VILLIN2 regulates cotton defense against Verticillium dahliae by modulating actin cytoskeleton remodeling.

The active structural change of actin cytoskeleton is a general host response upon pathogen attack. This study characterized the function of the cotton (Gossypium hirsutum) actin-binding protein VILLIN2 (GhVLN2) in host defense against the soilborne fungus Verticillium dahliae. Biochemical analysis demonstrated that GhVLN2 possessed actin-binding, -bundling, and -severing activities. A low concentration of GhVLN2 could shift its activity from actin bundling to actin severing in the presence of Ca2+. Knockdown of GhVLN2 expression by virus-induced gene silencing reduced the extent of actin filament bundling and interfered with the growth of cotton plants, resulting in the formation of twisted organs and brittle stems with a decreased cellulose content of the cell wall. Upon V. dahliae infection, the expression of GhVLN2 was downregulated in root cells, and silencing of GhVLN2 enhanced the disease tolerance of cotton plants. The actin bundles were less abundant in root cells of GhVLN2-silenced plants than in control plants. However, upon infection by V. dahliae, the number of actin filaments and bundles in the cells of GhVLN2-silenced plants was raised to a comparable level as those in control plants, with the dynamic remodeling of the actin cytoskeleton appearing several hours in advance. GhVLN2-silenced plants exhibited a higher incidence of actin filament cleavage in the presence of Ca2+, suggesting that pathogen-responsive downregulation of GhVLN2 could activate its actin-severing activity. These data indicate that the regulated expression and functional shift of GhVLN2 contribute to modulating the dynamic remodeling of the actin cytoskeleton in host immune responses against V. dahliae.

The Cotton Apoplastic Protein CRR1 Stabilizes Chitinase 28 to Facilitate Defense against the Fungal Pathogen Verticillium dahliae.

The apoplast serves as the first battlefield between the plant hosts and invading microbes; therefore, work on plant-pathogen interactions has increasingly focused on apoplastic immunity. In this study, we identified three proteins in the apoplast of cotton (Gossypium sp) root cells during interaction of the plant with the fungal pathogen Verticillium dahliae Among these proteins, cotton host cells secrete chitinase 28 (Chi28) and the Cys-rich repeat protein 1 (CRR1), while the pathogen releases the protease VdSSEP1. Biochemical analysis demonstrated that VdSSEP1 hydrolyzed Chi28, but CRR1 protected Chi28 from cleavage by Verticillium dahliae secretory Ser protease 1 (VdSSEP1). In accordance with the in vitro results, CRR1 interacted with Chi28 in yeast and plant cells and attenuated the observed decrease in Chi28 level that occurred in the apoplast of plant cells upon pathogen attack. Knockdown of CRR1 or Chi28 in cotton plants resulted in higher susceptibility to V. dahliae infection, and overexpression of CRR1 increased plant resistance to V dahliae, the fungus Botrytis cinerea, and the oomycete Phytophthora parasitica var nicotianae By contrast, knockout of VdSSEP1 in V. dahliae destroyed the pathogenicity of this fungus. Together, our results provide compelling evidence for a multilayered interplay of factors in cotton apoplastic immunity.

The potato transcription factor StbZIP61 regulates dynamic biosynthesis of salicylic acid in defense against Phytophthora infestans infection.

Salicylic acid (SA) signalling plays an essential role in plant innate immunity. In this study, we identified a component in the SA signaling pathway in potato (Solanum tuberosum), the transcription factor StbZIP61, and characterized its function in defence against Phytophthora infestans. Expression of StbZIP61 was induced upon P. infestans infection and following exposure to the defense signaling hormones SA, ethylene and jasmonic acid. Overexpression of StbZIP61 increased the tolerance of potato plants to P. infestans while RNA interference (RNAi) increased susceptibility. Yeast two-hybrid and pull down experiments revealed that StbZIP61 could interact with an NPR3-like protein (StNPR3L) that inhibited its DNA-binding and transcriptional activation activities. Moreover, StNPR3L interacted with StbZIP61 in an SA-dependent manner. Among candidate genes involved in SA-regulated defense responses, StbZIP61 had a significant impact on expression of StICS1, which encodes a key enzyme for SA biosynthesis. StICS1 transcription was induced upon P. infestans infection and this responsive expression to the pathogen was reduced in StbZIP61 RNAi plants. Accordingly, StICS1 expression was remarkably enhanced in StbZIP61-overexpressing plants. Together, our data demonstrate that StbZIP61 functions in concert with StNPR3L to regulate the temporal activation of SA biosynthesis, which contributes to SA-mediated immunity against P. infestans infection in potato.

