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Backgorund: Environmental stresses including abiotic stresses and biotic stresses limit yield of plants. Stress-tolerant breeding is an efficient way to improve plant yield under stress conditions. Genome editing by CRISPR/Cas9 can be used in molecular breeding to improve agronomic traits in crops, but in most cases, with fitness costs. The plant hormone ABA regulates plant responses to abiotic stresses via signaling transduction. We previously identified AITRs as a family of novel transcription factors that play a role in regulating plant responses to ABA and abiotic stresses. We found that abiotic stress tolerance was increased in the single, double and triple aitr mutants. However, it is unclear if the increased abiotic stress tolerance in the mutants may have fitness costs., Results: We report here the characterization of AITRs as suitable candidate genes for CRISPR/Cas9 editing to improve plant stress tolerance. By using CRISPR/Cas9 to target AITR3 and AITR4 simultaneously in the aitr256 triple and aitr1256 quadruple mutants respectively, we generated Cas9-free aitr23456 quintuple and aitr123456 sextuple mutants. We found that reduced sensitivities to ABA and enhanced tolerance to drought and salt were observed in these mutants. Most importantly, plant growth and development was not affected even in the aitr123456 sextuple mutants, in whom the entire AITR family genes have been knocked out, and the aitr123456 sextuple mutants also showed a wild type response to the pathogen infection., Conclusions: Our results suggest that knockout of the AITR family genes in Arabidopsis enhanced abiotic stress tolerance without fitness costs. Considering that knock-out a few AITRs will lead to enhanced abiotic stress tolerance, that AITRs are widely distributed in angiosperms with multiple encoding genes, AITRs may be targeted for molecular breeding to improve abiotic stress tolerance in plants including crops.

Both seed size and abiotic stress tolerance are important agronomic traits in crops. In Arabidopsis, two closely related transcription repressors DPA4 (Development-Related PcG Target in the APEX4)/NGAL3 and SOD7 (Suppressor of da1-1 )/NGAL2 (NGATHA-like protein) function redundantly to regulate seed size, which was increased in the dpa4 sod7 double mutants. Whereas ABA-induced transcription repressors (AITRs) are involved in the regulation of ABA signaling and abiotic stress tolerance, Arabidopsis aitr2 aitr5 aitr6 ( aitr256 ) triple mutant showed enhanced tolerance to drought and salt. Here we performed CRISPR/Cas9 genome editing to disrupt DPA4 and SOD7 in aitr256 mutant, trying to integrate seed size and abiotic stress tolerance traits in Arabidopsis, and also to examine whether DPA4 and SOD7 may regulate other aspects of plant growth and development. Indeed, seed size was increased in the dpa4 sod7 aitr256 quintuple mutants, and enhanced tolerance to drought was observed in the mutants. In addition, we found that shoot branching was affected in the dpa4 sod7 aitr256 mutants. The mutant plants failed to produce secondary branches, and flowers/siliques were distributed irregularly on the main stems of the plants. Floral organ number and fertility were also affected in the dpa4 sod7 aitr256 mutant plants. To examine if these phenotypes were dependent on loss-of-function of AITRs , dpa4 sod7 double mutants were generated in Col wild type background, and we found that the dpa4 sod7 mutant plants showed a phenotype similar to the dpa4 sod7 aitr256 quintuple mutants. Taken together, our results indicate that the integration of seed size and abiotic stress tolerance traits by CRISPR/Cas9 editing was successful, and our results also revealed a role of DPA4 and SOD7 in the regulation of inflorescence architecture in Arabidopsis.

Ovate family proteins (OFPs) are valued as a family of transcription factors that are unique to plants, and they play a pluripotent regulatory role in plant growth and development, including secondary-cell-wall synthesis, DNA repair, gibberellin synthesis, and other biological processes, via their interaction with TALE family proteins. In this study, CHIP-SEQ was used to detect the potential target genes of AtOFP1 and its signal-regulation pathways. On the other hand, Y2H and BIFC were employed to prove that AtOFP1 can participate in ABA signal transduction by interacting with one of the TALE family protein called AtKNAT3. ABA response genes are not only significantly upregulated in the 35S::HAOFP1 OE line, but they also show hypersensitivity to ABA in terms of seed germination and early seedling root elongation. In addition, the AtOFP1-regulated target genes are mainly mitochondrial membranes that are involved in the oxidative–phosphorylation pathway. Further qRT-PCR results showed that the inefficient splicing of the respiratory complex I subunit genes NAD4 and NAD7 may lead to ROS accumulation in 35S::HA-AtOFP1 OE lines. In conclusion, we speculated that the overexpression of AtOFP1 may cause the ABA hypersensitivity response by increasing the intracellular ROS content generated from damage to the intima systems of mitochondria. [ABSTRACT FROM AUTHOR]

Expression of stress response genes can be regulated by abscisic acid (ABA) dependent and ABA independent pathways. Osmotic stresses promote ABA accumulation, therefore inducing the expression of stress response genes via ABA signaling. Whereas cold and heat stresses induce the expression of stress response genes via ABA independent pathway. ABA induced transcription repressors (AITRs) are a family of novel transcription factors that play a role in ABA signaling, and Drought response gene (DRG) has previously been shown to play a role in regulating plant response to drought and freezing stresses. We report here the identification of DRG as a novel transcription factor and a regulator of ABA response in Arabidopsis. We found that the expression of DRG was induced by ABA treatment. Homologs searching identified AITR5 as the most closely related Arabidopsis protein to DRG, and homologs of DRG, including the AITR-like (AITRL) proteins in bryophytes and gymnosperms, are specifically presented in embryophytes. Therefore we renamed DRG as AITRL. Protoplast transfection assays show that AITRL functioned as a transcription repressor. In seed germination and seedling greening assays, the aitrl mutants showed an increased sensitivity to ABA. By using qRT-PCR, we show that ABA responses of some ABA signaling component genes including some PYR1-likes (PYLs), PROTEIN PHOSPHATASE 2Cs (PP2Cs) and SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2s (SnRK2s) were reduced in the aitrl mutants. Taken together, our results suggest that AITRLs are a family of novel transcription repressors evolutionally conserved in embryophytes, and AITRL regulates ABA response in Arabidopsis by affecting ABA response of some ABA signaling component genes. [ABSTRACT FROM AUTHOR]