Pectic galactan affects cell wall architecture during secondary cell wall deposition.

MAIN CONCLUSION: ß-(1,4)-galactan determines the interactions between different matrix polysaccharides and cellulose during the cessation of cell elongation. Despite recent advances regarding the role of pectic ß-(1,4)-galactan neutral side chains in primary cell wall remodelling during growth and cell elongation, little is known about the specific function of this polymer in other developmental processes. We have used transgenic Arabidopsis plants overproducing chickpea ßI-Gal ß-galactosidase under the 35S CaMV promoter (35S::ßI-Gal) with reduced galactan levels in the basal non-elongating floral stem internodes to gain insight into the role of ß-(1,4)-galactan in cell wall architecture during the cessation of elongation and the beginning of secondary growth. The loss of galactan mediated by ßI-Gal in 35S::ßI-Gal plants is accompanied by a reduction in the levels of KOH-extracted xyloglucan and an increase in the levels of xyloglucan released by a cellulose-specific endoglucanase. These variations in cellulose-xyloglucan interactions cause an altered xylan and mannan deposition in the cell wall that in turn results in a deficient lignin deposition. Considering these results, we can state that ß-(1,4)-galactan plays a key structural role in the correct organization of the different domains of the cell wall during the cessation of growth and the early events of secondary cell wall development. These findings reinforce the notion that there is a mutual dependence between the different polysaccharides and lignin polymers to form an organized and functional cell wall.

A cupin domain is involved in α-amylase inhibitory activity.

Proteinaceous α-amylase inhibitors have specialized activities that make some strong inhibition of α-amylases. New α-amylase inhibitors continue to be discovered so far. A proteinaceous α-amylase inhibitor CL-AI was isolated and identified from chickpea seeds. CL-AI, encoded by Q9SMJ4, was a storage legumin precursor containing one α-chain and one ß-chain, and each chain possessed a same conserved cupin domain. Amino acid mutation and deficiency of cupin domain would lead to loss of α-amylase inhibitory activity, indicating that it was essential for inhibitory activity. CL-AI(α + ß) in its single stranded state in vivo had inhibitory activity. After it was processed into one α-chain and one ß-chain, the two chains were connected to each other via disulfide bond, which would cover the cupin domains and lead to the loss of inhibitory activity. The CL-AI(α + ß), α-chain and ß-chain could inhibit various α-amylases and delay the seed germination of wheat, rice and maize as well as the growth and development of potato beetle larva. Two cupin proteins, Glycinin G1 in soybean and Glutelinin in rice were also found to have inhibitory activity. Our results indicated that the cupin domain is involved in α-amylase inhibitory activity and the proteins with a cupin domain may be a new kind of proteinaceous α-amylase inhibitor.

Overexpression of Cicer arietinum ßIII-Gal but not ßIV-Gal in arabidopsis causes a reduction of cell wall ß-(1,4)-galactan compensated by an increase in homogalacturonan.

In Cicer arietinum, as in several plant species, the ß-galactosidases are encoded by multigene families, although the role of the different proteins is not completely elucidated. Here, we focus in 2 members of this family, ßIII-Gal and ßIV-Gal, with high degree of amino acid sequence identity (81%), but involved in different developmental processes according to previous studies. Our objective is to deepen in the function of these proteins by establishing their substrate specificity and the possible alterations caused in the cell wall polysaccharides when they are overproduced in Arabidopsis thaliana by constructing the 35S::ßIII-Gal and 35S::ßIV-Gal transgenic plants. ßIII-Gal does cause visible alterations of the morphology of the transgenic plant, all related to a decrease in growth at different stages of development. FTIR spectroscopy and immunological studies showed that ßIII-Gal causes changes in the structure of the arabidopsis cell wall polysaccharides, mainly a reduction of the galactan side chains which is compensated by a marked increase in homogalacturonan, which allows us to attribute to galactan a role in the control of the architecture of the cell wall, and therefore in the processes of growth. The 35S::ßIV-Gal plants do not present any phenotypic changes, neither in their morphology nor in their cell walls. In spite of the high sequence homology, our results show different specificity of substrate for these proteins, maybe due to other dissimilar characteristics, such as isoelectric points or the number of N-glycosylation sites, which could determine their enzymatic properties and their distinct action in the cell walls.

