Abiotic stresses such as drought, cold, salt and heat cause reduction of plant growth and loss of crop yield worldwide. Reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide anions (O2•‾), hydroxyl radical (OH•) and singlet oxygen (1O2) are by-products of physiological metabolisms, and are precisely controlled by enzymatic and non-enzymatic antioxidant defense systems. ROS are significantly accumulated under abiotic stress conditions, which cause oxidative damage and eventually resulting in cell death. Recently, ROS have been also recognized as key players in the complex signaling network of plants stress responses. The involvement of ROS in signal transduction implies that there must be coordinated function of regulation networks to maintain ROS at non-toxic levels in a delicate balancing act between ROS production, involving ROS generating enzymes and the unavoidable production of ROS during basic cellular metabolism, and ROS-scavenging pathways. Increasing evidence showed that ROS play crucial roles in abiotic stress responses of crop plants for the activation of stress-response and defense pathways. More importantly, manipulating ROS levels provides an opportunity to enhance stress tolerances of crop plants under a variety of unfavorable environmental conditions. This review presents an overview of current knowledge about homeostasis regulation of ROS in crop plants. In particular, we summarize the essential proteins that are involved in abiotic stress tolerance of crop plants through ROS regulation. Finally, the challenges toward the improvement of abiotic stress tolerance through ROS regulation in crops are discussed.

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ROS Regulation During Abiotic Stress Responses in Crop Plants

Abscisic acid (ABA) is an important phytohormone regulating plant growth, development, and stress responses. It has an essential role in multiple physiological processes of plants, such as stomatal closure, cuticular wax accumulation, leaf senescence, bud dormancy, seed germination, osmotic regulation, and growth inhibition among many others. Abscisic acid controls downstream responses to abiotic and biotic environmental changes through both transcriptional and posttranscriptional mechanisms. During the past 20 years, ABA biosynthesis and many of its signaling pathways have been well characterized. Here we review the dynamics of ABA metabolic pools and signaling that affects many of its physiological functions.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/jipb.12899

Abscisic acid dynamics, signaling, and functions in plants

Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants.

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MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

Protein kinases are major players in various signal transduction pathways. Understanding the molecular mechanisms behind plant responses to biotic and abiotic stresses has become critical for developing and breeding climate-resilient crops. In this review, we summarize recent progress on understanding plant drought, salt, and cold stress responses, with a focus on signal perception and transduction by different protein kinases, especially sucrose nonfermenting1 (SNF1)-related protein kinases (SnRKs), mitogen-activated protein kinase (MAPK) cascades, calcium-dependent protein kinases (CDPKs/CPKs), and receptor-like kinases (RLKs). We also discuss future challenges in these research fields.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jipb.13061

Protein kinases in plant responses to drought, salt, and cold stress.

Phytohormone abscisic acid (ABA) induces anthocyanin biosynthesis; however, the underlying molecular mechanism is less known. In this study, we found that the apple MYB transcription factor MdMYB1 activated anthocyanin biosynthesis in response to ABA. Using a yeast screening technique, we isolated MdbZIP44, an ABA-induced bZIP transcription factor in apple, as a co-partner with MdMYB1. MdbZIP44 promoted anthocyanin accumulation in response to ABA by enhancing the binding of MdMYB1 to the promoters of downstream target genes. Furthermore, we identified MdBT2, a BTB protein, as an MdbZIP44-interacting protein. A series of molecular, biochemical, and genetic analysis suggested that MdBT2 degraded MdbZIP44 protein through the Ubiquitin-26S proteasome system, thus inhibiting MdbZIP44-modulated anthocyanin biosynthesis. Taken together, we reveal a novel working mechanism of MdbZIP44-mediated anthocyanin biosynthesis in response to ABA.

Apple bZIP transcription factor MdbZIP44 regulates abscisic acid-promoted anthocyanin accumulation.

Sugars play important roles in plant growth and development, crop yield and quality, as well as responses to abiotic stresses. Abscisic acid (ABA) is a multifunctional hormone. However, the exact mechanism by which ABA regulates sugar accumulation is largely unknown in plants. Here, we tested the expression profile of several sugar transporter and amylase genes in response to ABA treatment. MdSUT2 and MdAREB2 were isolated and genetically transformed into apple (Malus domestica) to investigate their roles in ABA-induced sugar accumulation. The MdAREB2 transcription factor was found to bind to the promoters of the sugar transporter and amylase genes and activate their expression. Both MdAREB2 and MdSUT2 transgenic plants produced more soluble sugars than controls. Furthermore, MdAREB2 promoted the accumulation of sucrose and soluble sugars in an MdSUT2-dependent manner. Our results demonstrate that the ABA-responsive transcription factor MdAREB2 directly activates the expression of amylase and sugar transporter genes to promote soluble sugar accumulation, suggesting a mechanism by which ABA regulates sugar accumulation in plants.

