Shamima Akhtar Sharmin

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.

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.

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.

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.

Agrobacterium‐mediated transformation of reed (Phragmites communis Trinius) using mature seed‐derived calli

Reed (Phragmites communis) is a potential bioenergy plant. We report on its first Agrobacterium‐mediated transformation using mature seed‐derived calli. The Agrobacterium strains LBA4404, EHA105, and GV3101, each harboring the binary vector pIG121Hm, were used to optimize T‐DNA delivery into the reed genome. Bacterial strain, cocultivation period and acetosyringone concentration significantly influenced the T‐DNA transfer. About 48% transient expression and 3.5% stable transformation were achieved when calli were infected with strain EHA105 for 10 min under 800 mbar negative pressure and cocultivated for 3 days in 200 μm acetosyringone containing medium. Putative transformants were selected in 25 mg l−1 hygromycin B. PCR, and Southern blot analysis confirmed the presence of the transgenes and their stable integration. Independent transgenic lines contained one to three copies of the transgene. Transgene expression was validated by RT‐PCR and GUS staining of stems and leaves.

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.

Advances in the molecular breeding of forage crops for abiotic stress tolerance

Forages are the backbone of sustainable agriculture. They includes a wide variety of plant species ranging from grasses, such as tall fescue and bermudagrass, to herbaceous legumes, such as alfalfa and white clover. Abiotic stresses, especially salinity, drought, temperature extremes, high photon irradiance, and levels of inorganic solutes, are the limiting factors in the growth and productivity of major cultivated forage crops. Given the great complexity of forage species and the associated difficulties encountered in traditional breeding methods, the potential from molecular breeding in improving forage crops has been recognized. Plant engineering strategies for abiotic stress tolerance largely rely on the gene expression for enzymes involved in pathways leading to the synthesis of functional and structural metabolites, proteins that confer stress tolerance, or proteins in signaling and regulatory pathways. Genetic engineering allows researchers to control timing, tissue-specificity, and expression level for optimal function of the introduced genes. Thus, the use of either a constitutive or stress-inducible promoter may be useful in certain cases. In this review, we summarize the recent progress made towards the development of transgenic forage plants with improved tolerance to abiotic stresses.

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.