The ubiquitin E3 ligase RZFP1 affects drought tolerance in poplar by mediating the degradation of the protein phosphatase PP2C-9.

Abscisic acid signaling has been implicated in plant responses to water deficit-induced osmotic stress. However, the underlying molecular mechanism remains unelucidated. This study identified the RING-type E3 ubiquitin ligase RING ZINC FINGER PROTEIN1 (PtrRZFP1) in poplar (Populus trichocarpa), a woody model plant. PtrRZFP1 encodes a ubiquitin E3 ligase that participates in protein ubiquitination. PtrRZFP1 mainly functions in the nucleus and endoplasmic reticulum and is activated by drought and abscisic acid. PtrRZFP1-overexpressing transgenic poplars (35S:PtrRZFP1) showed greater tolerance to drought, whereas PtrRZFP1-knockdown lines (KD-PtrRZFP1) showed greater sensitivity to drought. Under treatment with polyethylene glycol and abscisic acid, PtrRZFP1 promoted the production of NO and H2O2 in stomatal guard cells, ultimately enhancing stomatal closure and improving drought tolerance. Additionally, PtrRZFP1 physically interacted with the clade A Protein Phosphatase 2C protein PtrPP2C-9, a core regulator of abscisic acid signaling, and mediated its ubiquitination and eventual degradation through the ubiquitination-26S proteasome system, indicating that PtrRZFP1 positively regulates the abscisic acid signaling pathway. Furthermore, the PtrPP2C-9-overexpression line was insensitive to abscisic acid and more sensitive to drought than the wild-type plants, whereas the opposite phenotype was observed in 35S:PtrRZFP1 plants. In general, PtrRZFP1 negatively regulates the stability of PtrPP2C-9 to mediate poplar drought tolerance. The results of this study provide a theoretical framework for the targeted breeding of drought-tolerant traits in perennial woody plants.

The JA-to-ABA signaling relay promotes lignin deposition for wound healing in Arabidopsis.

Plants are frequently exposed to herbivory and mechanical damage that results in wounding. Two fundamental strategies, regeneration and healing, are employed by plants upon wounding. It is not fully understood how plants make different decisions, and how wound healing is sustained until the damaged tissues recover. In this study, we find that the local auxin accumulation patterns, determined by wounding modes, may activate different recovery programs in wounded tissues. Wounding triggers a transient jasmonic acid (JA) signaling that promotes lignin deposition in the first few hours after wounding occurs. This early response is subsequently relayed to ABA signaling via MYC2. The induced JA signaling promotes ABA biosynthesis to maintain the expression of RAP2.6, a key factor for sustained lignin biosynthesis and the later wound healing process. Our findings provide mechanistic insights into how plants heal from wounding and elucidate the molecular mechanisms underlying the prolonged healing process following wounding.

TaMYB44-5A reduces drought tolerance by repressing transcription of TaRD22-3A in the abscisic acid signaling pathway.

MAIN CONCLUSION: TaMYB44-5A identified as a transcription factor negatively regulates drought tolerance in transgenic Arabidopsis. Drought can severely reduce yields throughout the wheat-growing season. Many studies have shown that R2R3-MYB transcription factors are involved in drought stress responses. In this study, the R2R3-MYB transcription factor MYB44-5A was identified in wheat (Triticum aestivum L.) and functionally analyzed. Three homologs of TaMYB44 were isolated, all of which localized to the nucleus. Overexpression of TaMYB44-5A reduced drought tolerance in Arabidopsis thaliana. Further analysis showed that TaMYB44-5A reduced the sensitivity of transgenic Arabidopsis to ABA. Genetic and transcriptional regulation analyses demonstrated that the expression levels of drought- and ABA-responsive genes were downregulated by TaMYB44-5A, and TaMYB44-5A directly bound to the MYB-binding site on the promoter to repress the transcription level of TaRD22-3A. Our results provide insights into a novel molecular pathway in which the R2R3-MYB transcription factor negatively regulates ABA signaling in response to drought stress.

