A Comparison of the Effect of Lead (Pb) on the Slow Vacuolar (SV) and Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles.

Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.

iTRAQ-Based Proteomics Analysis of Response to Solanum tuberosum Leaves Treated with the Plant Phytotoxin Thaxtomin A.

Thaxtomin A (TA) is a phytotoxin secreted by Streptomyces scabies that causes common scab in potatoes. However, the mechanism of potato proteomic changes in response to TA is barely known. In this study, the proteomic changes in potato leaves treated with TA were determined using the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique. A total of 693 proteins were considered as differentially expressed proteins (DEPs) following a comparison of leaves treated with TA and sterile water (as a control). Among the identified DEPs, 460 and 233 were upregulated and downregulated, respectively. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, many DEPs were found to be involved in defense and stress responses. Most DEPs were grouped in carbohydrate metabolism, amino acid metabolism, energy metabolism, and secondary metabolism including oxidation-reduction process, response to stress, plant-pathogen interaction, and plant hormone signal transduction. In this study, we analyzed the changes in proteins to elucidate the mechanism of potato response to TA, and we provided a molecular basis to further study the interaction between plant and TA. These results also offer the option for potato breeding through analysis of the resistant common scab.

Nitric oxide-mediated alternative pathway alleviates aluminum-induced programmed cell death in soybean root tips.

Alternative pathway (AP) plays essential roles in plant adaptation to environmental stress. However, the exact role of AP in response to aluminum (Al) toxicity remains elusive. We here provide solid evidences that the activated AP capacity in root tips of soybean alleviated Al toxicity. Furthermore, inhibition of AP by pharmacological or transgenic approach aggravated Al-induced programmed cell death (PCD) occurrence mediated through reactive oxygen species (ROS)-dependent mitochondrial pathway. Our results also demonstrated that nitric oxide (NO) plays a negative role in PCD occurrence caused by Al in soybean root tips. Interestingly, the alleviating effect of NO on Al-induced PCD could be blocked by AP inhibition. Further investigation showed that NO mediates the induction of AP resulting from the upregulation of AOX expression and pyruvate content in Al-treated root tips of soybean. Taken together, our results clearly suggest that AP participates in the alleviation of Al toxicity and also plays a critical role in the alleviating effect of NO on Al-induced PCD occurrence, which will open up new avenues for the improvement of plant growth in acidic soils.

Dynamic regulation of small RNAs in anthocyanin accumulation during blueberry fruit maturation.

Blueberry is rich in anthocyanins which accumulate during fruit maturation. Previous studies mostly focus on their translational/transcriptional regulation, but usually underestimate their post-transcriptional regulation, e.g. small RNAs. This study aimed to identify sRNAs and their potential pathways associated with anthocyanin biosynthesis. During three typical phases of fruit maturation (green, pink, and blue), we investigated dynamic changes of sRNA by deep sequencing sRNA and examined the interaction of sRNAs with their target genes by degradome and RLM-PCR. During maturation, up-regulation of VcmiRNA156 and VcmiR393 resulted in down-regulation of VcSPLs and VcTIR1/AFBs, respectively. An important gene of anthocyanin biosynthesis, VcDFR, was substantially down-regulated at both the mRNA and protein levels, and potentially responded to regulation of VcSPLs and VcTIR1/AFBs. Additionally, indole acetic acid (IAA) and abscisic acid (ABA) were involved in the regulation of anthocyanin biosynthesis by interacting with VcmiR393-TIR1/AFBs and VcmiRNA319-VcMYBs respectively. This information provides another insight into blueberry anthocyanin biosynthesis.

Structural characterization of a polysaccharide from dry mycelium of Penicillium chrysogenum that induces resistance to Tobacco mosaic virus in tobacco plants.