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Transcription Factor AREB2 Is Involved in Soluble Sugar Accumulation by Activating Sugar Transporter and Amylase Genes

Soil salinity hinders the growth of most higher plants and becomes a gradually increasing threat to the agricultural production of such crops as the woody plant apple. In this study, a calcineurin B-like protein (CBL)-interacting protein kinase, MdCIPK24-LIKE1 (named as MdSOS2L1), was identified. Quantitative real-time polymerase chain reaction (qRT-PCR) assay revealed that the expression of MdSOS2L1 was upregulated by CaCl2 . Yeast two-hybrid (Y2H) assay and transiently transgenic analysis demonstrated that the MdSOS2L1 protein kinase physically interacted with MdCBL1, MdCBL4 and MdCBL10 proteins to increase salt tolerance in apple. Furthermore, iTRAQ proteome combined with liquid chromatography-tandem mass spectrometry (LC/MS) analysis found that several proteins, which are involved in reactive oxygen species (ROS) scavenging, procyanidin biosynthesis and malate metabolism, were induced in MdSOS2L1-overexpressing apple plants. Subsequent studies have shown that MdSOS2L1 increased antioxidant metabolites such as procyanidin and malate to improve salt tolerance in apple and tomato. In summary, our studies provide a mechanism in which SOS2L1 enhances the salt stress tolerance in apple and tomato.

Overexpression of MdSOS2L1, a CIPK protein kinase, increases the antioxidant metabolites to enhance salt tolerance in apple and tomato

Sugars increase with drought stress in plants and accumulate in the vacuole. However, the exact molecular mechanism underlying this process is not clear yet. In this study, protein interaction and phosphorylation experiments were conducted for sucrose transporter and CIPK kinase in apple. The specific phosphorylation site of sucrose transporter was identified with mass spectrometry. Transgenic analyses were performed to characterize their biological function. It was found that overexpression of sucrose transporter gene MdSUT2.2 in apple plants promoted sugar accumulation and drought tolerance. MdSUT2.2 protein was phosphorylated at Ser381 site in response to drought. A DUALmembrane system using MdSUT2.2 as bait through an apple cDNA library got a protein kinase MdCIPK22. Bimolecular fluorescence complementary (BiFC), pull-down and co-immunoprecipitation (Co-IP) assays further demonstrated that MdCIPK22 interacted with MdSUT2.2. A series of transgenic analysis showed that MdCIPK22 was required for the drought-induced phosphylation at Ser381 site of MdSUT2.2 protein, and that it enhanced the stability and transport activity of MdSUT2.2 protein. Finally, it was found that MdCIPK22 overexpression promoted sugar accumulation and improved drought tolerance in an MdSUT2.2-dependent manner in transgenic apple plants. MdCIPK22-MdSUT2.2 regulatory module shed light on the molecular mechanism by which plant accumulates sugars and enhances tolerance in response to drought stress.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13003

An apple sucrose transporter MdSUT2.2 is a phosphorylation target for protein kinase MdCIPK22 in response to drought

Phytohormone abscisic acid (ABA) regulates many important processes in plants. It is a major molecule facilitating signal transduction during the abiotic stress response. In this study, an ABA-inducible transcription factor gene, MdAREB2, was identified in apple. Transgenic analysis was performed to characterize its function in ABA sensitivity. Overexpression of the MdAREB2 gene increased ABA sensitivity in the transgenic apple compared with the wild-type (WT) control. In addition, it was found that the protein MdAREB2 was phosphorylated at a novel site Thr411 in response to ABA. A yeast two-hybridization screen of an apple cDNA library demonstrated that a protein kinase, MdCIPK22, interacted with MdAREB2. Their interaction was further verified with Pull Down and Co-IP assays. A series of transgenic analyses in apple calli and plantlets showed that MdCIPK22 was required for ABA-induced phosphorylation at Thr411 of the MdAREB2 protein and enhanced its stability and transcriptional activity. Finally, it was found that MdCIPK22 increased ABA sensitivity in an MdAREB2-dependent manner. Our findings indicate a novel phosphorylation site in CIPK-AREB regulatory module for the ABA signalling pathway, which would be helpful for researchers to identify the functions of uncharacterized homologs in the future.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/pce.13013

An apple CIPK protein kinase targets a novel residue of AREB transcription factor for ABA-dependent phosphorylation.

Soil salinity is one of the major abiotic stressors that negatively affect crop growth and yield. Salt stress can regulate antioxidants and the accumulation of osmoprotectants. In the study, a sucrose transporter MdSUT2.2 was identified in apple. Overexpression of MdSUT2.2 gene increased salt tolerance in the transgenic apple, compared with the WT control "Gala." In addition, it was found that protein MdSUT2.2 was phosphorylated at Ser254 site in response to salt. A DUAL membrane yeast hybridization system through an apple cDNA library demonstrated that a protein kinase MdCIPK13 interacted with MdSUT2.2. A series of transgenic analysis in apple calli showed that MdCIPK13 was required for the salt-induced phosphorylation of MdSUT2.2 protein and enhanced its stability and transport activity. Finally, it was found that MdCIPK13 improved salt resistance in an MdSUT2.2-dependent manner. These findings had enriched our understanding of the molecular mechanisms underlying abiotic stress.

Mei-Hong Sun

Papers

Transcription Factor AREB2 Is Involved in Soluble Sugar Accumulation by Activating Sugar Transporter and Amylase Genes

Overexpression of MdSOS2L1, a CIPK protein kinase, increases the antioxidant metabolites to enhance salt tolerance in apple and tomato

An apple sucrose transporter MdSUT2.2 is a phosphorylation target for protein kinase MdCIPK22 in response to drought

An apple CIPK protein kinase targets a novel residue of AREB transcription factor for ABA-dependent phosphorylation.

A CIPK protein kinase targets sucrose transporter MdSUT2.2 at Ser254 for phosphorylation to enhance salt tolerance