Reactive oxygen species (ROS) are signalling molecules that promote programmed cell death in animal and plant systems. However, their role in rice ( Oryza sativa L.) anther development is unclear. In this study, we show that lower transcript levels of the metallothionein gene OsMT2b in japonica rice plants obtained by RNA interference (RNAi) resulted in a serious reduction in the seed setting rate. Observations of semi-thin sections of anthers indicated that tapetum degradation initiates early and ends late in OsMT2b -RNAi plants relative to the wild type (WT). Nitroblue tetrazolium staining and measurements of hydrogen peroxide contents showed that ROS contents are higher in OsMT2b -RNAi plants than in WT. Terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assays showed that abnormal programmed cell death in the tapetum results in sterile microspores. In addition, the OsMT2b -RNAi plants were sensitive to photoperiod; they were sterile under natural long-day conditions but almost fully fertile under natural short-day conditions, indicating that OsMT2b integrates photoperiod information into pollen development. The discovery of this rice material may enrich germplasm resources for two-line hybrid rice breeding, and further research may enable its application in two-line hybrid rice breeding.

Pentatricopeptide repeat (PPR) proteins are essential regulators of plant growth and development, playing pivotal roles in plant cytoplasmic male sterility restoration, and the responses to both biotic and abiotic stresses. The vast majority of PPR proteins are targeted to mitochondria or chloroplasts. Mitochondrion-localized PPR proteins are primarily associated with defects in seed development and overall plant growth, whereas those localized to chloroplasts are mainly involved in modulating photosynthetic processes. However, an increasing number of PPR proteins has recently been identified outside of these organelles. Although research into their subcellular localization and underlying mechanisms is still in its early stages, emerging evidence indicates that these non-organellar PPR proteins perform critical functions in plant development. In this review, we provide a systematic overview of the structural features, classification, and functional mechanisms of PPR proteins. Furthermore, we further summarize and categorize currently known non-organellar PPR proteins, with an emphasis on the relationship between their diverse subcellular localizations and functional specificity. This synthesis contributes to a more comprehensive understanding of the functional versatility and regulatory complexity of PPR proteins in plant growth and development. Finally, we highlight key challenges and open questions in the study of non-organellar PPR proteins, aiming to guide future research in this emerging field.

Photosynthetic electron transport occurs via linear (LET) and cyclic (CET) pathways. The balance between LET and CET is essential for sustaining photosynthetic efficiency, as these pathways regulate ATP and NADPH production. In rice, the NDH complex mediates CET; however, the mechanisms regulating the CET proportion remain largely unclear. Here, we show that rice RNase ZS2, which localizes to the chloroplast, degrades mRNAs encoding the NDH complex subunits NdhD and NdhF, thereby negatively regulating the accumulation of NDH-PSI supercomplexes and affecting the CET/LET ratio. In transgenic plants with RNase ZS2 downregulation, increased NDH accumulation alters chloroplast ultrastructure and photosynthetic electron transport, resulting in a higher CET proportion. We demonstrate physical interactions between NdhD and cytochrome f (Cyt f), which may recruit additional Cyt b6f complexes to support CET, thereby regulating the CET/LET ratio to meet different metabolic demands at various developmental stages. These results reveal a regulatory mechanism in which the nuclear-encoded RNase ZS2 degrades chloroplast-encoded NDH mRNAs to modulate the CET/LET balance.

Protein acetylation, a conserved post-translational modification, is collaboratively catalyzed by acetyltransferases and deacetylases and is widespread in plants. This study reviews recent research regarding two key types of acetylation: histone acetylation and non-histone acetylation. Histone acetylation, occurring primarily in the nucleus, regulates the structure of chromatin to control gene transcription on a large scale. This process is crucial for the precise regulation of the plant organ formation and development. Non-histone protein acetylation is widely distributed across various organelles and can finely regulate almost all key cellular processes and functions. Histone and non-histone acetylation work together to construct a complex and precise acetylation-modification regulatory network in plants. Finally, this study also analyzes current research challenges and prospects related to acetylation modifications. Elucidating the regulatory mechanisms of acetylation modifications in plants not only enables us to better understand the molecular mechanisms of plant growth and development but also provides a theoretical basis and potential targets for the genetic improvement and enhancement of stress resistance in crops, with significant scientific and practical value.