Ectopic expression of SsPETE2, a plastocyanin from Suaeda salsa, improves plant tolerance to oxidative stress.

Accumulating evidence indicates that plant plastocyanin is involved in copper homeostasis, yet the physiological relevance remains elusive. In this study, we found that a plastocyanin gene (SsPETE2) from euhalophyte Suaeda salsa possessed a novel antioxidant function, which was associated with the copper-chelating activity of SsPETE2. In S. salsa, expression of SsPETE2 increased in response to oxidative stress and ectopic expression of SsPETE2 in Arabidopsis enhanced the antioxidant ability of the transgenic plants. SsPETE2 bound Cu ion and alleviated formation of hydroxyl radicals in vitro. Accordingly, SsPETE2 expression lowered the free Cu content that was associated with reduced H2O2 level under oxidative stress. Arabidopsis pete1 and pete2 mutants showed ROS-sensitive phenotypes that could be restored by expression of SsPETE2 or AtPETEs. In addition, SsPETE2-expressing plants exhibited more potent tolerance to oxidative stress than plants overexpressing AtPETEs, likely owing to the stronger copper-binding activity of SsPETE2 than AtPETEs. Taken together, these results demonstrated that plant PETEs play a novel role in oxidative stress tolerance by regulating Cu homeostasis under stress conditions, and SsPETE2, as an efficient copper-chelating PETE, potentially could be used in crop genetic engineering.

iTRAQ-based proteomics analysis of autophagy-mediated immune responses against the vascular fungal pathogen Verticillium dahliae in Arabidopsis.

The mechanisms underlying the functional link between autophagy and plant innate immunity remain largely unknown. In this study, we investigated the autophagy-mediated plant defense responses against Verticillium dahliae (V. dahliae) infection by comparative proteomics and cellular analyses. An assessment of the autophagy activity and disease development showed that autophagic processes were tightly related to the tolerance of Arabidopsis plant to Verticillium wilt. An isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics analysis was performed, and we identified a total of 780 differentially accumulated proteins (DAPs) between wild-type and mutant atg10-1 Arabidopsis plants upon V. dahliae infection, of which, 193 ATG8-family-interacting proteins were identified in silico and their associations with autophagy were verified for several selected proteins. Three important aspects of autophagy-mediated defense against V. dahliae infection were revealed: 1) autophagy is required for the activation of upstream defense responses; 2) autophagy-mediated mitochondrial degradation (mitophagy) occurs and is an important player in the defense process; and 3) autophagy promotes the transdifferentiation of perivascular cells and the formation of xylem hyperplasia, which are crucial for protection against this vascular disease. Together, our results provide several novel insights for understanding the functional association between autophagy and plant immune responses.

Overexpression of GhFIM2 propels cotton fiber development by enhancing actin bundle formation.

Cell elongation and secondary wall deposition are two consecutive stages during cotton fiber development. The mechanisms controlling the progression of these two developmental phases remain largely unknown. Here, we report the functional characterization of the actin-bundling protein GhFIM2 in cotton fiber. Overexpression of GhFIM2 increased the abundance of actin bundles, which was accompanied with accelerated fiber growth at the fast-elongating stage. Meanwhile, overexpression of GhFIM2 could propel the onset of secondary cell wall biogenesis. These results indicate that the dynamic rearrangement of actin higher structures involving GhFIM2 plays an important role in the development of cotton fiber cells.

iTRAQ-based proteomic analysis of defence responses triggered by the necrotrophic pathogen Rhizoctonia solani in cotton.

