Genome-Wide Identification of AMT2-Type Ammonium Transporters Reveal That CsAMT2.2 and CsAMT2.3 Potentially Regulate NH4+ Absorption among Three Different Cultivars of Camellia sinensis.

Ammonium (NH4+), as a major inorganic source of nitrogen (N) for tea plant growth, is transported and distributed across membranes by the proteins of ammonium transporters (AMTs). However, the AMT2-type AMTs from tea plants remain poorly understood. In this study, five CsAMT2 subfamily genes were identified in tea plant genomes, and their full-length coding sequences (CDS) were isolated from roots. Then, a NH4+ uptake kinetic comparison of Fudingdabaicha (FD), Huangdan (HD), and Maoxie (MX) showed that FD was a high N efficiency (HNE) cultivar that had a wide range of adaptability to NH4+, HD was a high N efficiency under high N conditions (HNEH) cultivar, in which it was easy to obtain higher yield in a high N environment, and MX was a high N efficiency under low N conditions (HNEL) cultivar, which had a higher affinity for NH4+ than the other two. Tissue-specific expression analysis suggested that CsAMT2.2 and CsAMT2.3 were highly expressed in the roots, indicating that these two members may be unique in the CsAMT2 subfamily. This is further supported by our findings from the temporal expression profiles in the roots among these three different N adaptation cultivars. Expression levels of CsAMT2.2 and CsAMT2.3 in FD and HD were upregulated by a short time (2 h) under high NH4+ treatment, while under low NH4+ treatment, CsAMT2.2 and CsAMT2.3 were highly expressed at 0 h and 2 h in the HNEL-type cultivar-MX. Furthermore, the functional analysis illustrated that CsAMT2.2 and CsAMT2.3 could make a functional complementation of NH4+-defective mutant yeast cells at low NH4+ levels, and the transport efficiency of CsAMT2.3 was higher than that of CsAMT2.2. Thus, we concluded that CsAMT2.2 and CsAMT2.3 might play roles in controlling the NH4+ uptake from the soil to the roots. These results will further the understanding of the NH4+ signal networks of AMT2-type proteins in tea plants.

Quantitative Measurement Reveals Dynamic Volatile Changes and Potential Biochemical Mechanisms during Green Tea Spreading Treatment.

Quantitative data provide clues for biochemical reactions or regulations. The absolute quantification of volatile compounds in tea is complicated by their low abundance, volatility, thermal liability, matrix complexity, and instrumental sensitivity. Here, by integrating solvent-assisted flavor evaporation extraction with a gas chromatography-triple quadrupole mass spectrometry platform, we successfully established a method based on multiple reaction monitoring (MRM). The method was validated by multiple parameters, including the linear range, limit of detection, limit of quantification, precision, repeatability, stability, and accuracy. This method was then applied to measure temporal changes of endogenous volatiles during green tea spreading treatment. In total, 38 endogenous volatiles were quantitatively measured, which are derived from the shikimic acid pathway, mevalonate pathway, 2-C-methylerythritol-4-phosphate pathway, and fatty acid derivative pathway. Hierarchical clustering and heat-map analysis demonstrated four different changing patterns during green tea spreading treatment. Pathway analysis was then conducted to explore the potential biochemistry underpinning these dynamic change patterns. Our data demonstrated that the established MRM method showed high selectivity and sensitivity for quantitative tea volatile measurement and offered novel insights about volatile formation during green tea spreading.

Dynamic Variation of Amino Acid Contents and Identification of Sterols in Xinyang Mao Jian Green Tea.

As important biomolecules in Camellia sinensis L., amino acids (AAs) are considered to contribute to the overall green tea sensory quality and undergo dynamic changes during growth. However, limited by analytical capacity, detailed AAs composition in different growth stages remains unclear. To address this question, we analyzed the dynamic changes of 23 AAs during leaf growth in Xinyang Mao Jian (XYMJ) green tea. Using amino acid analyzer, we demonstrated that most AAs are abundant on Pure Brightness Day and Grain Rain Day. After Grain Rain, 23 AAs decreased significantly. Further analysis shows that theanine has a high level on the day before Spring Equinox and Grain Rain, accounting for 44-61% of the total free AAs content in tea leaves. Glu, Pro, and Asp are the second most abundant AAs. Additionally, spinasterol and 22,23-dihydrospinasterol are first purified and identified in ethanol extract of XYMJ by silica gel column chromatography method. This study reveals the relationship between plucking days and the dynamic changes of AAs during the growth stage and proves the rationality of the traditional plucking days of XYMJ green tea.