Site-specific DNA integration is an important tool in plant genetic engineering. Traditionally, this process relies on homologous recombination (HR), which is known for its low efficiency in plant cells. In contrast, Agrobacterium-mediated T-DNA integration is highly efficient for plant transformation. However, T-DNA is typically inserted randomly into double-strand breaks within the plant genome via the non-homologous end-joining (NHEJ) DNA repair pathway. In this study, we developed an approach of CRISPR/Cas9-mediated targeted T-DNA integration in Arabidopsis, which was more rapid and efficient than the HR-mediated method. This targeted T-DNA integration aided in gene activation and male germline-specific gene tagging. Gene activation was accomplished by positioning the CaMV35S promoter at the left border of T-DNA, thereby activating specific downstream genes. The activation of FT and MYB26 significantly increased their transcriptional expression, which resulted in early flowering and an altered pattern of cell wall thickening in the anther endothelium, respectively. Male germline-specific gene tagging incorporates two reporters, namely, NeoR and MGH3::mCherry, within the T-DNA. This design facilitates the creation of insertional mutants, simplifies the genetic analysis of mutated alleles, and allows for cellular tracking of male germline cells during fertilization. We successfully applied this system to target the male germline-specific gene GEX2. In conclusion, our results demonstrated that site-specific integration of DNA fragments in the plant genome can be rapidly and efficiently achieved through the NHEJ pathway, making this approach broadly applicable in various contexts.

The AP2/ERF transcription factor family plays a vital role in regulating rice growth and development. Recent years have seen notable progress in understanding the functions of AP2/ERF transcription factors in rice. Studies indicate that these factors not only control the differentiation of rice inflorescence meristems but also participate in developing organs such as roots, stems, and leaves. However, the specific molecular mechanisms of AP2/ERF transcription factors, their interactions with other proteins, and how they precisely regulate the expression of particular genes still require further research. This paper systematically reviews recent advances in the functional studies of AP2/ERF transcription factors in rice growth and development, focusing on their roles in inflorescence development, grain formation, and the development of roots, stems, and leaves. It also discusses their potential applications in molecular breeding. By compiling recent research findings, this review aims to provide both theoretical insights and practical guidance for a better understanding of the regulatory networks involving AP2/ERF transcription factors and their use in rice genetic improvement.

High-temperature stress severely threatens rice (Oryza sativa L.) growth and productivity, particularly during the seedling stage, making heat tolerance an essential breeding target. In this study, we identified the heat shock transcription factor OsHsfc1a as a positive regulator of thermotolerance in rice seedlings. OsHsfc1a expression was strongly induced by heat stress, and overexpression lines exhibited enhanced heat tolerance, whereas knockout mutants were hypersensitive. Integrative transcriptome and DAP-seq analyses revealed that OsHsfc1a directly binds to and represses the transcription of OsMFT1, a gene previously implicated in stress responses. Molecular and genetic assays confirmed that OsHsfc1a-mediated suppression of OsMFT1 helps maintain the chloroplast structural integrity, reduces reactive oxygen species accumulation, and alleviates programmed cell death under heat stress. In contrast, OsMFT1 overexpression impaired chloroplast organization and decreased seedling survival, whereas its knockout enhanced heat tolerance. Furthermore, comparative transcriptome analysis revealed that the OsHsfc1a-OsMFT1 regulatory module maintains the chloroplast structural integrity during heat stress by modulating the expression of multiple chloroplast structure-related genes. Analysis of natural haplotypes revealed that OsHsfc1a allelic variation is associated with rice subspecies differentiation, providing useful genetic resources for breeding heat-resilient varieties. In summary, we demonstrate that OsHsfc1a enhances heat tolerance by repressing OsMFT1 to preserve chloroplast stability, providing both mechanistic insights and practical genetic resources for rice improvement under global warming.

