Wancong Yu

The leaf morphology is an important agronomic trait in crop production. Our study identified a maple leaf type (mlt) cucumber mutant and located the regulatory gene for leaf shape changes through BSA results. Hybrid F1 and F2 populations were generated by F1 self-crossing, and the candidate mlt genes were identified within the 2.8 Mb region of chromosome 2 using map cloning. Through the sequencing and expression analysis of genes within the bulk segregant analysis (BSA) region, we identified the target gene for leaf shape regulation as CsWOX4 (CsaV3_2G026510). The change from base C to T in the original sequence led to frameshift mutations and the premature termination of translation, resulting in shortened encoded proteins and conserved WUSCHEL (WUS) box sequence loss. The specific expression analysis of the CsWOX4/Cswox4 genes in the roots, stems, leaves and other tissue types of wild-type (WT) and mutant plants revealed that CsWOX4 was higher in the root, but Cswox4 (mutant gene) was significantly higher in the leaf. Subcellular localization analysis revealed that CsWOX4 was localized in the nucleus. RNA-seq analysis revealed that the differentially expressed genes were mainly enriched in the mitochondrial cell cycle phase transition, nucleosome and microtubule binding pathways. Simultaneously, the quantitative analysis of the expression trends of 25 typical genes regulating the leaf types revealed the significant upregulation of CsPIN3. In our study, we found that the conserved domain of CsWOX4 was missing in the mutant, and the transcriptome data revealed that the expression of some genes, such as CsPIN3, changed simultaneously, thereby jointly regulating changes in the cucumber leaf type.

Because rice is native to tropical and subtropical regions, it is generally more sensitive to cold stress compared to other cereals. Low-temperature stress has a strong influence on the growth, development, and yield of rice. Plant NAM, ATAF1/2 and CUC(NAC) transcription factors (TFs) play significant regulatory roles in stress response. In our research, we found that OsNAC050 (LOC_Os03g60080) is mainly expressed in leaves. Cold (4 °C), heat (42 °C), PEG 6000 (20%, w/v), NaCl (200 mM), H₂O₂ (1%), IAA (100 μM), ABA (100 μM) and GA3 (100 μM) were used to treat wild type (WT) plants. A low temperature further up-regulated OsNAC050 expression. OsNAC050 mutants created using CRISPR-Cas9 gene editing technology showed significantly enhanced tolerance to the low-temperature treatment. The measurement of enzyme activities related to the redox pathway also showed that mutants have stronger viability under low-temperature stress. Comparative transcriptome analysis showed that photosynthesis and soluble sugar metabolism were significantly affected in OsNAC050 mutant lines, suggesting that OsNAC050 may participate in the above molecular pathways in response to low-temperature stress. The results expand our understanding of the molecular mechanisms underlying the responses to cold stress in rice and can provide new strategies for engineering cold tolerance in high-yielding rice varieties.

Because rice is native to tropical and subtropical regions, it is generally more sensitive to cold stress compared to other cereals. Low temperature stress has become one of the most important conditions that affects the growth, development, and yield of rice. Plant NAC (NAM, ATAF1/2, and CUC) transcription factors are known to play significant regulatory roles in the stress response. In our research, we found that OsNAC050 (LOC_Os03g60080） is mainly expressed in leaves, and low temperature can further up-regulate its expression. OsNAC050 mutants created using CRISPR-Cas9 gene editing technology showed significantly enhanced tolerance to low temperature treatment. Detection of enzyme activities related to the redox pathway also showed that the mutants had stronger viability under low temperature stress. Comparative transcriptome analysis showed that photosynthesis and soluble sugar metabolism were significantly affected in the osnac050 mutant lines, suggesting that OsNAC050 may participate in the above molecular pathways in the response to low temperature stress. The results will enhance our understanding of molecular mechanisms underlying the responses to cold stress in rice and can provide new strategies for engineering cold-tolerance in high-yielding rice varieties.

Environmental drought stress threatens rice production. Previous studies have reported that related NAC (NAM, ATAF1/2, and CUC) transcription factors play an important role in drought stress. Herein, we identified and characterized OsNAC092, encoding an NAC transcription factor that is highly expressed and induced during drought tolerance. OsNAC092 knockout lines created using the clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) system exhibited increased drought resistance in rice. RNA sequencing showed that the knockout of OsNAC092 caused a global expression change, and differential gene expression is chiefly associated with “response to light stimulus,” “MAPK signaling pathway,” “plant hormone signal transduction,” “response to oxidative stress,” “photosynthesis,” and “water deprivation.” In addition, the antioxidants and enzyme activities of the redox response were significantly increased. OsNAC092 mutant rice exhibited a higher ability to scavenge more ROS and maintained a high GSH/GSSG ratio and redox level under drought stress, which could protect cells from oxidant stress, revealing the importance of OsNAC092 in the rice’s response to abiotic stress. Functional analysis of OsNAC092 will be useful to explore many rice resistance genes in molecular breeding to aid in the development of modern agriculture.