Teresa Jiménez

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.

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.

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.

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.

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.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

BackgroundMany proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats.ResultsST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development.ConclusionsWe describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.