The soil-borne necrotrophic pathogen fungus Rhizoctonia solani is destructive, causing disease in various important crops. To date, little is known about the host defence mechanism in response to invasion of R. solani. Here, an iTRAQ-based proteomic analysis was employed to investigate pathogen-responsive proteins in the disease tolerant/resistant cotton cultivar CRI35. A total of 174 differentially accumulated proteins (DAPs) were identified after inoculation of cotton plants with R. solani. Functional categorization analysis indicated that these DAPs can be divided into 12 subclasses. Notably, a large portion of DAPs are known to function in reactive oxygen species (ROS) metabolism and the expression of several histone-modifying and DNA methylating proteins were significantly induced upon challenge with the fungus, indicating that the redox homeostasis and epigenetic regulation are important for cotton defence against the pathogen. Additionally, the expression of proteins involved in phenylpropanoid biosynthesis was markedly changed in response to pathogen invasion, which may reflect a particular contribution of secondary metabolism in protection against the fungal attack in cotton. Together, our results indicate that the defence response of cotton plants to R. solani infection is active and multifaceted and involves the induction of proteins from various innate immunity-related pathways. SIGNIFICANCE: Cotton damping-off is a destructive disease caused by the necrotrophic fungus Rhizoctonia solani. To date, the host defence mechanism involved in the disease protection remains largely unknown. Here, we reported the first proteomic analysis on cotton immune responses against R. solani infection. Employing iTRAQ technique, we obtained a total of 174 differentially accumulated proteins (DAPs) that can be classified into 12 functional groups. Further analysis indicated that ROS homeostasis, epigenetic regulation and phenylpropanoid biosynthesis were tightly associated with the innate immune responses against R. solani infection in cotton. The obtained data provide not only important information for understanding the molecular mechanism involved in plant-R. solani interaction but also application clues for genetic breeding of crops with improved R. solani resistance.

iTRAQ Protein Profile Differential Analysis of Dormant and Germinated Grassbur Twin Seeds Reveals that Ribosomal Synthesis and Carbohydrate Metabolism Promote Germination Possibly Through the PI3K Pathway.

Grassbur is a destructive and invasive weed in pastures, and its burs can cause gastric damage to animals. The strong adaptability and reproductive potential of grassbur are partly due to a unique germination mechanism whereby twin seeds develop in a single bur: one seed germinates, but the other remains dormant. To investigate the molecular mechanism of seed germination in twin seeds, we used isobaric tags for relative and absolute quantitation (iTRAQ) to perform a dynamic proteomic analysis of germination and dormancy. A total of 1,984 proteins were identified, 161 of which were considered to be differentially accumulated. The differentially accumulated proteins comprised 102 up-regulated and 59 down-regulated proteins. These proteins were grouped into seven functional categories, ribosomal proteins being the predominant group. The authenticity and accuracy of the results were confirmed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time reverse transcription-PCR (qPCR). A dynamic proteomic analysis revealed that ribosome synthesis and carbohydrate metabolism affect seed germination possibly through the phosphoinositide 3-kinase (PI3K) pathway. As the PI3K pathway is generally activated by insulin, analyses of seeds treated with exogenous insulin by qPCR, ELISA and iTRAQ confirmed that the PI3K pathway can be activated, which suppresses dormancy and promotes germination in twin grassbur seeds. Together, these results show that the PI3K pathway may play roles in stimulating seed germination in grassbur by modulating ribosomal synthesis and carbohydrate metabolism.

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

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

Accumulating evidence indicates that plant MYB transcription factors participate in defense against pathogen attack, but their regulatory targets and related signaling processes remain largely unknown. Here, we identified a defense-related MYB gene (GhMYB108) from upland cotton (Gossypium hirsutum) and characterized its functional mechanism. Expression of GhMYB108 in cotton plants was induced by Verticillium dahliae infection and responded to the application of defense signaling molecules, including salicylic acid, jasmonic acid, and ethylene. Knockdown of GhMYB108 expression led to increased susceptibility of cotton plants to V. dahliae, while ecotopic overexpression of GhMYB108 in Arabidopsis thaliana conferred enhanced tolerance to the pathogen. Further analysis demonstrated that GhMYB108 interacted with the calmodulin-like protein GhCML11, and the two proteins form a positive feedback loop to enhance the transcription of GhCML11 in a calcium-dependent manner. Verticillium dahliae infection stimulated Ca(2+) influx into the cytosol in cotton root cells, but this response was disrupted in both GhCML11-silenced plants and GhMYB108-silenced plants in which expression of several calcium signaling-related genes was down-regulated. Taken together, these results indicate that GhMYB108 acts as a positive regulator in defense against V. dahliae infection by interacting with GhCML11. Furthermore, the data also revealed the important roles and synergetic regulation of MYB transcription factor, Ca(2+), and calmodulin in plant immune responses.

Functional Characterization of a Dihydroflavanol 4-Reductase from the Fiber of Upland Cotton (Gossypium hirsutum).

