly as a result of decreased expression of auxin synthesis genes OsYUCCAs and auxin efflux carriers OsPINs and OsPGPs. The knockout of OsARF12 also altered the abundance of mitochondrial iron-regulated (OsMIR), iron (Fe)-regulated transporter1 (OsIRT1) and short postembryonic root1 (OsSPR1) in roots of rice, and resulted in lower Fe content. • The data provide evidence for the biological function of OsARF12, which is implicated in regulating root elongation. Our investigation contributes a novel insight for uncovering regulation of RSA and the relationship between auxin response and Fe acquisition.

Mi-2 protein, the central component of the NuRD nucleosome remodeling and histone deacetylase complex, plays a role in transcriptional repression in animals. Mi-2-like genes have been reported in Arabidopsis, though their function in monocots remains largely unknown. In the present study, a rice Mi-2-like gene, OsCHR4 (Oryza sativa Chromatin Remodeling 4, LOC_Os07g03450), was cloned from a rice mutant with adaxial albino leaves. The Oschr4 mutant exhibited defective chloroplasts in adaxial mesophyll, but not in abaxial mesophyll. Ultrastructural observations indicated that proplastid growth and/or thylakoid membrane formation in adaxial mesophyll cells was blocked in the Oschr4 mutant. Subcellular localization revealed that OsCHR4::GFP fusion protein was targeted to the nuclei. OsCHR4 was mainly expressed in the root meristem, flower, vascular bundle, and mesophyll cells by promoter::GUS analysis in transgenic rice. The transcripts of some nuclear- and plastid-encoded genes required for early chloroplast development and photosynthesis were decreased in the adaxial albino mesophyll of the Oschr4 mutant. These observations provide evidence that OsCHR4, the rice Mi-2-like protein, plays an important role in early chloroplast development in adaxial mesophyll cells. The results increase our understanding of the molecular mechanism underlying tissue-specific chloroplast development in plants.