Polysaccharides are essential macromolecules that are present in all living organisms. They have a range of biological activities, such as antiviral, antioxidant, immunity-enhancing, and anticancer activities. In this study, a polysaccharide (PCPS) was separated and extracted from dry mycelium of Penicillium chrysogenum by a boiling water step and gel-filtration chromatography. Its structure was characterized by high performance gel-permeation chromatography, chemical derivative, and nuclear magnetic resonance analyses. The results showed that PCPS is a neutral galactomannan with an apparent molecular weight of 19.5 kDa. We evaluated the antiviral activity of PCPS. In half-leaf assays of tobacco plants, the protective effect of PCPS against Tobacco mosaic virus (TMV) was stronger than the protective effects of ningnanmycin and oligosaccharins. Electron microscopy analyses showed that PCPS can directly inactivate viral particles. The mechanism of the antiviral activity of PCPS was explored in a preliminary study. PCPS induced the production of NO and H2O2 to initiate an early defense response. Treatment with PCPS resulted in increased transcript levels of the genes PAL, 4CL, LPO, and increased activities of phenylalanine lyase and peroxidase, which improved the TMV resistance of Nicotiana glutinosa. Expression of the PR-1b gene was also activated during the defense response.

Effects of oxidative modification by 13-hydroperoxyoctadecadienoic acid on the structure and functional properties of rice protein.

13-hydroperoxyoctadecadienoic acid (13-HPODE) was selected as lipid peroxidation primary products during rice ageing to investigate the effects of lipid hydroperoxide modification on the structural characteristics and functional properties of rice protein. Incubation of rice protein with 13-HPODE resulted in protein carbonylation and loss of protein sulfhydryl groups in a concentration-dependent manner. FT-IR evaluated the effects of 13-HPODE on rice protein, and the results indicated increasing concentration of 13-HPODE resulted in continuous loss of α-helix, ß-turn and random coil structure, and increase in ß-sheet content and amino acid side chains. The content of soluble protein aggregates gradually increased, accompanied by protein crosslink via disulfide bonds. 13-HPODE had a negative impact on the solubility, water holding capacity, foaming capacity, and foam stability of rice protein. 13-HPODE could promote oil holding capacity of rice protein. With the concentration of 13-HPODE below 0.1 mmol/L, emulsifying activity index (EAI) and emulsifying stability index (ESI) of rice protein gradually increased, which due to gradual increase in rice protein surface hydrophobicity and flexibility. However, at relatively high concentration of 13-HPODE (above 1 mmol/L), the adverse effect in EAI and ESI was observed, which might be attributed to a decrease in surface hydrophobicity and protein aggregates formation.

Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress.

BACKGROUND: The family of NAC proteins (NAM, ATAF1/2, and CUC2) represent a class of large plant-specific transcription factors. However, identification and functional surveys of NAC genes of tomato (Solanum lycopersicum) remain unstudied, despite the tomato genome being decoded for several years. This study aims to identify the NAC gene family and investigate their potential roles in responding to Al stress. RESULTS: Ninety-three NAC genes were identified and named in accordance with their chromosome location. Phylogenetic analysis found SlNACs are broadly distributed in 5 groups. Gene expression analysis showed that SlNACs had different expression levels in various tissues and at different fruit development stages. Cycloheximide treatment and qRT-PCR analysis indicated that SlNACs may aid regulation of tomato in response to Al stress, 19 of which were significantly up- or down-regulated in roots of tomato following Al stress. CONCLUSION: This work establishes a knowledge base for further studies on biological functions of SlNACs in tomato and will aid in improving agricultural traits of tomato in the future.

Divergent receptor proteins confer responses to different karrikins in two ephemeral weeds.

