Control of Reactive Oxygen Species through Antioxidant Enzymes Plays a Pivotal Role during the Cultivation of Neopyropia yezoensis

Neopyropia yezoensis is an economically important marine crop that can survive dehydrating conditions when nets are lifted from seawater. During this process, production of oxygen radicals and the resulting up-regulation of antioxidant enzymes mediated by the abscisic acid (ABA) signaling pathway played an important role. However, there were no reports about the significance regarding the protection of seaweed throughout the entire production season. Especially, in new aquatic farms in Shandong Province that were formed when traditional N. yezoensis cultivation moved north. Here, we determined the levels of ABA, hydrogen peroxide (H2O2), soluble protein, chlorophyll, and cell wall polysaccharides in samples collected at different harvest periods from Jimo aquatic farm, Shandong Province. The activities and expression of NADPH oxidase (NOX) and antioxidant enzymes in the corresponding samples were also determined. Combined with the monitoring data of sea surface temperature and solar light intensity, we proposed that the cultivation of N. yezoensis in Shandong Province was not affected by high-temperature stress. However, photoinhibition in N. yezoensis usually occurs at noon, especially in March. Both the activities and the expression of NOX and antioxidant enzymes were up-regulated continuously. It is reasonable to speculate that the reactive oxygen species (ROS) produced by NOX induced the up-regulation of antioxidant enzymes through the ABA signaling pathway. Although antioxidant enzymes play a pivotal role during the cultivation of N. yezoensis, the production of ROS also caused a shift in gene expression, accumulation of secondary metabolites, and even decreased the chlorophyll pool size, which eventually led to a decrease in algae assimilation. Accordingly, we suggest that the dehydration of N. yezoensis nets should be adopted when necessary and the extent of dehydration should be paid special consideration to avoid an excessive cellular response caused by ROS.

Synthesis of Abscisic Acid in Neopyropia yezoensis and Its Regulation of Antioxidase Genes Expressions Under Hypersaline Stress

Abscisic acid (ABA) is regarded as crucial for plant adaptation to water-limited conditions and it functions evolutionarily conserved. Thus, insights into the synthesis of ABA and its regulation on downstream stress-responsive genes in Neopyropia yezoensis, a typical Archaeplastida distributed in intertidal zone, will improve the knowledge about how ABA signaling evolved in plants. Here, the variations in ABA contents, antioxidant enzyme activities and expression of the target genes were determined under the presence of exogenous ABA and two specific inhibitors of the ABA precursor synthesis. ABA content was down-regulated under the treatments of each or the combination of the two inhibitors. Antioxidant enzyme activities like SOD, CAT and APX were decreased slightly with inhibitors, but up-regulated when the addition of exogenous ABA. The quantitative assays using real-time PCR (qRT-PCR) results were consistent with the enzyme activities. All the results suggested that ABA can also alleviate oxidative stress in N. yezoensis as it in terrestrial plant. Combined with the transcriptome assay, it was hypothesized that ABA is synthesized in N. yezoensis via a pathway that is similar to the carotenoid pathway in higher plants, and both the MVA and that the MEP pathways for isoprenyl pyrophosphate (IPP) synthesis likely exist simultaneously. The ABA signaling pathway in N. yezoensis was also analyzed from an evolutionary standpoint and it was illustrated that the emergence of the ABA signaling pathway in this alga is an ancestral one. In addition, the presence of the ABRE motif in the promoter region of antioxidase genes suggested that the antioxidase system is regulated by the ABA signaling pathway.

