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Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Crop roots are colonized by large numbers of microorganisms, collectively known as the root-microbiome, which modulate plant growth, development and contribute to elemental nutrient uptake. In conditions of nitrogen limitation, the over-expressed Calcineurin B-like interacting protein kinase 2 (OsCIPK2) gene with root-specific promoter (RC) has been shown to enhance growth and nitrogen uptake in rice. Analysis of root-associated bacteria through high-throughput sequencing revealed that OsCIPK2 has a significant impact on the diversity of the root microbial community under low nitrogen stress. The quantification of nifH gene expression demonstrated a significant enhancement in nitrogen-fixing capabilities in the roots of RC transgenetic rice. Synthetic microbial communities (SynCom) consisting of six nitrogen-fixing bacterial strains were observed to be enriched in the roots of RC, leading to a substantial improvement in rice growth and nitrogen uptake in nitrogen-deficient soils. Forty and twenty-three metabolites exhibiting differential abundance were identified in the roots and rhizosphere soils of RC transgenic rice compared to wild-type (WT) rice. These findings suggest that OSCIPK2 plays a role in restructuring the microbial community in the roots through the regulation of metabolite synthesis and secretion. Further experiments involving the exogenous addition of citric acid revealed that an optimal concentration of this compound facilitated the growth of nitrogen-fixing bacteria and substantially augmented their population in the soil, highlighting the importance of citric acid in promoting nitrogen fixation under conditions of low nitrogen availability. These findings suggest that OsCIPK2 plays a role in enhancing nitrogen uptake by rice plants from the soil by influencing the assembly of root microbial communities, thereby offering valuable insights for enhancing nitrogen utilization in rice cultivation., (© 2024. The Author(s).)

Improving chilling tolerance at the seedling stage in rice is essential for agricultural research. We combined a physiological analysis with transcriptomics in a variety Dular subjected to chilling followed by recovery at normal temperature to better understand the chilling tolerance mechanisms of rice. Chilling inhibited the synthesis of chlorophyll and non-structural carbohydrate (NSC) and disrupted the ion balance of the plant, resulting in the impaired function of rice leaves. The recovery treatment can effectively reverse the chilling-related injury. Transcriptome results displayed that 21,970 genes were identified at three different temperatures, and 11,732 genes were differentially expressed. According to KEGG analysis, functional categories for differentially expressed genes (DEGs) mainly included ribosome (8.72%), photosynthesis-antenna proteins (7.38%), phenylpropanoid biosynthesis (11.41%), and linoleic acid metabolism (10.07%). The subcellular localization demonstrated that most proteins were located in the chloroplasts (29.30%), cytosol (10.19%), and nucleus (10.19%). We proposed that some genes involved in photosynthesis, ribosome, phenylpropanoid biosynthesis, and linoleic acid metabolism may play key roles in enhancing rice adaptation to chilling stress and their recovery capacity. These findings provide a foundation for future research into rice chilling tolerance mechanisms.

Background: The asynchronous filling between superior spikelets (SS) and inferior spikelets (IS) in rice has become a research hotspot. The stagnant development and poor grain filling of IS limit yields and the formation of good quality rice. A large number of studies on this phenomenon have been carried out from the genome, transcriptome and proteome level, indicating that asynchronous filling of SS and IS filling is a complex, but orderly physiological and biochemical process involving changes of a large number of genes, protein expression and modification. However, the analysis of metabolomics differences between SS and IS is rarely reported currently., Results: This study utilized untargeted metabolomics and identified 162 metabolites in rice spikelets. Among them, 17 differential metabolites associated with unsynchronized grain filling between SS and IS, 27 metabolites were related to the stagnant development of IS and 35 metabolites related to the lower maximum grain-filling rate of IS compared with the SS. We found that soluble sugars were an important metabolite during grain filling for SS and IS. Absolute quantification was used to further analyze the dynamic changes of 4 types of soluble sugars (sucrose, fructose, glucose, and trehalose) between SS and IS. The results showed that sucrose and trehalose were closely associated with the dynamic characteristics of grain filling between SS and IS. The application of exogenous sugar showed that trehalose functioned as a key sugar signal during grain filling of IS. Trehalose regulated the expression of genes related to sucrose conversion and starch synthesis, thereby promoting the conversion of sucrose to starch. The difference in the spatiotemporal expression of TPS-2 and TPP-1 between SS and IS was an important reason that led to the asynchronous change in the trehalose content between SS and IS., Conclusions: The results from this study are helpful for understanding the difference in grain filling between SS and IS at the metabolite level. In addition, the present results can also provide a theoretical basis for the next step of using metabolites to regulate the filling of IS., (© 2021. The Author(s).)

