Specific tissue proteins 1 and 6 are involved in root biology during normal development and under symbiotic and pathogenic interactions in Medicago truncatula.

MAIN CONCLUSION: ST1 and ST6 are possibly involved in primary and lateral root and symbiotic nodule development, but only ST6 participates in the interaction with hemibiotrophic fungi. Specific tissue (ST) proteins have been shown to be involved in several processes related to plant nutritional status, development, and responses to biotic agents. In particular, ST1 and ST6 are mainly expressed in roots throughout plant development. Here, we analyze where and how the expression of the genes encoding both proteins are modulated in the legume model plant Medicago truncatula in response to the plant developmental program, nodulation induced by a beneficial nitrogen-fixing bacterium (Sinorhizobium meliloti) and the defense response triggered by a pathogenic hemibiotrophic fungus (Fusarium oxysporum). Gene expression results show that ST1 and ST6 participate in the vasculature development of both primary and lateral roots, although only ST6 is related to meristem activity. ST1 and ST6 clearly display different roles in the biotic interactions analyzed, where ST1 is activated in response to a N2-fixing bacterium and ST6 is up-regulated after inoculation with F. oxysporum. The role of ST1 and ST6 in the nodulation process may be related to nodule organogenesis rather than to the establishment of the interaction itself, and an increase in ST6 correlates with the activation of the salicylic acid signaling pathway during the infection and colonization processes. These results further support the role of ST6 in response to hemibiotrophic fungi. This research contributes to the understanding of the complex network that controls root biology and strengthens the idea that ST proteins are involved in several processes such as primary and lateral root development, nodule organogenesis, and the plant-microbe interaction.

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.

ß-(1,4)-Galactan remodelling in Arabidopsis cell walls affects the xyloglucan structure during elongation.

MAIN CONCLUSION: Galactan turnover occurs during cell elongation and affects the cell wall xyloglucan structure which is involved in the interaction between cellulose and xyloglucan. ß-(1,4)-Galactan is one of the main side chains of rhamnogalacturonan I. Although the specific function of this polymer has not been completely established, it has been related to different developmental processes. To study ß-(1,4)-galactan function, we have generated transgenic Arabidopsis plants overproducing chickpea ßI-Gal ß-galactosidase under the 35S CaMV promoter (35S::ßI-Gal) to reduce galactan side chains in muro. Likewise, an Arabidopsis double loss-of-function mutant for BGAL1 and BGAL3 Arabidopsis ß-galactosidases (bgal1/bgal3) has been obtained to increase galactan levels. The characterization of these plants has confirmed the role of ß-(1,4)-galactan in cell growth, and demonstrated that the turnover of this pectic side chain occurs during cell elongation, at least in Arabidopsis etiolated hypocotyls and floral stem internodes. The results indicate that BGAL1 and BGAL3 ß-galactosidases act in a coordinate way during cell elongation. In addition, this work indicates that galactan plays a role in the maintenance of the cell wall architecture during this process. Our results point to an involvement of the ß-(1,4)-galactan in the xyloglucan structure and the interaction between cellulose and xyloglucan.

Three members of Medicago truncatula ST family are ubiquitous during development and modulated by nutritional status (MtST1) and dehydration (MtST2 and MtST3).

BACKGROUND: ShooT specific/Specific Tissue (ST) belong to a protein family of unknown function characterized by the DUF2775 domain and produced in specific taxonomic plant families, mainly Fabaceae and Asteraceae, with the Medicago truncatula ST family being the largest. The putative roles proposed for this family are cell elongation, biotic interactions, abiotic stress and N reserve. The aim of this work was to go deeper into the role of three M. truncatula ST proteins, namely ST1, ST2 and ST3. Our starting hypothesis was that each member of the family could perform a specific role, and hence, each ST gene would be subjected to a different type of regulation. RESULTS: The search for cis-acting regulatory elements (CREs) in silico in pST1, pST2 and pST3 promoters showed prevalence of tissue/organ specific motifs, especially root- and seed-specific ones. Light, hormone, biotic and abiotic related motifs were also present. None of these pSTs showed the same combination of CREs, or presented the same activity pattern. In general, pST activity was associated with the vascular cylinder, mainly in roots. Promoter activation was highly specific and dissimilar during reproductive development. The ST1, ST2 and ST3 transcripts accumulated in most of the organs and developmental stages analysed - decreasing with age - and expression was higher in the roots than in the aerial parts and more abundant in light-grown plants. The effect of the different treatments on transcript accumulation indicated that ST1 behaved differently from ST2 and ST3, mainly in response to several hormones and dehydration treatments (NaCl or mannitol), upon which ST1 transcript levels decreased and ST2 and ST3 levels increased. Finally, the ST1 protein was located in the cell wall whereas ST2 and ST3 were present both in the cytoplasm and in the cell wall. CONCLUSIONS: The ST proteins studied are ubiquitous proteins that could perform distinct/complementary roles in plant biology as they are encoded by differentially regulated genes. Based on these differences we have established two functional groups among the three STs. ST1 would participate in processes affected by nutritional status, while ST2 and ST3 seem to act when plants are challenged with abiotic stresses related to water stress and in physiologically controlled desiccation processes such as the seed maturation.

