Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

MAIN CONCLUSION: Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H+-ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H+-ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H+-ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H+-ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

Mapping the leaf proteome of Miscanthus sinensis and its application to the identification of heat-responsive proteins.

Miscanthus sinensis is a promising bioenergy crop; however, its genome is poorly represented in sequence databases. As an initial step in the comprehensive analysis of the M. sinensis proteome, we report a reference 2-DE protein map of the leaf. A total of 316 protein spots were excised from the gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or MALDI-TOF/TOF MS. Two hundred and thirty-two protein spots were identified, which are involved in a variety of cellular functions through distinct metabolic pathways. Functional annotation of the proteins revealed a nearly complete C3 and C4 cycle, starch and sugar synthesis pathway, glycolysis pathway, a significant portion of the pentose phosphate pathway, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, kaurene, chalcone, sesquiterpene and lignin biosynthesis. Other proteins belong to primary metabolism, transcription, protein synthesis, protein destination/storage, disease/defense, cell growth/division, transportation and signal transduction. To test the applicability of the constructed map, we studied the effect of heat stress on M. sinensis leaf proteome. Twenty-five protein spots were upregulated, five were newly induced and twenty-five spots were downregulated by heat treatment. The differentially accumulated proteins were involved in photosynthesis, energy metabolism, gene transcription, protein kinases and phosphatases, signal transduction, protein synthesis and heat shock responses. C4-specific pyruvate orthophosphate dikinase, Rubisco large subunit, Rubisco activase and some associated proteins were upregulated during heat stress and tend to restore upon recovery. Identification of these proteins provides some important clues regarding the way M. sinensis copes with hot climate. This work represents the first extensive proteomic description of M. sinensis and provides a reference map and heat-responsive candidates for future molecular and physiological studies of this bioenergy crop.

Engineering Nicotiana tabacum trichomes for triterpenic acid production.

In this work, we aimed at implementing the biosynthesis of triterpenic acids in Nicotiana tabacum glandular trichomes. Although endogenous genes coding for enzymes involved in such biosynthetic pathway are found in the Nicotiana tabacum genome, implementing such pathway specifically in glandular trichomes required to boost endogenous enzymatic activities. Five transgenes coding for a farnesyl-diphosphate synthase, a squalene synthase, a squalene epoxidase, a beta-amyrin synthase and a beta-amyrin 28-monooxygenase were introduced in N.tabacum, their expression being driven by pMALD1, a trichome-specific transcriptional promoter. This study aimed at testing whether sinking isoprenoid precursors localized in plastids, by exploiting potential cross-talks allowing the exchange of terpenoid pools from the chloroplast to the cytosol, could be a way to improve overall yield. By analyzing metabolites extracted from entire leaves, a low amount of ursolic acid was detected in plants expressing the five transgenes. Our study shows that the terpene biosynthetic pathway could be, in part, redirected in N.tabacum glandular trichomes with no deleterious phenotype at the whole plant level (chlorosis, dwarfism,…). In light of our results, possible ways to improve the final yield are discussed.

Overexpression of a chloroplast-localized small heat shock protein OsHSP26 confers enhanced tolerance against oxidative and heat stresses in tall fescue.

Small heat shock proteins are involved in stress tolerance. We previously isolated and characterized a rice cDNA clone, Oshsp26, encoding a chloroplast-localized small heat shock protein that is expressed following oxidative or heat stress. In this study, we transferred this gene to tall fescue plants by an Agrobacterium-mediated transformation system. The integration and expression of the transgene was confirmed by PCR, Southern, northern, and immunoblot analyzes. Compared to the control plants, the transgenic plants had significantly lower electrolyte leakage and accumulation of thiobarbituric acid-reactive substances when exposed to heat or methyl viologen. The photochemical efficiency of photosystem II (PSII) (Fv/Fm) in the transgenic tall fescue plants was higher than that in the control plants during heat stress (42°C). These results suggest that the OsHSP26 protein plays an important role in the protection of PSII during heat and oxidative stress in vivo.