BUMPY STEM Is an Arabidopsis Choline/Ethanolamine Kinase Required for Normal Development and Chilling Responses.

Phospholipid biosynthesis is a core metabolic pathway that affects all aspects of plant growth and development. One of the earliest step in this pathway is mediated by choline/ethanolamine kinases (CEKs), enzymes in the Kennedy pathway that catalyze the synthesis of the polar head groups found on the most abundant plant phospholipids. The Arabidopsis genome encodes four CEKs. CEK1-3 have been well characterized using viable mutants while CEK4 encodes an essential gene, making it difficult to characterize its effects on plant development and responses to the environment. We have isolated an EMS-induced allele of CEK4 called bumpy stem (bst). bst plants are viable, allowing the effects of decreased CEK4 function to be characterized throughout the Arabidopsis life cycle. bst mutants have a range of developmental defects including ectopic stem growths at the base of their flowers, reduced fertility, and short roots and stems. They are also sensitive to cold temperatures. Supplementation with choline, phosphocholine, ethanolamine, and phosphoethanolamine rescues bst root phenotypes, highlighting the flow of metabolites between the choline and ethanolamine branches of the Kennedy pathway. The identification of bst and characterization of its phenotypes defines new roles for CEK4 that go beyond its established biochemical function as an ethanolamine kinase.

Macro-composition quantification combined with metabolomics analysis uncovered key dynamic chemical changes of aging white tea.

It is a common belief in China that aging could improve the quality of white tea. However, the stored-induced compositional changes remain elusive. In this study, ten subsets of white tea samples, which had been stored for 1-, 2-, 3-, 4-, 5-, 6-, 7-, 10-, 11- and 13- years, were selected. Macro-compositions were quantified firstly. As the results showed, it was interesting to find total flavonoids, thearubigins (TRs), and theabrownines (TBs) increasing, accompanied with a gradual decrease of total polyphenols, which suggest a conversion of phenolic component in the aging process. Then, nontargeted metabolomics was further conducted on selected subsets of samples, including 1-, 7- and 13- years stored to profile their conversion. As a result, most different metabolites were related to flavonol glycosides and flavone glycosides, suggesting dynamic phenolic component changes were vital in aging. The partial least-squares-discriminant analysis (PLS-DA) also identified them as markers in distinguishing.

Drought stress modify cuticle of tender tea leaf and mature leaf for transpiration barrier enhancement through common and distinct modes.

Cuticle is the major transpiration barrier that restricts non-stomatal water loss and is closely associated with plant drought tolerance. Although multiple efforts have been made, it remains controversial what factors shape up the cuticular transpiration barrier. Previously, we found that the cuticle from the tender tea leaf was mainly constituted by very-long-chain-fatty-acids and their derivatives while alicyclic compounds dominate the mature tea leaf cuticle. The presence of two contrasting cuticle within same branch offered a unique system to investigate this question. In this study, tea seedlings were subjected to water deprivation treatment, cuticle structures and wax compositions from the tender leaf and the mature leaf were extensively measured and compared. We found that cuticle wax coverage, thickness, and osmiophilicity were commonly increased from both leaves. New waxes species were specifically induced by drought; the composition of existing waxes was remodeled; the chain length distributions of alkanes, esters, glycols, and terpenoids were altered in complex manners. Drought treatment significantly reduced leaf water loss rates. Wax biosynthesis-related gene expression analysis revealed dynamic expression patterns dependent on leaf maturity and the severity of drought. These data suggested that drought stress-induced structural and compositional cuticular modifications improve cuticle water barrier property. In addition, we demonstrated that cuticle from the tender leaf and the mature leaf were modified through both common and distinct modes.

Establishing a System for Functional Characterization of Full-Length cDNAs of Camellia sinensis.

