SUPPRESSOR OF MAX2-LIKE 6, 7, and 8 (SMXL6,7,8) function as repressors and transcription factors of the strigolactone (SL) signaling pathway, playing an important role in the development and stress tolerance in Arabidopsis thaliana. However, the molecular mechanism by which SMXL6,7,8 negatively regulate drought tolerance and ABA response remains largely unexplored. In the present study, the interacting protein and downstream target genes of SMXL6,7,8 were investigated. Our results showed that the substrate receptor for the CUL4-based E3 ligase DDB1-BINDING WD-REPEAT DOMAIN (DWD) HYPERSENSITIVE TO ABA DEFICIENT 1 (ABA1) (DWA1) physically interacted with SMXL6,7,8. The degradation of SMXL6,7,8 proteins were partially dependent on DWA1. Disruption of SMXL6,7,8 resulted in increased drought tolerance and could restore the drought-sensitive phenotype of the dwa1 mutant. In addition, SMXL6,7,8 could directly bind to the promoter of SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASE 2.3 (SnRK2.3) to repress its transcription. The mutations in SnRK2.2/2.3 significantly suppressed the hypersensitivity of smxl6/7/8 to ABA-mediated inhibition of seed germination. Conclusively, SMXL6,7,8 interact with DWA1 to negatively regulate drought tolerance and target ABA-response genes. These data provide insights into drought tolerance and ABA response in Arabidopsis via the SMXL6,7,8-mediated SL signaling pathway.

KEY MESSAGE:AtHSPR forms a complex with KNAT5 and OFP1 to regulate primary root growth through GA-mediated root meristem activity. KNAT5-OFP1 functions as a negative regulator of AtHSPR in response to GA. Plant root growth is modulated by gibberellic acid (GA) signaling and depends on root meristem maintenance. ARABIDOPSIS THALIANA HEAT SHOCK PROTEIN-RELATED (AtHSPR) is a vital regulator of flowering time and salt stress tolerance. However, little is known about the role of AtHSPR in the regulation of primary root growth. Here, we report that athspr mutant exhibits a shorter primary root compared to wild type and that AtHSPR interacts with KNOTTED1-LIKE HOMEOBOX GENE 5 (KNAT5) and OVATE FAMILY PROTEIN 1 (OFP1). Genetic analysis showed that overexpression of KNAT5 or OFP1 caused a defect in primary root growth similar to that of the athspr mutant, but knockout of KNAT5 or OFP1 rescued the short root phenotype in the athspr mutant by altering root meristem activity. Further investigation revealed that KNAT5 interacts with OFP1 and that AtHSPR weakens the inhibition of GIBBERELLIN 20-OXIDASE 1 (GA20ox1) expression by the KNAT5-OFP1 complex. Moreover, root meristem cell proliferation and root elongation in 35S::KNAT5athspr and 35S::OFP1athspr seedlings were hypersensitive to GA3 treatment compared to the athspr mutant. Together, our results demonstrate that the AtHSPR-KNAT5-OFP1 module regulates root growth and development by impacting the expression of GA biosynthetic gene GA20ox1, which could be a way for plants to achieve plasticity in response to the environment.

KEY MESSAGE:Hydrogen sulfide positively regulates autophagy and the expression of hypoxia response-related genes under submergence to enhance the submergence tolerance of Arabidopsis. Flooding seriously endangers agricultural production, and it is quite necessary to explore the mechanism of plant response to submergence for improving crop yield. Both hydrogen sulfide (H2S) and autophagy are involved in the plant response to submergence. However, the mechanisms by which H2S and autophagy interact and influence submergence tolerance have not been thoroughly elucidated. Here, we reported that exogenous H2S pretreatment increased the level of endogenous H2S and alleviated plant cell death under submergence. And transgenic lines decreased in the level of endogenous H2S, L-cysteine desulfurase 1 (des1) mutant and 35S::GFP-O-acetyl-L-serine(thiol)lyase A1 (OASA1)/des1-#56/#61, were sensitive to submergence, along with the lower transcript levels of hypoxia response genes, LOB DOMAIN 41 (LBD41) and HYPOXIA RESPONSIVE UNKNOWN PROTEIN 43 (HUP43). Submergence induced the formation of autophagosomes, and the autophagy-related (ATG) mutants (atg4a/4b, atg5, atg7) displayed sensitive phenotypes to submergence. Simultaneously, H2S pretreatment repressed the autophagosome producing under normal conditions, but enhanced this process under submergence by regulating the expression of ATG genes. Moreover, the mutation of DES1 aggravated the sensitivity of des1/atg5 to submergence by reducing the formation of autophagosomes under submergence. Taken together, our results demonstrated that H2S alleviated cell death through regulating autophagy and the expression of hypoxia response genes during submergence in Arabidopsis.

