Membrane protein MHZ3 regulates the on-off switch of ethylene signaling in rice.

Ethylene regulates plant growth, development, and stress adaptation. However, the early signaling events following ethylene perception, particularly in the regulation of ethylene receptor/CTRs (CONSTITUTIVE TRIPLE RESPONSE) complex, remains less understood. Here, utilizing the rapid phospho-shift of rice OsCTR2 in response to ethylene as a sensitive readout for signal activation, we revealed that MHZ3, previously identified as a stabilizer of ETHYLENE INSENSITIVE 2 (OsEIN2), is crucial for maintaining OsCTR2 phosphorylation. Genetically, both functional MHZ3 and ethylene receptors prove essential for OsCTR2 phosphorylation. MHZ3 physically interacts with both subfamily I and II ethylene receptors, e.g., OsERS2 and OsETR2 respectively, stabilizing their association with OsCTR2 and thereby maintaining OsCTR2 activity. Ethylene treatment disrupts the interactions within the protein complex MHZ3/receptors/OsCTR2, reducing OsCTR2 phosphorylation and initiating downstream signaling. Our study unveils the dual role of MHZ3 in fine-tuning ethylene signaling activation, providing insights into the initial stages of the ethylene signaling cascade.

Refinement of rice blast disease resistance QTLs and gene networks through meta-QTL analysis.

Rice blast disease is the most devastating disease constraining crop productivity. Vertical resistance to blast disease is widely studied despite its instability. Clusters of genes or QTLs conferring blast resistance that offer durable horizontal resistance are important in resistance breeding. In this study, we aimed to refine the reported QTLs and identify stable meta-QTLs (MQTLs) associated with rice blast resistance. A total of 435 QTLs were used to project 71 MQTLs across all the rice chromosomes. As many as 199 putative rice blast resistance genes were identified within 53 MQTL regions. The genes included 48 characterized resistance gene analogs and related proteins, such as NBS-LRR type, LRR receptor-like kinase, NB-ARC domain, pathogenesis-related TF/ERF domain, elicitor-induced defense and proteins involved in defense signaling. MQTL regions with clusters of RGA were also identified. Fifteen highly significant MQTLs included 29 candidate genes and genes characterized for blast resistance, such as Piz, Nbs-Pi9, pi55-1, pi55-2, Pi3/Pi5-1, Pi3/Pi5-2, Pikh, Pi54, Pik/Pikm/Pikp, Pb1 and Pb2. Furthermore, the candidate genes (42) were associated with differential expression (in silico) in compatible and incompatible reactions upon disease infection. Moreover, nearly half of the genes within the MQTL regions were orthologous to those in O. sativa indica, Z. mays and A. thaliana, which confirmed their significance. The peak markers within three significant MQTLs differentiated blast-resistant and susceptible lines and serve as potential surrogates for the selection of blast-resistant lines. These MQTLs are potential candidates for durable and broad-spectrum rice blast resistance and could be utilized in blast resistance breeding.

OsPUB9 Gene Edited by CRISPR/Cas9 Enhanced Resistance to Bacterial Leaf Blight in Rice (Oryza sativa L.).

Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.

Activation and Autoinhibition Mechanisms of NLR Immune Receptor Pi36 in Rice.

Nucleotide-binding and leucine-rich repeat receptors (NLRs) are the most important and largest class of immune receptors in plants. The Pi36 gene encodes a canonical CC-NBS-LRR protein that confers resistance to rice blast fungal infections. Here, we show that the CC domain of Pi36 plays a role in cell death induction. Furthermore, self-association is required for the CC domain-mediated cell death, and the self-association ability is correlated with the cell death level. In addition, the NB-ARC domain may suppress the activity of the CC domain through intramolecular interaction. The mutations D440G next to the RNBS-D motif and D503V in the MHD motif autoactivated Pi36, but the mutation K212 in the P-loop motif inhibited this autoactivation, indicating that nucleotide binding of the NB-ARC domain is essential for Pi36 activation. We also found that the LRR domain is required for D503V- and D440G-mediated Pi36 autoactivation. Interestingly, several mutations in the CC domain compromised the CC domain-mediated cell death without affecting the D440G- or D503V-mediated Pi36 autoactivation. The autoactivate Pi36 variants exhibited stronger self-associations than the inactive variants. Taken together, we speculated that the CC domain of Pi36 executes cell death activities, whereas the NB-ARC domain suppressed CC-mediated cell death via intermolecular interaction. The NB-ARC domain releases its suppression of the CC domain and strengthens the self-association of Pi36 to support the CC domain, possibly through nucleotide exchange.

