Rice, being a major staple food crop and sensitive to salinity conditions, bears heavy yield losses due to saline soil. Although some salt responsive genes have been identified in rice, their applications in developing salt tolerant cultivars have resulted in limited achievements. Herein, we used bioinformatic approaches to perform a meta-analysis of three transcriptome datasets from salinity and control conditions in order to reveal novel genes and the molecular pathways underlying rice response to salt. From a total of 28,432 expressed genes, we identify 457 core differentially expressed genes (DEGs) constitutively responding to salt, regardless of the stress duration, genotype, or the tissue. Gene co-expression analysis divided the core DEGs into three different modules, each of them contributing to salt response in a unique metabolic pathway. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses highlighted key biological processes and metabolic pathways involved in the salt response. We identified important novel hub genes encoding proteins of different families including CAM, DUF630/632, DUF581, CHL27, PP2-13, LEA4-5, and transcription factors, which could be functionally characterized using reverse genetic experiments. This novel repertoire of candidate genes related to salt response in rice will be useful for engineering salt tolerant varieties.

Several plant pathogens severely affect crop yield and quality, thereby threatening global food security. In order to cope with this challenge, genetic improvement of plant disease resistance is required for sustainable agricultural production, for which conventional breeding is unlikely to do enough. Luckily, genome editing systems that particularly clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has revolutionized crop improvement by enabling robust and precise targeted genome modifications. It paves the way towards new methods for genetic improvement of plant disease resistance and accelerates resistance breeding. In this review, the challenges, limitations and prospects for conventional breeding and the applications of CRISPR/Cas9 system for the development of transgene-free disease-resistant crops are discussed.

Summary: Starch is the main form of energy storage in higher plants. Although several enzymes and regulators of starch biosynthesis have been defined, the complete molecular machinery remains largely unknown. Screening for irregularities in endosperm formation in rice represents valuable prospect for studying starch synthesis pathway. Here, we identified a novel rice white‐core endosperm and defective grain filling mutant, ospk2, which displays significantly lower grain weight, decreased starch content and alteration of starch physicochemical properties when compared to wild‐type grains. The normal starch compound granules were drastically reduced and more single granules filled the endosperm cells of ospk2. Meanwhile, the germination rate of ospk2 seeds after 1‐year storage was observably reduced compared with wild‐type. Map‐based cloning of OsPK2 indicated that it encodes a pyruvate kinase (PK, ATP: pyruvate 2‐O‐phosphotransferase, EC 2.7.1.40), which catalyses an irreversible step of glycolysis. OsPK2 has a constitutive expression in rice and its protein localizes in chloroplasts. Enzyme assay showed that the protein product from expressed OsPK2 and the crude protein extracted from tissues of wild‐type exhibits strong PK activity; however, the mutant presented reduced protein activity. OsPK2 (PKpα1) and three other putative rice plastidic isozymes, PKpα2, PKpβ1 and PKpβ2, can interact to form heteromer. Moreover, the mutation leads to multiple metabolic disorders. Altogether, these results denote new insights into the role of OsPK2 in plant seed development, especially in starch synthesis, compound granules formation and grain filling, which would be useful for genetic improvement of high yield and rice grain quality.

Two-line hybrid rice with high yield potential is increasingly popular and the photo- and temperature-sensitive male sterile line is one of the basic components for two-line hybrid rice breeding. The development of male sterile lines through conventional breeding is a lengthy and laborious process, whereas developing thermo-sensitive genic male sterile (TGMS) lines for two-line hybrid breeding by editing a temperature-sensitivity gene by CRISPR/Cas9 is efficient and convenient. Here, thermo-sensitive genic male sterility (TGMS) was induced by employing the CRISPR/Cas9 gene editing technology to modify the gene TMS5. Two TGMS mutants, tms5–1 and tms5–2, both lacking any residual T-DNA, were generated in the indica rice cultivar Zhongjiazao17 (cv. YK17) background. When grown at a sub-optimal temperature (22 °C), both mutants produced viable pollen and successfully produced grain through self-fertilization, but at temperatures 24 and 26 °C, their pollen was sterile and no grain was set. F1 hybrids derived from the crosses between YK17S (tms5–1) and three different restorer lines outperformed both parental lines with respect to grain yield and related traits. The YK17S generated by CRISPR/Cas9 system was proved to be a new TGMS line with superior yield potential and can be widely utilized in two-line hybrid breeding of indica rice.

Aroma is a key grain quality trait that directly influences the market price of rice globally. Loss of function of betaine aldehyde dehydrogenase 2 (OsBADH2) affects the biosynthesis of 2‐acetyl‐1‐pyrroline (2‐AP), which is responsible for aroma in fragrant rice. The current study was aimed at creating new alleles of BADH2 using CRISPR/Cas9 gene editing technology under the genetic background of the japonica Ningjing 1 (NJ1) and indica Huang Huazhan (HHZ) varieties. Sensory evaluation and analysis using headspace solid‐phase microextraction gas chromatography–mass spectrometry (HS‐SPME‐GC‐MS) showed that the grains of the four homozygous T₁ lines with new alleles of BADH2 (nj1‐cr ^BADH2‐1, nj1‐cr ^BADH2‐2, hhz‐cr ^BADH2‐1 and hhz‐cr ^BADH2‐2) produced moderate fragrance and had significantly increased 2‐AP content compared with wild‐types. Moreover, there were no significant differences in the amylose content and gelatinization temperature among the four lines with new alleles of BADH2 to the wild‐types. Thereafter, we crossed the HHZ background new alleles of BADH2 with CMS line Taonong 1A (TN1A) to produce a three‐line hybrid variety B‐Tao‐You‐Xiangzhan (BTYXZ) with increased grain aroma. The 2‐AP content in grains of the improved BTYXZ‐1 and BTYXZ‐2 reached at 26.16 and 18.74 μg/kg, and the gel consistency of BTYXZ‐1 and BTYXZ‐2 increased significantly by 9.1% and 6.5%, respectively, compared with the wild‐type Tao‐You‐Xiangzhan (TYXZ). However, the γ‐aminobutyric acid (GABA) content in the improved three‐line hybrid rice BTYXZ‐1 (5.6 mg/100 g) and BTYXZ‐2 (10.7 mg/100 g) was significantly lower than that of the TYXZ. These results demonstrated that CRISPR/Cas9 gene editing technology could be successfully utilized in improving aroma in non‐fragrant japonica and indica varieties. In addition, the newly developed BADH2 alleles provided important genetic resources for grain aroma improvement in three‐line hybrid rice.

Starch and storage proteins are the main components of rice (Oryza sativa L.) grains. Despite their importance, the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive. Here, we identified a rice opaque endosperm mutant, opaque3 (o3), that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type. The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells. OPAQUE3 (O3) encodes a transmembrane basic leucine zipper (bZIP) transcription factor (OsbZIP60) and is localized in the endoplasmic reticulum (ER) and the nucleus, but it is localized mostly in the nucleus under ER stress. We demonstrated that O3 could activate the expression of several starch synthesis-related genes (GBSSI, AGPL2, SBEI, and ISA2) and storage protein synthesis-related genes (OsGluA2, Prol14, and Glb1). O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1, two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells. High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants. We also revealed that OsbZIP50 can assist O3 in response to ER stress, especially under high-temperature conditions. We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.