Dihydroflavanol 4-reductase (DFR) is a key later enzyme involved in two polyphenols' (anthocyanins and proanthocyanidins (PAs)) biosynthesis, however it is not characterized in cotton yet. In present reports, a DFR cDNA homolog (designated as GhDFR1) was cloned from developing fibers of upland cotton. Silencing GhDFR1 in cotton by virus-induced gene silencing led to significant decrease in accumulation of anthocyanins and PAs. More interestingly, based on LC-MS analysis, two PA monomers, (-)-epicatachin and (-)-epigallocatachin, remarkably decreased in content in fibers of GhDFR1-silenced plants, but two new monomers, (-)-catachin and (-)-gallocatachin were present compared to the control plants infected with empty vector. The ectopic expression of GhDFR1 in an Arabidopsis TT3 mutant allowed for reconstruction of PAs biosynthesis pathway and led to accumulation of PAs in seed coat. Taken together, these data demonstrate that GhDFR1 contributes to the biosynthesis of anthocyanins and PAs in cotton.

The mitochondrial malate dehydrogenase 1 gene GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton.

Cotton, an important commercial crop, is cultivated for its natural fibers, and requires an adequate supply of soil nutrients, including phosphorus, for its growth. Soil phosporus exists primarily in insoluble forms. We isolated a mitochondrial malate dehydrogenase (MDH) gene, designated as GhmMDH1, from Gossypium hirsutum L. to assess its effect in enhancing P availability and absorption. An enzyme kinetic assay showed that the recombinant GhmMDH1 possesses the capacity to catalyze the interconversion of oxaloacetate and malate. The malate contents in the roots, leaves and root exudates was significantly higher in GhmMDH1-overexpressing plants and lower in knockdown plants compared with the wild-type control. Knockdown of GhmMDH1 gene resulted in increased respiration rate and reduced biomass whilst overexpression of GhmMDH1 gave rise to decreased respiration rate and higher biomass in the transgenic plants. When cultured in medium containing only insoluble phosphorus, Al-phosphorus, Fe-phosphorus, or Ca-phosphorus, GhmMDH1-overexpressing plants produced significantly longer roots and had a higher biomass and P content than WT plants, however, knockdown plants showed the opposite results for these traits. Collectively, our results show that GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton, owing to its functions in leaf respiration and P acquisition.

The RING finger protein NtRCP1 is involved in the floral transition in tobacco (Nicotiana tabacum).

The transition from the vegetative phase to the reproductive phase is a major developmental process in flowering plants. The underlying mechanism controlling this cellular process remains a research focus in the field of plant molecular biology. In the present work, we identified a gene encoding the C3H2C3-type RING finger protein NtRCP1 from tobacco BY-2 cells. Enzymatic analysis demonstrated that NtRCP1 is a functional E3 ubiquitin ligase. In tobacco plants, expression level of NtRCP1 was higher in the reproductive shoot apices than in the vegetative ones. NtRCP1-overexpressing plants underwent a more rapid transition from the vegetative to the reproductive phase and flowered markedly earlier than the wild-type control. Histological analysis revealed that the shoot apical meristem of NtRCP1-overexpressing plants initiated inflorescence primordia precociously compared to the wild-type plant due to accelerated cell division. Overexpression of NtRCP1 in BY-2 suspension cells promoted cell division, which was a consequence of the shortened G2 phase in the cell cycle. Together, our data suggest that NtRCP1 may act as a regulator of the phase transition, possibly through its role in cell cycle regulation, during vegetative/reproductive development in tobacco plant.

Cotton major latex protein 28 functions as a positive regulator of the ethylene responsive factor 6 in defense against Verticillium dahliae.

In this study, we identified a defense-related major latex protein (MLP) from upland cotton (designated GhMLP28) and investigated its functional mechanism. GhMLP28 transcripts were ubiquitously present in cotton plants, with higher accumulation in the root. Expression of the GhMLP28 gene was induced by Verticillium dahliae inoculation and was responsive to defense signaling molecules, including ethylene, jasmonic acid, and salicylic acid. Knockdown of GhMLP28 expression by virus-induced gene silencing resulted in increased susceptibility of cotton plants to V. dahliae infection, while ectopic overexpression of GhMLP28 in tobacco improved the disease tolerance of the transgenic plants. Further analysis revealed that GhMLP28 interacted with cotton ethylene response factor 6 (GhERF6) and facilitated the binding of GhERF6 to GCC-box element. Transient expression assay demonstrated that GhMLP28 enhanced the transcription factor activity of GhERF6, which led to the augmented expression of some GCC-box genes. GhMLP28 proteins were located in both the nucleus and cytoplasm and their nuclear distribution was dependent on the presence of GhERF6. Collectively, these results demonstrate that GhMLP28 acts as a positive regulator of GhERF6, and synergetic actions of the two proteins may contribute substantially to protection against V. dahliae infection in cotton plants.