OsNAC121 regulates root development, tillering, panicle morphology, and grain filling in rice plant.

Transcription factors in coordination with phytohormones form an intricate regulatory network modulating vital cellular mechanisms like development, growth and senescence in plants. In this study, we have functionally characterized the transcription factor OsNAC121 by developing gene silencing and overexpressing transgenic rice plants, followed by detailed analyses of the plant architecture. Transgenic lines exhibited remodelling in crown root development, lateral root structure and density, tiller height and number, panicle and grain morphologies, underpinning the imbalanced auxin: cytokinin ratio due to perturbed auxin transportation. Application of cytokinin, auxin and abscisic acid increased OsNAC121 gene expression nearly 17-, 6- and 91-folds, respectively. qRT-PCR results showed differential expressions of auxin and cytokinin pathway genes, implying their altered levels. A 47-fold higher expression level of OsNAC121 during milky stage in untransformed rice, compared to 14-day old shoot tissue, suggests its crucial role in grain filling; as evidenced by a large number of undeveloped grains produced by the gene silenced lines. Crippled gravitropic response by the transgenic plants indicates their impaired auxin transport. Bioinformatics revealed that OsNAC121 interacts with co-repressor (TOPLESS) proteins and forms a part of the inhibitor complex OsIAA10, an essential core component of auxin signalling pathway. Therefore, OsNAC121 emerges as an important regulator of various aspects of plant architecture through modulation of crosstalk between auxin and cytokinin, altering their concentration gradient in the meristematic zones, and consequently modifying different plant organogenesis processes.

Creation of rice doubled haploids with low amylose content using in vitro anther culture.

In vitro androgenesis is a unique model for producing homozygous doubled haploid plants. The use of haploid biotechnology accelerates to obtain of doubled haploid plants, which is very important in rice breeding. The purpose of this work is to improve the production of doubled haploids in rice anther culture in vitro and selection of doubled haploid plants with valuable traits. The study the influence of nutrient media on the production of calli and plant regeneration processes in anther culture of 35 rice genotypes was revealed a significant influence of nutrient media on callus production. It was shown that the addition to culture medium phytohormones ratio with high level of cytokinin (5.0 mg/L BAP) and a low level of auxin (0.5 mg/L NAA), supplemented with amino acid composition promotes high production of green regenerated plants (68.75%) compared to albino plants (31.25%). As a result, doubled haploid lines of the glutinous variety Violetta were selected, which characterized by a low amylose content variation (from 1.86 to 2.80%). These doubled haploids are superior to the original variety in some yield traits and represent valuable breeding material.

Genetic Analysis and Fine Mapping of a New Rice Mutant, Leaf Tip Senescence 2.

Premature leaf senescence significantly reduces rice yields. Despite identifying numerous factors influencing these processes, the intricate genetic regulatory networks governing leaf senescence demand further exploration. We report the characterization of a stably inherited, ethyl methanesulfonate(EMS)-induced rice mutant with wilted leaf tips from seedling till harvesting, designated lts2. This mutant exhibits dwarfism and early senescence at the leaf tips and margins from the seedling stage when compared to the wild type. Furthermore, lts2 displays a substantial decline in both photosynthetic activity and chlorophyll content. Transmission electron microscopy revealed the presence of numerous osmiophilic granules in chloroplast cells near the senescent leaf tips, indicative of advanced cellular senescence. There was also a significant accumulation of H2O2, alongside the up-regulation of senescence-associated genes within the leaf tissues. Genetic mapping situated lts2 between SSR markers Q1 and L12, covering a physical distance of approximately 212 kb in chr.1. No similar genes controlling a premature senescence leaf phenotype have been identified in the region, and subsequent DNA and bulk segregant analysis (BSA) sequencing analyses only identified a single nucleotide substitution (C-T) in the exon of LOC_Os01g35860. These findings position the lts2 mutant as a valuable genetic model for elucidating chlorophyll metabolism and for further functional analysis of the gene in rice.