Wildfires can encourage the establishment of invasive plants by releasing potent germination stimulants, such as karrikins. Seed germination of Brassica tournefortii, a noxious weed of Mediterranean climates, is strongly stimulated by KAR1, the archetypal karrikin produced from burning vegetation. In contrast, the closely-related yet non-fire-associated ephemeral Arabidopsis thaliana is unusual because it responds preferentially to KAR2. The α/ß-hydrolase KARRIKIN INSENSITIVE 2 (KAI2) is the putative karrikin receptor identified in Arabidopsis. Here we show that B. tournefortii expresses three KAI2 homologues, and the most highly-expressed homologue is sufficient to confer enhanced responses to KAR1 relative to KAR2 when expressed in Arabidopsis. We identify two amino acid residues near the KAI2 active site that explain the ligand selectivity, and show that this combination has arisen independently multiple times within dicots. Our results suggest that duplication and diversification of KAI2 proteins could confer differential responses to chemical cues produced by environmental disturbance, including fire.

H2O2Accumulation, Host Cell Death and Differential Levels of Proteins Related to Photosynthesis, Redox Homeostasis, and Required for Viral Replication Explain the Resistance of EMS-mutagenized Cowpea to Cowpea Severe Mosaic Virus.

Infection with Cowpea severe mosaic virus (CPSMV) represents one of the main limitations for cowpea (Vigna unguiculata L. Walp.) productivity due to the severity of the disease symptoms, frequency of incidence, and difficulties in dissemination control. This study aimed to identify the proteins and metabolic pathways associated with the susceptibility and resistance of cowpea plants to CPSMV. Therefore, we treated the seeds of a naturally susceptible cowpea genotype (CE-31) with the mutagenic agent ethyl methane sulfonate (EMS) and compared the secondary leaf proteomic profile of the mutagenized resistant plants inoculated with CPSMV (MCPI plant group) to those of the naturally susceptible cowpea genotype CE-31 inoculated (CPI) and noninoculated (CPU) with CPSMV. MCPI responded to CPSMV by accumulating proteins involved in the oxidative burst, increasing H2O2 generation, promoting leaf cell death (LCD), increasing the synthesis of defense proteins, and decreasing host factors important for the establishment of CPSMV infection. In contrast, CPI accumulated several host factors that favor CPSMV infection and did not accumulate H2O2 or present LCD, which allowed CPSMV replication and systemic dissemination. Based on these results, we propose that the differential abundance of defense proteins and proteins involved in the oxidative burst, LCD, and the decrease in cowpea protein factors required for CPSMV replication are associated with the resistance trait acquired by the MCPI plant group.

Comparative proteomic analysis reveals that exogenous 6-benzyladenine (6-BA) improves the defense system activity of waterlogged summer maize.

BACKGROUND: Exogenous 6-benzyladenine (6-BA) could improve leaf defense system activity. In order to better understand the regulation mechanism of exogenous 6-benzyladenine (6-BA) on waterlogged summer maize, three treatments including control (CK), waterlogging at the third leaf stage for 6 days (V3-6), and application of 100 mg dm- 3 6-BA after waterlogging for 6 days (V3-6-B), were employed using summer maize hybrid DengHai 605 (DH605) as the experimental material. We used a labeling liquid chromatography-based quantitative proteomics approach with tandem mass tags to determine the changes in leaf protein abundance level at the tasseling stage. RESULTS: Waterlogging significantly hindered plant growth and decreased the activities of SOD, POD and CAT. In addition, the activity of LOX was significantly increased after waterlogging. As a result, the content of MDA and H2O2 was significantly increased which incurred serious damages on cell membrane and cellular metabolism of summer maize. And, the leaf emergence rate, plant height and grain yield were significantly decreased by waterlogging. However, application of 6-BA effectively mitigated these adverse effects induced by waterlogging. Compared with V3-6, SOD, POD and CAT activity of V3-6-B were increased by 6.9, 12.4, and 18.5%, LOX were decreased by 13.6%. As a consequence, the contents of MDA and H2O2 in V3-6-B were decreased by 22.1 and 17.2%, respectively, compared to that of V3-6. In addition, the leaf emergence rate, plant height and grain yield were significantly increased by application of 6-BA. Based on proteomics profiling, the proteins involved in protein metabolism, ROS scavenging and fatty acid metabolism were significantly regulated by 6-BA, which suggested that application of 6-BA exaggerated the defensive response of summer maize at proteomic level. CONCLUSIONS: These results demonstrated that 6-BA had contrastive effects on waterlogged summer maize. By regulating key proteins related to ROS scavenging and fatty acid metabolism, 6-BA effectively increased the defense system activity of waterlogged summer maize, then balanced the protein metabolism and improved the plant physiological traits and grain yield.