A Novel Embryo Phenotype Associated With Interspecific Hybrid Weakness in Rice Is Controlled by the MADS-Domain Transcription Factor OsMADS8

A novel hybrid weakness gene, DTE9, associated with a dark tip embryo (DTE) trait, was observed in CR6078, an introgression line derived from a cross between the Oryza sativa spp. japonica “Hwayeong” (HY) and the wild relative Oryza rufipogon. CR6078 seeds exhibit protruding embryos and flowers have altered inner floral organs. DTE9 was also associated with several hybrid weakness symptoms including decreased grain weight. Map-based cloning and transgenic approaches revealed that DTE9 is an allele of OsMADS8, a MADS-domain transcription factor. Genetic analysis indicated that two recessive complementary genes were responsible for the expression of the DTE trait. No sequence differences were observed between the two parental lines in the OsMADS8 coding region; however, numerous single nucleotide polymorphisms were detected in the promoter and intronic regions. We generated overexpression (OX) and RNA interference (RNAi) transgenic lines of OsMADS8 in HY and CR6078, respectively. The OsMADS8-OX lines showed the dark tip embryo phenotype, whereas OsMADS8-RNAi recovered the normal embryo phenotype. Changes in gene expression, including of ABCDE floral homeotic genes, were observed in the OsMADS8-OX and OsMADS8-RNAi lines. Overexpression of OsMADS8 led to decreased expression of OsEMF2b and ABA signaling-related genes including OsVP1/ABI3. HY seeds showed higher ABA content than CR6078 seeds, consistent with OsMADS8/DTE9 regulating the expression of genes related ABA catabolism in CR6078. Our results suggest that OsMADS8 is critical for floral organ determination and seed germination and that these effects are the result of regulation of the expression of OsEMF2b and its role in ABA signaling and catabolism.

AtTrxh3, a Thioredoxin, Is Identified as an Abscisic AcidBinding Protein in Arabidopsis thaliana

Abscisic acid (ABA, 1) is a plant hormone that regulates various plant physiological processes such as seed developing and stress responses. The ABA signaling system has been elucidated; binding of ABA with PYL proteins triggers ABA signaling. We have previously reported a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, a protein cross-linker, and a bioactive small molecule with an azido group (azido probe). This method was used to identify the unknown ABA binding protein of Arabidopsis thaliana. As a result, AtTrxh3, a thioredoxin, was isolated as an ABA binding protein. Our developed method can be applied to the identification of binding proteins of bioactive compounds.

A dynamic model of the ABA Signaling pathway with its core components: translation rate of PP2C determines the kinetics of ABA-induced gene expression

The abscisic acid (ABA) signaling pathway is the key defense mechanism against drought stress in plants, yet the connectivity of cellular molecules related to gene expression in response to ABA is little understood. A dynamic model of the core components of the ABA signaling pathway was built using ordinary differential equations to understand the connectivity. Parameter values of protein-protein interactions and enzymatic reactions in the model were implemented from the data obtained by previously conducted experiments. On the other hand, parameter values of gene expression and translation were determined by comparing the kinetics of gene expression in the model to those of ABA-induced RD29A (response to desiccation 29A) in actual plants. Based on the analyses of the optimized model, we hypothesized that the translation rate of PP2C (protein phosphatase type 2C) is downregulated by ABA to increase the ABRE (ABA-responsive element) promoter activity. The hypotheses were preliminarily supported by newly conducted experiments using transgenic Arabidopsis plants that carry a luciferase expression cassette driven by the RD29A promoter (RD29A::LUC). The model suggests that identifying a mechanism that alters PP2C translation rate would be one of the next research frontiers in the ABA signaling pathway.

Mutation of GmAITR Genes by CRISPR/Cas9 Genome Editing Results in Enhanced Salinity Stress Tolerance in Soybean

