Tobacco Transcription Factor NtWRKY70b Facilitates Leaf Senescence via Inducing ROS Accumulation and Impairing Hydrogen Sulfide Biosynthesis.

Leaf senescence is the terminal stage of leaf development, and its initiation and progression are closely controlled by the integration of a myriad of endogenous signals and environmental stimuli. It has been documented that WRKY transcription factors (TFs) play essential roles in regulating leaf senescence, yet the molecular mechanism of WRKY-mediated leaf senescence still lacks detailed elucidation in crop plants. In this study, we cloned and identified a tobacco WRKY TF gene, designated NtWRKY70b, acting as a positive regulator of natural leaf senescence. The expression profile analysis showed that NtWRKY70b transcript levels were induced by aging and hydrogen peroxide (H2O2) and downregulated upon hydrogen sulfide (H2S) treatment. The physiological and biochemical assays revealed that overexpression of NtWRKY70b (OE) clearly promoted leaf senescence, triggering increased levels of reactive oxygen species (ROS) and decreased H2S content, while disruption of NtWRKY70b by chimeric repressor silencing technology (SRDX) significantly delayed the onset of leaf senescence, leading to a decreased accumulation of ROS and elevated concentration of H2S. The quantitative real-time PCR analysis showed that the expression levels of various senescence-associated genes and ROS biosynthesis-related genes (NtRbohD and NtRbohE) were upregulated in OE lines, while the expression of H2S biosynthesis-related genes (NtDCD and NtCYSC1) were inhibited in OE lines. Furthermore, the Yeast one-hybrid analysis (Y1H) and dual luciferase assays showed that NtWRKY70b could directly upregulate the expression of an ROS biosynthesis-related gene (NtRbohD) and a chlorophyll degradation-related gene (NtPPH) by binding to their promoter sequences. Accordingly, these results indicated that NtWYKY70b directly activated the transcript levels of NtRbohD and NtPPH and repressed the expression of NtDCD and NtCYCS1, thereby promoting ROS accumulation and impairing the endogenous H2S production, and subsequently accelerating leaf aging. These observations improve our knowledge of the regulatory mechanisms of WRKY TFs controlling leaf senescence and provide a novel method for ensuring high agricultural crop productivity via genetic manipulation of leaf senescence in crops.

Hydrogen Sulfide Alleviates Manganese Stress in Arabidopsis.

Hydrogen sulfide (H2S) has been shown to participate in various stress responses in plants, including drought, salinity, extreme temperatures, osmotic stress, and heavy metal stress. Manganese (Mn), as a necessary nutrient for plant growth, plays an important role in photosynthesis, growth, development, and enzymatic activation of plants. However, excessive Mn2+ in the soil can critically affect plant growth, particularly in acidic soil. In this study, the model plant Arabidopsis thaliana was used to explore the mechanism of H2S participation and alleviation of Mn stress. First, using wild-type Arabidopsis with excessive Mn2+ treatment, the following factors were increased: H2S content, the main H2S synthetase L-cysteine desulfhydrase enzyme (AtLCD) activity, and the expression level of the AtLCD gene. Further, using the wild-type, AtLCD deletion mutant (lcd) and overexpression lines (OE5 and OE32) as materials, the phenotype of Arabidopsis seedlings was observed by exogenous application of hydrogen sulfide donor sodium hydrosulfide (NaHS) and scavenger hypotaurine (HT) under excessive Mn2+ treatment. The results showed that NaHS can significantly alleviate the stress caused by Mn2+, whereas HT aggravates this stress. The lcd mutant is more sensitive to Mn stress than the wild type, and the overexpression lines are more resistant. Moreover, the mechanism of H2S alleviating Mn stress was determined. The Mn2+ content and the expression of the Mn transporter gene in the mutant were significantly higher than those of the wild-type and overexpression lines. The accumulation of reactive oxygen species was significantly reduced in NaHS-treated Arabidopsis seedlings and AtLCD overexpression lines, and the activities of various antioxidant enzymes (SOD, POD, CAT, APX) also significantly increased. In summary, H2S is involved in the response of Arabidopsis to Mn stress and may alleviate the inhibition of Mn stress on Arabidopsis seedling growth by reducing Mn2+ content, reducing reactive oxygen species content, and enhancing antioxidant enzyme activity. This study provides an important basis for further study of plant resistance to heavy metal stress.

A NAC Transcription Factor from 'Sea Rice 86' Enhances Salt Tolerance by Promoting Hydrogen Sulfide Production in Rice Seedlings.