Improved drought and salt tolerance of Arabidopsis thaliana by transgenic expression of a novel DREB gene from Leymus chinensis.

Dehydration-responsive element-binding (DREB) proteins are important transcription factors in plant stress responses and signal transduction. Based on high-throughput sequencing results, a new cDNA sequence encoding an LcDREB3a transcription factor from the drought-resistant forage grass, Leymus chinensis, was isolated by RACE PCR. Sequence similarity analysis indicates that the gene product is active in the ABA-responsive pathway, and real-time PCR-based expression analysis shows the transcript accumulates in response to a variety of stress treatments. These results indicate that LcDREB3a is involved in both ABA-dependent and -independent signal transduction in the stress-responsive process of L. chinensis. The identity of the gene product as a DREB transcription factor is supported by observations of its nuclear localization when transiently expressed as a GFP fusion in onion epidermal cells. Furthermore, LcDREB3a is able to activate reporter gene expression, and the protein is shown to specifically bind to the conserved DRE element in a yeast one-hybrid assay. The transgenic expression of LcDREB3a in Arabidopsis causes no growth retardation and induces the increased expression of stress tolerance genes compared to control, resulting in improved drought and salt stress tolerance. Thus, LcDREB3a, encoding a stress-inducible DREB transcription factor, could enhance the abiotic stress tolerance of plants.

Mitochondria-Targeted SmsHSP24.1 Overexpression Stimulates Early Seedling Vigor and Stress Tolerance by Multi-Pathway Transcriptome-Reprogramming.

Among the diverse array of heat shock proteins across the three domains of life, mitochondria-targeted small heat shock proteins (sHSPs) are evolved in the plant lineage. However, they remained mysterious and understudied. In this study, we reported a systematic study of a novel mitochondria-targeted nuclear sHSP from eggplant (Solanum melongena L.; SmsHSP24.1). Differential expression of SmsHSP24.1 indicated its positive role exerted during stress conditions. Escherichia coli-BL21 cell line overexpressing the SmsHSP24.1 showed excellent thermo-tolerance ability, tolerating up to 52°C. Spectrometry and electron microscopy revealed a multimeric structure of the protein which acted as a molecular chaperone at high temperatures. Overexpression of SmsHSP24.1 significantly enhanced resistance against heat, drought, and salt stresses and showed rapid germination in constitutively overexpressed eggplant lines. RNA-seq analysis reveals an apparent upregulation of a set of reactive oxygen species (ROS) scavenging enzymes of the glutathione (GHS) pathway and mitochondrial electron transport chain (ETC). Significant upregulation was also observed in auxin biosynthesis and cell-wall remodeling transcripts in overexpressed lines. qPCR, biochemical and physiological analysis further aligned with the finding of transcriptome analysis and suggested an essential role of SmsHSP24.1 under various stress responses and positive physiological influence on the growth of eggplants. Therefore, this gene has immense potential in engineering stress-resilient crop plants.

Enhanced tolerance of transgenic tall fescue plants overexpressing 2-Cys peroxiredoxin against methyl viologen and heat stresses.

Plant 2-Cys peroxiredoxins (2-Cys Prx) has both peroxidase and chaperon function. We overexpressed an Arabidopsis 2-Cys Prx in transgenic tall fescue (Festuca arundinacea) plants to confer tolerance against heat and methyl viologen (MV) stress. Transgenic plants were generated by Agrobacterium-mediated genetic transformation, and integration and expression of the transgene was confirmed by Southern, northern and western blot analyses. Compared to control plants, transgenic plants had significantly less electrolyte leakage and thiobarbituric acid-reactive substances (TBARS) when exposed to heat or MV. Under heat stress (42 degrees C), transgenic plants maintained their chlorophyll fluorescence (Fv/Fm) for 24 h while control plants lost chlorophyll fluorescence very quickly. We conclude that the high levels of 2-Cys Prx proteins in transgenic plants protect leaves from oxidative damage probably due to chaperon activity.

Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress.

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

Glyphosate-induced oxidative stress in rice leaves revealed by proteomic approach.

