Photosynthetic activity and metabolic profiling of bread wheat cultivars contrasting in drought tolerance.

The rapid increase in population growth under changing climatic conditions causes drought stress, threatening world food security. The identification of physiological and biochemical traits acting as yield-limiting factors in diverse germplasm is pre-requisite for genetic improvement under water-deficit conditions. The major aim of the present study was the identification of drought-tolerant wheat cultivars with a novel source of drought tolerance from local wheat germplasm. The study was conducted to screen 40 local wheat cultivars against drought stress at different growth stages. Barani-83, Blue Silver, Pak-81, and Pasban-90 containing shoot and root fresh weight >60% of control and shoot and root dry weight >80% and 70% of control, respectively, P (% of control >80 in shoot and >88 in root), K+ (>85% of control), and quantum yield of PSII > 90% of control under polyethylene glycol (PEG)-induced drought stress at seedling stage can be considered as tolerant, while more reduction in these parameters make FSD-08, Lasani-08, Punjab-96, and Sahar-06 as drought-sensitive cultivars. FSD-08 and Lasani-08 could not maintain growth and yield due to protoplasmic dehydration, decreased turgidity, cell enlargement, and cell division due to drought treatment at adult growth stage. Stability of leaf chlorophyll content (<20% decrease) reflects photosynthetic efficiency of tolerant cultivars, while ~30 µmol/g fwt concentration of proline, 100%-200% increase in free amino acids, and ~50% increase in accumulation of soluble sugars were associated with maintaining leaf water status by osmotic adjustment. Raw OJIP chlorophyll fluorescence curves revealed a decrease in fluorescence at O, J, I, and P steps in sensitive genotypes FSD-08 and Lasani-08, showing greater damage to photosynthetic machinery and greater decrease in JIP test parameters, performance index (PIABS), maximum quantum yield (Fv/Fm) associated with increase in Vj, absorption (ABS/RC), and dissipation per reaction center (DIo/RC) while a decrease in electron transport per reaction center (ETo/RC). During the present study, differential modifications in morpho-physiological, biochemical, and photosynthetic attributes that alleviate the damaging effects of drought stress in locally grown wheat cultivars were analyzed. Selected tolerant cultivars could be explored in various breeding programs to produce new wheat genotypes with adaptive traits to withstand water stress.

Identification of novel source of salt tolerance in local bread wheat germplasm using morpho-physiological and biochemical attributes.

Salt tolerant wheat cultivars may be used as genetic resource for wheat breeding to ensure yield stability in future. The study was aimed to select salt tolerant cultivar(s) to identify novel source of salt tolerance in local wheat germplasm. Initially, 40 local wheat cultivars were screened at 150 mM NaCl stress at seedling stage. Selected salt-tolerant (three; S-24, LU-26S and Pasban-90) and salt-sensitive (four; MH-97, Kohistan-97, Inqilab-91 and Iqbal-2000) wheat cultivars were further evaluated using growth, yield, biochemical and physiological attributes. Growth and yield of selected cultivars were reduced under salt stress due to decline in plant water status, limited uptake of macronutrients (N, P and K), reduced K+/Na+ ratio, photosynthetic pigments and quantum yield of PSII. Wheat plants tried to acclimate salt stress by osmotic adjustment (accumulation of total soluble sugars, proline and free amino acids). Degree of salinity tolerance in cvs. S-24 and LU-26S found to be associated with maintenance of K+/Na+ ratio, osmo-protectant and photosynthetic activity and can be used as donor for salt tolerance in wheat breeding program at least in Pakistan. These cultivars can be further characterized using molecular techniques to identify QTLs/genes for salt exclusion, osmo-protectant and photosynthetic activity for molecular breeding.

Foliar fertigation of ascorbic acid and zinc improves growth, antioxidant enzyme activity and harvest index in barley (Hordeum vulgare L.) grown under salt stress.