Breeding of stress-tolerant plants is able to improve crop yield under stress conditions, whereas CRISPR/Cas9 genome editing has been shown to be an efficient way for molecular breeding to improve agronomic traits including stress tolerance in crops. However, genes can be targeted for genome editing to enhance crop abiotic stress tolerance remained largely unidentified. We have previously identified abscisic acid (ABA)-induced transcription repressors (AITRs) as a novel family of transcription factors that are involved in the regulation of ABA signaling, and we found that knockout of the entire family of AITR genes in Arabidopsis enhanced drought and salinity tolerance without fitness costs. Considering that AITRs are conserved in angiosperms, AITRs in crops may be targeted for genome editing to improve abiotic stress tolerance. We report here that mutation of GmAITR genes by CRISPR/Cas9 genome editing leads to enhanced salinity tolerance in soybean. By using quantitative RT-PCR analysis, we found that the expression levels of GmAITRs were increased in response to ABA and salt treatments. Transfection assays in soybean protoplasts show that GmAITRs are nucleus proteins, and have transcriptional repression activities. By using CRISPR/Cas9 to target the six GmAITRs simultaneously, we successfully generated Cas9-free gmaitr36 double and gmaitr23456 quintuple mutants. We found that ABA sensitivity in these mutants was increased. Consistent with this, ABA responses of some ABA signaling key regulator genes in the gmaitr mutants were altered. In both seed germination and seedling growth assays, the gmaitr mutants showed enhanced salt tolerance. Most importantly, enhanced salinity tolerance in the mutant plants was also observed in the field experiments. These results suggest that mutation of GmAITR genes by CRISPR/Cas9 is an efficient way to improve salinity tolerance in soybean.

HbSnRK2.6 Functions in ABA-Regulated Cold Stress Response by Promoting HbICE2 Transcriptional Activity in Hevea brasiliensis

Low temperature remarkably limits rubber tree (Hevea brasiliensis Muell. Arg.) growth, latex production, and geographical distribution, but the underlying mechanisms of Hevea brasiliensis cold stress response remain elusive. Here, we identified HbSnRK2.6 as a key component in ABA signaling functions in phytohormone abscisic acid (ABA)-regulated cold stress response in Hevea brasiliensis. Exogenous application of ABA enhances Hevea brasiliensis cold tolerance. Cold-regulated (COR) genes in the CBF pathway are upregulated by ABA. Transcript levels of all five HbSnRK2.6 members are significantly induced by cold, while HbSnRK2.6A, HbSnRK2.6B, and HbSnRK2.6C can be further activated by ABA under cold conditions. Additionally, HbSnRK2.6s are localized in the cytoplasm and nucleus, and can physically interact with HbICE2, a crucial positive regulator in the cold signaling pathway. Overexpression of HbSnRK2.6A or HbSnRK2.6B in Arabidopsis extensively enhances plant responses to ABA and expression of COR genes, leading to increased cold stress tolerance. Furthermore, HbSnRK2.6A and HbSnRK2.6B can promote transcriptional activity of HbICE2, thus, increasing the expression of HbCBF1. Taken together, we demonstrate that HbSnRK2.6s are involved in ABA-regulated cold stress response in Hevea brasiliensis by regulating transcriptional activity of HbICE2.

Downregulation of lncRNA PpL-T31511 and Pp-miRn182 Promotes Hydrogen Cyanamide-Induced Endodormancy Release through the PP2C-H2O2 Pathway in Pear (Pyrus pyrifolia)

Bud endodormancy is an important, complex process subject to both genetic and epigenetic control, the mechanism of which is still unclear. The endogenous hormone abscisic acid (ABA) and its signaling pathway play important roles in the endodormancy process, in which the type 2C protein phosphatases (PP2Cs) is key to the ABA signal pathway. Due to its excellent effect on endodormancy release, hydrogen cyanamide (HC) treatment is considered an effective measure to study the mechanism of endodormancy release. In this study, RNA-Seq analysis was conducted on endodormant floral buds of pear (Pyrus pyrifolia) with HC treatment, and the HC-induced PP2C gene PpPP2C1 was identified. Next, software prediction, expression tests and transient assays revealed that lncRNA PpL-T31511-derived Pp-miRn182 targets PpPP2C1. The expression analysis showed that HC treatment upregulated the expression of PpPP2C1 and downregulated the expression of PpL-T31511 and Pp-miRn182. Moreover, HC treatment inhibited the accumulation of ABA signaling pathway-related genes and hydrogen peroxide (H2O2). Furthermore, overexpression of Pp-miRn182 reduced the inhibitory effect of PpPP2C1 on the H2O2 content. In summary, our study suggests that downregulation of PpL-T31511-derived Pp-miRn182 promotes HC-induced endodormancy release in pear plants through the PP2C-H2O2 pathway.