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.

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

BACKGROUND: Many 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. RESULTS: ST 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. CONCLUSIONS: We 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.

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.

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.

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.

Subcellular location of Arabidopsis thaliana subfamily a1 ß-galactosidases and developmental regulation of transcript levels of their coding genes.

The aim of this work is to gain insight into the six members of the a1 subfamily of the ß-galactosidases (BGAL) from Arabidopsis thaliana. First, the subcellular location of all these six BGAL proteins from a1 subfamily has been established in the cell wall by the construction of transgenic plants producing the enhanced green fluorescent protein (eGFP) fused to the BGAL proteins. BGAL12 is also located in the endoplasmic reticulum. Our study of the AtBGAL transcript accumulation along plant development indicated that all AtBGAL transcript appeared in initial stages of development, both dark- and light-grown seedlings, being AtBGAL1, AtBGAL2 and AtBGAL3 transcripts the predominant ones in the latter condition, mainly in the aerial part and with levels decreasing with age. The high accumulation of transcript of AtBGAL4 in basal internodes and in leaves at the end of development, and their strong increase after treatment both with BL and H3BO3 point to an involvement of BGAL4 in cell wall changes leading to the cease of elongation and increased rigidity. The changes of AtBGAL transcript accumulation in relation to different stages and conditions of plant development, suggest that each of the different gene products have a plant-specific function and provides support for the proposed function of the subfamily a1 BGAL in plant cell wall remodelling for cell expansion or for cell response to stress conditions.

The gene for a xyloglucan endotransglucosylase/hydrolase from Cicer arietinum is strongly expressed in elongating tissues.

We have isolated a Cicer arietinum cDNA clone (CaXTH1) encoding a protein that belongs to the family 16 of glycosyl hydrolases and has all the conserved features of xyloglucan endotransglucosylase/hydrolases (XTH) proteins, including the presence of a highly conserved domain (DEIDFEFLG) and four Cys which suggest the potential for forming disulfide bonds. These facts indicate that CaXTH1 encodes a putative XTH. This chickpea protein showed a high level of sequence identity with group 1 XTHs that have xyloglucan endotransglucosylase (XET) activity. CaXTH1 was selected by differential screening of a cDNA library constructed using mRNA from C. arietinum polyethylene glycol (PEG) treated epicotyls, as a clone whose expression decreased when epicotyl growth was inhibited by PEG. CaXTH1 shows an expression pattern that seems to be specific for growing tissue, mostly epicotyls and the growing internodes of adult stems. CaXTH1 mRNA was not detected in any other organs of either seedlings or adult plants. CaXTH1 mRNA was abundant when epicotyls are actively growing; there was almost no expression after PEG-treatment. CaXTH1 was up-regulated by indole acetic acid (IAA) and brassinolides (BR), showing the highest transcript levels after IAA plus BR treatment. In situ hybridization study revealed that CaXTH1 is mainly expressed in epidermal cells, the target of the cell expansion process, and also in vascular tissues. The present results suggest an involvement of the putative XTH encoded by CaXTH1 in the chickpea cell expansion process.

A seedling specific vegetative lectin gene is related to development in Cicer arietinum.