: Tea (Camellia sinensis) is enriched with bioactive secondary metabolites, and is one of the most popular nonalcoholic beverages globally. Two tea reference genomes have been reported; however, the functional analysis of tea genes has lagged, mainly due to tea's recalcitrance to genetic transformation and the absence of alternative high throughput heterologous expression systems. A full-length cDNA collection with a streamlined cloning system is needed in this economically important woody crop species. RNAs were isolated from nine different vegetative tea tissues, pooled, then used to construct a normalized full-length cDNA library. The titer of unamplified and amplified cDNA library was 6.89 × 106 and 1.8 × 1010 cfu/mL, respectively; the library recombinant rate was 87.2%. Preliminary characterization demonstrated that this collection can complement existing tea reference genomes and facilitate rare gene discovery. In addition, to streamline tea cDNA cloning and functional analysis, a binary vector (pBIG2113SF) was reengineered, seven tea cDNAs isolated from this library were successfully cloned into this vector, then transformed into Arabidopsis. One FL-cDNA, which encodes a putative P1B-type ATPase 5 (CsHMA5), was characterized further as a proof of concept. We demonstrated that overexpression of CsHMA5 in Arabidopsis resulted in copper hyposensitivity. Thus, our data demonstrated that this represents an efficient system for rare gene discovery and functional characterization of tea genes. The integration of a tea FL-cDNA collection with efficient cloning and a heterologous expression system would facilitate functional annotation and characterization of tea genes.

Identification and comparison of oligopeptides during withering process of White tea by ultra-high pressure liquid chromatography coupled with quadrupole-orbitrap ultra-high resolution mass spectrometry.

Peptides could have specific tastes or bioactivities depending on the length and sequence of amino acids. Till date it remains unknown what peptides are formed during the white tea manufacturing process and whether they contribute to the flavor or bio-activities of white tea. As a first step to address these questions, we applied ultra-high pressure liquid chromatography coupled with quadrupole-orbitrap ultra-high resolution mass spectrometry (UPLC-Quadrupole-Orbitrap-UHRMS) to monitor peptides dynamic changes during the withering process. A total of 196 abundant peptides were identified. Most of them were oligopeptides within a molecular weight of 1000 Da. Four of them were randomly selected, synthesized peptides were applied for further confirmation and quantification. Sequence analysis suggested that some of them were potential taste contributors. Proteinase cleave site analysis identified two separate periods of active proteins degradation at 0-12 h and 30-42 h of the withering processes. Further analysis of cleavage sites also suggested that protein degradation during withering steps were random rather than a stepwise reaction.

Non-targeted metabolomics reveals distinct chemical compositions among different grades of Bai Mudan white tea.

So far, the chemical quality of different grades of white tea has largely remained unexplored. The objective of this study was to establish a model for quality evaluation of different grades of Bai Mudan white tea. We applied non-targeted ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry in combination with multivariate analysis and analyzed four different grades of Bai Mudan white tea. We found that the metabolite composition from the super-grade and the first-grade shared higher similarity compared to the second-grade or the third-grade white tea, and the Partial Least Square-Discriminant Analysis model showed high capability to explain the sample variation (R2Y = 0.998, Q2 = 0.95 in negative ionization modes). In total, 93 metabolites were structurally identified, wherein 21 low abundant metabolites showed distinct changes in abundance that were closely correlated with tea grade variation. These findings suggested their potential as markers to discriminate different grades of Bai Mudan white tea.

Hydrophilic interaction liquid chromatography coupled with quadrupole-orbitrap ultra high resolution mass spectrometry to quantitate nucleobases, nucleosides, and nucleotides during white tea withering process.

Nucleotides, nucleosides, and nucleobases are important bioactive compounds. Recent studies suggested that they possess taste activity. However, it remains unknown about their presence in white tea and how they change during white tea manufacture. Here, we first established method based on hydrophilic interaction liquid chromatography coupled with quadrupole-orbitrap ultra high resolution mass spectrometry (HILIC-Quadrupole-Orbitrap-UHRMS) platform, then applied it to study the dynamic changes of nucleotides, nucleosides, and nucleobases during white tea withering process. Five compounds, including adenosine 5'-monophosphate monohydrate (AMP), guanosine 5'-monophosphate disodium salt hydrate (GMP), adenosine, cytidine, thymine and uracil, were detected from withering samples. They showed a general decline trend during white tea withering process, however, considerable amount of them was retained after withering for 48 h except adenosine which was below detection limit after withering for 21 h. This study provided a complete picture about nucleotides, nucleosides and nucleobases changes during white tea withering process.

Tender leaf and fully-expanded leaf exhibited distinct cuticle structure and wax lipid composition in Camellia sinensis cv Fuyun 6.