Soil salinity severely threatens plant growth and crop performance. Hydrogen sulfide (H2S), a plant signal molecule, has been implicated in the regulation of plant responses to salinity stress. However, it is unclear how the transcriptional network regulates H2S biosynthesis during salt stress response. In this study, we identify a rice NAC (NAM, ATAF and CUC) transcription factor, OsNAC35-like (OsNACL35), from a salt-tolerant cultivar 'Sea Rice 86' (SR86) and further show that it may have improved salt tolerance via enhanced H2S production. The expression of OsNACL35 was significantly upregulated by high salinity and hydrogen peroxide (H2O2). The OsNACL35 protein was localized predominantly in the nucleus and was found to have transactivation activity in yeast. The overexpression of OsNACL35 (OsNACL35-OE) in japonica cultivar Nipponbare ramatically increased resistance to salinity stress, whereas its dominant-negative constructs (SUPERMAN repression domain, SRDX) conferred hypersensitivity to salt stress in the transgenic lines at the vegetative stage. Moreover, the quantitative real-time PCR analysis showed that many stress-associated genes were differentially expressed in the OsNACL35-OE and OsNACL35-SRDX lines. Interestingly, the ectopic expression of OsNACL35 triggered a sharp increase in H2S content by upregulating the expression of a H2S biosynthetic gene, OsDCD1, upon salinity stress. Furthermore, the dual luciferase and yeast one-hybrid assays indicated that OsNACL35 directly upregulated the expression of OsDCD1 by binding to the promoter sequence of OsDCD1. Taken together, our observations illustrate that OsNACL35 acts as a positive regulator that links H2S production to salt stress tolerance, which may hold promising utility in breeding salt-tolerant rice cultivar.

Brassinosteroids regulate root growth by controlling reactive oxygen species homeostasis and dual effect on ethylene synthesis in Arabidopsis.

The brassinosteroids (BRs) represent a class of phytohormones, which regulate numerous aspects of growth and development. Here, a det2-9 mutant defective in BR synthesis was identified from an EMS mutant screening for defects in root length, and was used to investigate the role of BR in root development in Arabidopsis. The det2-9 mutant displays a short-root phenotype, which is result from the reduced cell number in root meristem and decreased cell size in root maturation zone. Ethylene synthesis is highly increased in the det2-9 mutant compared with the wild type, resulting in the hyper-accumulation of ethylene and the consequent inhibition of root growth. The short-root phenotype of det2-9 was partially recovered in the det2-9/acs9 double mutant and det2-9/ein3/eil1-1 triple mutant which have defects either in ethylene synthesis or ethylene signaling, respectively. Exogenous application of BR showed that BRs either positively or negatively regulate ethylene biosynthesis in a concentration-dependent manner. Different from the BR induced ethylene biosynthesis through stabilizing ACSs stability, we found that the BR signaling transcription factors BES1 and BZR1 directly interacted with the promoters of ACS7, ACS9 and ACS11 to repress their expression, indicating a native regulation mechanism under physiological levels of BR. In addition, the det2-9 mutant displayed over accumulated superoxide anions (O2-) compared with the wild-type control, and the increased O2- level was shown to contribute to the inhibition of root growth. The BR-modulated control over the accumulation of O2- acted via the peroxidase pathway rather than via the NADPH oxidase pathway. This study reveals an important mechanism by which the hormone cross-regulation between BRs and ethylene or/and ROS is involved in controlling root growth and development in Arabidopsis.

A Rice NAC Transcription Factor Promotes Leaf Senescence via ABA Biosynthesis.

It is well known that abscisic acid (ABA)-induced leaf senescence and premature leaf senescence negatively affect the yield of rice (Oryza sativa). However, the molecular mechanism underlying this relationship, especially the upstream transcriptional network that modulates ABA level during leaf senescence, remains largely unknown. Here, we demonstrate a rice NAC transcription factor, OsNAC2, that participates in ABA-induced leaf senescence. Overexpression of OsNAC2 dramatically accelerated leaf senescence, whereas its knockdown lines showed a delay in leaf senescence. Chromatin immunoprecipitation-quantitative PCR, dual-luciferase, and yeast one-hybrid assays demonstrated that OsNAC2 directly activates expression of chlorophyll degradation genes, OsSGR and OsNYC3 Moreover, ectopic expression of OsNAC2 leads to an increase in ABA levels via directly up-regulating expression of ABA biosynthetic genes (OsNCED3 and OsZEP1) as well as down-regulating the ABA catabolic gene (OsABA8ox1). Interestingly, OsNAC2 is upregulated by a lower level of ABA but downregulated by a higher level of ABA, indicating a feedback repression of OsNAC2 by ABA. Additionally, reduced OsNAC2 expression leads to about 10% increase in the grain yield of RNAi lines. The novel ABA-NAC-SAGs regulatory module might provide a new insight into the molecular action of ABA to enhance leaf senescence and elucidates the transcriptional network of ABA production during leaf senescence in rice.

