Nai-Qin Zhong

Cloning and functional characterization of PpDBF1 gene encoding a DRE-binding transcription factor from Physcomitrella patens.

The dehydration-responsive element binding (DREB) transcription factors play central roles in regulating expression of stress-inducible genes under abiotic stresses. In the present work, PpDBF1 (Physcomitrella patensDRE-binding Factor1) containing a conserved AP2/ERF domain was isolated from the moss P. patens. Sequence comparison and phylogenetic analysis revealed that PpDBF1 belongs to the A-5 group of DREB transcription factor subfamily. The transcriptional activation activity and DNA-binding specificity of PpDBF1 were verified by yeast one-hybrid and electrophoretic mobility shift assay experiments, and its nuclear localization was demonstrated by particle biolisitics. PpDBF1 transcripts were accumulated under various abiotic stresses and phytohormones treatments in P. patens, and transgenic tobacco plants over-expressing PpDBF1 gained higher tolerance to salt, drought and cold stresses. These results suggest that PpDBF1 may play a role in P. patens as a DREB transcription factor, implying that similar regulating systems are conserved in moss and higher plants.

The Cotton Transcription Factor TCP14 Functions in Auxin-Mediated Epidermal Cell Differentiation and Elongation

GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, and it may act as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells. Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells.

SsTypA1, a chloroplast-specific TypA/BipA-type GTPase from the halophytic plant Suaeda salsa, plays a role in oxidative stress tolerance

Suaeda salsa is a leaf-succulent euhalophytic plant capable of surviving under seawater salinity. Here, we report the isolation and functional analysis of a novel Suaeda gene (designated as SsTypA1) encoding a member of the TypA/BipA GTPase gene family. The steady-state transcript level of SsTypA1 in S. salsa was up-regulated in response to various external stressors. Expression of SsTypA1 was restricted to the epidermal layers of the leaf and stem in S. salsa, and SsTypA1-green fluorescence protein (GFP) fusion proteins were targeted to the chloroplasts of tobacco leaves. Ectopic over-expression of SsTypA1 rendered the transgenic tobacco plants with significantly increased tolerance to oxidative stress, and this was accompanied by a reduction in H(2)O(2) content. Enzymatic and Western blot analyses revealed that the activity and amount of the thylakoid-bound NAD(P)H dehydrogenase (NDH) complex in the chloroplasts of leaf cells were enhanced. Additionally, an in vitro assay demonstrated that SsTypA1 bound to GTP and possessed GTPase activity that was stimulated by the presence of chloroplast 70S ribosomes. Together, these results suggest that SsTypA1 may play a critical role in the development of oxidative stress tolerance, perhaps as a translational regulator of the stress-responsive proteins involved in reactive oxygen species (ROS) suppression in chloroplast.

The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton

The root apoplast secretome of Gossypium barbadense actively changes upon infection, and a thioredoxin plays an important role in apoplastic ROS balance. Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae. Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response.

The Tobacco BLADE-ON-PETIOLE2 Gene Mediates Differentiation of the Corolla Abscission Zone by Controlling Longitudinal Cell Expansion

The BLADE-ON-PETIOLE (BOP) genes of Arabidopsis (Arabidopsis thaliana) have been shown to play an essential role in floral abscission by specializing the abscission zone (AZ) anatomy. However, the molecular and cellular mechanisms that underlie differentiation of the AZ are largely unknown. In this study, we identified a tobacco (Nicotiana tabacum) homolog of BOP (designated NtBOP2) and characterized its cellular function. In tobacco plants, the NtBOP2 gene is predominantly expressed at the base of the corolla in an ethylene-independent manner. Both antisense suppression of NtBOP genes and overexpression of NtBOP2 in tobacco plants caused a failure in corolla shedding. Histological analysis revealed that the differentiation of the corolla AZ was blocked in the transgenic flowers. This blockage was due to uncontrolled cell elongation at the region corresponding to wild-type AZ. The role of NtBOP2 in regulating cell elongation was further demonstrated in Bright Yellow 2 single cells: perturbation of NtBOP2 function by a dominant negative strategy led to the formation of abnormally elongated cells. Subcellular localization analysis showed that NtBOP2-green fluorescent protein fusion proteins were targeted to both the nucleus and cytoplasm. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that NtBOP2 proteins interacted with TGA transcription factors. Taken together, these results indicated that NtBOP2 mediated the differentiation of AZ architecture by controlling longitudinal cell growth. Furthermore, NtBOP2 may achieve this outcome through interaction with the TGA transcription factors and via an ethylene-independent signaling pathway.