Comparative Cytological and Gene Expression Analysis Reveals That a Common Wild Rice Inbred Line Showed Stronger Drought Tolerance Compared with the Cultivar Rice.

Common wild rice (Oryza rufipogon Griff.) is an important germplasm resource containing valuable genes. Our previous analysis reported a stable wild rice inbred line, Huaye3, which derives from the common wild rice of Guangdong Province. However, there was no information about its drought tolerance ability. Here, we assessed the germination characteristics and seedling growth between the Dawennuo and Huaye3 under five concentrations of PEG6000 treatment (0, 5%, 10%, 15%, and 20%). Huaye3 showed a stronger drought tolerance ability, and its seed germination rate still reached more than 52.50% compared with Dawennuo, which was only 25.83% under the 20% PEG6000 treatment. Cytological observations between the Dawennuo and Huaye3 indicated the root tip elongation zone and buds of Huaye3 were less affected by the PEG6000 treatment, resulting in a lower percentage of abnormalities of cortical cells, stele, and shrinkage of epidermal cells. Using the re-sequencing analysis, we detected 13,909 genes that existed in the genetic variation compared with Dawennuo. Of these genes, 39 were annotated as drought stress-related genes and their variance existed in the CDS region. Our study proved the strong drought stress tolerance ability of Huaye3, which provides the theoretical basis for the drought resistance germplasm selection in rice.

Automated Phenotypic Trait Extraction for Rice Plant Using Terrestrial Laser Scanning Data.

To quickly obtain rice plant phenotypic traits, this study put forward the computational process of six rice phenotype features (e.g., crown diameter, perimeter of stem, plant height, surface area, volume, and projected leaf area) using terrestrial laser scanning (TLS) data, and proposed the extraction method for the tiller number of rice plants. Specifically, for the first time, we designed and developed an automated phenotype extraction tool for rice plants with a three-layer architecture based on the PyQt5 framework and Open3D library. The results show that the linear coefficients of determination (R2) between the measured values and the extracted values marked a better reliability among the selected four verification features. The root mean square error (RMSE) of crown diameter, perimeter of stem, and plant height is stable at the centimeter level, and that of the tiller number is as low as 1.63. The relative root mean squared error (RRMSE) of crown diameter, plant height, and tiller number stays within 10%, and that of perimeter of stem is 18.29%. In addition, the user-friendly automatic extraction tool can efficiently extract the phenotypic features of rice plant, and provide a convenient tool for quickly gaining phenotypic trait features of rice plant point clouds. However, the comparison and verification of phenotype feature extraction results supported by more rice plant sample data, as well as the improvement of accuracy algorithms, remain as the focus of our future research. The study can offer a reference for crop phenotype extraction using 3D point clouds.

Candidate Genes and Favorable Haplotypes Associated with Iron Toxicity Tolerance in Rice.

Iron (Fe) toxicity is a major issue adversely affecting rice production worldwide. Unfortunately, the physiological and genetic mechanisms underlying Fe toxicity tolerance in rice remain relatively unknown. In this study, we conducted a genome-wide association study using a diverse panel consisting of 551 rice accessions to identify genetic mechanisms and candidate genes associated with Fe toxicity tolerance. Of the 29 quantitative trait loci (QTL) for Fe toxicity tolerance detected on chromosomes 1, 2, 5, and 12, five (qSH_Fe5, qSFW_Fe2.3, qRRL5.1, qRSFW1.1, and qRSFW12) were selected to identify candidate genes according to haplotype and bioinformatics analyses. The following five genes were revealed as promising candidates: LOC_Os05g40160, LOC_Os05g40180, LOC_Os12g36890, LOC_Os12g36900, and LOC_Os12g36940. The physiological characteristics of rice accessions with contrasting Fe toxicity tolerance reflected the importance of reactive oxygen species-scavenging antioxidant enzymes and Fe homeostasis for mitigating the negative effects of Fe toxicity on rice. Our findings have clarified the genetic and physiological mechanisms underlying Fe toxicity tolerance in rice. Furthermore, we identified valuable genetic resources for future functional analyses and the development of Fe toxicity-tolerant rice varieties via marker-assisted selection.