As the global climate changes, the frequency and severity of flood disasters have increased significantly, seriously affecting agricultural production. Hydrogen sulfide (H2S), as a gaseous signal molecule, plays an important role in plant development and stress adaptation. Previous studies suggested that H2S acts as an oxygen sensor when animals experience hypoxia. However, the role of H2S in the response and adaptation to flooding-induced hypoxia stress in plants is poorly understood. Here, we report that pretreatment with exogenous H2S or Cys significantly improved tolerance to submergence in Arabidopsis. Total submergence of soil-grown Arabidopsis increased the levels of endogenous H2S and its main endogenously generated product, cysteine (Cys) over time, and altered the expression of H2S or Cys biosynthesis- or metabolism-related genes, implying that the H2S-Cys homeostasis may contribute to regulation of tolerance to flooding. Transcriptomic and metabolomic analyses demonstrated that exogenous H2S-pretreatment activated hypoxia-responsive transcription factors and hypoxia sensing-related genes in a timely manner and limited cellular activities associated with growth through regulation of hormonal signaling, but had less influence on transcripts associated with photosynthesis. H2S-pretreatment also altered the levels of metabolites, particularly of amino acids and plant hormones, during submergence. Taken together, this study provides new insight into how H2S regulates tolerance to submergence-induced hypoxia stress and lays the foundation to design new strategies for the improvement of submergence resistance in crops.

Hydrogen sulfide (H2S), once recognized only as a poisonous gas, is now considered the third endogenous gaseous transmitter, along with nitric oxide (NO) and carbon monoxide (CO). Multiple lines of emerging evidence suggest that H2S plays positive roles in plant growth and development when at appropriate concentrations, including seed germination, root development, photosynthesis, stomatal movement, and organ abscission under both normal and stress conditions. H2S influences these processes by altering gene expression and enzyme activities, as well as regulating the contents of some secondary metabolites. In its regulatory roles, H2S always interacts with either plant hormones, other gasotransmitters, or ionic signals, such as abscisic acid (ABA), ethylene, auxin, CO, NO, and Ca2+. Remarkably, H2S also contributes to the post-translational modification of proteins to affect protein activities, structures, and sub-cellular localization. Here, we review the functions of H2S at different stages of plant development, focusing on the S-sulfhydration of proteins mediated by H2S and the crosstalk between H2S and other signaling molecules.

The NAC (NAM, ATAF1/2, and CUC2) family of proteins is one of the largest plant-specific transcription factor (TF) families and its members play varied roles in plant growth, development, and stress responses. In recent years, NAC TFs have been demonstrated to participate in crop-pathogen interactions, as positive or negative regulators of the downstream defense-related genes. NAC TFs link signaling pathways between plant hormones, including salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA), or other signals, such as reactive oxygen species (ROS), to regulate the resistance against pathogens. Remarkably, NAC TFs can also contribute to hypersensitive response and stomatal immunity or can be hijacked as virulence targets of pathogen effectors. Here, we review recent progress in understanding the structure, biological functions and signaling networks of NAC TFs in response to pathogens in several main food crops, such as rice, wheat, barley, and tomato, and explore the directions needed to further elucidate the function and mechanisms of these key signaling molecules.