The goal of the present study was to compare the structural and compositional differences of cuticle between tender leaf and fully-expanded leaf in Camellia sinensis, and provide metabolic base for the further characterization of wax biosynthesis in this economically important crop species. The tender second leaf and the fully-expanded fifth leaf from new twig were demonstrated to represent two different developmental stages, their cuticle thickness were measured by transmission electron microscopy. The thickness of the adaxial cuticle on the second and fifth leaf was 1.15 µm and 2.48 µm, respectively; the thickness of the abaxial cuticle on the second and fifth leaf was 0.47 µm and 1.05 µm, respectively. The thickness of the epicuticular wax layer from different leaf position or different sides of same leaf were similar. However, the intracuticular wax layer of the fifth leaf was much thicker than that of the second leaf. Total wax lipids were isolated from the second leaf and the fifth leaf, respectively. Gas chromatography-mass spectrometry analysis identified 51 wax constituents belonging to 13 chemical classes, including esters, glycols, terpenoids, fatty acids and their derivatives. Wax coverage on the second and fifth leaf was 4.76 µg/cm2 and 15.38 µg/cm2, respectively. Primary alcohols dominated in the tender second leaf. However, triterpenoids were the major components from the fully-expanded fifth leaf. The predominant carbon chains varied depending on chemical class. These data showed that the wax profiles of Camellia sinensis leaves are development stage dependent, suggesting distinct developmental dependent metabolic pathways and regulatory mechanisms.

ITS1/5.8S/ITS2, a Good Marker for Initial Classification of Shiitake Culinary-Medicinal Lentinus edodes (Agaricomycetes) Strains in China.

China is home to rich wild and cultivated strains of Lentinus edodes, an important edible and medicinal mushroom. Artificial selection of L. edodes has a long history, and the widely cultivated strains belong to populations different from those of most wild strains. Internal transcribed spacer (ITS) regions have been used as good markers to identify L. edodes populations. But because ITS regions exhibit incomplete concerted evolution, the use of an ITS to identify L. edodes populations has been questioned. The objective of this study was to determine whether the ITS region is suitable for identifying L. edodes populations and which populations the widely cultivated strains and the most wild strains belong to by investigating intraindividual and differential ITS polymorphisms between 44 cultivars and 44 wild strains of L. edodes in China. Intraindividual ITS polymorphism is common in L. edodes strains, and most strains possessed 2 different ITS sequences, which came from their heterokaryons. The genetic polymorphisms of ITS1, 5.8S, and ITS2 in L. edodes strains are distinct. All strains were divided into one 5.8S type (5.8S-A), 2 ITS1 types (ITS1-A and ITS1-B), and 2 ITS2 types (ITS2-A and ITS2-B), which were subdivided into 2 branches (ITS2-A1 and ITS2-A2; ITS2-B1 and ITS2-B2). ITS1/5.8S/ITS2 could be used as a good marker in preliminary classification of L. edodes strains in China. It not only exhibited classified information of ITS1, 5.8S, and ITS2 for each strain at the same time, it also indicated whether the strain was heterozygous. The 44 cultivated strains were mainly the A/A/A1 type, and the 44 wild strains were mainly the A/A/A2 and other mixed types.

Mechanism of Glucose Regulates the Fruiting Body Formation in the Beech Culinary-Medicinal Mushroom, Hypsizygus marmoreus (Agaricomycetes).

To understand the fruiting process of Hypsizygus marmoreus, a synthetic liquid medium (SLM) was optimized to induce fruiting body initiation. Dependent on the SLM, the effect of a monofactor (glucose) on the fruiting bodies of H. marmoreus was studied at different concentrations (10 and 40 g/L). Primordia appeared approximately 10 days earlier in low-glucose media (LGM) than in high-glucose media (HGM), whereas mature fruiting bodies formed on mushrooms approximately 7 days earlier and more primordia developed into mature fruiting bodies when cultured in HGM. In addition, the morphogenesis of the primordia was clustered in HGM, which was different than what was observed in LGM. Furthermore, differentially expressed genes (DEGs) that encoded various proteins involved in cell structure, general metabolism, signal transduction, and transcription and translation were analyzed by transcriptome sequencing. Six DEGs were detected by quantitative reverse-transcriptase polymerase chain reaction, and the results were consistent with the altered patterns of gene expression revealed by the transcriptome. This study not only identifies new candidate genes involved in the development of H. marmoreus but also provides a new research platform for studying the development of other edible mushrooms.

The potato tuber mitochondrial proteome.

Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum 'Folva') and their proteome investigated. Proteins were resolved by one-dimensional gel electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap XL. Using four different search programs, a total of 1,060 nonredundant proteins were identified in a quantitative manner using normalized spectral counts including as many as 5-fold more "extreme" proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome (possibly as high as 85%). The dynamic range of protein expression spanned 1,800-fold and included nearly all components of the electron transport chain, tricarboxylic acid cycle, and protein import apparatus. Additionally, we identified 71 pentatricopeptide repeat proteins, 29 membrane carriers/transporters, a number of new proteins involved in coenzyme biosynthesis and iron metabolism, the pyruvate dehydrogenase kinase, and a type 2C protein phosphatase that may catalyze the dephosphorylation of the pyruvate dehydrogenase complex. Systematic analysis of prominent posttranslational modifications revealed that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy for unbiased, near-comprehensive identification of mitochondrial proteins and their modified forms.

Sesamin induces cell cycle arrest and apoptosis through the inhibition of signal transducer and activator of transcription 3 signalling in human hepatocellular carcinoma cell line HepG2.

Sesamin, one of the most abundant lignans in sesame seeds, has been shown to exhibit various pharmacological effects. The aim of this study was to elucidate whether sesamin promotes cell cycle arrest and induces apoptosis in HepG2 cells and further to explore the underlying molecular mechanisms. Here, we found that sesamin inhibited HepG2 cell growth by inducing G2/M phase arrest and apoptosis. Furthermore, sesamin suppressed the constitutive and interleukin (IL)-6-induced signal transducer and activator of transcription 3 (STAT3) signalling pathway in HepG2 cells, leading to regulate the downstream genes, including p53, p21, cyclin proteins and the Bcl-2 protein family. Our studies showed that STAT3 signalling played a key role in sesamin-induced G2/M phase arrest and apoptosis in HepG2 cells. These findings provided a molecular basis for understanding of the effects of sesamin in hepatocellular carcinoma tumour cell proliferation. Therefore, sesamin may thus be a potential chemotherapy drug for liver cancer.

Hypoglycemic activities of commonly-used traditional Chinese herbs.

The α-amylase and α-glucosidase inhibition activity of 92 Traditional Chinese Medicinal (TCM) herbs, which are permitted to be used as food ingredients, were evaluated using the high throughput assay developed in our laboratory. Among these herbs, twenty-seven of them possessed significant α-amylase inhibition activities ranging from 2.4 to 349.2 µmol AE/g (AE = acarbose equivalent) with inhibition concentrations at 50% inhibition (IC50) from 16.0 to 2342.2 µg/mL, respectively. In addition, they showed α-glucosidase inhibition activities ranging from 0.5 to 31.6 µmolAE/g (IC50 from 49.0 to 3385.5 µg/mL). The extracts of Rhizoma fagopyri dibotryis (Jinqiáomài), Rosa rugosa (Méiguihua), Caulis polygoni multiflori (Shǒuwuténg), Fructus amomi (Sharén), Rhizoma alpiniae officinarum (Gaoliángjiang), Folium ginkgo (Yínxìngyè) and Cortex cinnamomi (Ròuguì) showed the better inhibitory activities against both α-amylase and α-glucosidase. Our results illustrated that these food grade herbs are potent natural hypoglycemic agents and can be used as active ingredients for low glycemic index food production or TCM herbal formulations for controlling hyperglycemia.

Germacrone induces apoptosis in human hepatoma HepG2 cells through inhibition of the JAK2/STAT3 signalling pathway.

Previous studies have shown that STAT3 plays a vital role in the genesis and progression of cancer. In this study, we investigated the relationship between the JAK2/STAT3 signalling pathway and germacrone-induced apoptosis in HepG2 cells. HepG2 cells were incubated with germacrone for 24 h, the protein expression of p-STAT3, STAT3, p-JAK2 and JAK2 was detected by Western Blotting, and RT-PCR was used to determine the expression of STAT3, p53, Bcl-2 and Bax at transcriptional levels. Besides that, HepG2 cells were pre-treated with AG490 or IL-6 for 2 h, and then incubated with germacrone for 24 h. The expression of p-JAK2, JAK2, p-STAT3, STAT3, p53, Bax and Bcl-2 was detected by Western blotting. The activity of HepG2 cells was tested by MTT assay. The apoptosis of HepG2 cells and levels of reactive oxygen species (ROS) were flow cytometrically measured. The results showed that germacrone exposure decreased p-STAT3 and p-JAK2 and regulated expression of p53 and Bcl-2 family members at the same time. Moreover, IL-6 enhanced the activation of the JAK2/STAT3 signalling pathway and therefore attenuated the germacrone-induced apoptosis. Suppression of JAK2/STAT3 signalling pathway by AG490, an inhibitor of JAK2, resulted in apoptosis and an increase in ROS in response to germacrone exposure. We therefore conclude that germacrone induces apoptosis through the JAK2/STAT3 signalling pathway.