Root and vascular tissue-specific expression of glycine-rich protein AtGRP9 and its interaction with AtCAD5, a cinnamyl alcohol dehydrogenase, in Arabidopsis thaliana

The vascular tissue of roots performs essential roles in the physical support and transport of water, nutrients, and signaling molecules in higher plants. The molecular mechanisms underlying the function of root vascular tissue are poorly understood. In this study, we analyzed the expression pattern of AtGRP9, a salt stress-responsive gene encoding a glycine-rich protein, and its interacting partner, in Arabidopsisthaliana. Analysis of GUS or GFP expression under the control of the AtGRP9 promoter showed that AtGRP9 was expressed in the vascular tissue of the root; subcellular localization analysis further demonstrated that AtGRP9 proteins were localized in the cell wall and in the cytoplasm. Yeast two-hybrid analysis revealed that AtGRP9 interacted with AtCAD5, a major cinnamyl alcohol dehydrogenase (CAD) involved in lignin biosynthesis, for which tissue-specific distribution was comparable with that of AtGRP9. These results suggest that AtGRP9 may be involved in lignin synthesis in response to salt stress as a result of its interaction with AtCAD5 in A. thaliana.

The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection

Highlight Cotton MYB108 interacts with CML11 and acts as a positive regulator in defense against V. dahliae infection.

Ectopic expression of a bacterium NhaD-type Na+/H+ antiporter leads to increased tolerance to combined salt/alkali stresses.

AaNhaD, a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica, encodes a Na(+) /H(+) antiporter crucial for the bacteriums resistance to salt/alkali stresses. However, it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses. To investigate the use of extremophile genetic resources in higher plants, transgenic tobacco BY-2 cells and plants harboring AaNhaD were generated and their stress tolerance was evaluated. Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner. Compared to wild-type controls, the transgenic cells exhibited increased Na(+) concentrations and pH levels in the vacuoles. Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts. Similar to the transgenic BY-2 cells, AaNhaD-overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil. These results indicate that AaNhaD functions as a pH-dependent tonoplast Na(+) /H(+) antiporter in plant cells, thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.

Evaluation of the resistance of transgenic potato plants expressing various levels of Cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say) in the laboratory and field.

BACKGROUND The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, is a destructive pest. The CPB is a quarantine pest in China, but has now invaded the Xinjiang Uygur Autonomous Region and is continuing to spread eastwards. To control the damage and overspreading, transgenic potato plants expressing Cry3A toxin were developed, and their resistance to CPB was evaluated by bioassays in the laboratory and field in 2009, 2010 and 2011. RESULTS The insect resistance of the high-dose (HD) transgenic lines was significantly greater than the middle-dose (MD) and low-dose (LD) transgenic lines regarding leaf consumption, biomass accumulation and mortality. The HD and MD transgenic lines showed 100% mortality when inoculated with first- and second-instar larvae; however, the LD transgenic lines showed about 50% mortality. The HD transgenic lines exhibited a significantly higher yield than the MD and LD transgenic lines owing to their high CPB resistance. CONCLUSION Commercially available transgenic potato plants with above 0.1% Cry3A of total soluble protein and NT control refugia could control damage, delay adaptation and halt dispersion eastwards. The two HD transgenic lines developed in this study, PAH1 and PAH2, are ideal for use as cultivars or germplasm to breed new cultivars.

Heterologous expression of a chloroplast outer envelope protein from Suaeda salsa confers oxidative stress tolerance and induces chloroplast aggregation in transgenic Arabidopsis plants

Suaeda salsa is a euhalophytic plant that is tolerant to coastal seawater salinity. In this study, we cloned a cDNA encoding an 8.4 kDa chloroplast outer envelope protein (designated as SsOEP8) from S. salsa and characterized its cellular function. Steady-state transcript levels of SsOEP8 in S. salsa were up-regulated in response to oxidative stress. Consistently, ectopic expression of SsOEP8 conferred enhanced oxidative stress tolerance in transgenic Bright Yellow 2 (BY-2) cells and Arabidopsis, in which H(2) O(2) content was reduced significantly in leaf cells. Further studies revealed that chloroplasts aggregated to the sides of mesophyll cells in transgenic Arabidopsis leaves, and this event was accompanied by inhibited expression of genes encoding proteins for chloroplast movements such as AtCHUP1, a protein involved in actin-based chloroplast positioning and movement. Moreover, organization of actin cytoskeleton was found to be altered in transgenic BY-2 cells. Together, these results suggest that SsOEP8 may play a critical role in oxidative stress tolerance by changing actin cytoskeleton-dependent chloroplast distribution, which may consequently lead to the suppressed production of reactive oxygen species (ROS) in chloroplasts. One significantly novel aspect of this study is the finding that the small chloroplast envelope protein is involved in oxidative stress tolerance.