Flowering is a dynamic and synchronized process, the timing of which is finely tuned by various environmental signals. A T-DNA insertion mutant in Arabidopsis HEAT SHOCK PROTEIN-RELATED (AtHSPR) exhibited late-flowering phenotypes under both long-day (LD) and short-day (SD) conditions compared to the wild-type, while over-expression of AtHSPR promoted flowering. Exogenous application of gibberellin (GA) partially rescued the late-flowering mutant phenotype under both LD and SD conditions, suggesting that AtHSPR is involved in GA biosynthesis and/or the GA signaling that promotes flowering. Under SD or low-light conditions, the Athspr mutant exhibited late flowering together with reduced pollen viability and seed set, defective phenotypes that were partially rescued by GA treatment. qRT-PCR assays confirmed that GA biosynthetic genes were down-regulated, that GA catabolic genes were up-regulated, and that the levels of bioactive GA and its intermediates were decreased in Athspr under both SD and low-light/LD, further suggesting that AtHSPR could be involved in the GA pathway under SD and low-light conditions. Furthermore, AtHSPR interacted in vitro with OFP1 and KNAT5, which are transcriptional repressors of GA20ox1 in GA biosynthesis. Taken together, our findings demonstrate that AtHSPR plays a positive role in GA- and light intensity-mediated regulation of flowering and seed set.

Drought represents a major threat to crop growth and yields. Strigolactones (SLs) contribute to regulating shoot branching by targeting the SUPPRESSOR OF MORE AXILLARY GROWTH2 (MAX2)-LIKE6 (SMXL6), SMXL7 and SMXL8 for degradation in a MAX2-dependent manner in Arabidopsis. Although SLs are implicated in plant drought response, the functions of the SMXL6, 7 and 8 in the SL-regulated plant response to drought stress have remained unclear. Here, we performed transcriptomic, physiological and biochemical analyses of smxl6, 7, 8 and max2 plants to understand the basis for SMXL6/7/8-regulated drought response. We found that three D53 (DWARF53)-Like SMXL members, SMXL6, 7 and 8, are involved in drought response as the smxl6smxl7smxl8 triple mutants showed markedly enhanced drought tolerance compared to wild type (WT). The smxl6smxl7smxl8 plants exhibited decreased leaf stomatal index, cuticular permeability and water loss, and increased anthocyanin biosynthesis during dehydration. Moreover, smxl6smxl7smxl8 were hypersensitive to ABA-induced stomatal closure and ABA responsiveness during and after germination. In addition, RNA-sequencing analysis of the leaves of the D53-like smxl mutants, SL-response max2 mutant and WT plants under normal and dehydration conditions revealed an SMXL6/7/8-mediated network controlling plant adaptation to drought stress via many stress- and/or ABA-responsive and SL-related genes. These data further provide evidence for crosstalk between ABA- and SL-dependent signaling pathways in regulating plant responses to drought. Our results demonstrate that SMXL6, 7 and 8 are vital components of SL signaling and are negatively involved in drought responses, suggesting that genetic manipulation of SMXL6/7/8-dependent SL signaling may provide novel ways to improve drought resistance.

Strigolactones (SLs) and karrikins (KARs) are both butenolide molecules that play essential roles in plant growth and development. SLs are phytohormones, with SLs having known functions within the plant they are produced in, while KARs are found in smoke emitted from burning plant matter and affect seeds and seedlings in areas of wildfire. It has been suggested that SL and KAR signaling may share similar mechanisms. The α/β hydrolases DWARF14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2), which act as receptors of SL and KAR, respectively, both interact with the F-box protein MORE AXILLARY GROWTH 2 (MAX2) in order to target SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE/D53 family members for degradation via the 26S proteasome. Recent reports suggest that SLs and/or KARs are also involved in regulating plant responses and adaptation to various abiotic stresses, particularly nutrient deficiency, drought, salinity, and chilling. There is also crosstalk with other hormone signaling pathways, including auxin, gibberellic acid (GA), abscisic acid (ABA), cytokinin (CK), and ethylene (ET), under normal and abiotic stress conditions. This review briefly covers the biosynthetic and signaling pathways of SLs and KARs, compares their functions in plant growth and development, and reviews the effects of any crosstalk between SLs or KARs and other plant hormones at various stages of plant development. We also focus on the distinct responses, adaptations, and regulatory mechanisms related to SLs and/or KARs in response to various abiotic stresses. The review closes with discussion on ways to gain additional insights into the SL and KAR pathways and the crosstalk between these related phytohormones.

