Josefina Hernández-Nistal

Cold and salt stress regulates the expression and activity of a chickpea cytosolic Cu/Zn superoxide dismutase

Abstract A cDNA clone encoding a cytosolic superoxide dismutase (SOD) was isolated from a cDNA library constructed from poly(A) + RNA from epicotyls of 5-day-old Cicer arietinum L. etiolated seedlings after a differential screening to select clones whose expression decreases with epicotyl growth. Analysis of its deduced amino acid sequence showed all the typical structural motifs of plant cytosolic SODs (EC 1.15.1.1.). The expression of this clone is always higher in young and growing tissues than in old and storage ones, and diminishes throughout the development of the seedlings. Cytosolic Cu/Zn-SOD activity is also higher in radicles and younger internodes. Under stress conditions only cold increases the gene expression whereas the activity is clearly raised up by a saline medium. The results are discussed in relation to the gene regulation and enzyme activity control that crop plants use to resolve the different stresses that reduce their productivity.

Two cell wall Kunitz trypsin inhibitors in chickpea during seed germination and seedling growth.

Two Kunitz trypsin inhibitors TPI-1 and TPI-2, encoded by CaTPI-1 and CaTPI-2, previously identified and characterized, have been detected in chickpea (Cicer arietinum L.) embryonic axes from seeds imbibed up to 48 h. Their gene transcription commenced before germination sensu stricto was completed. The transcript amount of CaTPI-1 remained high until 24 h after imbibition, when the epicotyls started to grow, while CaTPI-2 mRNA, which appeared later, reached a maximum at 48 h. Both the temporal and the spatial distribution of TPI-1 and TPI-2 proteins in the embryonic axes suggest that they perform different functions. The early appearance of TPI-1 in imbibed seeds suggests that it plays a protective role, preventing the premature degradation of the proteins stored in the embryonic axes. Its pattern of distribution suggests that the protein is involved in the regulation of vascular tissue differentiation, protecting the cells from some proteinases involved in programmed cell death. With regard to TPI-2, its later synthesis after imbibition, together with its tissue distribution, indicates that it is mainly active following germination, during elongation of the embryonic axes.

The accumulation of a Kunitz trypsin inhibitor from chickpea (TPI-2) located in cell walls is increased in wounded leaves and elongating epicotyls.

Here, we report the identification and characterization of CaTPI-2, which is a member of a Cicer arietinum gene family encoding Kunitz-type proteinase inhibitors with at least two members -CaTPI-1 and CaTPI-2. The widespread mRNA accumulation of CaTPI-2 in all the different organs of 4-day-old etiolated seedlings and in stem internodes differs from the more specific Cicer arietinum Trypsin Proteinase Inhibitor-1 (CaTPI-1) transcription. After the generation of polyclonal antibodies against the recombinant Trypsin Proteinase Inhibitor-2 (TPI-2) protein, the protein was located in the cell walls of vegetative organs. The decrease found in both transcription and TPI-2 protein levels when the epicotyls aged, together with the wider and more intensive immunostaining of the protein in apical zones of epicotyls and radicles, in consonance with their higher elongation rate, indicated a relationship of the TPI-2 protein with the elongation process. CaTPI-2 mRNA levels were increased by wounding in both epicotyls and leaves. The accumulation of CaTPI-2 mRNA in seedlings, which was further amplified by mechanical wounding in epicotyls and leaves, suggests the involvement of TPI-2 in the response to wounds. Our results indicate that TPI-2 protein has features different from those of the former characterized Trypsin Proteinase Inhibitor-1 (TPI-1), such as its different gene regulation under light, a different cellular location and its upregulation by wounding, which implies a function different from that of TPI-1 in chickpea metabolism.