Identification of Purple Acid Phosphatases in Chickpea and Potential Roles of CaPAP7 in Seed Phytate Accumulation.

Purple acid phosphatases (PAPs) play important roles in phosphate (Pi) acquisition and utilization. These PAPs hydrolyze organic Phosphorus (P) containing compounds in rhizosphere as well as inside the plant cell. However, roles of PAPs in one of the most widely cultivated legumes, chickpea (Cicer arietnum L.), have not been unraveled so far. In the present study, we identified 25 putative PAPs in chickpea (CaPAPs) which possess functional PAP motifs and domains. Differential regulation of CaPAPs under different nutrient deficiencies revealed their roles under multiple nutrient stresses including Pi deficiency. Interestingly, most of the CaPAPs were prominently expressed in flowers and young pods indicating their roles in flower and seed development. Association mapping of SNPs underlying CaPAPs with seed traits revealed significant association of low Pi inducible CaPAP7 with seed weight and phytate content. Biochemical characterization of recombinant CaPAP7 established it to be a functional acid phosphatase with highest activity on most abundant organic-P substrate, phytate. Exogenous application of recombinant CaPAP7 enhanced biomass and Pi content of Arabidopsis seedlings supplemented with phytate as sole P source. Taken together, our results uncover the PAPs in chickpea and potential roles of CaPAP7 in seed phytate accumulation.

Assessment of Antioxidant Enzyme Activity and Mineral Nutrients in Response to NaCl Stress and its Amelioration Through Glutathione in Chickpea.

Salinity stress has been reckoned as one of the major threat towards crop productivity as it causes significant decline in the yield. The impact of NaCl stress (0, 1, 10, 50, 100 and 200 mg L(-1)) as well as glutathione (10 mg L(-1)) either alone or in combination has been evaluated on the induction of multiple shoots, antioxidant enzymes' activity, lipid peroxidation, relative permeability, concentration of nutrients, photosynthetic pigments, protein and proline content of nodal segments of chickpea after 14 days of culture. The antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) were found to be increased under salt stress as well as glutathione-supplemented medium. A significant decrease in the concentrations of chlorophylls a, b, total chlorophyll and carotenoid was observed under salt stress. Concentrations of nitrogen, phosphorus, potassium, calcium, carbon, magnesium and sulphur showed an initial increase up to 10 mg L(-1) NaCl, but a decline was seen at higher NaCl levels. Proline content and malondialdehyde concentration were found to be increased under salt stress. Three isoforms of SOD, one of CAT and four of GPX were expressed during native polyacrylamide gel electrophoresis (PAGE) analysis. However, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the stressed nodal explants revealed the over-expression of several polypeptide bands related to NaCl stress. These findings for the first time suggest that glutathione (GSH) helps in ameliorating NaCl stress in nodal explants of chickpea by manipulating various biochemical and physiological responses of plants.

Biodegradable polycaprolactone (PCL) nanosphere encapsulating superoxide dismutase and catalase enzymes.

Biodegradable polycaprolactone (PCL) nanosphere encapsulating superoxide dismutase (SOD) and catalase (CAT) were successfully synthesized using double emulsion (w/o/w) solvent evaporation technique. Characterization of the nanosphere using dynamic light scattering, field emission scanning electron microscope, and Fourier transform infrared spectroscopy revealed a spherical-shaped nanosphere in a size range of 812 ± 64 nm with moderate protein encapsulation efficiency of 55.42 ± 3.7 % and high in vitro protein release. Human skin HaCat cells were used for analyzing antioxidative properties of SOD- and CAT-encapsulated PCL nanospheres. Oxidative stress condition in HaCat cells was optimized with exposure to hydrogen peroxide (H2O2; 1 mM) as external stress factor and verified through reactive oxygen species (ROS) analysis using H2DCFDA dye. PCL nanosphere encapsulating SOD and CAT together indicated better antioxidative defense against H2O2-induced oxidative stress in human skin HaCat cells in comparison to PCL encapsulating either SOD or CAT alone as well as against direct supplement of SOD and CAT protein solution. Increase in HaCat cells SOD and CAT activities after treatment hints toward uptake of PCL nanosphere into the human skin HaCat cells. The result signifies the role of PCL-encapsulating SOD and CAT nanosphere in alleviating oxidative stress.