Hydrogen peroxide (H2O2) is the key factor in many physiological and metabolic processes in plants. During seed germination, exogenous H2O2 application influences gravitropism and induces curvature of the primary root in grass pea and pea seedlings. However, it remains unclear whether and how this happens in the model plant Arabidopsis thaliana. In the present study, the effect of exogenous H2O2 on the gravitropic response of primary roots during Arabidopsis seed germination was studied using histology and molecular biology approaches. Appropriate H2O2 treatment not only restrained primary root growth, but also disrupted gravitropism and induced root curvature. Histological staining and molecular analysis demonstrated that exogenous H2O2 correlated with lack of starch-dense amyloplasts in root tip columella cells, which ultimately results in the lack of gravisensing. Detection of calcium ion (Ca2+) by a fluorescent probe showed that Ca2+ distribution changed and intracellular Ca2+ concentration increased in H2O2-treated primary root, which was consistent with alterations in auxin distribution and concentration triggered by H2O2 treatment. Furthermore, the normally polar localization of Pin-formed 1 (PIN1) and PIN2 became uniformly distributed on root tip cell membranes after treatment with H2O2. This leads to speculation that the IAA signaling pathway was affected by exogenous H2O2, causing asymmetrical distribution of IAA on both sides of the primary root, which would influence the gravitropic response.

To understand the functions of Populus euphratica CPD (PeCPD) and DWF4 (PeDWF4), the responses to exogenous phytohormone in Arabidopsis-PeCPD and -PeDWF4 transgenic lines (PeCPD-TL and PeDWF4-TL) and corresponding wild type (WT) seedlings were investigated. Results showed that all of PeCPD-TL, PeDWF4-TL and WT seedlings cultured on the mediums containing 2,4-dichlorophenoxyacetic acid (2,4-D) + 6-benzylaminopurin (6-BA) or 2,4-D + 6-BA + brassinolide (BL) could be dedifferentiated to callus with 100% frequency, but they displayed strong differences in callus formation sites, callus growth rates (CGRs) and tissue dedifferentiation degrees. On the medium containing 2,4-D alone, the seedlings of all the plants could formed callus, but callus formation times (CFTs) were delayed, and callus formation rates (CFRs) were differentially decreased. After adding lower concentrations of BL, their CFRs were all restored to 100%, but tissue dedifferentiation degrees were obviously lower than these on the mediums with 2,4-D + 6-BA or 2,4-D + 6-BA + BL. On the mediums containing 6-BA or 6-BA + BL, the seedlings of all the plants failed to produce callus. Semi-quantitative RT-PCR analysis also showed that the transcription levels of PeCPD, PeDWF4, AtCPD and AtDWF4 in PeCPD-TL, PeDWF4-TL and WT were evidently different. These results suggest that PeCPD and PeDWF4 play similar but not exactly the same roles in the regulation of callus morphogenesis of Arabidopsis seedlings, and that BL can partially replace the role of cytokinin to induce callus formation through interacting with auxin.

Our data present H 2 S in a new role, serving as a multi-faceted transducer to different response mechanisms during NO-induced acquisition of tolerance to flooding-induced hypoxia in maize seedling roots.