Genome-wide identification of small heat-shock protein (HSP20) gene family in grape and expression profile during berry development.

BACKGROUND: Studies have shown that HSP20 (heat-shock protein 20) genes play important roles in regulating plant growth, development, and stress response. However, the grape HSP20 gene family has not been well studied. RESULTS: A total of 48 VvHSP20 genes were identified from the grape genome, which were divided into 11 subfamilies (CI, CII, CIII, CV, CVI, CVII, MI, MII, ER, CP and PX/Po) based on a phylogenetic analysis and subcellular localization. Further structural analysis showed that most of the VvHSP20 genes (93.8%) had no intron or only one intron, while genes that clustered together based on a phylogenetic tree had similar motifs and evolutionarily conserved structures. The HSP20s share a conservedα-crystalline domain (ACD) and the different components of the ACD domain suggest the functional diversity of VvHSP20s. In addition, the 48 VvHSP20 genes were distributed on 12 grape chromosomes and the majority of VvHSP20 genes were located at the proximal or distal ends of chromosomes. Chromosome mapping indicated that four groups of VvHSP20 genes were identified as tandem duplication genes. Phytohormone responsive, abiotic and biotic stress-responsive, and plant development-related cis-elements were identified from the cis-regulatory elements analysis of VvHSP20s. The expression profiles of VvHSP20s genes (VvHSP20-1, 11, 14, 17, 18, 19, 20, 24, 25, 28, 31, 39, 42, and 43) were largely similar between RNA-Seq and qRT-PCR analysis after hydrogen peroxide (H2O2) treatment. The results showed that most VvHSP20s were down-regulated by H2O2 treatment during fruit development. VvHSP20s genes were indeed found to be involved in the grape berry development and differences in their transcriptional levels may be the result of functional differentiation during evolution. CONCLUSIONS: Our results provide valuable information on the evolutionary relationship of genes in the VvHSP20 family, which is useful for future studies on the functional characteristics of VvHSP20 genes in grape.

Inactivation of Potato Polyphenol Oxidase Using Microwave Cold Plasma Treatment.

This study was conducted to examine the effects of microwave cold plasma (CP) treatment on inactivation of polyphenol oxidase (PPO) of potato. The PPO activity and treatment variables were fit to first-order kinetics, the Weibull model, and the second-order model. The optimum CP-generation power and treatment time for inactivating PPO in the PPO extract were found to be 900 W and 40 min, respectively, which resulted in the highest inactivation of PPO (49.5%). PPO activity after CP treatment of potato slices decreased from 72.4% to 59.0% as the sample surface-to-volume ratio increased from 7.1 to 9.0. CP treatment delayed the browning of potato slices. Microwave CP treatment effectively inactivated PPO in potatoes, demonstrating the potential of CP treatment for controlling PPO activity in foods. PRACTICAL APPLICATION: This study demonstrated that microwave CP treatment, a nonthermal food processing technology, inactivates PPO activity in potatoes. The results showed that the inactivation effect of CP treatment on PPO corresponded to the surface-to-volume ratio of potato slices. Furthermore, this study proposed an enzyme inactivation model that is suitable for predicting the inactivation of PPO activity and confirmed that CP treatment delayed browning in potatoes.

Structure and function of class III pistil-specific extensin-like protein in interspecific reproductive barriers.