Crop productivity is limited by several environmental constraints. Among these, salt stress plays a key role in limiting the growth and yield production of economically important agricultural crops. However, the exogenous fertigation of vitamins and minerals could serve as a "shot-gun" approach for offsetting the deleterious effects of salts present in the rhizosphere. Therefore, an experiment was conducted to quantify the efficacy of foliar fertigation of ascorbic acid (vitamin-C) and zinc (Zn) on the physio-biochemical attributes of barley (Hordeum vulgare L. Genotype B-14011) grown in a saline environment. The salt stress resulted in a reduced biological yield associated with a decrease in chlorophyll pigment, while a significant enhancement in Na+ and Zn2+ was observed under salinity stress. Similarly, the contents of total soluble proteins, total free amino acids, lipid peroxidation, and H2O2 and the activities of antioxidative enzymes (SOD, POD, CAT, APX and proline) were significantly enhanced under salinity stress. Moreover, salinity negatively affected the yield attributes and ion uptake of plants. However, foliar fertigation with AsA +0.03% Zn enhanced vegetative growth, photosynthetic pigments, synchronized ion uptake, the synthesis of enzymatic and non-enzymatic antioxidants, and the harvest index. It is inferred from this study that among all treatments, the effect of foliar fertigation with the AsA+0.03% Zn combination not only improved the salt stress tolerance but also improved the yield attributes, which will aid in the improvement in barley seed yield and is a step to solve the problem of malnutrition through biofortification of vitamin-C and zinc.

Enzyme inhibition and antibacterial potential of 4-Hydroxycoumarin derivatives

The 4-Hydroxycoumarin derivatives are known to show a broad spectrum of pharmacological applications. In this paper we are reporting the synthesis of a new series of 4-Hydroxycoumarin derivatives synthesized through Knovenegal condensation; they were characterized by using UV-Vis, FT-IR, NMR spectroscopies. The synthesized compounds were evaluated for antibacterial activity against Staphylococcus aureus and Salmonella typhimurium strains. The compounds (2), (3) and (8) showed favorable antibacterial activity with zone of inhibitions 26.5± 0.84, 26.0 ± 0.56 and 26.0 ± 0.26 against Staphylococcus aureus (Gram-positive) respectively. However, the compounds (5) and (9) were found more active with 19.5 ± 0.59 and 19.5 ± 0.32 zone of inhibitions against Salmonella typhimurium (Gram-negative). Whereas, in urease inhibition assay, none of the synthesized derivatives showed significant anti-urease activity; although, in carbonic anhydrase-II inhibition assay, the compound (2) and (6) showed enzyme inhibition activity with IC50 values 263±0.3 and 456±0.1, respectively.

Arabidopsis thionin-like genes are involved in resistance against the beet-cyst nematode (Heterodera schachtii).

Plants express various antimicrobial peptides including thionins to protect themselves against pathogens. It was recently found that, in addition to four thionin genes, Arabidopsis contains 67 thionin-like (ThiL) genes including six pseudogenes. It is known that thionins have antimicrobial activity and are part of the plant defense system, however, nothing is known about ThiL genes. In this study, we present a bioinformatic analysis of the (ThiL) gene family in Arabidopsis. We identified 15 different motifs which positioned the ThiL peptides in four groups. A comparison of amino acid sequences showed that the ThiL peptides are actually more similar to the acidic domain of thionin proproteins than to the thionin domain. We selected 10 ThiL genes to study the expression and possible function in the Arabidopsis plant. RT-PCR and promoter:GUS fusions showed that most genes were expressed at a very low level but in several organs and at different developmental stages. Some genes were also expressed in syncytia induced by the beet cyst nematode Heterodera schachti in roots while others were downregulated in syncytia. Some overexpression lines supported lower number of nematodes that developed on the roots after inoculation. Two of the genes resulted in a strong hypersensitive response when infiltrated into leaves of Nicotiana benthamiana. These results indicate that ThiL genes might be involved in the response to biotic stress. ThiL genes have been expanded in the Brassicales and specifically the Brassicaceae. The most extreme example is the CRP2460 subfamily that contains 28 very closely related genes from Arabidopsis which are mostly the result of tandem duplications.

