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In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.

Rice is an important staple food crop for over half of the world’s population. However, abiotic stresses seriously threaten rice yield improvement and sustainable production. Breeding and planting rice varieties with high environmental stress tolerance are the most cost-effective, safe, healthy, and environmentally friendly strategies. In-depth research on the molecular mechanism of rice plants in response to different stresses can provide an important theoretical basis for breeding rice varieties with higher stress resistance. This review presents the molecular mechanisms and the effects of various abiotic stresses on rice growth and development and explains the signal perception mode and transduction pathways. Meanwhile, the regulatory mechanisms of critical transcription factors in regulating gene expression and important downstream factors in coordinating stress tolerance are outlined. Finally, the utilization of omics approaches to retrieve hub genes and an outlook on future research are prospected, focusing on the regulatory mechanisms of multi-signaling network modules and sustainable rice production.

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.

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.

Sucrose controls various developmental and metabolic processes in plants. It also functions as a signaling molecule in the synthesis of carbohydrates, storage proteins, and anthocyanins, as well as in floral induction and defense response. We found that sucrose preferentially induced OsWRKY7 , whereas other sugars (such as mannitol, glucose, fructose, galactose, and maltose) did not have the same effect. A hexokinase inhibitor mannoheptulose did not block the effect of sucrose, which is consequently thought to function directly. MG132 inhibited sucrose induction, suggesting that a repressor upstream of OsWRKY7 is degraded by the 26S proteasome pathway. The 3-kb promoter sequence of OsWRKY7 was preferentially induced by sucrose in the luciferase system. Knockout mutants of OsWRKY7 were more sensitive to the rice blast fungus Magnaporthe oryzae , whereas the overexpression of OsWRKY7 enhanced the resistance, indicating that this gene is a positive regulator in the plant defense against this pathogen. The luciferase activity driven by the OsPR10a promoter was induced by OsWRKY7 and this transcription factor bound to the promoter region of OsPR10a , suggesting that OsWRKY7 directly controls the expression of OsPR10a . We conclude that sucrose promotes the transcript level of OsWRKY7 , thereby increasing the expression of OsPR10a for the defense response in rice.

Understanding the behavior of pollen during pollination is important for food security in the future. The elucidation of pollen development and growth regulation largely relies on the study of the dicotyledonous model plant Arabidopsis thaliana. However, rice (Oryza sativa) pollen exhibits different characteristics to that of Arabidopsis. The latter undergoes programmed dehydration and withstands adverse environmental conditions, whereas rice pollen is sensitive to desiccation. Moreover, the short longevity of rice pollen significantly hampers hybrid seed production. Although the “omics” data for mature rice pollen have been accumulated, few genes that control pollination and pollen hydration have been identified. Therefore, to facilitate future studies, it is necessary to summarize the developmental processes involved in pollen production in rice and to consolidate the underlying mechanisms discovered in previous studies. In this review, we describe the pollen developmental processes and introduce gametophytic mutants, which form defective pollen in Arabidopsis and rice. In addition, we discuss the perspectives on the research on pollen longevity, adhesion and hydration.

The fine-tuning of inorganic phosphate (Pi) for enhanced use efficiency has long been a challenging subject in agriculture, particularly in regard to rice as a major crop plant. Among ribonucleases (RNases), the RNase T2 family is broadly distributed across kingdoms, but little has been known on its substrate specificity compared to RNase A and RNase T1 families. Class I and class II of the RNase T2 family are defined as the S-like RNase (RNS) family and have showed the connection to Pi recycling in Arabidopsis. In this study, we first carried out a phylogenetic analysis of eight rice and five Arabidopsis RNS genes and identified mono-specific class I and dicot-specific class I RNS genes, suggesting the possibility of functional diversity between class I RNS family members in monocot and dicot species through evolution. We then compared the in silico expression patterns of all RNS genes in rice and Arabidopsis under normal and Pi-deficient conditions and further confirmed the expression patterns of rice RNS genes via qRT-PCR analysis. Subsequently, we found that most of the OsRNS genes were differentially regulated under Pi-deficient treatment. Association of Pi recycling by RNase activity in rice was confirmed by measuring total RNA concentration and ribonuclease activity of shoot and root samples under Pi-sufficient or Pi-deficient treatment during 21 days. The total RNA concentrations were decreased by < 60% in shoots and < 80% in roots under Pi starvation, respectively, while ribonuclease activity increased correspondingly. We further elucidate the signaling pathway of Pi starvation through upregulation of the OsRNS genes. The 2-kb promoter region of all OsRNS genes with inducible expression patterns under Pi deficiency contains a high frequency of P1BS cis-acting regulatory element (CRE) known as the OsPHR2 binding site, suggesting that the OsRNS family is likely to be controlled by OsPHR2. Finally, the dynamic transcriptional regulation of OsRNS genes by overexpression of OsPHR2 , ospho2 mutant, and overexpression of OsPT1 lines involved in Pi signaling pathway suggests the molecular basis of OsRNS family in Pi recycling via RNA decay under Pi starvation.

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.

Epitranscriptomics has added a new layer of regulatory machinery to eukaryotes, and the advancement of sequencing technology has revealed more than 170 post-transcriptional modifications in various types of RNAs, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and long non-coding RNA (lncRNA). Among these, N6-methyladenosine (m6A) and N5-methylcytidine (m5C) are the most prevalent internal mRNA modifications. These regulate various aspects of RNA metabolism, mainly mRNA degradation and translation. Recent advances have shown that regulation of RNA fate mediated by these epitranscriptomic marks has pervasive effects on a plant’s development and responses to various biotic and abiotic stresses. Recently, it was demonstrated that the removal of human-FTO-mediated m6A from transcripts in transgenic rice and potatoes caused a dramatic increase in their yield, and that the m6A reader protein mediates stress responses in wheat and apple, indicating that regulation of m6A levels could be an efficient strategy for crop improvement. However, changing the overall m6A levels might have unpredictable effects; therefore, the identification of precise m6A levels at a single-base resolution is essential. In this review, we emphasize the roles of epitranscriptomic modifications in modulating molecular, physiological, and stress responses in plants, and provide an outlook on epitranscriptome engineering as a promising tool to ensure food security by editing specific m6A and m5C sites through robust genome-editing technology.

Plants are susceptible to infection by various pathogens with high epidemic potential. Xanthomonas oryzae pv . oryzae ( Xoo ) causes bacterial blight in rice, one of the most significant diseases in both temperate and tropical regions. In this study, we report the identification and characterization of OsWRKY26 , a sucrose-inducible transcription factor, that plays a role in the plant defense responses following Xoo infection. We found that mutant plants with defective OsWRKY26 showed enhanced defense response specifically to Xoo , indicating that this transcription factor acts as a negative defense regulator. In contrast, mutant plants did not exhibit higher resistance compared to wild-type (WT) plants when infected with the rice blast fungal pathogen Magnaporthe oryzae . Transcriptomic analysis of mutant and WT plants revealed that several pathogen resistance genes were upregulated in mutants. Of these, we selected OsXa39 for further analysis. Transient expression experiments in rice protoplasts showed that OsWRKY26 repressed the expression of a Luciferase reporter gene driven by the OsXa39 promoter. Chromatin immunoprecipitation analysis revealed that OsWRKY26 binds directly to the promoter region of OsXa39 . These findings suggest that OsWRKY26 negatively regulates the defense response during Xoo infection by repressing OsXa39 as well as other pathogen-related genes such as OsXa47 , OsBBR1 , OsRSR1 , OsPR1a , OsPR1-11 , OsPR2 , and OsPR4c.