DWF4 and CPD are key brassinosteroids (BRs) biosynthesis enzyme genes. To explore the function of Populus euphratica DWF4 (PeDWF4) and CPD (PeCPD), Arabidopsis thaliana transgenic lines (TLs) expressing PeDWF4, PeCPD or PeDWF4 plus PeCPD, namely PeDWF4-TL, PeCPD-TL and PeCP/DW-TL, were characterized. Compared with wild type (WT), the changes of both PeDWF4-TL and PeCPD-TL in plant heights, silique and hypocotyls lengths and seed yields were similar, but in bolting time and stem diameters, they were opposite. PeCP/DW-TL was more in plant heights and the lengths of primary root, silique, and fruit stalk, but less in silique numbers and seed yields than either PeDWF4-TL or PeCPD-TL. PeDWF4 and PeCPD specially expressed in PeDWF4-TL or PeCPD-TL, and the transcription level of PeDWF4 was higher than that of PeCPD. In PeCP/DW-TL, their expressions were all relatively reduced. Additionally, the expression of PeDWF4 and PeCPD differentially made the expression levels of AtDWF4, AtCPD, AtBR6OX2, AtFLC, AtTCP1 and AtGA5 change in the TLs. The total BRs contents were PeDWF4-TL > PeCP/DW-TL > WT > PeCPD-TL. These results imply that PeDWF4 is functionally not exactly the same as PeCPD and there may be a synergistic and antagonistic effects in physiology between both of them in the regulation of plant growth and development.

Flooding stress constrains crop growth and yield because most agricultural species are flood-sensitive. However, many of the plant species that live in permanently or temporarily flooded habitats have evolved specific traits to cope with these harsh conditions. Grass pea (Lathyrus sativus L.) is a legume that tolerates stresses such as drought, diseases, and pests; however, it is unclear whether grass pea has a tolerance mechanism for flooding stress. To understand if grass pea tolerates hypoxia and how it deals with hypoxic stress, the effects of hypoxia on root tip death, physiological, and morpho-anatomical alterations in grass pea and pea (Pisum sativum), which is sensitive to hypoxia, were compared. The results showed that activities of antioxidant enzymes, namely superoxide dismutase, catalase, ascorbate peroxidase, and glutathione content in grass pea were greater than in pea during hypoxia, which protected the root tip from oxidative damage and reduced ion leakage, which helped maintain membrane integrity. Furthermore, aerenchyma and lateral root development accompanied by ethylene production, moderate ROS accumulation-mediated cell death, and Ca2+ spatial-temporal heterogeneity developed well in grass pea compared to pea, which may not only facilitate internal gas diffusion but also promote removal of toxic by-products under hypoxic conditions. These results demonstrate that grass pea is more tolerant to hypoxic stress induced by flooding than garden pea seedlings. This discovery not only provides significant information for understanding the hypoxia-tolerant mechanisms in plants, but also promotes the usability of grass pea in flood-prone areas.

Salt stress is a harmful and global abiotic stress to plants and has an adverse effect on all physiological processes of plants. Recently, we cloned and identified a novel AtHSPR (Arabidopsis thaliana Heat Shock Protein Related), which encodes a nuclear-localized protein with ATPase activity, participates in salt and drought tolerance in Arabidopsis. Transcript profiling analysis revealed a differential expression of genes involved in accumulation of reactive oxygen species (ROS), abscisic acid (ABA) signaling, stress response and photosynthesis between athspr mutant and WT under salt stress. Here, we provide further analysis of the data showing the regulation of salt-induced cell death and endoplasmic reticulum (ER) stress response in Arabidopsis and propose a hypothetical model for the role of AtHSPR in the regulation of the salt tolerance in Arabidopsis.

PttKN1 (Populus tremula × tremuloides KNOTTED1) gene plays an important role in plant morphological formation. To investigate the gene functions, it was introduced into tobacco using the leaf disc method. The transgenic tobacco lines displayed various morphologies different from those of wild type. The results of RT-PCR suggested that these morphological alterations were caused by the ectopic expression of the PttKN1 gene. Notably, almost all the transgenic lines displayed phenotypes sporadically except for one, which appeared with wrinkled leaves throughout the plant growth. The plant grow two round stems compared to one of the wild type, and have mild, intermediate or severe wrinkled leaves distributed on both of the stems. The results of RT-PCR, hiTAIL-PCR and Southern blot analysis indicated that the foreign PttKN1 gene was integrated into the genome of the plant by the way of direct repeats with two copies. Further study on endogenous hormonal levels from HPLC showed that the CK (cytokinin) level was increased and GA (gibberellins) level was decreased in the transgenic line. Correspondingly, the expression of GA 20-oxidase gene that related to GA biosynthesis was down-regulated. These data demonstrated that the ectopic expression of the PttKN1 gene induced aberrant leaf morphology and endogenous hormonal levels in transgenic tobacco, which would enhance our understanding on the functions of PttKN1 gene in leaf development.