Glyphosate is one of the most widely used herbicides in cereal-growing regions worldwide. In the present work, the protein expression profile of rice leaves exposed to glyphosate was analyzed in order to investigate the alternative effects of glyphosate on plants. Two-week-old rice leaves were subjected to glyphosate or a reactive oxygen species (ROS) inducing herbicide paraquat, and total soluble proteins were extracted and analyzed by two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. A total of 25 differentially expressed proteins were identified from the glyphosate treated sample, wherein 18 proteins were up-regulated and 7 proteins were down-regulated. These proteins had shown a parallel expression pattern in response to paraquat. Results from the 2-DE analysis, combined with immunoblotting, clearly revealed that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit was significantly decreased by the treatment of both herbicides. An increased accumulation of antioxidant enzymes including ascorbate peroxidase, glutathione S-transferase, thioredoxin h-type, nucleoside diphosphate kinase 1, peroxiredoxin and a superoxide dismutase [Cu-Zn] chloroplast precursor in the glyphosate-treated sample suggests that a glyphosate treatment possibly generates oxidative stress in plants. Moreover, a gene expression analysis of five antioxidant enzymes by Northern blot confirmed their mRNA levels in the rice leaves. A histo-cytochemical investigation with DAB (3,3-diaminobenzidine) to localize H(2)O(2) and increases of the thiobarbituric acid reactive substances (TBARS) concentration revealed that the glyphosate application generates ROS, which resulted in the peroxidation and destruction of lipids in the rice leaves.

Pathway based therapeutic targets identification and development of an interactive database CampyNIBase of Campylobacter jejuni RM1221 through non-redundant protein dataset.

The bacterial species Campylobacter jejuni RM1221 (CjR) is the primary cause of campylobacteriosis which poses a global threat for human health. Over the years the efficacy of antibiotic treatment is becoming more fruitless due to the development of multiple drug resistant strains. Therefore, identification of new drug targets is a valuable tool for the development of new treatments for affected patients and can be obtained by targeting essential protein(s) of CjR. We conducted this in silico study in order to identify therapeutic targets by subtractive CjR proteome analysis. The most important proteins of the CjR proteome, which includes chokepoint enzymes, plasmid, virulence and antibiotic resistant proteins were annotated and subjected to subtractive analyses to filter out the CjR essential proteins from duplicate or human homologous proteins. Through the subtractive and characterization analysis we have identified 38 eligible therapeutic targets including 1 potential vaccine target. Also, 12 potential targets were found in interactive network, 5 targets to be dealt with FDA approved drugs and one pathway as potential pathway based drug target. In addition, a comprehensive database 'CampyNIBase' has also been developed. Besides the results of this study, the database is enriched with other information such as 3D models of the identified targets, experimental structures and Expressed Sequence Tag (EST) sequences. This study, including the database might be exploited for future research and the identification of effective therapeutics against campylobacteriosis. URL: (http://nib.portal.gov.bd/site/page/4516e965-8935-4129-8c3f-df95e754c562#Banner).

Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter.

Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission.

Screening for salt-responsive proteins in two contrasting alfalfa cultivars using a comparative proteome approach.

A comparative proteomic approach was carried out between two contrasting alfalfa cultivars, nonomu (NM-801; salt tolerant) and vernal (VN; salt intolerant) in terms of salt tolerance. Seedlings were subjected to salt stress (50 and 100 mM NaCl) for three days. Several physiological parameters (leaf water, chlorophyll, root Na(+), K(+), and Ca(2+)) and root proteome profile were analyzed. Comparison of physiological status revealed that NM-801 is more tolerant to salt than VN. Eighty three differentially expressed proteins were found on 2-DE maps, of which 50 were identified by MALDI-TOF or MALDI-TOF/TOF mass spectrometry. These proteins were involved in ion homeostasis, protein turnover and signaling, protein folding, cell wall components, carbohydrate and energy metabolism, reactive oxygen species regulation and detoxification, and purine and fatty acid metabolism. The comparative proteome analysis showed that 33 salt-responsive proteins were significantly changed in both cultivars, while 17 (14 in VN and 3 in NM-801) were cultivar-specific. Peroxidase, protein disulfide-isomerase, NAD synthetase, and isoflavone reductase were up-regulated significantly only in NM-801 in all salt concentrations. In addition, we identified novel proteins including NAD synthetase and biotin carboxylase-3 that were not reported previously as salt-responsive. Taken together, these results provide new insights of salt stress tolerance in alfalfa.