Deciphering molecular regulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) signalling networks in Oryza genus amid environmental stress.

The Oryza genus, containing Oryza sativa L., is quintessential to sustain global food security. This genus has a lot of sophisticated molecular mechanisms to cope with environmental stress, particularly during vulnerable stages like flowering. Recent studies have found key involvements and genetic modifications that increase resilience to stress, including exogenous application of melatonin, allantoin, and trehalose as well as OsSAPK3 and OsAAI1 in the genetic realm. Due to climate change and anthropogenic reasons, there is a rise in sea level which raises a concern of salinity stress. It is tackled through osmotic adjustment and ion homeostasis, mediated by genes like P5CS, P5CR, GSH1, GSH2, and SPS, and ion transporters like NHX, NKT, and SKC, respectively. Oxidative damage is reduced by a complex action of antioxidants, scavenging RONS. A complex action of genes mediates cold stress with studies highlighting the roles of OsWRKY71, microRNA2871b, OsDOF1, and OsICE1. There is a need to research the mechanism of action of proteins like OsRbohA in ROS control and the action of regulatory genes in stress response. This is highly relevant due to the changing climate which will raise a lot of environmental changes that will adversely affect production and global food security if certain countermeasures are not taken. Overall, this study aims to unravel the molecular intricacies of ROS and RNS signaling networks in Oryza plants under stress conditions, with the ultimate goal of informing strategies for enhancing stress tolerance and crop performance in this important agricultural genus.

Stress-induced nuclear translocation of ONAC023 improves drought and heat tolerance through multiple processes in rice.

Drought and heat are major abiotic stresses frequently coinciding to threaten rice production. Despite hundreds of stress-related genes being identified, only a few have been confirmed to confer resistance to multiple stresses in crops. Here we report ONAC023, a hub stress regulator that integrates the regulations of both drought and heat tolerance in rice. ONAC023 positively regulates drought and heat tolerance at both seedling and reproductive stages. Notably, the functioning of ONAC023 is obliterated without stress treatment and can be triggered by drought and heat stresses at two layers. The expression of ONAC023 is induced in response to stress stimuli. We show that overexpressed ONAC23 is translocated to the nucleus under stress and evidence from protoplasts suggests that the dephosphorylation of the remorin protein OSREM1.5 can promote this translocation. Under drought or heat stress, the nuclear ONAC023 can target and promote the expression of diverse genes, such as OsPIP2;7, PGL3, OsFKBP20-1b, and OsSF3B1, which are involved in various processes including water transport, reactive oxygen species homeostasis, and alternative splicing. These results manifest that ONAC023 is fine-tuned to positively regulate drought and heat tolerance through the integration of multiple stress-responsive processes. Our findings provide not only an underlying connection between drought and heat responses, but also a promising candidate for engineering multi-stress-resilient rice.

Effects of functional defects in the NMD pathway on rice phenotype and transcriptome.

Nonsense-mediated mRNA decay (NMD) is an important RNA quality control pathway. It aids in degrading harmful erroneous mRNA, thereby preserving a stable and healthy internal environment. In this study, we employed CRISPR/Cas9 and amiRNA technology to generate knock out or knock down mutants of realted genes in the rice NMD pathway. Through transcriptome sequencing and observing phenotype changes, the study explored the impact of NMD pathway defects on rice gene expression and alternative splicing. The results suggest that even partial defects will induce phenotypic changes such as plant height and pollen vitality to different degrees, showing necessity of NMD factors. Gene expression analysis reveals that most differentially expressed genes are upregulated in the mutants, with ko-upf1-like and kd-upf1 defects having a more significant impact than kd-upf2 and kd-upf3. Specifically, NMD pathway defects result in increased expression levels of rice defense response-related genes and decreased expression levels of secondary metabolism-related genes, with a wider range of affected genes observed in 60-day-old senescence mutants. Transcript analysis indicates that different NMD related genes defects alter hundreds of alternative splicing events, mostly enriched in genes involving alternative splicing regulatory pathways. Approximately half of these events are shared among different mutants, and a substantial number of affected transcripts show NMD target features. NMD could affect both the transcript abundance and their splicing subtypes to regulate the defense response and early-senescence associated pathways, which plays a vital role in rice growth and reproduction.