The Location of the Chickpea Cell Wall ßV-Galactosidase Suggests Involvement in the Transition between Cell Proliferation and Cell Elongation

We report the generation of antibodies against a ß-galactosidase (ßV-Gal) from Cicer arietinum and the subsequent immunolocalization of the protein in different parts and developmental stages of the plant. ßV-Gal is a cell wall protein encoded by the CanBGal-5 gene, which belongs to a family of at least four ß-galactosidase genes in chickpea. We have previously reported that CanBGal-5 transcripts are located in organs with high elongation and cell division rates, such as meristematic hooks, very young epicotyls, and apical internodes. ßV-Gal protein is the only studied chickpea ß-galactosidase widely present in meristematic hooks, mainly in the meristematic apical zone. These results agree with the previously reported transcription pattern of CanBGal-5 and may reflect its involvement in cell wall modifications during the final stages of cell proliferation, leading to the establishment of an expanding cell wall. The location of ßV-Gal in the cell wall of procambium cells and in pericycle cells of the developing lateral roots also supports the involvement of ßV-Gal in this process. During seedling and plant growth, the highest levels of βV-Gal protein were detected in the youngest actively growing epicotyls and in the apical growing internodes. Thus, protein levels pointed to a relationship between βV-Gal and the events occurring in the cell wall during the early stages of development. Immunolocalization studies in different zones of epicotyls and radicles suggest a role for ßV-Gal in cell elongation.

Limestone particle size and liming scheduling influence soil properties and pasture production.

Liming is a common practice in Galician (NW Spain) soils devoted to pasture production. Although many studies have established the right liming rates, there is a lack of information concerning the ideal particle size for optimal agronomic results. This study aims to evaluate the effects of particle size (2-4, 0.5-2, 0.25-0.5, <0.25 mm) of magnesium limestone as well as the application schedule (in a single application or split in 3 yearly applications) on the proprieties of an acid soil in Galicia and on the yield and quality of pasture growing on the soil during the 2 years after liming. The soil proprieties were monitored seasonally, and the pasture yield and nutritional contents were determined in summer and autumn. The soil analysis showed that the plots treated with a single application of the finest limestone exhibited the highest pH (pH water 5.05-5.53), and the lowest exchangeable Al (<10% Al saturation throughout the period of study), the highest concentrations of exchangeable Ca (8.40-10.18 cmol(+) kg−1) and Mg (1.39-1.71 cmol(+) kg−1) and the highest effective cation exchange capacity (11.2-13.7 cmol(+) kg−1). In contrast, plots treated with the coarsest limestone had values similar to control plots. The highest production of total dry matter and, especially, the highest yield of sown species were found in the plots receiving the finest limestone (0.75-1.10 t ha−1 dry matter in summer harvest versus 0.30-0.75 t ha−1 in control plots). Available P, exchangeable cations (K and Ca), and pH explain a high percentage of the variance of these parameters. The Mg concentrations and the total contents of Ca and Mg in plant tissues were significantly higher in the plots treated with the finest limestone.

Effect of particle size of limestone on Ca, Mg and K contents in soil and in sward plants

ABSTRACT: Liming increases crop production through improved soil conditions in acidic soils. Among theeffects of liming, increased availabilities of alkaline and alkaline-earth cations are worth mention. Theseavailabilities may be affected by the particle size of applied limestone, which influences lime reactivity. Theeffects of particle size and application schedule of magnesium limestone were investigated on extractable Ca,Mg and K in soil, their concentrations in sward plants and dry-matter yield. Magnesium limestone of variousparticle sizes was applied to experimental plots at a rate of 3 t ha –1 , a grass-clover sward was sown, and the plotswere monitored during three years. The finest limestone (< 0.25 mm) in a single application yielded thehighest soil Ca and Mg concentrations extracted by Mehlich-3 and NH 4 Cl. The same limestone split in threeannual doses was less effective. Plots treated with the coarsest limestone (2-4 mm) did not differ from controlplots. Liming had no effect on potassium, either in soil or plants. Soil concentrations of Ca, Mg and Kextracted by Mehlich-3 and NH

βIII-Gal is Involved in Galactan Reduction During Phloem Element Differentiation in Chickpea Stems

