Md. Amdadul Huq, Shahina Akter, III Sup Nou, Hoy Taek Kim, Yu Jin Jung, and Kwon Kyoo Kang. J Plant Biotechnol 2016;43:1-11. https://doi.org/10.5010/JPB.2016.43.1.1

Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T₂ OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein–protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.

The utilization of male sterility into hybrid seed production reduces its cost and ensures high purity of tomato varieties because it does not produce pollen and has exserted stigmas. Here, we report on the generation of gene edited lines into male sterility phenotype by knockout of SlMS10 gene (Solyc02g079810) encoding the bHLH transcription factor that regulates meiosis and cell death of the tapetum during microsporogenesis in the tomato. Twenty-eight gene edited lines out of 60 transgenic plants were selected. Of these, eleven different mutation types at the target site of the SlMS10 gene were selected through deep sequencing analysis. These mutations were confirmed to be transmitted to subsequent generations. The null lines without the transferred DNA (T-DNA) were obtained by segregation in the T₁ and T₂ generations. In addition, we showed that the cr-ms10-1-4 mutant line exhibited dysfunctional meiosis and abnormal tapetum during flower development, resulting in no pollen production. RT-PCR analysis showed that the most genes associated with pollen and tapetum development in tomatoes had lower expression in the cr-ms10-1-4 mutant line compared to wild type. We demonstrate that modification of the SlMS10 gene via CRISPR/Cas9-mediated genome editing results in male sterility of tomato plants. Our results suggest an alternative approach to generating male sterility in crops.

Plants exhibit high regenerative capacity, which is controlled by various genetic factors. Here, we report that ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2) controls de novo shoot organogenesis by regulating auxin-cytokinin interaction. The auxin-inducible ATXR2 Trithorax Group (TrxG) protein temporally interacts with the cytokinin-responsive type-B ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) at early stages of shoot regeneration. The ATXR2-ARR1 complex binds to and deposits the H3K36me3 mark in the promoters of a subset of type-A ARR genes, ARR5 and ARR7, thus activating their expression. Consequently, the ATXR2/ARR1-type-A ARR module transiently represses cytokinin signaling and thereby de novo shoot regeneration. The atxr2-1 mutant calli exhibit enhanced shoot regeneration with low expression of ARR5 and ARR7, which ultimately upregulates WUSCHEL (WUS) expression. Thus, ATXR2 regulates cytokinin signaling and prevents premature WUS activation to ensure proper cell fate transition, and the auxin-cytokinin interaction underlies the initial specification of shoot meristem in callus.

Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography–tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.