Our study explores the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the rpt5a mutant characterized by persistent reddish-brown leaf spots from seedling stage to maturity, leading to extensive leaf necrosis. Through map-based cloning, we localized the responsible locus to a 330Kb region on chromosome 2 and identified LOC_Os02g56000 (named OsRPT5A) as the causative gene. A point mutation in OsRPT5A, substituting valine for glutamic acid, was identified as the critical factor for the phenotype. Functional complementation and generation of knockout lines in the IR64 background confirmed the central role of OsRPT5A in controlling this trait. Our study also revealed that OsRPT5A is constitutively expressed across various tissues, with its subcellular localization unaffected by mutations. Notably, we observed an abnormal accumulation of reactive oxygen species (ROS) in rpt5a mutants, suggesting a disruption in the ROS system. Complementation studies indicated OsRPT5A's involvement in ROS homeostasis and catalase activity regulation. Moreover, the rpt5a mutant exhibited enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), highlighting OsRPT5A's role in rice's pathogen resistance mechanisms. These findings elucidate the genetic basis of the spotted leaf phenotype and emphasize the intricate interplay between plant defense responses and developmental pathways.

Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a major bacterial disease in rice. Transcription activator-like effectors (TALEs) from Xanthomonas can induce host susceptibility (S) genes and facilitate infection. However, knowledge of the function of Xoc TALEs in promoting bacterial virulence is limited. In this study, we demonstrated the importance of Tal10a for the full virulence of Xoc. Through computational prediction and gene expression analysis, we identified the hexokinase gene OsHXK5 as a host target of Tal10a. Tal10a directly binds to the gene promoter region and activates the expression of OsHXK5. CRISPR/Cas9-mediated gene editing in the effector binding element (EBE) of OsHXK5 significantly increases rice resistance to Xoc, while OsHXK5 overexpression enhances the susceptibility of rice plants and impairs rice defense responses. Moreover, simultaneous editing of the promoters of OsSULTR3;6 and OsHXK5 confers robust resistance to Xoc in rice. Taken together, our findings highlight the role of Tal10a in targeting OsHXK5 to promote infection and suggest that OsHXK5 represents a potential target for engineering rice resistance to Xoc.

Diurnal flower-opening time (DFOT), the time of spikelet opening during the day, is an important trait for hybrid rice (Oryza sativa L.) seed production. Hybrids between indica and japonica rice varieties have strong heterosis, but the parental lines usually have different, nonoverlapping DFOTs. This reduces the success of hybrid seed production in crosses between indica and japonica subspecies, thus hindering the utilization of indica and japonica inter-subspecies heterosis. However, little is known about the molecular mechanisms regulating DFOT in rice. Here, we obtained japonica rice lines with a DFOT 1.5 h earlier than the wild type by overexpressing OsMYC2, a gene encoding a key transcription factor in the jasmonate (JA) signaling pathway. OsMYC2 is activated by JA signaling and directly regulates the transcription of genes related to JA biosynthesis and cell wall metabolism. Overexpressing OsMYC2 led to significantly increased JA contents and decreased cellulose and hemicellulose contents in lodicule cells, as well as the softening of lodicule cell walls. This may facilitate the swelling of lodicules, resulting in early diurnal flower-opening. These results suggest that the OsMYC2-JA feedback loop regulates DFOT in rice via cell wall remodeling. These findings shed light on the understanding of regulatory mechanism of the DFOT of plants, which should promote the development of indica and japonica varieties suitable for hybrid rice breeding.