In vivo expression of a Cicer arietinum beta-galactosidase in potato tubers leads to a reduction of the galactan side-chains in cell wall pectin.

We report the generation of Solanum tuberosum transformants expressing Cicer arietinum betaIII-Gal. betaIII-Gal is a beta-galactosidase able to degrade cell wall pectins during cell wall loosening that occurs prior to cell elongation. cDNA corresponding to the gene encoding this protein was identified among several chickpea beta-galactosidase cDNAs, and named CanBGal-3. CanBGal-3 cDNA was expressed in potato under the control of the granule-bound starch synthase promoter. Three betaIII-Gal transformants with varying levels of expression were chosen for further analysis. The transgenic plants displayed no significant altered phenotype compared to the wild type. However, beta-galactanase and beta-galactosidase activities were increased in the transgenic tuber cell walls and this affected the potato tuber pectins. A reduction in the galactosyl content of up to 50% compared to the wild type was observed in the most extreme transformant, indicating a reduction of 1,4-beta-galactan side-chains, as revealed by analysis with LM5 specific antibodies. Our results confirm the notion that the pectin-degrading activity of chickpea betaIII-Gal reported in vitro also occurs in vivo and in other plants, and confirm the involvement of betaIII-Gal in the cell wall autolysis process. An increase in the homogalacturonan content of transgenic tuber cell walls was also observed by Fourier transform infrared spectroscopy (FTIR) analysis.

Acid phosphatase role in chickpea/maize intercropping.

BACKGROUND AND AIMS: Organic P comprises 30-80 % of the total P in most agricultural soils. It has been proven that chickpea facilitates P uptake from an organic P source by intercropped wheat. In this study, acid phosphatase excreted from chickpea roots is quantified and the contribution of acid phosphatase to the facilitation of P uptake by intercropped maize receiving phytate is examined. METHODS: For the first experiment using hydroponics, maize (Zea mays 'Zhongdan No. 2') and chickpea (Cicer arietinum 'Sona') were grown in either the same or separate containers, and P was supplied as phytate, KH2PO4 at 0.25 mmol P L(-1), or not at all. The second experiment involved soil culture with three types of root separation between the two species: (1) plastic sheet, (2) nylon mesh, and (3) no barrier. Maize plants were grown in one compartment and chickpea in the other. Phosphorus was supplied as phytate, Ca(H2PO4)2 at 50 mg P kg(-1), or no P added. KEY RESULTS: In the hydroponics study, the total P uptake by intercropped maize supplied with phytate was 2.1-fold greater than when it was grown as a monoculture. In the soil experiment, when supplied with phytate, total P uptake by maize with mesh barrier and without root barrier was 2.2 and 1.5 times, respectively, as much as that with solid barrier. In both experiments, roots of both maize and chickpea supplied with phytate and no P secreted more acid phosphatase than those with KH2PO4 or Ca(H2PO4)2. However, average acid phosphatase activity of chickpea roots supplied with phytate was 2-3-fold as much as maize. Soil acid phosphatase activity in the rhizosphere of chickpea was also significantly higher than maize regardless of P sources. CONCLUSIONS: Chickpea can mobilize organic P in both hydroponic and soil cultures, leading to an interspecific facilitation in utilization of organic P in maize/chickpea intercropping.

Cloning of a Cicer arietinum beta-galactosidase with pectin-degrading function.

The cDNA clone (CanBGal-3) encoding a cell wall pectin-degrading beta-galactosidase (beta III-Gal) from Cicer arietinum L. cv. Castellana has been identified. The identification was carried out by comparing the deduced amino acid sequences of several isolated chickpea beta-galactosidase clones with the purified beta III-Gal protein sequence. The expression pattern of the gene corresponding to CanBGal-3 was in concordance with the fluctuations of the enzyme beta III-Gal in different seedling organs, being specific to elongating organs such as epicotyls and roots. Transformation of Solanum tuberosum plants with the chickpea CanBGal-3 clone indicated that the beta-galactosidase encoded by this clone is a pectin-degrading enzyme. The authors propose an important role for chickpea beta III-Gal in pectin degradation in cell walls of vegetative organs such as epicotyls and roots. The degradation of galactan carried out by this enzyme may determine structural changes and affect cell wall porosity. It is suggested that the increase in the size of cell wall pores could permit access of other cell wall-modifying enzymes to their substrate.