OsNAC2 encoding a NAC transcription factor that affects plant height through mediating the gibberellic acid pathway in rice.

Plant height and flowering time are key agronomic traits affecting yield in rice (Oryza sativa). In this study, we investigated the functions in rice growth and development of OsNAC2, encoding a NAC transcription factor in rice. Transgenic plants that constitutively expressed OsNAC2 had shorter internodes, shorter spikelets, and were more insensitive to gibberellic acid (GA(3)). In addition, the levels of GAs decreased in OsNAC2 overexpression plants, compared with the wild-type. Moreover, flowering was delayed for approximately 5 days in transgenic lines. The transcription of Hd3a, a flowering-time related gene, was suppressed in transgenic lines. In addition, transgenic Arabidopsis plants expressing OsNAC2 were also more insensitive to GA(3). The expression levels of GA biosynthetic genes OsKO2 and OsKAO were repressed. The expression of OsSLRL, encoding a repressor in the GA signal pathway, and OsEATB, which encodes a repressor of GA biosynthesis, were both enhanced. Western blotting indicated that DELLA also accumulated at the protein level. Dual-luciferase reporter analyses, yeast one-hybrid assays and ChIP-qPCR suggested that OsNAC2 directly interacted with the promoter of OsEATB and OsKO2. Taken together, we proposed that OsNAC2 is a negative regulator of the plant height and flowering time, which acts by directly regulating key genes of the GA pathway in rice.

RcLEA, a late embryogenesis abundant protein gene isolated from Rosa chinensis, confers tolerance to Escherichia coli and Arabidopsis thaliana and stabilizes enzyme activity under diverse stresses.

The late embryogenesis abundant (LEA) protein family is a large protein family that is closely associated with resistance to abiotic stresses in many organisms, such as plants, bacteria and animals. In this study, we isolated a LEA gene, RcLEA, which was cytoplasm-localized, from Rosa chinensis. RcLEA was found to be induced by high temperature through RT-PCR. Overexpression of RcLEA in Escherichia coli improved its growth performance compared with the control under high temperature, low temperature, NaCl and oxidative stress conditions. RcLEA was also overexpressed in Arabidopsis thaliana. The transgenic Arabidopsis showed better growth after high and low temperature treatment and exhibited less peroxide according to 3, 3-diaminobenzidine staining. However, RcLEA did not improve the tolerance to NaCl or osmotic stress in Arabidopsis. In vitro analysis showed that RcLEA was able to prevent the freeze-thaw-induced inactivation or heat-induced aggregation of various substrates, such as lactate dehydrogenase and citrate synthase. It also protected the proteome of E. coli from denaturation when the proteins were heat-shocked or subjected to acidic conditions. Furthermore, bimolecular fluorescence complementation assays suggested that RcLEA proteins function in a complex manner by making the form of homodimers.

The NAC family transcription factor OsNAP confers abiotic stress response through the ABA pathway.

Plants respond to environmental stresses by altering gene expression, and several genes have been found to mediate stress-induced expression, but many additional factors are yet to be identified. OsNAP is a member of the NAC transcription factor family; it is localized in the nucleus, and shows transcriptional activator activity in yeast. Analysis of the OsNAP transcript levels in rice showed that this gene was significantly induced by ABA and abiotic stresses, including high salinity, drought and low temperature. Rice plants overexpressing OsNAP did not show growth retardation, but showed a significantly reduced rate of water loss, enhanced tolerance to high salinity, drought and low temperature at the vegetative stage, and improved yield under drought stress at the flowering stage. Microarray analysis of transgenic plants overexpressing OsNAP revealed that many stress-related genes were up-regulated, including OsPP2C06/OsABI2, OsPP2C09, OsPP2C68 and OsSalT, and some genes coding for stress-related transcription factors (OsDREB1A, OsMYB2, OsAP37 and OsAP59). Our data suggest that OsNAP functions as a transcriptional activator that plays a role in mediating abiotic stress responses in rice.

WOX5 is Shining in the Root Stem Cell Niche.

The WUS-RELATED HOMEOBOX 5 (WOX5) gene is expressed in the quiescent center (QC) to regulate the columella stem cell (CSC) identity. Three recent reports not only show how WOX5 is controlled but also highlight the key role of WOX5 in root stem cell niche maintenance.