Inter-subspecific indica-japonica hybrid rice (Oryza sativa) has the potential for increased yields over traditional indica intra-subspecies hybrid rice, but limited yield of F-1 hybrid seed production (FHSP) hinders the development of indica-japonica hybrid rice breeding. Diurnal flower-opening time (DFOT) divergence between indica and japonica rice has been a major contributing factor to this issue, but few DFOT genes have been cloned. Here, we found that manipulating the expression of jasmonate (JA) pathway genes can effectively modulate DFOT to improve the yield of FHSP in rice. Treating japonica cultivar Zhonghua 11 (ZH11) with methyl jasmonate (MeJA) substantially advanced DFOT. Furthermore, overexpressing the JA biosynthesis gene OPDA REDUCTASE 7 (OsOPR7) and knocking out the JA inactivation gene CHILLING TOLERANCE 1 (OsHAN1) in ZH11 advanced DFOT by 1- and 2-h respectively; and knockout of the JA signal suppressor genes JASMONATE ZIM-DOMAIN PROTEIN 7 (OsJAZ7) and OsJAZ9 resulted in 50-min and 1.5-h earlier DFOT respectively. The yields of FHSP using japonica male-sterile lines GAZS with manipulated JA pathway genes were significantly higher than that of GAZS wildtype. Transcriptome analysis, cytological observations, measurements of elastic modulus and determination of cell wall components indicated that the JA pathway could affect the loosening of the lodicule cell walls by regulating their composition through controlling sugar metabolism, which in turn influences DFOT. This research has vital implications for breeding japonica rice cultivars with early DFOT to facilitate indica-japonica hybrid rice breeding.

Leaves play a crucial role in the growth and development of rice (Oryza sativa) as sites for the production of photosynthesis. Early leaf senescence leads to substantial drops in rice yields. Whether and how DNA methylation regulates gene expression and affects leaf senescence remains elusive. Here, we demonstrate that mutations in rice ARGONAUTE 2 (OsAGO2) lead to premature leaf senescence, with chloroplasts in Osago2 having lower chlorophyll content and an abnormal thylakoid structure compared with those from wild-type plants. We show that OsAGO2 associates with a 24-nt microRNA and binds to the promoter region of OsNAC300, which causes DNA methylation and suppressed expression of OsNAC300. Overexpressing OsNAC300 causes the similar premature leaf senescence as Osago2 mutants and knocking out OsNAC300 in the Osago2 mutant background suppresses the early senescence of Osago2 mutants. Based on yeast one-hybrid, dual-luciferase, and electrophoresis mobility shift assays, we propose that OsNAC300 directly regulates transcription of the key rice aging gene NAC-like, activated by APETALA3/PISTILLATA (OsNAP) to control leaf senescence. Our results unravel a previously unknown epigenetic regulatory mechanism underlying leaf senescence in which OsAGO2-OsNAC300-OsNAP acts as a key regulatory module of leaf senescence to maintain leaf function.

Summary Thermo‐sensitive genic male sterile (TGMS) lines are the core of two‐line hybrid rice ( Oryza sativa ). However, elevated or unstable critical sterility‐inducing temperatures (CSITs) of TGMS lines are bottlenecks that restrict the development of two‐line hybrid rice. However, the genes and molecular mechanisms controlling CSIT remain unknown. Here, we report the CRITICAL STERILITY‐INDUCING TEMPERATURE 2 ( CSIT2 ) that encodes a really interesting new gene (RING) type E3 ligase, controlling the CSIT of thermo‐sensitive male sterility 5 ( tms5 )‐based TGMS lines through ribosome‐associated protein quality control (RQC). CSIT2 binds to the large and small ribosomal subunits and ubiquitinates 80S ribosomes for dissociation, and may also ubiquitinate misfolded proteins for degradation. Mutation of CSIT2 inhibits the possible damage to ubiquitin system and protein translation, which allows more proteins such as catalases to accumulate for anther development and inhibits abnormal accumulation of reactive oxygen species (ROS) and premature programmed cell death (PCD) in anthers, partly rescuing male sterility and raised the CSIT of tms5 ‐based TGMS lines. These findings reveal a mechanism controlling CSIT and provide a strategy for solving the elevated or unstable CSITs of tms5 ‐based TGMS lines in two‐line hybrid rice.