Pepper SBP-box transcription factor, CaSBP13, plays a negatively role in drought response.

The SBP-box gene significantly influences plant growth, development, and stress responses, yet its function in pepper plants during drought stress remains unexplored. Using virus-induced gene silencing and overexpression strategies, we examined the role of CaSBP13 during drought stress in plants. The results revealed that the expression of CaSBP13 can be induced by drought stress. Silencing of CaSBP13 in pepper notably boosted drought resistance, as evident by decreased active oxygen levels. Furthermore, the water loss rate, relative electrical conductivity, malondialdehyde content, and stomatal density were reduced in CaSBP13-silenced plants compared to controls. In contrast, CaSBP13 overexpression in Nicotiana benthamiana decreased drought tolerance with elevated reactive oxygen levels and stomatal density. Additionally, ABA signaling pathway genes (CaPP2C, CaAREB) exhibited reduced expression levels in CaSBP13-silenced plants post drought stress, as compared to control plants. On the contrary, CaPYL9 and CaSNRK2.4 showed heightened expression in CaSBP13-sienced plants under the same conditions. However, a converse trend for NbAREB, NbSNRK2.4, and NbPYL9 was observed post-four day drought in CaSBP13-overexpression plants. These findings suggest that CaSBP13 negatively regulates drought tolerance in pepper, potentially via ROS and ABA signaling pathways.

Modulation of abscisic acid signaling via endosomal TOL proteins.

The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Collectively our findings show that discrete TOM1-LIKE (TOL) proteins, which are functional ESCRT-0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery. TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters. TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA-controlled stomatal aperture. Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought-tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance.

SOS2-AFP2 module regulates seed germination by inducing ABI5 degradation in response to salt stress in Arabidopsis.

Soil salinity pose a significant challenge to global agriculture, threatening crop yields and food security. Understanding the salt tolerance mechanisms of plants is crucial for improving their survival under salt stress. AFP2, a negative regulator of ABA signaling, has been shown to play a crucial role in salt stress tolerance during seed germination. Mutations in AFP2 gene lead to increased sensitivity to salt stress. However, the underline mechanisms by which AFP2 regulates seed germination under salt stress remain elusive. In this study, we identified a protein interaction between AFP2 and SOS2, a Ser/Thr protein kinase known to play a critical role in salt stress response. Using a combination of genetic, biochemical, and physiological approaches, we investigated the role of the SOS2-AFP2 module in regulating seed germination under salt stress. Our findings reveal that SOS2 physically interacts with AFP2 and stabilizes it, leading to the degradation of the ABI5 protein, a negative transcription factor in seed germination under salt stress. This study sheds light on previously unknown connections within salt stress and ABA signaling, paving the way for novel strategies to enhance plant resilience against environmental challenges.

ZmASR1 negatively regulates drought stress tolerance in maize.

Abscisic acid-, stress-, and ripening-induced (ASR) proteins in plants play a significant role in plant response to diverse abiotic stresses. However, the functions of ASR genes in maize remain unclear. In the present study, we identified a novel drought-induced ASR gene in maize (ZmASR1) and functionally characterized its role in mediating drought tolerance. The transcription of ZmASR1 was upregulated under drought stress and abscisic acid (ABA) treatment, and the ZmASR1 protein was observed to exhibit nuclear and cytoplasmic localization. Moreover, ZmASR1 knockout lines generated with the CRISPR-Cas9 system showed lower ROS accumulation, higher ABA content, and a higher degree of stomatal closure than wild-type plants, leading to higher drought tolerance. Transcriptome sequencing data indicated that the significantly differentially expressed genes in the drought treatment group were mainly enriched in ABA signal transduction, antioxidant defense, and photosynthetic pathway. Taken together, the findings suggest that ZmASR1 negatively regulates drought tolerance and represents a candidate gene for genetic manipulation of drought resistance in maize.

