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Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist’s mind, as it allows genome editing in multiple biological systems.

The biosynthesis of anthocyanins is still questionable in regulating the quantities of anthocyanins biosynthesized in rice seeds and the expression levels of transcription factors and the structural genes involved in the biosynthetic pathway of anthocyanins. We herein investigated the relationship between the accumulated anthocyanin contents and the expression levels of genes related to the biosynthesis of anthocyanins in rice seeds. Liquid chromatography/mass spectrometry-mass spectrometry analysis of cyanidin 3-glucoside (C3G) in rice seeds showed no accumulation of C3G in white and red rice cultivars, and the differential accumulation of C3G among black rice cultivars. RNA-seq analysis in rice seeds, including white, red, and black rice cultivars, at twenty days after heading (DAH) further exhibited that the genes involved in the biosynthesis of anthocyanins were differentially upregulated in developing seeds of black rice. We further verified these RNA-seq results through gene expression analysis by a quantitative real-time polymerase chain reaction in developing seeds of white, red, and black rice cultivars at 20 DAH. Of these genes related to the biosynthesis of anthocyanins, bHLH s, MYB s, and WD40 , which are regulators, and the structural genes, including chalcone synthase ( CHS ), flavanone 3-hydroxylase ( F3H ), flavonoid 3´-hydroxylase ( F3 ´ H ), dihydroflavonol 4-reductase ( DFR ), and anthocyanidin synthase ( ANS ), were differentially upregulated in black rice seeds. The correlation analysis revealed that the quantities of C3G biosynthesized in black rice seeds were positively correlated to the expression levels of bHLH s, MYB s and WD40 , CHS , F3H , F3 ´ H , DFR , and ANS . In addition, we present bHLH2 ( LOC_Os04g47040 ) and MYB s ( LOC_Os01g49160 , LOC_Os01g74410 , and LOC_Os03g29614 ) as new putative transcription factor genes for the biosynthesis of anthocyanins in black rice seeds. It is expected that this study will help to improve the understanding of the molecular levels involved in the biosynthesis of anthocyanins in black rice seeds.

Plant peptides, synthesized from larger precursor proteins, often undergo proteolytic cleavage and post-translational modifications to form active peptide hormones. This process involves several proteolytic enzymes (proteases). Among these, SBTs (serine proteases) are a major class of proteolytic enzymes in plants and play key roles in various regulatory mechanisms, including plant immune response, fruit development and ripening, modulating root growth, seed development and germination, and organ abscission. However, current knowledge about SBTs is largely limited to ‘in vitro cleavage assays ,’ with few studies exploring loss of function analyses for more in depth characterization. Research focused on economically significant horticultural crops, like tomato and pepper, remains scarce. Given this, leveraging SBTs for horticultural crop improvement through advanced gene-editing tools is critical for enhancing crop resilience to stress and pathogens. Over the past five years, research on proteolytic enzymes, especially SBTs, has increased markedly, yet reports involving loss- or gain-of function analyses aimed at improving crop yield and quality are still limited. This review summarizes recent findings on SBT enzymes, which act as ‘protein scissors’ in activating peptide hormones, and discusses the potential for using selected SBTs in CRISPR-Cas9 gene editing to enhance the growth and resilience of economically important Solanaceae crops, with a focus on pepper.

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.

Background In plants, the key roles played by RopGEF-mediated ROP signaling in diverse processes, including polar tip growth, have been identified. Despite their important roles in reproduction, a comprehensive analysis of RopGEF members has not yet been performed in rice ( Oryza sativa ). To determine whether RopGEF regulators are involved in rice pollen tube growth, we performed genome-wide analysis of this family in rice. Results Phylogenomic and meta-expression analysis of eleven RopGEFs in rice showed that four genes were preferentially expressed in mature pollen. These four genes contain the plant-specific Rop nucleotide exchanger (PRONE) domain and possible phosphorylated residues, suggesting a conserved role in polar tip growth with Arabidopsis thaliana . In subcellular localization analysis of the four RopGEFs through tobacco ( Nicotiana benthamiana ) infiltration, four proteins were predominantly identified in plasma membrane. Moreover, double mutants of RopGEF2/8 exhibited reduced pollen germination, causing partial male sterility. These genes possess unique cis-acting elements in their promoters compared with the other RopGEF genes. Conclusions In this study, four RopGEF genes were identified as pollen-specific gene in eleven members of rice, and the expression pattern, promoter analysis, and evolutionary relationship of the RopGEF family were studied compared with Arabidopsis. Our study indicated that four RopGEF genes might function during pollen germination in distinct subcellular localization. Our study could provide valuable information on the functional study of RopGEF in rice.

Summary Vertical farming offers the advantage of providing a stable environment for plant cultivation, shielding them from adverse conditions such as climate change. For fruit‐harvesting plants like tomato, vertical farming necessitates the optimization of plant growth and architecture. The gibberellin 3‐oxidase (GA3ox) genes encode gibberellin 3‐oxidases responsible for activating GA within the pathway and modulating stem length. Among the five SlGA3ox genes, we targeted the coding regions of three SlGA3ox genes (named SlGA3ox3, SlGA3ox4 and SlGA3ox5) using multiplex CRISPR genome editing. The slga3ox4 single mutants exhibited a slight reduction in primary shoot length, leading to a smaller stature. In contrast, the slga3ox3 and slga3ox5 single mutants showed subtle phenotypic changes. Notably, the slga3ox3 slga3ox4 double mutants developed a more compact shoot architecture with minor physiological differences, potentially making them suitable for vertical farming applications. We observed a correlation between total yield and plant size across all genotypes through multiple yield trials. Observations from vertical farm cultivation revealed that slga3ox3 slga3ox4 plants possess a markedly compact plant size, offering potential benefits for space‐efficient cultivation. Our research suggests that targeted manipulation of hormone biosynthetic genes can effectively tailor plant architecture for vertical farming.