Our study investigates the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the spl43 mutant. This mutant is characterized by persistent reddish-brown leaf spots from the seedling stage to maturity, leading to extensive leaf necrosis. Using map-based cloning, we localized the responsible locus to a 330 Kb region on chromosome 2. We identified LOC_Os02g56000, named OsRPT5A, as the causative gene. A point mutation in OsRPT5A, substituting valine for glutamic acid, was identified as the critical factor for the phenotype. Functional complementation and the generation of CRISPR/Cas9-mediated knockout lines in the IR64 background confirmed the central role of OsRPT5A in controlling this trait. The qPCR results from different parts of the rice plant revealed that OsRPT5A is constitutively expressed across various tissues, with its subcellular localization unaffected by the mutation. Notably, we observed an abnormal accumulation of reactive oxygen species (ROS) in spl43 mutants by examining the physiological indexes of leaves, suggesting a disruption in the ROS system. Complementation studies indicated OsRPT5A’s involvement in ROS homeostasis and catalase activity regulation. Moreover, the spl43 mutant exhibited enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), highlighting OsRPT5A’s role in rice pathogen resistance mechanisms. Overall, our results suggest that OsRPT5A plays a critical role in regulating ROS homeostasis and enhancing pathogen resistance in rice.

Organ size shapes plant architecture during rice (Oryza sativa) growth and development, affecting key factors influencing yield, such as plant height, leaf size, and seed size. Here, we report that the rice Enhancer of Zeste [E(z)] homolog SET DOMAIN GROUP 711 (OsSDG711) regulates organ size in rice. Knockout of OsSDG711 produced shorter plants with smaller leaves, thinner stems, and smaller grains. We demonstrate that OsSDG711 affects organ size by reducing cell length and width and increasing cell number in leaves, stems, and grains. The result of chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) using an antitrimethylation of histone H3 lysine 27 (H3K27me3) antibody showed that the levels of H3K27me3 associated with cytokinin oxidase/dehydrogenase genes (OsCKXs) were lower in the OsSDG711 knockout line Ossdg711. ChIP-qPCR assays indicated that OsSDG711 regulates the expression of OsCKX genes through H3K27me3 histone modification. Importantly, we show that OsSDG711 directly binds to the promoters of these OsCKX genes. Furthermore, we measured significantly lower cytokinin contents in Ossdg711 plants than in wild-type plants. Overall, our results reveal an epigenetic mechanism based on OsSDG711-mediated modulation of H3K27me3 levels to regulate the expression of genes involved in the cytokinin metabolism pathway and control organ development in rice. OsSDG711 may be an untapped epigenetic resource for ideal plant type improvement.

Pentatricopeptide repeat (PPR) proteins represent one of the largest protein families in plants and typically localize to organelles like mitochondria and chloroplasts. By contrast, CYTOPLASM- LOCALIZED PPR1 (OsCPPR1) is a cytoplasm-localized PPR protein that can degrade OsGOLDEN- LIKE1 (OsGLK1) mRNA in the tapetum of rice anther. However, the mechanism, by which OsCPPR1 recognizes and binds to OsGLK1 transcripts, remains unknown. Through protein structure prediction and macromolecular docking experiments, we observed that distinct PPR motif structures of OsCPPR1 exhibited varying binding efficiencies to OsGLK1 RNA. Moreover, RNA-electrophoretic mobility shift assay experiment demonstrated that the recombinant OsCPPR1 can directly recognize and bind to OsGLK1 mRNA in vitro. This further confirmed that the mutations in the conserved amino acids in each PPR motif resulted in loss of activity, while truncation of OsCPPR1 decreased its binding efficiency. These findings collectively suggest that it may require some co-factors to assist in cleavage, a facet that warrants further exploration in subsequent studies.