Elimination and detection of viruses in meristem-derived plantlets of sweetpotato as a low-cost option toward commercialization.

Viral diseases affecting sweetpotato are the most devastating and cause up to 98 % yield loss. In this paper, we report, meristem culture, graft transmission and virus indexing for management of viral pathogens in seven elite sweetpotato cultivars. Plantlets were developed in vitro from the apical meristematic dome with one to two leaf primordia. Mericlones were grafted on virus-sensitive indicator plant Ipomoea setosa and no viral disease symptoms were seen on I. setosa leaves in most cases. This indicates that no viruses translocated from meristem-derived scions to the virus-sensitive root stock. On the other hand, most of the non-tested traditional planting material induced distinct disease symptoms upon grafting, which revealed the presence of one or more viruses in it. About 85 % of mericlones recovered from 0.3-0.5 mm size meristem were tested as virus free, whereas it is difficult to culture meristems smaller than 0.3 mm due to dissection damage and too small a size. Virus-tested mericlones were further micropropagated and transferred to the field. Only few plants were found to be diseased in the R1 field trial. Root yield in the R2 generation was increased significantly when compared with non-tested control plants. During field exposure, only a low percentage of healthy plants were found infected with viruses when managed in a net house. This implies that viral vectors were present during the growing season and reinfection could be effectively reduced by net house management. We concluded that this low-cost technique of producing virus-tested planting material would significantly boost the yield through efficient removal of yield-reducing pathogens.

Chromium-induced physiological and proteomic alterations in roots of Miscanthus sinensis.

Despite the widespread occurrence of chromium toxicity, its molecular mechanism is poorly documented in plants compared to other heavy metals. To investigate the molecular mechanisms that regulate the response of Miscanthus sinensis roots to elevated level of chromium, seedlings were grown for 4 weeks and exposed to potassium dichromate for 3 days. Physiological, biochemical and proteomic changes in roots were investigated. Lipid peroxidation and H2O2 content in roots were significantly increased. Protein profiles analyzed by two-dimensional gel electrophoresis revealed that 36 protein spots were differentially expressed in chromium-treated root samples. Of these, 13 protein spots were up-regulated, 21 protein spots were down-regulated and 2 spots were newly induced. These differentially displayed proteins were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. The identified proteins included known heavy metal-inducible proteins such as carbohydrate and nitrogen metabolism, molecular chaperone proteins and novel proteins such as inositol monophosphatase, nitrate reductase, adenine phosphoribosyl transferase, formate dehydrogenase and a putative dihydrolipoamide dehydrogenase that were not known previously as chromium-responsive. Taken together, these results suggest that Cr toxicity is linked to heavy metal tolerance and senescence pathways, and associated with altered vacuole sequestration, nitrogen metabolism and lipid peroxidation in Miscanthus roots.

Analysis of arsenic stress-induced differentially expressed proteins in rice leaves by two-dimensional gel electrophoresis coupled with mass spectrometry.

In the present study, we have investigated the protein expression profile of rice leaves under arsenic (As) stress. Two-week-old rice seedlings were exposed to two concentrations of arsenate (50 or 100 microM), and leaf samples were collected 4d after treatment. To elucidate the As stress-induced differentially expressed proteins in rice leaves, proteins were extracted from the control and treated samples, separated by two-dimensional gel electrophoresis (2-DE), and visualized by staining with Coomassie Brilliant Blue (CBB). A total of 14 protein spots showed reproducible changes in expression of at least 1.5-fold when compared to the control and showed a similar expression pattern in both treatments. Of these 14 spots, 8 were up-regulated and 6 were down-regulated following exposure to As. These proteins were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The increased expression of several proteins associated with energy production and metabolism suggests that higher energy is required for activation of the metabolic processes in leaves exposed to As. On the other hand, results from the 2-DE analysis, combined with immunoblotting, clearly revealed that the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit was significantly decreased under As stress. Thus, the down-regulation of RuBisCO and chloroplast 29 kDa ribonucleoproteins might be the possible causes of the decreased photosynthesis rate under As stress.

Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage.

To gain better insight into how soybean roots respond to waterlogging stress, we carried out proteomic profiling combined with physiological analysis at two time points for soybean seedlings in their early vegetative stage. Seedlings at the V2 stage were subjected to 3 and 7 days of waterlogging treatments. Waterlogging stress resulted in a gradual increase of lipid peroxidation and in vivo H2O2 level in roots. Total proteins were extracted from root samples and separated by two-dimensional gel electrophoresis (2-DE). A total of 24 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue (CBB) staining were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. Of these, 14 proteins were upregulated; 5 proteins were decreased; and 5 were newly induced in waterlogged roots. The identified proteins include well-known classical anaerobically induced proteins as well as novel waterlogging-responsive proteins that were not known previously as being waterlogging responsive. The novel proteins are involved in several processes, i.e. signal transduction, programmed cell death, RNA processing, redox homeostasis and metabolisms of energy. An increase in abundance of several typical anaerobically induced proteins, such as glycolysis and fermentation pathway enzymes, suggests that plants meet energy requirement via the fermentation pathway due to lack of oxygen. Additionally, the impact of waterlogging on the several programmed cell death- and signal transduction-related proteins suggest that they have a role to play during stress. RNA gel blot analysis for three programmed cell death-related genes also revealed a differential mRNA level but did not correlate well with the protein level. These results demonstrate that the soybean plant can cope with waterlogging through the management of carbohydrate consumption and by regulating programmed cell death. The identification of novel proteins such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrinogen oxidase in response to waterlogging stress may provide new insight into the molecular basis of the waterlogging-stress response of soybean.

Somatic embryogenesis and plant regeneration in Wedelia calendulacea Less. an endangered medicinal plant

In this work, plant regeneration via somatic embryogenesis was achieved from leaf and internode derived callus of Wedelia calendulacea, an endangered medicinal plant. Primary callus was induced by culturing leaf disc and internode explant on Murashige and Skoog medium supplemented with 2.0 mg L-1 of 2,4-D under light condition. Transfer of embryogenic callus on a reduced concentration of 2,4-D facilitated somatic embryo development while calluses remained unorganized at the same 2,4-D level. A histological analysis confirmed somatic embryo by revealing the presence of a closed vascular system in the developing embryos and lack of a vascularconnection with surrounding callus tissues. Somatic embryos germinated into plantlets upon transfer on MS medium containing 1.0 mg L-1 BAP plus 0.5 mg L-1 GA3. Plantlets were acclimatized successfully and survived under soil condition. This is the first on somatic embryogenesis of W.calendulacea. This result could facilitate genetic transformation of this important medicinal plant.

Micropropagation of an elite F1 watermelon (Citrullus lanatus) hybrid from the shoot tip of field grown plants

The aim of this work was to develop a protocol for rapid micropropagation of an elite F1 hybrid watermelon cultivar using shoot tip of field-grown plants. Maximum frequency (73%) of shoot tip showed growth response in MS medium supplemented with 5 mg l-1 benzyl adenine (BA) and 0.1 mg l-1 indole-3 acetic acid (IAA). Upon transfer to cytokinin-enriched medium, the cultures produced multiple shoots and 2.0 mg l-1 BA was optimum in this respect. Addition of gibberellic acid (GA3) in the multiplication medium resulted in better growth of shoots. Rooting rate was 100% when shoots were obtained from second subculture were cultured in medium with 1.0 mg l-1 indole-3 butyric acid (IBA). The shoots produced more roots with increasing number of subcultures. About 72% of the regenerated plantlets acclimatized successfully and survived in the soil condition.