Plant lectins are a group of glycoproteins with the ability to recognize and bind carbohydrate ligands. Seed lectins function as storage and defense proteins, but the specific function of vegetative lectins is uncertain. In this paper we describe the characterization of a clone, CanVLEC, encoding a vegetative lectin from chickpea (Cicer arietinum L. cv. Castellana). The expression of the CanVLEC gene was specific in seedlings, mostly in hooks and elongating epicotyls, and no expression was detected in adult plants. The level of chickpea vegetative lectin transcripts in epicotyls decreased through the epicotyl growth suggesting a relationship to development. Treatment with indoleacetic acid (IAA) and brassinolides (BR), hormones that promoted elongation in chickpea epicotyl, increased the level of CanVLEC mRNA, supporting a relationship to growth. CanVLEC is drastically down regulated by water deficit ruling out its possible involvement in plant response to water stress, unlike other vegetative lectins. CanVLEC protein may be targeted to an extracellular location owing to the presence of a signal peptide.

Brassinolides and IAA induce the transcription of four alpha-expansin genes related to development in Cicer arietinum.

Four different cDNAs encoding alpha-expansins have been identified in Cicer arietinum (Ca-EXPA1, Ca-EXPA2, Ca-EXPA3 and Ca-EXPA4). The shared amino acid sequence similarity among the four alpha-expansin proteins ranged from 67 to 89%. All of them display common characteristics such as molecular mass (around 24 kDa), amino acid numbers, and also the presence of a signal peptide. The transcription pattern of chickpea alpha-expansin genes in seedlings and plants suggests a specific role for each of the four alpha-expansins in different phases of development or in different plant organs. High levels of Ca-EXPA2 transcripts coincide with maximum epicotyl and stem growth, indicating an important involvement of this particular alpha-expansin in elongating tissues. Ca-EXPA3 would be related to radicle development, while Ca-EXPA4 seems to be involved in pod development. A considerable increase in the level of all Ca-EXPA transcripts accompanied the indole acetic acid (IAA) plus brassinolide (BR)-induced elongation of excised epicotyl segments. This IAA + BR induction was seen even for the chickpea expansin genes whose transcription was not affected by IAA or BR alone.

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.

Promoter activities of genes encoding ß-galactosidases from Arabidopsis a1 subfamily.

Promoter regions of each of the six AtBGAL gene of the subfamily a1 of Arabidopsis thaliana were used to drive the expression of the ß-glucuronidase gene. The pattern of promoters (pAtBGAL) activity was followed by histological staining during plant development. pAtBGAL1, pAtBGAL3 and pAtBGAL4 showed a similar activity pattern, being stronger in cells and organs in expansion, and the staining decreasing when cell expansion decreased with age. That indicates a consistent involvement of the encoded ß-galactosidases in cells undergoing cell wall extension or remodelling in cotyledons, leaves and flower buds. These promoters were also active in the calyptra cells and in pollen grains. pAtBGAL2 activity showed a clear relationship with hypocotyl elongation in both light and dark conditions and, like pAtBGAL1, pAtBGAL3 and pAtBGAL4, it was detected during the expansion of cotyledons, rosette leaves and cauline leaves. Its activity was also intense in the early stages of flower and fruit development. pAtBGAL5 was the only one among those from the subfamily a1 that was active in the trichomes that appear throughout the plant, indicating a high specificity of the AtBGAL5 protein and its involvement in the cell wall changes that accompany the formation of the trichome. The activity of pAtBGAL5 was also high in radicles and roots, except in the meristematic area of these organs, and during seed formation. Finally, the activity of pAtBGAL12 was mainly detected in meristematic zones of the plant: the leaf primordium, emerging secondary roots and developing seeds, which indicates an involvement in the differentiation process.

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.

The immunolocation of a xyloglucan endotransglucosylase/hydrolase specific to elongating tissues in Cicer arietinum suggests a role in the elongation of vascular cells.

In a previous work, a Cicer arietinum cDNA clone (CaXTH1) encoding a xyloglucan endotransglucosylase/hydrolase (XTH1) protein was isolated and characterized. CaXTH1 showed an expression pattern specific to growing tissue: mostly epicotyls and the upper growing internodes of adult stems. CaXTH1 mRNA was not detected in any other organs of either seedlings or adult plants, suggesting an involvement of the putative XTH encoded by CaXTH1 in the chickpea cell expansion process. After the generation of polyclonal antibodies by using the XTH1 recombinant protein and the analysis of the specificity of the antibodies for XTH proteins, here the specific location of the chickpea XTH1-cross-reacting protein in cell walls of epicotyls, radicles, and stems is reported, evaluated by western blot and immunocytochemical studies. The results indicate a function for this protein in the elongation of parenchyma cells of epicotyls and also in developing vascular tissue, suggesting a role in the elongation of vascular cells.

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.