Drought stress is a major environmental factor that limits the growth, development, and yield of rice (Oryza sativa L.). Histone deacetylases (HDACs) are involved in the regulation of drought stress responses. HDA704 is an RPD3/HDA1 class HDAC that mediates the deacetylation of H4K8 (lysine 8 of histone H4) for drought tolerance in rice. In this study, we show that plants overexpressing HDA704 (HDA704-OE) are resistant to drought stress and sensitive to abscisic acid (ABA), whereas HDA704 knockout mutant (hda704) plants displayed decreased drought tolerance and ABA sensitivity. Transcriptome analysis revealed that HDA704 regulates the expression of ABA-related genes in response to drought stress. Moreover, HDA704 was recruited by a drought-resistant transcription factor, WAX SYNTHESIS REGULATORY 2 (OsWR2), and co-regulated the expression of the ABA biosynthesis genes NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), NCED4, and NCED5 under drought stress. HDA704 also repressed the expression of ABA-INSENSITIVE 5 (OsABI5) and DWARF AND SMALL SEED 1 (OsDSS1) by regulating H4K8ac levels in the promoter regions in response to polyethylene glycol 6000 treatment. In agreement, the loss of OsABI5 function increased resistance to dehydration stress in rice. Our results demonstrate that HDA704 is a positive regulator of the drought stress response and offers avenues for improving drought resistance in rice.

Mulberry is a valuable woody plant with significant economic importance. It can be propagated through two main methods: cutting and grafting. Waterlogging can have a major impact on mulberry growth and can significantly reduce production. In this study, we examined gene expression patterns and photosynthetic responses in three waterlogged mulberry cultivars propagated through cutting and grafting. Compared to the control group, waterlogging treatments reduced levels of chlorophyll, soluble protein, soluble sugars, proline, and malondialdehyde (MDA). Additionally, the treatments significantly decreased the activities of ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT) in all three cultivars, except for superoxide dismutase (SOD). Waterlogging treatments also affected the rate of photosynthesis (Pn), stomatal conductance (Gs), and transpiration rate (Tr) in all three cultivars. However, no significant difference in physiological response was observed between the cutting and grafting groups. Gene expression patterns in the mulberry changed dramatically after waterlogging stress and varied between the two propagation methods. A total of 10,394 genes showed significant changes in expression levels, with the number of differentially expressed genes (DEGs) varying between comparison groups. GO and KEGG analysis revealed important DEGs, including photosynthesis-related genes that were significantly downregulated after waterlogging treatment. Notably, these genes were upregulated at day 10 in the cutting group compared to the grafting group. In particular, genes involved in carbon fixation were significantly upregulated in the cutting group. Finally, cutting propagation methods displayed better recovery capacity from waterlogging stress than grafting. This study provides valuable information for improving mulberry genetics in breeding programs.

Two-line hybrid breeding can fully utilize heterosis in crops. In thermo-sensitive genic male sterile (TGMS) lines, low critical sterility-inducing temperature (CSIT) is vital to safeguard the production of two-line hybrid seeds in rice (Oryza sativa), but the molecular mechanism determining CSIT is unclear. Here, we report the cloning of CSIT1, which encodes an E3 ubiquitin ligase, and show that CSIT1 modulates the CSIT of thermosensitive genic male sterility 5 (tms5)-based TGMS lines through ribosome-associated quality control (RQC). Biochemical assays demonstrated that CSIT1 binds to the 80S ribosomes and ubiquitinates abnormal nascent polypeptides for degradation in the RQC process. Loss of CSIT1 function inhibits the possible damage of tms5 to the ubiquitination system and protein translation, resulting in enhanced accumulation of anther-related proteins such as catalase to suppress abnormal accumulation of reactive oxygen species and premature programmed cell death in the tapetum, thereby leading to a much higher CSIT in the tms5-based TGMS lines. Taken together, our findings reveal a regulatory mechanism of CSIT, providing new insights into RQC and potential targets for future two-line hybrid breeding.

Photosynthesis is the largest mass- and energy-conversion process on Earth, and it is the material basis for almost all biological activities. The efficiency of converting absorbed light energy into energy substances during photosynthesis is very low compared to theoretical values. Based on the importance of photosynthesis, this article summarizes the latest progress in improving photosynthesis efficiency from various perspectives. The main way to improve photosynthetic efficiency is to optimize the light reactions, including increasing light absorption and conversion, accelerating the recovery of non-photochemical quenching, modifying enzymes in the Calvin cycle, introducing carbon concentration mechanisms into C3 plants, rebuilding the photorespiration pathway, de novo synthesis, and changing stomatal conductance. These developments indicate that there is significant room for improvement in photosynthesis, providing support for improving crop yields and mitigating changes in climate conditions.