Efficient production of hybrid rice seeds requires male sterility systems to overcome the challenges of self‐pollination. In this study, we identified male gametic transfer defect ( GTrD ) 5 and GTrD9 as essential genes for the process of male gamete transmission in rice. Mutations in these genes cause partial male sterility by impairing pollen tube elongation, thereby reducing the fertilisation rate. Using CRISPR/Cas9 technology, we developed gtrd5flo5 and gtrd9flo5 double mutants that combined the gtrd5 and gtrd9 genes responsible for partial male sterility with the floury endosperm (FLO)5 gene, whose defects result in seeds with an opaque endosperm, allowing us to easily differentiate between hybrid and self‐pollinated seeds. This two‐line hybrid system demonstrated a high rate of hybrid seed production with significantly reduced self‐pollination ratios. The hybrid seeds resulted in plants with increased height, panicle size and grain yield compared with those obtained from the parental lines and also displayed heterosis. Unlike the current two‐line hybrid systems based on photoperiod‐ and thermosensitive genic male‐sterile lines, our approach is independent of environmental factors, ensuring a stable and reliable hybrid seed production. This novel method simplifies seed production, enhances efficiency and offers a cost‐effective and environmentally sustainable solution for hybrid rice breeding.

Rice (Oryza sativa. L) has been intensively studied to ensure a stable global supply of this commodity in the face of rapid global climate change. A critical factor that decreases crop yield is drought, which has been analyzed in various ways through many researches. Microbiome-based studies of rice investigate the symbiosis between rice and bacteria, which has been proposed as a way to overcome problems caused by drought. Several rice-associated metagenomic profiles obtained under drought conditions have been reported since the advent of next generation sequencing (NGS) technology. To elucidate the future diversity of plants and microorganisms and to promote sustainable agriculture, we reanalyzed 64 of the publicly available 16S amplicon sequencing data produced under drought condition. In the process of integrating data sets, however, we found an inconsistency that serves as a bottleneck for microbiome-based sustainability research. While this report provides clues about the composition of the microbiome under the drought conditions, the results are affected by differences in the location of the experiments, sampling conditions, and analysis protocols. Re-analysis of amplicon sequencing data of the soil microbiome in rice fields suggests that microbial composition shifts in response to drought condition and the presence of plants. Among the bacteria involved, the phylum Proteobacteria appears to play the most important role in the survival of rice under drought condition.

Pollen in angiosperms plays a critical role in double fertilization by germinating and elongating pollen tubes rapidly in one direction to deliver sperm. In this process, the secretory vesicles deliver cell wall and plasma membrane materials, and excessive materials are sequestered via endocytosis. However, endocytosis in plants is poorly understood. AP180 N-terminal homology (ANTH) domain-containing proteins function as adaptive regulators for clathrin-mediated endocytosis in eukaryotic systems. Here, we identified 17 ANTH domain-containing proteins from rice based on a genome-wide investigation. Motif and phylogenomic analyses revealed seven asparagine-proline-phenylalanine (NPF)-rich and 10 NPF-less subgroups of these proteins, as well as various clathrin-mediated endocytosis-related motifs in their C-terminals. To investigate their roles in pollen germination, we performed meta-expression analysis of all genes encoding ANTH domain-containing proteins in Oryza sativa ( OsANTH genes) in anatomical samples, including pollen, and identified five mature pollen-preferred OsANTH genes. The subcellular localization of four OsANTH proteins that were preferentially expressed in mature pollen can be consistent with their role in endocytosis in the plasma membrane. Of them, OsANTH3 represented the highest expression in mature pollen. Functional characterization of OsANTH3 using T-DNA insertional knockout and gene-edited mutants revealed that a mutation in OsANTH3 decreased seed fertility by reducing the pollen germination percentage in rice. Thus, our study suggests OsANTH3-mediated endocytosis is important for rice pollen germination.

The resveratrol-producing rice ( Oryza sativa L.) inbred lines, Iksan 515 (I.515) and Iksan 526 (I.526), developed by the expression of the groundnut ( Arachis hypogaea ) resveratrol synthase 3 ( AhRS3 ) gene in the japonica rice cultivar Dongjin, accumulated both resveratrol and its glucoside, piceid, in seeds. Here, we investigated the effect of the AhRS3 transgene on the expression of endogenous piceid biosynthesis genes ( UGTs ) in the developing seeds of the resveratrol-producing rice inbred lines. Ultra-performance liquid chromatography (UPLC) analysis revealed that I.526 accumulates significantly higher resveratrol and piceid in seeds than those in I.515 seeds and, in I.526 seeds, the biosynthesis of resveratrol and piceid reached peak levels at 41 days after heading (DAH) and 20 DAH, respectively. Furthermore, RNA-seq analysis showed that the expression patterns of UGT genes differed significantly between the 20 DAH seeds of I.526 and those of Dongjin. Quantitative real-time PCR (RT-qPCR) analyses confirmed the data from RNA-seq analysis in seeds of Dongjin, I.515 and I.526, respectively, at 9 DAH, and in seeds of Dongjin and I.526, respectively, at 20 DAH. A total of 245 UGT s, classified into 31 UGT families, showed differential expression between Dongjin and I.526 seeds at 20 DAH. Of these, 43 UGT s showed more than 2-fold higher expression in I.526 seeds than in Dongjin seeds. In addition, the expression of resveratrol biosynthesis genes ( PAL , C4H and 4CL ) was also differentially expressed between Dongjin and I.526 developing seeds. Collectively, these data suggest that AhRS3 altered the expression pattern of UGT genes, and PAL , C4H and 4CL in developing rice seeds.