Salt stress from soil or irrigation water limits plant growth. A T-DNA insertion mutant in C24, named athspr (Arabidopsis thaliana heat shock protein-related), showed several phenotypes, including reduced organ size and enhanced sensitivity to environmental cues. The athspr mutant is severely impaired under salinity levels at which wild-type (WT) plants grow normally. AtHSPR encodes a nuclear-localized protein with ATPase activity, and its expression was enhanced by high salinity and abscisic acid (ABA). Overexpression (OE) of AtHSPR significantly enhanced tolerance to salt stress by increasing the activities of the antioxidant system and by maintaining K+/Na+ homeostasis. Quantitative RT-PCR analyses showed that OE of AtHSPR increased the expression of ABA/stress-responsive, salt overly sensitive (SOS)-related and antioxidant-related genes. In addition, ABA content was reduced in athspr plants with or without salt stress, and exogenous ABA restored WT-like salt tolerance to athspr plants. athspr exhibited increased leaf stomatal density and stomatal index, slower ABA-induced stomatal closure and reduced drought tolerance relative to the WT. AtHSPR OE enhanced drought tolerance by reducing leaf water loss and stomatal aperture. Transcript profiling in athspr showed a differential salt-stress response for genes involved in accumulation of reactive oxygen species (ROS), ABA signaling, cell death, stress response and photosynthesis. Taken together, our results suggested that AtHSPR is involved in salt tolerance in Arabidopsis through modulation of ROS levels, ABA-dependent stomatal closure, photosynthesis and K+/Na+ homeostasis.

The vascular system--xylem, phloem and the cambium--is essential for water supply, nutrient transport, and physical support in higher plants. Although it is known that vascular-specific gene expression is regulated by cis-acting regulatory sequences in promoters, it is largely unknown how many regulatory elements exist and what their roles are in promoters. To understand the regulatory elements of vascular-specific promoters and their roles in vascular development, a T-DNA insertion mutant showing delayed growth and diminished resistance to environmental stress was isolated using promoter trap strategy. The novel gene, Arabidopsis thaliana heat shock protein-related (Athspr), was cloned from Arabidopsis ecotype C24. Strong GUS (β-glucuronidase) staining in the original promoter trap line was found in the vascular tissues of all organs in the mutant. The Athspr promoter was cloned and fused with GUS and eGFP (enhanced green fluorescent protein) reporter genes to verify its vascular-specific expression in Arabidopsis. Further histochemical analysis in transgenic plants demonstrated a similar GUS expression pattern in the vascular tissues. In addition, ATHSPR-eGFP driven by Athspr promoter was observed in vascular bundles of the transgenic seedling roots. Finally, comparative analysis with promoter motifs from 37 genes involved in vascular development revealed that Athspr and all other promoters active in vascular tissues contained regulatory elements responding to phytohormones, light, biotic and abiotic stresses, as well as those regulating tissue-specific expression. These results demonstrated that the Athspr promoter has a vascular tissue-specific activity and Athspr may have multiple functions in vascular development and resistance against various stresses.

以山黧豆（ Lathyrus sativus L．）幼苗为试验材料，施加H2 O2处理山黧豆初生根，然后检测初生根中H2 O2含量变化，并采用双向电泳与质谱鉴定技术，对初生根中蛋白质表达的变化进行分析。结果显示：施加H2 O2处理引起了内源H2 O2水平的产生与积累；利用双向电泳技术，共获得了850～900个蛋白质点，并检测到70个蛋白质表现出显著的差异表达，其中43个蛋白发生上调，21个蛋白发生下调，新出现6个蛋白。对15个蛋白点MALDI-TOF-MS／MS质谱鉴定结果表明，呼吸代谢、蛋白折叠、信号转导及细胞防御等方面的蛋白参与了山黧豆初生根对H2 O2的应答反应。这些蛋白的发现将有助于进一步揭示根对氧化胁迫的应答及H2 O2作用机制。