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.

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.

Three members of Medicago truncatula ST family (MtST4, MtST5 and MtST6) are specifically induced by hormones involved in biotic interactions.

In this work, we study the function of the Medicago truncatula ST4, ST5 and ST6 proteins that belong to a protein family of unknown function characterized by the DUF2775 domain. Thus, we analyse their promoter sequence and activity, their transcript accumulation, and their subcellular location. The analysis of the three promoters showed different combination of cis-acting regulatory elements and they presented different activity pattern. Throughout development only ST6 mRNAs have been detected in most of the stages analysed, while ST4 was faintly detected in the roots and in the flowers and ST5 was always absent. The addition of MeJA, ET and SA revealed specific responses of the STs, the ST4 transcript accumulation increased by MeJA; the ST5 by MeJA and ET when applied together; and the ST6 by ET and by SA. Finally, the ST4 and ST5 proteins were in the cell wall whereas the ST6 had a dual location. From these results, we can conclude that the ST4, ST5 and ST6 RNAs are specifically and differentially up-regulated by MeJA, ET and SA, plant regulators also involved in the plant defence, pointing that ST4, ST5 and ST6 proteins might be involved in specific biotic interactions through different signalling pathways.

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.

ßIII-Gal is involved in galactan reduction during phloem element differentiation in chickpea stems.

ßIII-Gal, a member of the chickpea ß-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 ßIII-Gal in different seedling and plant organs was analyzed by using specific anti-ßIII-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 ßIII-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, ßIII-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 ß-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 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.

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.