BACKGROUND: The transmitting tissue of the style is the pathway for pollen tube growth to the ovules and has components that function in recognizing and discriminating appropriate pollen genotypes. In Nicotiana tabacum, the class III pistil extensin-like (PELPIII) arabinogalactan protein is essential for the inhibition of N. obtusifolia pollen tube growth. The transmitting tissue-specific (TTS) arabinogalactan protein amino acid sequence and expression pattern is similar to PELPIII, but it facilitates self-pollinated N. tabacum. The TTS and PELPIII arabinogalactan protein can be divided into the less conserved N-terminal (NTD) and the more conserved C-terminal (CTD) domains. This research tested whether the NTD is the key domain in determining PELPIII function in the inhibition of interspecific pollen tube growth. Three variant PELPIII gene constructs were produced where the PELPIII NTD was exchanged with the TTS NTD and a single amino acid change (cysteine to alanine) was introduced into the PELPIII NTD. The PELPIII variants of N. tabacum were tested for activity by measuring the inhibition N. obtusifolia pollen tube growth by using them to complement a 3'UTR RNAi transgenic line with reduced PELPIII mRNA. RESULTS: The RNAi N. tabacum line had reduced PELPIII mRNA accumulation and reduced inhibition of N. obtusifolia pollen tube growth, but had no effect on self-pollen tube growth or pollen tube growth of 12 other Nicotiana species. The NTD of PELPIII with either the PELPIII or TTS CTDs complemented the loss PELPIII activity in the RNAi transgenic line as measured by inhibition of N. obtusifolia pollen tube growth. The TTS NTD with the PELPIII CTD and a variant PELPIII with a cysteine to alanine mutation in its NTD failed to complement the loss of PELPIII activity and did not inhibit N. obtusifolia pollen tube growth. CONCLUSION: The NTD is a key determinant in PELPIII's function in regulating interspecific pollen tube growth and is a first step toward understanding the mechanism of how PELPIII NTD regulates pollen tube growth.

The role of SIPK signaling pathway in antioxidant activity and programmed cell death of tobacco cells after exposure to cadmium.

Cadmium (Cd) toxicity induces oxidative burst and leads to programmed cell death (PCD) in plant cells. The role of salicylic acid-induced protein kinase (SIPK) signaling pathway in Cd-induced oxidative stress was investigated in suspension-cultured tobacco (Nicotiana tabacum L. cv. Barley 21). The cells were pretreated with 40 µM PD98059 (inhibitor of MAPKK) and then exposed to 50 µM Cd for 24 h. The percentages of cell viability, apoptosis, necrosis, and the content of reactive oxygen species (ROS) were monitored by flow cytometry. Expression of PCD related gene (Hsr203J) and the contents of certain signaling molecules were measured as well. The results showed that Cd increased the expression of SIPK, Hsr203J, and CAT genes, the activities of catalase and caspase-3-like enzymes. Addition of PD98059 inhibitor reduced the expression of Hsr203J and CAT genes, decreased CAT activity, but increased ROS and SA contents, and caspase-3-like activity and apoptosis rate. The highest apoptosis level was accompanied by the highest level of Hsr203J gene expression. From the results it can be suggested that upon treatment of tobacco cells with Cd, internal SA content increased and induced the SIPK signaling pathway, thereby inhibited the antioxidant system and led to PCD.

Intermittent lead-induced stress on antioxidant enzyme activity and subcellular distribution of Pb in Pogonatherum crinitum seedlings.

Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator due to its high Pb tolerance and accumulation ability. However, the mechanisms that support Pb accumulation and tolerance in P. crinitum are not yet clearly understood. An indoor hydroponic experiment was conducted by cultivating P. crinitum seedlings exposed to intermittent Pb stress for 60 days, divided into four stages (T1, T2, T3 and T4), with a 15-day duration per stage. The following concentrations of Pb were used: 0, 500, 0, 500 mg·l-1 and 0, 1000, 0, 1000 mg·l-1 ). Antioxidant enzyme activity, Pb concentration and subcellular distribution of Pb were measured at each of the above stages. The results showed that superoxide dismutase (SOD) activity in shoots, and SOD, peroxidase (POD) and malondialdehyde (MDA) activity in shoots and roots significantly increased from T1 (no Pb stress) to T2 (Pb stress) in both 500 mg·l-1 and 1000 mg·l-1 treatments; however, no significant difference was noted between stages T3 (no Pb stress) and T4 (Pb stress). There was no obvious effect of Pb stress on catalase (CAT) activity in shoots and roots among different stages. The Pb concentration in shoots was up to 5090.90 mg·kg-1 and 7573.57 mg·kg-1 , and the bioconcentration factor (BFC) was 10.18 and 7.57 for the 500 mg·l-1 and 1000 mg·l-1 treatments, respectively, which confirmed the Pb hyperaccumulator characteristics of P. crinitum. For plants under Pb stress, most of the Pb was fixed in the cell walls, with a smaller amount in leaves and root vacuoles. Both SOD and POD scavenging of reactive oxygen radicals and fixing and compartmentalisation of Pb in the cell wall might play important roles in detoxification of P. crinitum seedlings in response to Pb stress. There was no phased response of P. crinitum to intermittent Pb stress and the physiological response to Pb stress may be contiguous.

Waterlogging-induced adventitious root formation in cucumber is regulated by ethylene and auxin through reactive oxygen species signalling.

Development of adventitious roots (ARs) at the base of the shoot is an important adaptation of plants to waterlogging stress; however, its physiological mechanisms remain unclear. Here, we investigated the regulation of AR formation under waterlogged conditions by hormones and reactive oxygen species (ROS) in Cucumis sativus L., an agriculturally and economically important crop in China. We found that ethylene, auxin, and ROS accumulated in the waterlogged cucumber plants. On the other hand, application of the ethylene receptor inhibitor 1-methylcyclopropene (1-MCP), the auxin transport inhibitor 1-naphthylphthalamic acid (NPA), or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) decreased the number of ARs induced by waterlogging. Auxin enhanced the expression of ethylene biosynthesis genes, which led to ethylene entrapment in waterlogged plants. Both ethylene and auxin induced the generation of ROS. Auxin-induced AR formation was inhibited by 1-MCP, although ethylene-induced AR formation was not inhibited by NPA. Both ethylene- and auxin-induced AR formation were counteracted by DPI. These results indicate that auxin-induced AR formation is dependent on ethylene, whereas ethylene-induced AR formation is independent of auxin. They also show that ROS signals mediate both ethylene- and auxin-induced AR formation in cucumber plants.

Effect of Methyl Jasmonate on the Expression of Wcs Genes and the Activity of Antioxidant Enzymes at Wheat Cold Adaptation.

We studied the effect of phytohormone methyl jasmonate (MJ) on the expression of WCS (wheat cold specific) family genes and the activity of the key antioxidant enzymes in wheat seedling leaves at a low hardening temperature (4°C). Incubation at 4°C induced an increase in the cold tolerance of seedlings, which was accompanied by an increase in the levels of transcripts of WCS120 and WCS19 genes as well as an increase in the activity of superoxide dismutase (SOD) and peroxidase (PO). The pretreatment of seedlings with MJ significantly increased the mRNA content of WCS120 and WCS19 genes and, at the same time, caused an increase in the activity of SOD and PO, thus contributing to further increase in the cold tolerance of the plants (i.e., enhanced the hardening effect induced by low temperature).

G proteins sculp root architecture in response to nitrogen in rice and Arabidopsis.