Genetic background influences mineral accumulation in rice straw and grains under different soil pH conditions.

Mineral element accumulation in plants is influenced by soil conditions and varietal factors. We investigated the dynamic accumulation of 12 elements in straw at the flowering stage and in grains at the mature stage in eight rice varieties with different genetic backgrounds (Japonica, Indica, and admixture) and flowering times (early, middle, and late) grown in soil with various pH levels. In straw, Cd, As, Mn, Zn, Ca, Mg, and Cu accumulation was influenced by both soil pH and varietal factors, whereas P, Mo, and K accumulation was influenced by pH, and Fe and Ni accumulation was affected by varietal factors. In grains, Cd, As, Mn, Cu, Ni, Mo, Ca, and Mg accumulation was influenced by both pH and varietal factors, whereas Zn, Fe, and P accumulation was affected by varietal factors, and K accumulation was not altered. Only As, Mn, Ca and Mg showed similar trends in the straw and grains, whereas the pH responses of Zn, P, K, and Ni differed between them. pH and flowering time had synergistic effects on Cd, Zn, and Mn in straw and on Cd, Ni, Mo, and Mn in grains. Soil pH is a major factor influencing mineral uptake in rice straw and grains, and genetic factors, flowering stage factors, and their interaction with soil pH contribute in a combined manner.

Multifunctional Roles of Zinc in Cadmium Transport in Soil-Rice Systems: Novel Insights from Stable Isotope Fractionation and Gene Expression.

The effect of Zn on Cd accumulation in rice varies under flooding and drainage conditions, and the underlying mechanism during uptake and transport from the soil to grains remains unclear. Isotope fractionation and gene expression were investigated using pot experiments under distinct water regimes and with Zn addition to gain a deeper understanding of the molecular effects of Zn on Cd uptake and transport in rice. The higher OsHMA2 expression but constitutively lower expression of zinc-regulated, iron-regulated transporter-like protein (ZIP) family genes in roots under the drainage regime than the flooding regime caused the enrichment of nonheavy Zn isotopes in the shoots relative to roots but minimally affected Cd isotopic fractionation. Drainage regime seem to exert a striking effect on the root-to-shoot translocation of Zn rather than Cd, and increased Zn transport via OsHMA2. The changes in expression patterns in response to Zn addition were similar to those observed upon switching from the flooding to drainage regime, except for OsNRAMP1 and OsNRAMP5. However, soil solution-to-rice plants and root-to-shoot fractionation toward light Zn isotopes with Zn addition (Δ66Znrice plant-soil solution = -0.49 to -0.40‰, Δ66Znshoot-root = -0.36 to -0.27‰) indicated that Zn transport occurred via nonspecific uptake pathways and OsHMA2, respectively. Accordingly, the less pronounced and minimally varied Cd isotope fractionation suggested that OsNRAMP5 and OsHMA2 are crucial for Cd uptake and root-to-shoot transport, respectively, facilitating Cd accumulation in grains. This study demonstrated that a high Zn supply promotes Cd uptake and root-to-shoot transport in rice by sharing distinct pathways, and by utilizing a non-Zn-sensitive pathway with a high affinity for Cd.

Genomic investigation of 18,421 lines reveals the genetic architecture of rice.

Understanding how numerous quantitative trait loci (QTL) shape phenotypic variation is an important question in genetics. To address this, we established a permanent population of 18,421 (18K) rice lines with reduced population structure. We generated reference-level genome assemblies of the founders and genotyped all 18K-rice lines through whole-genome sequencing. Through high-resolution mapping, 96 high-quality candidate genes contributing to variation in 16 traits were identified, including OsMADS22 and OsFTL1 verified as causal genes for panicle number and heading date, respectively. We identified epistatic QTL pairs and constructed a genetic interaction network with 19 genes serving as hubs. Overall, 170 masking epistasis pairs were characterized, serving as an important factor contributing to genetic background effects across diverse varieties. The work provides a basis to guide grain yield and quality improvements in rice.

