Berta Dopico

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.

Brassinolides promote the expression of a new Cicer arietinum β-tubulin gene involved in the epicotyl elongation

A cDNA clone, CanTUB, encoding a putative β-tubulin protein was isolated from a cDNA library constructed from 5-day old chickpea (Cicer arietinum) epicotyls. Analysis of its deduced amino acid sequence showed all the typical structural motifs of plant β-tubulins. Putative sequences for autoregulation and tubulin mRNA stability, GTP and Ca2+/MAPs (microtubule-associated proteins) binding sites were present. Southern blot analysis of chickpea genomic DNA revealed that there are multiple β-tubulin genes. The level of expression of β-tubulin genes was correlated with the rate of growth in either seedlings and adult plants. The transcript levels of β-tubulin genes were higher in actively elongating tissues such as etiolated epicotyls, roots and stem tissues of adult plants. Brassinolide-induced growth in chickpea epicotyls was accompanied by promotion of the expression of the gene coding for β-tubulin.

Two growth-related organ-specific cDNAs from Cicer arietinum epicotyls.

Two cDNAs, CanST-1 and CanST-2, encoding two different growth-related organ-specific sequences have been isolated from a cDNA library from growing epicotyls of Cicer arietinum. An intriguing property of these two clones is the presence in their coding region of a repeated sequence which is highly conserved except for the number of repeats. The corresponding genes of CanST-1 and CanST-2 encode for proteins related to elongation processes. CanST-1 and CanST-2 are up-regulated during epicotyl growth, the transcript levels of both clones decrease when the growth of epicotyls is inhibited by several treatments and their expression increases when epicotyls resume growth. Furthermore, clones CanST-1 and CanST-2 are tissue-specific and are only expressed in epicotyls, mesocotyls, roots and stem tissues whose cells undergo elongation processes. Neither clone was found to be expressed in other organs such as cotyledons, leaves, flowers, pods and immature seeds. The results of auxin (IAA) and brassinolides (BR) treatments suggest that the processes in which the proteins encoded by CanST-1 and CanST-2 are involved are not mediated by these hormones.

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.

Characterization and localization of the cell wall autolysis substrate in Pisum sativum epicotyls

Abstract The products released in cell wall autolysis from 4-day-old epicotyls of Pisum sativum elute in gel filtration chromatography (Bio Gel P.2) as two components, mono and polysaccharides, in a practically constant ratio over the time of incubation. The polysaccharides are mainly composed of arabinose and galactose, with smaller amounts of xylose and glucose, whereas the monosaccharide are almost exclusively composed of galactose. The same results were obtained when inactive cell walls were hydrolyzed by the enzymes extracted from the cell wall with LiCl. The hydrolysis of the different cell wall fractions by these enzymes shows that the autolytic substrates are preferentially located on the pectic fractions.

A chickpea Kunitz trypsin inhibitor is located in cell wall of elongating seedling organs and vascular tissue

Kunitz proteinase inhibitors in legumes have mainly been described as defence and storage proteins. Here, we report a Kunitz trypsin inhibitor, encoded by the CaTPI-1 gene from Cicer arietinum. The transcription of this gene mainly occurs in seedling vegetative organs, and is affected by the light and growth stages. The recombinant TPI-1 protein expressed in E. coli was seen to be an efficient inhibitor of trypsin. After the generation of polyclonal antibodies against recombinant TPI-1 protein, the protein was located in the cell wall of elongating epicotyls and radicles by Western-blot experiments, in agreement with the transcription pattern. These results, together with the fact that both CaTPI-1 mRNA and protein levels decreased with epicotyl growth, suggest a possible role in the elongation of seedling epicotyls and radicles. Immunolocalization analyses of the TPI-1 protein indicated that it is abundant in the cell walls of both immature primary xylem cells and surrounding parenchyma cells. This location has led us to explore potential functions for TPI-1 protein in vascular tissue during seedling elongation.

Remodelling pectin structure in potato

Figure options Download full-size image Download as PowerPoint slide Pectin is a collection of polysaccharides, which play an important role in controlling the pore size of the plant cell wall, regulating cell-cell adhesion, and providing a source of sig-nalling molecules that elicit a range of cellular responses. Apart from this, pectins are of interest because they are an attractive hydrocolloid for various food applications. The kind and distribution of decorative groups in the pectic molecules largely determines for which application a particular pectin is most suitable. After the extraction of starch from potato tubers, a by-product is obtained, which is relatively rich in pectin. However, the quality of these pectins is poor compared to that from other sources such as citrus and apple. Rather than trying to change the structural characteristics of potato pectin post-harvest, we have embarked on achieving this in the potato plant itself. This paper summarises the structural features of pectin, the distribution of various pectic epitopes in tuber cell walls, the enzymes involved in its biosynthesis and degradation, and strategies employed to alter its fine structure in planta.

Immunolocalization of a Cell Wall ß-Galactosidase Reveals its Developmentally Regulated Expression in Cicer arietinum and its Relationship to Vascular Tissue

We report the generation of antibodies against a ß-galactosidase from Cicer arietinum, ßIV-Gal, and the subsequent immunolocalization of the protein in different parts and developmental stages of the plant. The ßIV-Gal protein is encoded by the CanBGal-4 gene, which belongs to a family of at least four ß-galactosidase genes, transcripts of which were previously reported to be mainly present in seedling epicotyls and plant stem, its transcription pattern being inversely related to elongation rate of these organs. ßIV-Gal protein was detected in the cell walls of seedling epicotyls and plant stems. The immunodetection of ßIV-Gal protein in the cell wall protein extracts from aged epicotyls and basal stem internodes, both undergoing low rates of elongation, is in agreement with the trend of the CanBGal-4 transcript and indicates a relationship of this cell wall protein with the end of cell elongation. The specific main location of the ßIV-Gal protein in vascular tissue of epicotyls and stems and in a layer of sclerenchymatic cells surrounding the vascular cylinder (perivascular fibers) allows us to postulate a function for this ß-galactosidase in the modification of cell wall polymers during the development of cells of the vascular system. The localization of the ßIV-Gal protein also in the cell walls of collenchyma cells in internodes is consistent with the involvement of ßIV-Gal in cell wall modifications that lead to thick cell walls, such as in vascular cells.

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.

Changes during epicotyl growth of an autolysis-related β-galactosidase from the cell wall of Cicer arietinum

Abstract During the growth of Cicer arietinum epicotyls an increase in specific cell wall β-glucosidase and β-galactosidase activities were observed. In contrast, the amount of cell wall proteins extracted with 3 M LiCl decreased between the third and the fourth day of germination, remaining constant up to the seventh day. Partial purification of the cell wall protein extracts from different days by SP-Sephadex chromatography showed that one fraction increased both in the amount of protein and β-galactosidase activity throughout the growth of epicotyls. This protein fraction, which is the third β-galactosidase (βIII) eluted from a SP-Sephadex chromatography, has been previously characterized by us as the main enzyme involved in the autolytic process.