Nitrogen is a key nutrient for plant growth and development. Plants regulate nitrogen availability and uptake efficiency through controlling root architecture. While the heterotrimeric G protein complex is an important element to regulate root morphology, it remains unknown whether the G protein regulates the root architecture in response to nitrogen supply. We used rice and Arabidopsis G protein mutants to study the root architecture in response to different nitrogen concentrations. We found that nitrogen inhibits root horizontal projection area (network area), root perimeter, total length, but not root diameter (average root width). Nitrogen influenced bushiness and root spatial distribution by inhibiting horizontal growth and promoting vertical expansion. The dynamic changes of the rice G protein mutant DK22 at different concentrations of nitrogen from day 7 to day 9 were different from the wild type with regard to bushiness and spatial distribution. The agb1-2 mutant in Arabidopsis lacked the inhibitory effect of nitrate on root growth. The heterotrimeric G protein complex regulates the inhibitory effect on root growth caused by high nitrogen supply and root spatial distribution in response to different nitrogen concentrations.

Effects of manganese toxicity on the protein profile of tomato (Solanum lycopersicum) roots as revealed by two complementary proteomic approaches, two-dimensional electrophoresis and shotgun analysis.

The aim of this work was to assess the effects of manganese (Mn) toxicity on the proteome of tomato roots using two proteomic approaches, shotgun and two-dimensional electrophoresis. The shotgun approach yielded 367 reliable proteins, whereas the 2-DE approach detected 340 consistent spots. The 2-DE method found 54 proteins changing in relative abundance in the excess Mn treatment, whereas the shotgun detected changes in 118 proteins. Only 7% of the differential proteins were found by both methods, illustrating their complementary nature. Metabolic pathways most affected were protein metabolism, oxido-reductases and signaling. Results support that Mn toxicity alters the protein turnover and impairs energy production in roots, leading to changes in glycolysis, pyruvate metabolism, TCA and oxidative phosphorylation. Excess Mn also induced changes in peroxidases and hydrolases participating in cell wall lignification and suberization and activated plant defense mechanisms, with changes occurring via pathogenesis-related proteins as well as peroxidases. Finally, Mn toxicity elicited regulatory mechanisms and affected the abundance of root nutrient reservoir proteins. The overall analysis of the differential root proteome upon Mn toxicity suggests a general slowdown of metabolic activities, especially energy production, cell wall integrity and protein turnover, which occurs in parallel with increases in stress related proteins.

Molecular cloning and expression analysis of ammonium transporters in tea plants (Camellia sinensis (L.) O. Kuntze) under different nitrogen treatments.

Ammonium is a major inorganic nitrogen source for tea plant growth and is mainly taken up and transported by ammonium transporters (AMTs). Here, we analyzed the NH4+ uptake kinetics of three tea cultivars, Longjing43 (LJ43), Zhongcha108 (ZC108) and Zhongcha302 (ZC302). The results revealed that ZC302 had a higher NH4+ uptake efficiency than the other two cultivars. The full CDS sequences of three Camellia sinensis ammonium transporter (CsAMT) genes, i.e., CsAMT1.1, CsAMT1.2 and CsAMT3.1, were cloned. Analysis of tissue-specific expression showed that CsAMT1.2 followed a root-specific expression pattern, while transcripts of CsAMT1.1 and CsAMT3.1 were mainly accumulated in leaves. The temporal course experiment on gene expression levels showed CsAMT1.1 and CsAMT3.1 followed a reciprocal expression pattern in leaves as CsAMT1.1 was up-regulated by a short time (2 h, 6 h) nitrogen (N) supply both in the leaves and buds of LJ43 and ZC108; and the expression of CsAMT3.1 in leaves was increased by a long time (72 h) N supply, particularly in ZC302. Therefore, we inferred that CsAMT1.1 and CsAMT3.1 might play important roles in photorespiratory ammonium metabolism. The expression of CsAMT1.2 was extremely high in roots and can be greatly induced by N over a short period of time, especially in ZC302; thus, we concluded CsAMT1.2 might play an important role in ammonium uptake from soils in tea plant roots.