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus.

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl ß-d-N,N',N''-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (ß/α)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 µg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 µg/well Similarly, insect toxicity bioassays of GgChi (50 µg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase.

Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis.

Plant WRKY transcription factors play a vital role in abiotic stress tolerance and regulation of plant defense responses. This study examined AtWRKY11 and AtWRKY17 expression under ABA, salt, and osmotic stress at different developmental stages in Arabidopsis. We used reverse transcriptase PCR, quantitative real-time PCR, and promoter:GUS lines to analyze expression. Both genes were upregulated in response to abiotic stress. Next, we applied the same stressors to seedlings of T-DNA insertion wrky11 and 17 knock-out mutants (single and double). Under stress, the mutants exhibited slower germination and compromised root growth compared with the wild type. In most cases, double-mutant seedlings were more affected than single mutants. These results suggest that wrky11 and wrky17 are not strictly limited to plant defense responses but are also involved in conferring stress tolerance.

CHLOROGENIC ACID: A PHARMACOLOGICALLY POTENT MOLECULE.

Chlorogenic acid (CGA; (IS,3R,4R.5R)-3-{[(2Z)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid) is a naturally occurring polyphenol mostly present in vegetables and fruits. CGA is a prominent component of Traditional Chinese Medicines and is known for various pharmacological activities such as antioxidant, antimicrobial, anti-inflammatory and hepatoprotective etc. This mini-review is an attempt to summarize the available literature in the last decade and to point out future perspectives in this area of research.

Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli.

Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.

Comparison of Expression Vectors for Transient Expression of Recombinant Proteins in Plants.

Production of recombinant proteins in plants is of increasing importance for practical applications. However, the production of stable transformed transgenic plants is a lengthy procedure. Transient expression, on the other hand, can deliver recombinant proteins within a week, and many viral vectors have been constructed for that purpose. Each of them is reported to be highly efficient, robust and cost-effective. Here, a variety of expression vectors which were designed for transient and stable plant transformation, including pPZP3425, pPZP5025, pPZPTRBO, pJLTRBO, pEAQ-HT and pBY030-2R, was compared for the expression of green fluorescent protein and ß-glucuronidase in Nicotiana benthamiana by Agrobacterium-mediated transient expression. Our results show that pPZPTRBO, pJLTRBO and pEAQ-HT had comparable expression levels without co-infiltration of a RNA-silencing inhibitor. The other vectors, including the non-viral vectors pPZP5025 and pPZP3425, needed co-infiltration of the RNA-silencing inhibitor P19 to give good expression levels.

pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage.

BACKGROUND: The production of transgenic plants, either for the overproduction of the protein of interest, for promoter: reporter lines, or for the downregulation of genes is an important prerequisite in modern plant research but is also very time-consuming. RESULTS: We have produced additions to the pPZP family of vectors. Vector pPZP500 (derived from pPZP200) is devoid of NotI sites and vector pPZP600 (derived from pPZP500) contains a bacterial kanamycin resistance gene. Vector pMAA-Red contains a Pdf2.1: DsRed marker and a CaMV:: GUS cassette within the T-DNA and is useful for the production of promoter: GUS lines and overexpression lines. The Pdf2.1 promoter is expressed in seeds and syncytia induced by the beet cyst nematode Heterodera schachti in Arabidopsis roots. Transgenic seeds show red fluorescence which can be used for selection and the fluorescence level is indicative of the expression level of the transgene. The advantage is that plants can be grown on soil and that expression of the marker can be directly screened at the seed stage which saves time and resources. Due to the expression of the Pdf2.1: DsRed marker in syncytia, the vector is especially useful for the expression of a gene of interest in syncytia. CONCLUSIONS: The vector pMAA-Red allows for fast and easy production of transgenic Arabidopsis plants with a strong expression level of the gene of interest.