Novel nucleus-localized GRAM protein encoding OsGRAM57 gene enhances salt tolerance through ABA-dependent pathway and modulated carbohydrate metabolism.

GRAM (Glucosyltransferases-like GTPase activators and Myotubularin) domain-encoding proteins play pivotal roles in plant growth and responses to biotic stresses. Yet, their influence on abiotic stress responses has remained enigmatic. This study unveils a novel nucleus-localized OsGRAM57, a GRAM protein-encoding gene and its profound regulatory functions in enhancing salt stress tolerance using Arabidopsis thaliana as a model plant. OsGRAM57-OEX (OsGRAM57-OEX) lines displayed significant enhancement in salt tolerance, modulated physiological, biochemical, K+/Na+ ratios, and enzymatic indices as compared to their wild-type (WT). Furthermore, OsGRAM57-OEX seedlings demonstrate increased levels of endogenous abscisic acid (ABA) and other phytohormones, while metabolic profiling revealed enhanced carbohydrate metabolism. Delving into the ABA signaling pathway, OsGRAM57 emerged as a central regulator, orchestrating the expression of genes crucial for salt stress responses, carbohydrate metabolism, and ABA signaling. The observed interactions with target genes and transactivation assays provided additional support for OsGRAM57's pivotal role. These findings underscore OsGRAM57's positive influence on the ABA pathway and affirm its capacity to enhance salt tolerance through an ABA-dependent pathway and fine-tuned carbohydrate metabolism. In summary, this new study reveals the previously undiscovered regulatory roles of OsGRAM57 in Arabidopsis abiotic stress responses, offering promising ways for strengthening plant resilience in the face of adverse environmental conditions.

TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics.

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

Transcription Factor McHB7 Improves Ice Plant Drought Tolerance through ABA Signaling Pathway.

As global climate change continues, drought episodes have become increasingly frequent. Studying plant stress tolerance is urgently needed to ensure food security. The common ice plant is one of the model halophyte plants for plant stress biology research. This study aimed to investigate the functions of a newly discovered transcription factor, Homeobox 7 (HB7), from the ice plant in response to drought stress. An efficient Agrobacterium-mediated transformation method was established in the ice plant, where ectopic McHB7 expression may be sustained for four weeks. The McHB7 overexpression (OE) plants displayed drought tolerance, and the activities of redox enzymes and chlorophyll content in the OE plants were higher than the wild type. Quantitative proteomics revealed 1910 and 495 proteins significantly changed in the OE leaves compared to the wild type under the control and drought conditions, respectively. Most increased proteins were involved in the tricarboxylic acid cycle, photosynthesis, glycolysis, pyruvate metabolism, and oxidative phosphorylation pathways. Some were found to participate in abscisic acid signaling or response. Furthermore, the abscisic acid levels increased in the OE compared with the wild type. McHB7 was revealed to bind to the promoter motifs of Early Responsive to Dehydration genes and abscisic acid-responsive genes, and protein-protein interaction analysis revealed candidate proteins responsive to stresses and hormones (e.g., abscisic acid). To conclude, McHB7 may contribute to enhance plant drought tolerance through abscisic acid signaling.

Arabidopsis class II TPS controls root development and confers salt stress tolerance through enhanced hydrophobic barrier deposition.

