Autophagy receptor OsNBR1 modulates salt stress tolerance in rice.

KEY MESSAGE: Autophagy receptor OsNBR1 modulates salt stress tolerance by affecting ROS accumulation in rice. The NBR1 (next to BRCA1 gene 1), as important selective receptors, whose functions have been reported in animals and plants. Although the function of NBR1 responses to abiotic stress has mostly been investigated in Arabidopsis thaliana, the role of NBR1 under salt stress conditions remains unclear in rice (Oryza sativa). In this study, by screening the previously generated activation-tagged line, we identified a mutant, activation tagging 10 (AC10), which exhibited salt stress-sensitive phenotypes. TAIL-PCR (thermal asymmetric interlaced PCR) showed that the AC10 line carried a loss-of-function mutation in the OsNBR1 gene. OsNBR1 was found to be a positive regulator of salt stress tolerance and was localized in aggregates. A loss-of-function mutation in OsNBR1 increased salt stress sensitivity, whereas overexpression of OsNBR1 enhanced salt stress resistance. The osnbr1 mutants showed higher ROS (reactive oxygen species) production, whereas the OsNBR1 overexpression (OsNBR1OE) lines showed lower ROS production, than Kitaake plants under normal and salt stress conditions. Furthermore, RNA-seq analysis revealed that expression of OsRBOH9 (respiratory burst oxidase homologue) was increased in osnbr1 mutants, resulting in increased ROS accumulation in osnbr1 mutants. Together our results established that OsNBR1 responds to salt stress by influencing accumulation of ROS rather than by regulating transport of Na+ and K+ in rice.

Piezo-photocatalysis for efficient charge separation to promote CO2 photoreduction in nanoclusters.

The substantial emissions of CO2 greenhouse gases have resulted in severe environmental problems, and research on the implementation of semiconductor materials to minimize CO2 is currently a highly discussed subject. Effective separation of interface charges is a major challenge for efficient piezo-photocatalytic systems. Meanwhile, the ultrasmall-sized metal nanoclusters can shorten the distance of electron transport. Herein, we synthesized Au25(p-MBA)18 nanoclusters (Au25 NCs) modified red graphitic carbon nitride (RCN) nanocatalysts with highly exposed Au active sites by in-situ seed growth method. The loading of Au25 NCs on the RCN surface provides more active sites and creates a long-range ordered electric field. It allows for the direct utilization of the piezoelectric field to separate photogenerated carriers during photo-piezoelectric excitation. Based on the above advantages, the rate of CO2 reduction to CO over Au25 NCs/RCN (111.95 µmol g-1 h-1) was more than triple compared to that of pristine RCN. This paper has positive implication for further application of metal clusters loaded semiconductor for piezo-photocatalytic CO2 reduction.

Effect of ultrasound treatment on interactions of whey protein isolate with rutin.

Rutin is a biologically active polyphenol, but its poor water solubility and low bioavailability limit its application to the food industry. We investigated the effect of ultrasound treatment on the properties of rutin (R) and whey protein isolate (WPI) using spectral and physicochemical analysis. The results revealed that there was covalent interaction between whey protein isolate with rutin, and the binding degree of whey isolate protein with rutin increased with ultrasound treatment. Additionally, solubility and surface hydrophobicity of WPI-R complex improved with ultrasonic treatment, and a maximum solubility of 81.9 % at 300 W ultrasonic power. The ultrasound treatment caused the complex to develop a more ordered secondary structure, resulting in a three-dimensional network structure with small and uniform pore sizes. This research could provide a theoretical reference for studying protein-polyphenol interactions and their applications in food delivery systems.

Transcription Factor AtOFP1 Involved in ABA-Mediated Seed Germination and Root Growth through Modulation of ROS Homeostasis 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.

Integration of biochar into Ag3PO4/α-Fe2O3 heterojunction for enhanced reactive oxygen species generation towards organic pollutants removal.

A biochar (BC) harbored Ag3PO4/α-Fe2O3 type-Ⅰ heterojunction (Ag-Fe-BC) was prepared by a hydrothermal-impregnation method to transfer active center of heterojunctions. The electrochemical and spectroscopic tests demonstrated that BC enhanced the catalytic performance of the heterojunction by enhancing photocurrent, reducing fluorescence intensity, and facilitating separation of electron-hole pairs. The photocatalytic activity showed the Ag-Fe-BC (5:1:3) could degrade Rhodamine B (20 mg/L) by up to 92.7%, which was 3.35 times higher than Ag3PO4/α-Fe2O3. Tetracycline and ciprofloxacin (20 mg/L) were degraded efficiently by 58.3% and 79.4% within 2 h, respectively. Electron paramagnetic resonance and scavenging experiments confirmed the major reactive oxygen species (ROS) consisted of singlet oxygen (1O2) and superoxide (·O2-). Excellent RhB adsorption and electrons capturing capacity of BC facilitated electron-hole pairs separation and ROS transferring to target organics followed by elevated degradation. Thus, a facile method was proposed to synthesize a highly efficient visible-light responsive photocatalyst for degradation of various organics in water.

Knockout of the entire family of AITR genes in Arabidopsis leads to enhanced drought and salinity tolerance without fitness costs.

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.

AITRL, an evolutionarily conserved plant specific transcription repressor regulates ABA response in Arabidopsis.

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.

SlEAD1, an EAR motif-containing ABA down-regulated novel transcription repressor regulates ABA response in tomato.

EAR motif-containing proteins are able to repress gene expression, therefore play important roles in regulating plants growth and development, plant response to environmental stimuli, as well as plant hormone signal transduction. ABA is a plant hormone that regulates abiotic stress tolerance in plants via signal transduction. ABA signaling via the PYR1/PYLs/RCARs receptors, the PP2Cs phosphatases, and SnRK2s protein kinases activates the ABF/AREB/ABI5-type bZIP transcription factors, resulting in the activation/repression of ABA response genes. However, functions of many ABA response genes remained largely unknown. We report here the identification of the ABA-responsive gene SlEAD1 (Solanum lycopersicum EAR motif-containing ABA down-regulated 1) as a novel EAR motif-containing transcription repressor gene in tomato. We found that the expression of SlEAD1 was down-regulated by ABA treatment, and SlEAD1 repressed reporter gene expression in transfected protoplasts. By using CRISPR gene editing, we generated transgene-free slead1 mutants and found that the mutants produced short roots. By using seed germination and root elongation assays, we examined ABA response of the slead1 mutants and found that ABA sensitivity in the mutants was increased. By using qRT-PCR, we further show that the expression of some of the ABA biosynthesis and signaling component genes were increased in the slead1 mutants. Taken together, our results suggest that SlEAD1 is an ABA response gene, that SlEAD1 is a novel EAR motif-containing transcription repressor, and that SlEAD1 negatively regulates ABA responses in tomato possibly by repressing the expression of some ABA biosynthesis and signaling genes.

Genome Editing to Integrate Seed Size and Abiotic Stress Tolerance Traits in Arabidopsis Reveals a Role for DPA4 and SOD7 in the Regulation of Inflorescence Architecture.

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.