Transglutaminases (TGases), which are widespread cross-linking enzymes in plants, play key roles in photosynthesis and abiotic/biotic stress responses; however, evidence concerning the genetics underlying how TGase improves the capability of photosynthesis and the mechanism of TGase-mediated photosynthesis are not clear in this crop species. In this study, we clarified the function of TGase in the regulation of photosynthesis in tomato by comparing wild-type (WT) plants, tgase mutants generated by the CRISPR/Cas9 system and TGase-overexpressing (TGaseOE) plants. Our results showed that increasing the transcript level of TGase resulted in an enhanced net photosynthetic rate (Pn), whereas the tgase mutants presented significantly inhibited Pns and CO2 assimilation compared with the WT. Although the total RuBisCO activity was not affected by TGase, the initial and activation status of RuBisCO and the activity of RuBisCO activase (RCA) and fructose-1,6-bisphosphatase (FBPase) in TGaseOE plants were significantly higher than that in WT plants. Except for RuBisCO small subunit (RbcS), the transcription levels of Benson–Calvin cycle-related genes were positively related to the endogenous TGase activity. Furthermore, TGaseOE plants had higher protein levels of RuBisCO large subunit (RbcL) and RCA than did WT plants and showed a reduced redox status by enhancing the activity of dehydroascorbate reductase (DHAR) and glutathione reductase (GR), which was compromised in TGase-deficient plants. Overall, TGase positively regulated photosynthesis by maintaining the activation states of the Benson–Calvin cycle and inducing changes in cellular redox homeostasis in tomato.

High temperature is considered a critical abiotic stressor that is increasing continuously, which is severely affecting plant growth and development. The use of heat-resistant rootstock grafting is a viable technique that is practiced globally to improve plant resistance towards abiotic stresses. In this experiment, we explored the efficacy of bitter melon rootstock and how it regulates photosynthesis and the antioxidant defense system to alleviate heat stress (42 °C/32 °C) in cucumber. Our results revealed that bitter-melon-grafted seedlings significantly relieved heat-induced growth inhibition and photoinhibition, maintained better photosynthesis activity, and accumulated a greater biomass than self-grafted seedlings. We measured the endogenous polyamine and hydrogen peroxide (H2O2) contents to determine the inherent mechanism responsible for these effects, and the results showed that heat stress induced a transient increase in polyamines and H2O2 in the inner courtyard of grafted seedlings. This increment was greater and more robust in bitter-melon-grafted seedlings. In addition, the use of polyamine synthesis inhibitors MGBG (methylglyoxal bis-guanylhydrazone) and D-Arg (D-arginine), further confirmed that the production of H2O2 under heat stress is mediated by the accumulation of endogenous polyamines. Moreover, compared with other treatments, the bitter-melon-grafted seedlings maintained high levels of antioxidant enzyme activity under high temperature conditions. However, these activities were significantly inhibited by polyamine synthesis inhibitors and H2O2 scavengers (dimethylthiourea, DMTU), indicating that bitter melon rootstock not only maintained better photosynthetic activity under conditions of high temperature stress but also mediated the production of H2O2 through the regulation of the high level of endogenous polyamines, thereby boosting the antioxidant defense system and comprehensively improving the heat tolerance of cucumber seedlings. Taken together, these results indicate that grafting with a resistant cultivar is a promising alternative tool for reducing stress-induced damage.

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Barhl1, a mouse homologous gene of Drosophila BarH class homeobox genes, is highly expressed within the inner ear and crucial for the long-term maintenance of auditory hair cells that mediate hearing and balance, yet little is known about the molecular events underlying Barhl1 regulation and function in hair cells. In this study, through data mining and in vitro report assay, we firstly identified Barhl1 as a direct target gene of Atoh1 and one E-box (E3) in Barhl1 3’ enhancer is crucial for Atoh1-mediated Barhl1 activation. Then we generated a mouse embryonic stem cell (mESC) line carrying disruptions on this E3 site E-box (CAGCTG) using CRISPR/Cas9 technology and this E3 mutated mESC line is further subjected to an efficient stepwise hair cell differentiation strategy in vitro. Disruptions on this E3 site caused dramatic loss of Barhl1 expression and significantly reduced the number of induced hair cell-like cells, while no affections on the differentiation toward early primitive ectoderm-like cells and otic progenitors. Finally, through RNA-seq profiling and gene ontology (GO) enrichment analysis, we found that this E3 box was indispensable for Barhl1 expression to maintain hair cell development and normal functions. We also compared the transcriptional profiles of induced cells from CDS mutated and E3 mutated mESCs, respectively, and got very consistent results except the Barhl1 transcript itself. These observations indicated that Atoh1-mediated Barhl1 expression could have important roles during auditory hair cell development. In brief, our findings delineate the detail molecular mechanism of Barhl1 expression regulation in auditory hair cell differentiation.

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