Identification of candidate genes controlling cold tolerance at the early seedling stage from Dongxiang wild rice by QTL mapping, BSA-Seq and RNA-Seq.

BACKGROUND: The cold tolerance of rice is closely related to its production and geographic distribution. The identification of cold tolerance-related genes is of important significance for developing cold-tolerant rice. Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is well-adapted to the cold climate of northernmost-latitude habitats ever found in the world, and is one of the most valuable rice germplasms for cold tolerance improvement. RESULTS: Transcriptome analysis revealed genes differentially expressed between Xieqingzao B (XB; a cold sensitive variety) and 19H19 (derived from an interspecific cross between DXWR and XB) in the room temperature (RT), low temperature (LT), and recovery treatments. The results demonstrated that chloroplast genes might be involved in the regulation of cold tolerance in rice. A high-resolution SNP genetic map was constructed using 120 BC5F2 lines derived from a cross between 19H19 and XB based on the genotyping-by-sequencing (GBS) technique. Two quantitative trait loci (QTLs) for cold tolerance at the early seedling stage (CTS), qCTS12 and qCTS8, were detected. Moreover, a total of 112 candidate genes associated with cold tolerance were identified based on bulked segregant analysis sequencing (BSA-seq). These candidate genes were divided into eight functional categories, and the expression trend of candidate genes related to 'oxidation-reduction process' and 'response to stress' differed between XB and 19H19 in the RT, LT and recovery treatments. Among these candidate genes, the expression level of LOC_Os12g18729 in 19H19 (related to 'response to stress') decreased in the LT treatment but restored and enhanced during the recovery treatment whereas the expression level of LOC_Os12g18729 in XB declined during recovery treatment. Additionally, XB contained a 42-bp deletion in the third exon of LOC_Os12g18729, and the genotype of BC5F2 individuals with a survival percentage (SP) lower than 15% was consistent with that of XB. Weighted gene coexpression network analysis (WGCNA) and modular regulatory network learning with per gene information (MERLIN) algorithm revealed a gene interaction/coexpression network regulating cold tolerance in rice. In the network, differentially expressed genes (DEGs) related to 'oxidation-reduction process', 'response to stress' and 'protein phosphorylation' interacted with LOC_Os12g18729. Moreover, the knockout mutant of LOC_Os12g18729 decreased cold tolerance in early rice seedling stage signifcantly compared with that of wild type. CONCLUSIONS: In general, study of the genetic basis of cold tolerance of rice is important for the development of cold-tolerant rice varieties. In the present study, QTL mapping, BSA-seq and RNA-seq were integrated to identify two CTS QTLs qCTS8 and qCTS12. Furthermore, qRT-PCR, genotype sequencing and knockout analysis indicated that LOC_Os12g18729 could be the candidate gene of qCTS12. These results are expected to further exploration of the genetic mechanism of CTS in rice and improve cold tolerance of cultivated rice by introducing the cold tolerant genes from DXWR through marker-assisted selection.

The elite haplotype OsGATA8-H coordinates nitrogen uptake and productive tiller formation in rice.

Excessive nitrogen promotes the formation of nonproductive tillers in rice, which decreases nitrogen use efficiency (NUE). Developing high-NUE rice cultivars through balancing nitrogen uptake and the formation of productive tillers remains a long-standing challenge, yet how these two processes are coordinated in rice remains elusive. Here we identify the transcription factor OsGATA8 as a key coordinator of nitrogen uptake and tiller formation in rice. OsGATA8 negatively regulates nitrogen uptake by repressing transcription of the ammonium transporter gene OsAMT3.2. Meanwhile, it promotes tiller formation by repressing the transcription of OsTCP19, a negative modulator of tillering. We identify OsGATA8-H as a high-NUE haplotype with enhanced nitrogen uptake and a higher proportion of productive tillers. The geographical distribution of OsGATA8-H and its frequency change in historical accessions suggest its adaption to the fertile soil. Overall, this study provides molecular and evolutionary insights into the regulation of NUE and facilitates the breeding of rice cultivars with higher NUE.

Compensatory Modulation of Seed Storage Protein Synthesis and Alteration of Starch Accumulation by Selective Editing of 13 kDa Prolamin Genes by CRISPR-Cas9 in Rice.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.