KEY MESSAGE: The mechanism of conferring salt tolerance by AtTPS9 involves enhanced deposition of suberin lamellae in the Arabidopsis root endodermis, resulting in reduction of Na+ transported to the leaves. Members of the class I trehalose-6-phosphate synthase (TPS) enzymes are known to play an important role in plant growth and development in Arabidopsis. However, class II TPSs and their functions in salinity stress tolerance are not well studied. We characterized the function of a class II TPS gene, AtTPS9, to understand its role in salt stress response and root development in Arabidopsis. The attps9 mutant exhibited significant reduction of soluble sugar levels in the leaves and formation of suberin lamellae (SL) in the endodermis of roots compared to the wild type (WT). The reduction in SL deposition (hydrophobic barriers) leads to increased apoplastic xylem loading, resulting in enhanced Na+ content in the plants, which explains salt sensitivity of the mutant plants. Conversely, AtTPS9 overexpression lines exhibited increased SL deposition in the root endodermis along with increased salt tolerance, showing that regulation of SL deposition is one of the mechanisms of action of AtTPS9 in conferring salt tolerance to Arabidopsis plants. Our data showed that besides salt tolerance, AtTPS9 also regulates seed germination and root development. qRT-PCR analyses showed significant downregulation of selected SNF1-RELATED PROTEIN KINASE2 genes (SnRK2s) and ABA-responsive genes in the mutant, suggesting that AtTPS9 may regulate the ABA-signaling intermediates as part of the mechanism conferring salinity tolerance.

Genome-Wide Identification of PYL/RCAR ABA Receptors and Functional Analysis of LbPYL10 in Heat Tolerance in Goji (Lycium barbarum).

The characterization of the PYL/RCAR ABA receptors in a great deal of plant species has dramatically advanced the study of ABA functions involved in key physiological processes. However, the genes in this family are still unclear in Lycium (Goji) plants, one of the well-known economically, medicinally, and ecologically valuable fruit crops. In the present work, 12 homologs of Arabidopsis PYL/RCAR ABA receptors were first identified and characterized from Lycium (L.) barbarum (LbPYLs). The quantitative real-time PCR (qRT-PCR) analysis showed that these genes had clear tissue-specific expression patterns, and most of them were transcribed in the root with the largest amount. Among the three subfamilies, while the Group I and Group III members were down-regulated by extraneous ABA, the Group II members were up-regulated. At 42 °C, most transcripts showed a rapid and violent up-regulation response to higher temperature, especially members of Group II. One of the genes in the Group II members, LbPYL10, was further functionally validated by virus-induced gene silencing (VIGS) technology. LbPYL10 positively regulates heat stress tolerance in L. barbarum by alleviating chlorophyll degradation, thus maintaining chlorophyll stability. Integrating the endogenous ABA level increase following heat stress, it may be concluded that LbPYL-mediated ABA signaling plays a vital role in the thermotolerance of L. barbarum plants. Our results highlight the strong potential of LbPYL genes in breeding genetically modified L. barbarum crops that acclimate to climate change.

OsSCYL2 is Involved in Regulating ABA Signaling-Mediated Seed Germination in Rice.

Seed germination represents a multifaceted biological process influenced by various intrinsic and extrinsic factors. In the present study, our investigation unveiled the regulatory role of OsSCYL2, a gene identified as a facilitator of seed germination in rice. Notably, the germination kinetics of OsSCYL2-overexpressing seeds surpassed those of their wild-type counterparts, indicating the potency of OsSCYL2 in enhancing this developmental process. Moreover, qRT-PCR results showed that OsSCYL2 was consistently expressed throughout the germination process in rice. Exogenous application of ABA on seeds and seedlings underscored the sensitivity of OsSCYL2 to ABA during both seed germination initiation and post-germination growth phases. Transcriptomic profiling following OsSCYL2 overexpression revealed profound alterations in metabolic pathways, MAPK signaling cascades, and phytohormone-mediated signal transduction pathways, with 15 genes related to the ABA pathways exhibiting significant expression changes. Complementary in vivo and in vitro assays unveiled the physical interaction between OsSCYL2 and TOR, thereby implicating OsSCYL2 in the negative modulation of ABA-responsive genes and its consequential impact on seed germination dynamics. This study elucidated novel insights into the function of OsSCYL2 in regulating the germination process of rice seeds through the modulation of ABA signaling pathways, thereby enhancing the understanding of the functional significance of the SCYL protein family in plant physiological processes.

BcABF1 Plays a Role in the Feedback Regulation of Abscisic Acid Signaling via the Direct Activation of BcPYL4 Expression in Pakchoi.