Characterization of proanthocyanidins in stems of Polygonum multiflorum Thunb as strong starch hydrolase inhibitors.

Characterization of polyphenolic compounds in the stems of P. multiflorum was conducted using HPLC, high resolution LC-MS and LC-MSn. Proanthocyanidins in particular were isolated in 4.8% yield using solvent extraction followed by Sephadex LH-20 fractionation. HPLC analysis using a diol column revealed oligomers (from dimer to nonamer) as minor components, with (epi)catechin monomeric units predominating, and oligomers with higher degree of polymerization being dominant. Thiolysis treatment of the proanthocyanidins using mercaptoacetic acid produced thioether derivatives of (epi)catechin as the major product and a mean value of the degree of polymerization of 32.6 was estimated from the ratio of terminal and extension units of the (epi)catechin. The isolated proanthocyanidins were shown to strongly inhibit α-amylase with an acarbose equivalence (AE) value of 1,954.7 µmol AE/g and inhibit α-glucosidase with an AE value of 211.1 µmol AE/g.

Anti-tumor effect of germacrone on human hepatoma cell lines through inducing G2/M cell cycle arrest and promoting apoptosis.

Germacrone is one of the main bioactive components in the traditional Chinese medicine Rhizoma curcuma. In this study, the anti-proliferative effect of germacrone on the human hepatoma cell lines and the molecular mechanism underlying the cytotoxicity of germacrone were investigated. Treatment of human hepatoma cell lines HepG2 and Bel7402 with germacrone resulted in cell cycle arrest and apoptosis in a dose-dependent manner as measured by MTT assay, flow cytometric and fluorescent microscopy analysis, while much lower effect on normal human liver cell L02 was observed. Flow cytometric analysis revealed that germacrone induced G2/M arrest in the cell cycle progression that was associated with an obvious decrease in the protein expression of cyclin B1 and its activating partner CDK1 with concomitant inductions of p21. Hoechst 33258 and Annexin V/PI staining results showed that the total cell number in apoptosis associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2/Bcl-xl was increased. In the meantime, the up-regulation of p53 and reactive oxygen species increase were observed, which suggested that germacrone might be a new potent chemopreventive drug candidate for liver cancer via regulating the expression of proteins related to G2/M cell cycle and apoptosis, and p53 and oxidative damage may play important roles in the inhibition of human hepatoma cells growth by germacrone.

Overexpression of a wheat aquaporin gene, TaAQP8, enhances salt stress tolerance in transgenic tobacco.

Aquaporin (AQP) proteins have been shown to transport water and other small molecules through biological membranes, which is crucial for plants to combat salt stress. However, the precise role of AQP genes in salt stress response is not completely understood in plants. In this study, a PIP1 subgroup AQP gene, designated TaAQP8, was cloned and characterized from wheat. Transient expression of TaAQP8-green fluorescent protein (GFP) fusion protein revealed its localization in the plasma membrane. TaAQP8 exhibited water channel activity in Xenopus laevis oocytes. TaAQP8 transcript was induced by NaCl, ethylene and H(2)O(2). Further investigation showed that up-regulation of TaAQP8 under salt stress involves ethylene and H(2)O(2) signaling, with ethylene causing a positive effect and H(2)O(2) acting as a negative factor. Overexpression of TaAQP8 in tobacco increased root elongation compared with controls under salt stress. The roots of transgenic plants also retained a high K(+)/Na(+) ratio and Ca(2+) content, but reduced H(2)O(2) accumulation by an enhancement of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under salt stress. Further investigation showed that whole seedlings from transgenic lines displayed higher SOD, CAT and POD activities, increased NtSOD and NtCAT transcript levels, and decreased H(2)O(2) accumulation and membrane injury under salt stress. Taken together, our results demonstrate that TaAQP8 confers salt stress tolerance not only by retaining high a K(+)/Na(+) ratio and Ca(2+) content, but also by reducing H(2)O(2) accumulation and membrane damage by enhancing the antioxidant system.