The poor grain filling of inferior spikelets (IS) situated on the lower secondary rachis branch leads to a remarkable decrease in rice yield and quality. The AGPase small subunit 2 (AGPS2) encodes a small subunit of adenosine diphosphate-glucose pyrophosphorylase (AGPase) enzyme, which plays an important role in sucrose-starch conversion and starch biosynthesis in the grain filling of rice. In the present study, qPCR analysis showed low expression abundance of AGPS2 in IS, compared to the superior spikelets (SS), which was consistent with the lower grain weight of IS. To evaluate the molecular mechanism of AGPS2, we first identified the AGPS2 interaction network through Co-immunoprecipitation (Co-IP). In total, 29 proteins of AGPS2 interaction network were characterized by LC-MS/MS. Bioinformatics analysis revealed that, the characterized proteins in the interaction network are likely to be related to starch synthesis, sugar conversion, energy pathway, and folding/modification, and most of them were involved in the grain filling of rice. The sequent Co-IP analysis showed that AGPS2 can bind to starch branching enzyme (SBE), pullulanase (PUL) and starch debranching enzyme (DBE) and assemble into starch synthesizing protein complex (SSPC). In addition, the 14-3-3 protein GF14e was also found to interact with AGPS2. Further analysis by qPCR showed that the expression of GF14e was much higher on IS than on SS. The qPCR results also showed that the expression of GF14e was relatively stable in SS, but changed significantly in IS under alternate wetting and moderate soil drying (WMD), which is consistent with the AGPS2 expression pattern. Our present work provides direct molecular evidence for the different expression patterns of AGPS2 in SS and IS, which could be greatly helpful for the molecular amelioration of the poor grain filling of IS in rice., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nitrogen (N) is one of the indispensable factors in rice growth and development. China holds a premier position in the production of rice and at the same time also faces higher N fertilizer costs along with serious damage to the environment. A better solution is much needed to address these issues, without disrupting the production of rice as an important cereal, while minimizing all the deleterious effects on the environment. Two isogenic lines Kitaake (WT) and its genetically modified line CIPK2 (RC), overexpressing the gene for Calcineurin B-like interacting protein kinase 2 (Os CIPK2 ) with better nitrogen use efficiency (NUE), were compared for their growth and development under low versus normal levels of N. NUE is a complex trait mainly related to a plant's efficiency in extraction, assimilation, and recycling of N from soil. The microbial population was analyzed using high-throughput Illumina Miseq 16S rRNA sequencing and found that RC with CIPK2, specifically expressed in rice root, not only performed better without nitrogen fertilizer (LN) but also increased the diversity of bacterial communities in rice rhizosphere compartments (rhizosphere, rhizoplane, and endosphere). The relative abundance of beneficial bacteria phyla increased, which are known to promote the circulation and transformation of N in rhizosphere soil. To further explore the potential of RC regarding better performance under LN, the ion fluxes in root apical were detected by non-invasive micro-test technique (NMT). We found that RC can absorb more Ca 2+ and NO 3 - under LN as compared to WT. Finally, compared to WT, RC plants exhibited better growth of root and shoot, and increased yield and N uptake under LN, whereas there was no significant difference in the growth of two rice lines under normal nitrogen (NN) treatment. We are able to get preliminary results, dealing with the OsCIPK2 overexpressed rice line, by studying the rice molecular, physiological, and chemical parameters related to NUE. The results laid the foundation for further research on N absorption and utilization in rice from the soil and the interaction with microbial communities.