βIII-Gal, a member of the chickpea β-galactosidase family, is the enzyme responsible for the cell wall autolytic process. This enzyme, whose activity increases during epicotyl growth, displays significant hydrolytic activity against cell wall pectins, and its natural substrate has been determined as an arabinogalactan from the pectic fraction of the cell wall. In the present work, the localization of βIII-Gal in different seedling and plant organs was analyzed by using specific anti-βIII-Gal antibodies. Our results revealed that besides its possible role in cell wall loosening and in early events during primary xylem and phloem fiber differentiation βIII-Gal acts on the development of sieve elements. Localization of the enzyme in this tissue, both in epicotyls and radicles from seedlings and in the different stem internodes, is consistent with the reduction in galactan during the maturation of phloem elements, as can be observed with LM5 antibodies. Thus, βIII-Gal could act on its natural substrate, the neutral side chains of rhamnogalacturonan I, contributing to cell wall reinforcement allowing phloem elements to differentiate, and conferring the necessary strengthening of the cell wall to fulfill its function. This work completes the immunolocation studies of all known chickpea β-galactosidases. Taken together, our results reflect the broad range of developmental processes covered by different members of this protein family, and confirm their crucial role in cell wall remodeling during tissue differentiation.

The βI‐galactosidase of Cicer arietinum is located in thickened cell walls such as those of collenchyma, sclerenchyma and vascular tissue

We report localisation of the chickpea βI-Gal, a member of the chickpea β-galactosidase family, which contains at least four members. After generation of specific antibodies, the distribution and cellular immunolocalisation of the protein in different organs and developmental stages of the plant was studied. βI-Gal protein is much longer than the other chickpea β-galactosidases because of the presence of a lectin-like domain in the carboxyl terminus of the protein. Western blot experiments indicated that the active βI-Gal retains this lectin-like domain for its function in the plant. The βI-Gal protein was mainly detected in cell walls of elongating organs, such as seedling epicotyls and stem internodes. An immunolocation study indicated a very good correlation between the presence of this βΙ-galactosidase and cells whose walls are thickening, not only in aged epicotyls and mature internodes in the final phase of elongation, but mostly in cells with a support function, such as collenchyma cells, xylem and phloem fibres and a layer of sclerenchyma cells surrounding the vascular cylinder (perivascular fibres). These results could suggest a function for the βI-Gal in modification of cell wall polymers, leading to thicker walls than the primary cell walls.

The immunolocation of XTH1 in embryonic axes during chickpea germination and seedling growth confirms its function in cell elongation and vascular differentiation

In a previous work, the immunolocation of the chickpea XTH1 (xyloglucan endotransglucosylase/hydrolase 1) protein in the cell walls of epicotyls, radicles, and stems was studied, and a role for this protein in the elongation of vascular cells was suggested. In the present work, the presence and the location of the XTH1 protein in embryonic axes during the first 48 h of seed imbibition, including radicle emergence, were studied. The presence of the XTH1 protein in the cell wall of embryonic axes as early as 3 h after imbibition, before radicle emergence, supports its involvement in germination, and the fact that the protein level increased until 24 h, when the radicle had already emerged, also suggests its participation in the elongation of embryonic axes. The localization of XTH1 clearly indicates that the protein is related to the development of vascular tissue in embryonic axes during the period studied, suggesting that the role of this protein in embryonic axes is the same as that proposed for seedlings and plants.

Organ accumulation and subcellular location of Cicer arietinum ST1 protein.

The ST (ShooT Specific) proteins are a new family of proteins characterized by a signal peptide, tandem repeats of 25/26 amino acids, and a domain of unknown function (DUF2775), whose presence is limited to a few families of dicotyledonous plants, mainly Fabaceae and Asteraceae. Their function remains unknown, although involvement in plant growth, fruit morphogenesis or in biotic and abiotic interactions have been suggested. This work is focused on ST1, a Cicer arietinum ST protein. We established the protein accumulation in different tissues and organs of chickpea seedlings and plants and its subcellular localization, which could indicate the possible function of ST1. The raising of specific antibodies against ST1 protein revealed that its accumulation in epicotyls and radicles was related to their elongation rate. Its pattern of tissue location in cotyledons during seed formation and early seed germination, as well as its localization in the perivascular fibres of epicotyls and radicles, indicated a possible involvement in seed germination and seedling growth. ST1 protein appears both inside the cell and in the cell wall. This double subcellular localization was found in every organ in which the ST1 protein was detected: seeds, cotyledons and seedling epicotyls and radicles.