Salinity stress is one of the most prominent abiotic stresses that negatively affect crop production. Transcription factors (TFs) are involved in the absorption, transport, or compartmentation of sodium (Na⁺) or potassium (K⁺) to resist salt stress. The basic helix–loop–helix (bHLH) is a TF gene family critical for plant growth and stress responses, including salinity. Herein, we used the CRISPR/Cas9 strategy to generate the gene editing mutant to investigate the role of OsbHLH024 in rice under salt stress. The A nucleotide base deletion was identified in the osbhlh024 mutant (A91). Exposure of the A91 under salt stress resulted in a significant increase in the shoot weight, the total chlorophyll content, and the chlorophyll fluorescence. Moreover, high antioxidant activities coincided with less reactive oxygen species (ROS) and stabilized levels of MDA in the A91. This better control of oxidative stress was accompanied by fewer Na⁺ but more K⁺, and a balanced level of Ca²⁺, Zn²⁺, and Mg²⁺ in the shoot and root of the A91, allowing it to withstand salt stress. Furthermore, the A91 also presented a significantly up-regulated expression of the ion transporter genes (OsHKT1;3, OsHAK7, and OsSOS1) in the shoot when exposed to salt stress. These findings imply that the OsbHLH024 might play the role of a negative regulator of salt stress, which will help to understand better the molecular basis of rice production improvement under salt stress.

Panicle architecture and grain size are two important agronomic traits which determine grain yield directly in rice. In the present study, a mutant named ltbsg1 (longer top branch and shorter grain 1) was isolated from the cultivar “Zhenong 34” (Oryza sativa L. ssp. indica) by ethyl methane sulfonate (EMS) mutagenesis. The target gene was studied through phenotype observation, genetic analysis, map-based cloning and functional analysis. The histocytological analysis indicated that the elongated top branch and shorter grain of mutant ltbsg1 were caused from the defects of cell elongation. The ltbsg1 gene in mutant revealed a single nucleotide substitution (G-A) in the exon 2 of LOC_Os10g25780, causing an amino acid variation (Glycine-Arginine) in the FAD (Flavin-adenine dinucleotide)-binding domain of delta (24)-sterol reductase, which was involved in the brassinosteroid (BR) biosynthesis. LTBSG1 was constitutively expressed and the protein was widely localized in chloroplast, nucleus and cytomembrane. The ltbsg1 seedlings had a lower endogenous BR level and could be restored to the phenotype of wild type by exogenous BR. The LTBSG1 knock-out lines showed similar phenotype defects as mutant ltbsg1, which confirmed that LTBSG1 was responsible for top branch elongation and grain size reduction. Furthermore, LTBSG1 along with other BR-related genes were feedback-regulated due to their obvious altered expression in mutant ltbsg1. This study demonstrated that LTBSG1 could play a new role in regulating panicle and grain development by BR biosynthetic pathway.

Isolating and characterizing mutants with altered senescence phenotypes is one of the ways to understand the molecular basis of leaf aging. Using ethyl methane sulfonate mutagenesis, a new rice (Oryza sativa) mutant, brown midrib leaf (bml), was isolated from the indica cultivar ‘Zhenong34’. The bml mutants had brown midribs in their leaves and initiated senescence prematurely, at the onset of heading. The mutants had abnormal cells with degraded chloroplasts and contained less chlorophyll compared to the wild type (WT). The bml mutant showed excessive accumulation of reactive oxygen species (ROS), increased activities of superoxide dismutase, catalase, and malondialdehyde, upregulation of senescence-induced STAY-GREEN genes and senescence-related transcription factors, and down regulation of photosynthesis-related genes. The levels of abscisic acid (ABA) and jasmonic acid (JA) were increased in bml with the upregulation of some ABA and JA biosynthetic genes. In pathogen response, bml demonstrated higher resistance against Xanthomonas oryzae pv. oryzae and upregulation of four pathogenesis-related genes compared to the WT. A genetic study confirmed that the bml trait was caused by a single recessive nuclear gene (BML). A map-based cloning using insertion/deletion markers confirmed that BML was located in the 57.32kb interval between the L5IS7 and L5IS11 markers on the short arm of chromosome 5. A sequence analysis of the candidate region identified a 1 bp substitution (G to A) in the 5′-UTR (+98) of bml. BML is a candidate gene associated with leaf senescence, ROS regulation, and disease response, also involved in hormone signaling in rice. Therefore, this gene might be useful in marker-assisted backcrossing/gene editing to improve rice cultivars.