In rice (Oryza sativa L.), later flowering inferior spikelets (IS), which are located on proximal secondary branches, fill slowly and produce smaller and lighter grains than earlier flowering superior spikelets (SS). Many genes have been reported to be involved in poor grain filling of IS, however the underlying molecular mechanisms remain unclear. The present study determined that GF14f, a member of the 14-3-3 protein family, showed temporal and spatial differences in expression patterns between SS and IS. Using GF14f-RNAi plants, we observed that a reduction in GF14f expression in the endosperm resulted in a significant increase in both grain length and weight, which in turn improved grain yield. Furthermore, pull-down assays indicated that GF14f interacts with enzymes that are involved in sucrose breakdown, starch synthesis, tricarboxylic acid (TCA) cycle and glycolysis. At the same time, an increase in the activity of sucrose synthase (SuSase), adenosine diphosphate-glucose pyrophosphorylase (AGPase), and starch synthase (StSase) was observed in the GF14f-RNAi grains. Comprehensive analysis of the proteome and metabolite profiling revealed that the abundance of proteins related to the TCA cycle, and glycolysis increased in the GF14f-RNAi grains together with several carbohydrate intermediates. These results suggested that GF14f negatively affected grain development and filling, and the observed higher abundance of the GF14f protein in IS compared with SS may be responsible for poor IS grain filling. The study provides insights into the molecular mechanisms underlying poor grain filling of IS and suggests that GF14f could serve as a potential tool for improving rice grain filling., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Plant 14-3-3 proteins belong to a large family of proteins involved in numerous physiological processes, and function by binding to phosphorylated client proteins to regulate their function. However, little is known about their regulatory mechanisms in rice root growth. In this study, four 14-3-3 isoforms (GF14b, GF14c, GF14e, GF14f) exhibiting prominent expression profiles in rice roots, were selected for further investigation. Through a pull-down assay using four 14-3-3 isoforms in rice roots, 87 client proteins were identified that are involved in metabolism, protein synthesis and trafficking, energy metabolism, cell structure and growth, and other cellular processes. Importantly, we found that 14-3-3 proteins may play an important role in the root stress response through interactions with proteins functioning in oxidative stress, pathogenesis and secondary metabolism. By using real-time RT-PCR, it was found that 14-3-3 proteins exhibited diverse patterns of gene expression in response to salinity and drought stresses in rice root. The results revealed the isoform-specific functions of root 14-3-3 proteins. Our current study provides insight into understanding the functional roles of 14-3-3 proteins during rice root growth., Significance: Rice (Oryza sativa L.) is one of the most important food crops in the world, as it is consumed by more than 3 billion people. Regulation of root growth plays an important role in rice adaption to biotic and abiotic, stress such as rhizosphere microbes and nutrient stress, and this process is directly related to the final yield. 14-3-3 proteins form a multi-gene family regulating developmental processes in plants. However, the correlation between the 14-3-3 protein family and its role in rice root growth has very little study. We applied an affinity chromatographic approach, in combination with LC-MS/MS, to explore the client proteins of four 14-3-3 isoforms that exhibit much more prominent gene expression than other members of the 14-3-3 family in rice roots. Assessments of the identified client proteins are able to obtain novel information toward understanding the functional mechanism of 14-3-3 proteins in rice root growth as expected., (Copyright © 2017. Published by Elsevier B.V.)

Cultivars of rice (Oryza sativa L.), especially of the type with large spikelets, often fail to reach the yield potential as expected due to the poor grain-filling on the later flowering inferior spikelets (in contrast to the earlier-flowering superior spikelets). The present study showed that the size and grain weight of superior spikelets (SS) was greater than those of inferior spikelets (IS), and the carbohydrate supply should not be the major problem for the poor grain-filling because there was adequate amount of sucrose in IS at the initial grain-filling stage. High resolution two-dimensional gel electrophoresis (2-DE) in combination with Coomassie-brilliant blue (CBB) and Pro-Q Diamond phosphoprotein fluorescence stain revealed that 123 proteins in abundance and 43 phosphoproteins generated from phosphorylation were significantly different between SS and IS. These proteins and phosphoproteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism and protein synthesis/destination. Expression analyses of the proteins and phosphoproteins associated with different functional categories/subcategories indicated that the starch synthesis, central carbon metabolism, N metabolism and cell growth/division were closely related to the poor grain-filling of IS. Functional and expression pattern studies also suggested that 14-3-3 proteins played important roles in IS poor grain-filling by regulating the activity of starch synthesis enzymes. The proteome and phosphoproteome obtained from this study provided a better understanding of the molecular mechanism of the IS poor grain-filling. They were also expected to be highly useful for improving the grain filling of rice.

Cultivars of rice (Oryza sativa L.), especially the large-spikelet-type, often fail to achieve the high yield potential due to poor grain-filling of their inferior (late-flowering) spikelets. The superior (early-flowering) spikelets normally contain more abscisic acid (ABA) than the inferior spikelets. It was speculated that ABA might play a pivotal role in the grain-filling of inferior spikelets. To understand the molecular regulation involved in this process, we employed the 2-D gel-based comparative proteomic and phosphoproteomic analyses to search for differentially expressed proteins in the inferior spikelets under exogenous ABA treatment. A total of 111 significantly differential proteins and 31 phosphoproteins were found in the inferior spikelets after treatment. Among them, 100 proteins and 23 phosphoproteins were identified by using MALDI-TOF/TOF MS. In addition, the gene expression patterns of the inferior spikelets were confirmed with RT-PCR. These differentially expressed proteins are active in defense response, carbohydrate, protein, amino acid, energy and secondary metabolisms, as well as cell development and photosynthesis. The results suggest that the grain-filling of rice inferior spikelets is regulated by ABA through some proteins and phosphoproteins participating in carbon, nitrogen and energy metabolisms., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)