Abscisic acid-responsive element-binding factor 1 (ABF1), a key transcription factor in the ABA signal transduction process, regulates the expression of downstream ABA-responsive genes and is involved in modulating plant responses to abiotic stress and developmental processes. However, there is currently limited research on the feedback regulation of ABF1 in ABA signaling. This study delves into the function of BcABF1 in Pakchoi. We observed a marked increase in BcABF1 expression in leaves upon ABA induction. The overexpression of BcABF1 not only spurred Arabidopsis growth but also augmented the levels of endogenous IAA. Furthermore, BcABF1 overexpression in Arabidopsis significantly decreased leaf water loss and enhanced the expression of genes associated with drought tolerance in the ABA pathway. Intriguingly, we found that BcABF1 can directly activate BcPYL4 expression, a critical receptor in the ABA pathway. Similar to BcABF1, the overexpression of BcPYL4 in Arabidopsis also reduces leaf water loss and promotes the expression of drought and other ABA-responsive genes. Finally, our findings suggested a novel feedback regulation mechanism within the ABA signaling pathway, wherein BcABF1 positively amplifies the ABA signal by directly binding to and activating the BcPYL4 promoter.

Plant growth-promoting rhizobacteria Pseudomonas aeruginosa HG28-5 improves salt tolerance by regulating Na+/K+ homeostasis and ABA signaling pathway in tomato.

Salinity stress badly restricts the growth, yield and quality of vegetable crops. Plant growth-promoting rhizobacteria (PGPR) is a friendly and effective mean to enhance plant growth and salt tolerance. However, information on the regulatory mechanism of PGPR on vegetable crops in response to salt stress is still incomplete. Here, we screened a novel salt-tolerant PGPR strain Pseudomonas aeruginosa HG28-5 by evaluating the tomatoes growth performance, chlorophyll fluorescence index, and relative electrolyte leakage (REL) under normal and salinity conditions. Results showed that HG28-5 colonization improved seedling growth parameters by increasing the plant height (23.7%), stem diameter (14.6%), fresh and dry weight in the shoot (60.3%, 91.1%) and root (70.1%, 92.5%), compared to salt-stressed plants without colonization. Likewise, HG28-5 increased levels of maximum photochemical efficiency of PSII (Fv/Fm) (99.3%), the antioxidant enzyme activities as superoxide dismutase (SOD, 85.5%), peroxidase (POD, 35.2%), catalase (CAT, 20.6%), and reduced the REL (48.2%), MDA content (41.3%) and ROS accumulation in leaves of WT tomatoes under salt stress in comparison with the plants treated with NaCl alone. Importantly, Na+ content of HG28-5 colonized salt-stressed WT plants were decreased by15.5% in the leaves and 26.6% in the roots in the corresponding non-colonized salt-stressed plants, which may be attributed to the higher K+ concentration and SOS1, SOS2, HKT1;2, NHX1 transcript levels in leaves of colonized plants under saline condition. Interestingly, increased abscisic acid (ABA) content and upregulation of ABA pathway genes (ABA synthesis-related genes NCED1, NCED2, NCED4, NECD6 and signal genes ABF4, ABI5, and AREB) were observed in HG28-5 inoculated salt-stressed WT plants. ABA-deficient mutant (not) with NCED1 deficiency abolishes the effect of HG28-5 on alleviating salt stress in tomato, as exhibited by the substantial rise of REL and ROS accumulation and sharp drop of Fv/Fm in the leaves of not mutant plants. Notably, HG28-5 colonization enhances tomatoes fruit yield by 54.9% and 52.4% under normal and saline water irrigation, respectively. Overall, our study shows that HG28-5 colonization can significantly enhance salt tolerance and improved fruit yield by a variety of plant protection mechanism, including reducing oxidative stress, regulating plant growth, Na+/K+ homeostasis and ABA signaling pathways in tomato. The findings not only deepen our understanding of PGPR regulation plant growth and salt tolerance but also allow us to apply HG28-5 as a microbial fertilizer for agricultural production in high-salinity areas.