The Thellungiella salsuginea tonoplast aquaporin TsTIP1;2 functions in protection against multiple abiotic stresses.

Examination of aquaporin (AQP) membrane channels in extremophile plants may increase our understanding of plant tolerance to high salt, drought or other conditions. Here, we cloned a tonoplast AQP gene (TsTIP1;2) from the halophyte Thellungiella salsuginea and characterized its biological functions. TsTIP1;2 transcripts accumulate to high levels in several organs, increasing in response to multiple external stimuli. Ectopic overexpression of TsTIP1;2 in Arabidopsis significantly increased plant tolerance to drought, salt and oxidative stresses. TsTIP1;2 had water channel activity when expressed in Xenopus oocytes. TsTIP1;2 was also able to conduct H2O2 molecules into yeast cells in response to oxidative stress. TsTIP1;2 was not permeable to Na(+) in Xenopus oocytes, but it could facilitate the entry of Na(+) ions into plant cell vacuoles by an indirect process under high-salinity conditions. Collectively, these data showed that TsTIP1;2 could mediate the conduction of both H2O and H2O2 across membranes, and may act as a multifunctional contributor to survival of T. salsuginea in highly stressful habitats.

The dual functions of WLIM1a in cell elongation and secondary wall formation in developing cotton fibers.

LIN-11, Isl1 and MEC-3 (LIM)-domain proteins play pivotal roles in a variety of cellular processes in animals, but plant LIM functions remain largely unexplored. Here, we demonstrate dual roles of the WLIM1a gene in fiber development in upland cotton (Gossypium hirsutum). WLIM1a is preferentially expressed during the elongation and secondary wall synthesis stages in developing fibers. Overexpression of WLIM1a in cotton led to significant changes in fiber length and secondary wall structure. Compared with the wild type, fibers of WLIM1a-overexpressing plants grew longer and formed a thinner and more compact secondary cell wall, which contributed to improved fiber strength and fineness. Functional studies demonstrated that (1) WLIM1a acts as an actin bundler to facilitate elongation of fiber cells and (2) WLIM1a also functions as a transcription factor to activate expression of Phe ammonia lyase-box genes involved in phenylpropanoid biosynthesis to build up the secondary cell wall. WLIM1a localizes in the cytosol and nucleus and moves into the nucleus in response to hydrogen peroxide. Taken together, these results demonstrate that WLIM1a has dual roles in cotton fiber development, elongation, and secondary wall formation. Moreover, our study shows that lignin/lignin-like phenolics may substantially affect cotton fiber quality; this finding may guide cotton breeding for improved fiber traits.

The cotton transcription factor TCP14 functions in auxin-mediated epidermal cell differentiation and elongation.

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.

A comparative proteomic analysis provides insights into pigment biosynthesis in brown color fiber.

A comparative proteomic analysis was performed to identify the differences between brown cotton fiber and a white near-isogenic line, and 78 differential spots were identified at three time points (12-, 18-, and 24-day post-anthesis [DPA]) using MALDI-TOF/TOF. Our data illustrate several aspects of pigment synthesis and fiber development in brown color fiber (BCF). First, 21 spots were associated with secondary metabolism; 15 of these with high abundance in BCF were involved in flavonoid biosynthesis. Second, several spots with lower abundance in BCF were found. Thirteen spots were related to energy/carbohydrate metabolism; in particular, spots related to the glycolytic pathway exhibited lower abundance in 12 DPA BCF. Several spots related to redox homeostasis, cytoskeleton, and protein metabolism also showed lower abundance in BCF, including proteins that are critical for fiber development, such as ascorbate peroxidase, superoxide dismutase, actin, annexin and heat shock protein. Third, several proteins such as leucine aminopeptidase preprotein and progesterone-5-beta-reductase were newly identified proteins in cotton fibers. These findings demonstrated the presence of a complicated metabolic network in BCF and advanced our understanding of the molecular mechanisms of pigment biosynthesis in colored cotton, which will provide new insight for the development of new color types by genetic manipulation.

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 bacterium's 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.