Multiomics unravels potential molecular switches in the C3 to CAM transition of Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum (common ice plant), a facultative CAM plant, shifts from C3 to CAM photosynthesis under salt stress, enhancing water use efficiency. Here we used transcriptomics, proteomics, and targeted metabolomics to profile molecular changes during the diel cycle of C3 to CAM transition. The results confirmed expected changes associated with CAM photosynthesis, starch biosynthesis and degradation, and glycolysis/gluconeogenesis. Importantly, they yielded new discoveries: 1) Transcripts displayed greater circadian regulation than proteins. 2) Oxidative phosphorylation and inositol methylation may play important roles in initiating the transition. 3) V-type H+-ATPases showed consistent transcriptional regulation, aiding in vacuolar malate uptake. 4) A protein phosphatase 2C, a major component in the ABA signaling pathway, may trigger the C3 to CAM transition. Our work highlights the potential molecular switches in the C3 to CAM transition, including the potential role of ABA signaling. SIGNIFICANCE: The common ice plant is a model facultative CAM plant, and under stress conditions it can shift from C3 to CAM photosynthesis within a three-day period. However, knowledge about the molecular changes during the transition and the molecular switches enabling the transition is lacking. Multi-omic analyses not only revealed the molecular changes during the transition, but also highlighted the importance of ABA signaling, inositol methylation, V-type H+-ATPase in initiating the shift. The findings may explain physiological changes and nocturnal stomatal opening, and inform future synthetic biology effort in improving crop water use efficiency and stress resilience.

Novel 4-Chromanone-Derived Compounds as Plant Immunity Inducers against CMV Disease in Passiflora spp. (Passion Fruit).

This study involved the design and synthesis of a series of novel 4-chromanone-derived compounds. Their in vivo anti-cucumber mosaic virus (CMV) activity in field trials against CMV disease in Passiflora spp. was then assessed. Bioassay results demonstrated that compounds 7c and 7g exhibited remarkable curative effects and protection against CMV, with inhibition rates of 57.69% and 51.73% and 56.13% and 52.39%, respectively, surpassing those of dufulin and comparable to ningnanmycin. Field trials results indicated that compound 7c displayed significant efficacy against CMV disease in Passiflora spp. (passion fruit) after the third spraying at a concentration of 200 mg/L, with a relative control efficiency of 47.49%, surpassing that of dufulin and comparable to ningnanmycin. Meanwhile, nutritional quality test results revealed that compound 7c effectively enhanced the disease resistance of Passiflora spp., as evidenced by significant increases in soluble protein, soluble sugar, total phenol, and chlorophyll contents in Passiflora spp. leaves as well as improved the flavor and taste of Passiflora spp. fruits, as demonstrated by notable increases in soluble protein, soluble sugar, soluble solid, and vitamin C contents in Passiflora spp. fruits. Additionally, a transcriptome analysis revealed that compound 7c primarily targeted the abscisic acid (ABA) signaling pathway, a crucial plant hormone signal transduction pathway, thereby augmenting resistance against CMV disease in Passiflora spp. Therefore, this study demonstrates the potential application of these novel 4-chromanone-derived compounds as effective inducers of plant immunity for controlling CMV disease in Passiflora spp. in the coming decades.

The GARP family transcription factor MtHHO3 negatively regulates salt tolerance in Medicago truncatula.

High salinity is one of the detrimental environmental factors restricting plant growth and crop production throughout the world. This study demonstrated that the GARP family transcription factor MtHHO3 is involved in response to salt stress and abscisic acid (ABA) signaling in Medicago truncatula. The transcription of MtHHO3 was repressed by salt, osmotic stress, and ABA treatment. The seed germination assay showed that, overexpression of MtHHO3 in Arabidopsis thaliana caused hypersensitivity to salt and osmotic stress, but increased resistance to ABA inhibition. Overexpression of MtHHO3 in M. truncatula resulted in decreased tolerance of salinity, while loss-of-function mutants mthho3-1 and mthho3-2 were more resistant to salt stress compared with wild-type plants. qRT-PCR analyses showed that MtHHO3 downregulated the expression of genes in stress and ABA responsive pathways. We further demonstrated that MtHHO3 repressed the transcription of the pathogenesis-related gene MtPR2 by binding to its promoter. Overall, these results indicate that MtHHO3 negatively regulates salt stress response in plants and deepen our understanding of the role of the GARP subfamily transcription factors in modulating salt stress and ABA signaling.

GhAGL16 (AGAMOUS-LIKE16) Negatively Regulates Tolerance to Water Deficit in Transgenic Arabidopsis and Cotton.

Cotton is one of the most economically important crops in the world, and drought is a key abiotic factor that can significantly reduce cotton yield. MADS-box transcription factors play essential roles in various aspects of plant growth and development as well as responses to biotic and abiotic stress. However, the use of MADS-box transcription factors to regulate water stress responses has not been fully explored in cotton. Here, we showed that GhAGL16 acts as a negative regulator of water deficit in cotton, at least in part by regulating ABA signaling. GhAGL16-overexpressing (GhAGL16-OE) transgenic Arabidopsis had lower survival rates and relative water contents (RWCs) under water stress. Isolated leaves of GhAGL16-OE Arabidopsis had increased water loss rates, likely attributable to their increased stomatal density. GhAGL16-OE Arabidopsis also showed reduced primary root lengths in response to mannitol treatment and decreased sensitivity of seed germination to ABA treatment. By contrast, silencing GhAGL16 in cotton enhanced tolerance to water deficit by increasing proline (Pro) content, increasing superoxide dismutase (SOD) and peroxidase (POD) activities, and reducing malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents under water stress. Subcellular localization and transcriptional activation assays confirmed that GhAGL16 is a nuclear protein that lacks transcriptional self-activation activity. The expression of ABA biosynthesis-related genes (GhNCED3/7/14), a catabolism-related gene (GhCYP707A), and a gene related to the ABA signaling pathway (GhABF4) was altered in GhAGL16-silenced plants. Taken together, our data demonstrate that GhAGL16 plays an important role in cotton resistance to water stress.

Overexpression of NtDOGL4 improves cadmium tolerance through abscisic acid signaling pathway in tobacco.

The DELAY OF GERMINATION1-LIKE (DOGL) genes play an essential role in diverse biological processes in plants. However, their exact involvement in the response to cadmium (Cd) stress via the ABA pathway remains unclear. Here, we focused on NtDOGL4, a tobacco DOGL gene whose expression is highly induced upon exposure to Cd. Overexpression of NtDOGL4 in tobacco resulted in elevated endogenous ABA levels, reduced Cd accumulation, and increased tolerance to Cd. Moreover, NtDOGL4 overexpression led to decreased accumulation of reactive oxygen species (ROS) and improved ROS scavenging capacity under Cd stress. Further analyses revealed the direct binding of the transcription factor ABSCISIC ACID-INSENSITIVE 5 (ABI5) to the NtDOGL4 promoter, positively regulating its expression in tobacco. Notably, NtDOGL4 overexpression promoted suberin formation and deposition, while suppressing the expression of Cd transporter genes in tobacco roots, as evidenced by histochemical staining, suberin fraction determination, and qRT-PCR assays. Collectively, our results demonstrate that NtDOGL4 overexpression reduces Cd accumulation, thereby improving Cd stress tolerance through the modulation of antioxidant system, transcription of Cd transporters, and suberin deposition. Notably, the NtABI5-NtDOGL4 module functions as a positive regulator in tobacco's Cd tolerance, underscoring its potential as a molecular target for developing low-Cd crops to ensure environmental safety.