Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues

Summary Most cell wall components are carbohydrate including the major matrix polysaccharides, pectins and hemicelluloses, and the arabinogalactan-protein proteoglycans. Both types of molecules are assembled in the Golgi apparatus and transported in secretory vesicles to the cell surface. We have employed antibodies specific to -(16) and -(14)-D-galactans, present in plant cell wall polysaccharides, in conjunction with immunofluorescence and electron microscopy to determine the location of the galactan-containing components in the cell wall and Golgi stacks of flax root tip tissues. Immunofluorescence data show that -(14)-D-galactan epitopes are restricted to peripheral cells of the root cap. These epitopes are not expressed in meristematic and columella cells. In contrast, -(16)-D-galactan epitopes are found in all cell types of flax roots. Immunogold labeling experiments show that both epitopes are specifically located within the wall immediately adjacent to the plasma membrane. They are also detected in Golgi cisternae and secretory vesicles, which indicates the involvement of the Golgi apparatus in their synthesis and transport. These findings demonstrate that the synthesis and localization of -(14)-D-galactan epitopes are highly regulated in developing flax roots and that different -linked D-galactans associated with cell wall polysaccharides are expressed in a cell type-specific manner.     

A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis.

Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.     

Comparative structure and ontogeny of the foliar domatia in three neotropical myrmecophytes

The origin and timing of the appearance of leaf domatia during the ontogeny of plants are important evolutionary traits driving the maintenance of ant-plant associations. In this study conducted in French Guiana on Hirtella physophora, Maieta guianensis, and Tococa guianensis, we focused on the formation and development of leaf domatia having different morphological origins. We modeled the timing of the onset of these domatia, then compared their morpho-anatomical structure. Although the ontogenetic development of the domatia differed between species, they developed very early in the plant's ontogeny so that we did not note differences in the timing of the onset of these domatia. For H. physophora seedlings, a transitional leaf forms before the appearance of fully developed domatia, whereas in M. guianensis and T. guianensis the domatia forms abruptly without transitional leaves. Moreover, in all cases, the morpho-anatomical structure of the domatia differed considerably from the lamina. All three species had similar morpho-anatomical characteristics for the domatia, indicating a convergence in their structural and functional characteristics. This convergence between taxonomically distant plant species bearing domatia having different morphological origins could be interpreted as a product of the plant's evolution toward the morphology and anatomy most likely to maximize ant recruitment and long-term residence.     

Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: identification by mass spectrometry and bioinformatics

Weakly bound cell wall proteins of Arabidopsis thaliana were identified using a proteomic and bioinformatic approach. An efficient protocol of extraction based on vacuum-infiltration of the tissues was developed. Several salts and a chelating agent were compared for their ability to extract cell wall proteins without releasing cytoplasmic contaminants. Of the 93 proteins that were identified, a large proportion (60%) was released by calcium chloride. From bioinformatics analysis, it may be predicted that most of them (87 out of 93) had a signal peptide, whereas only six originated from the cytoplasm. Among the putative apoplastic proteins, a high proportion (67 out of 87) had a basic pI. Numerous glycoside hydrolases and proteins with interacting domains were identified, in agreement with the expected role of the extracellular matrix in polysaccharide metabolism and recognition phenomena. Ten proteinases were also found as well as six proteins with unknown functions. Comparison of the cell wall proteome of rosettes with the previously published cell wall proteome of cell suspension cultures showed a high level of cell specificity, especially for the different members of several large multigenic families.     

Comparison between the anatomical and morphological structure of leaf blades and foliar domatia in the ant-plant Hirtella physophora (Chrysobalanaceae)

Background and Aims: Myrmecophytes, or ant-plants, are characterized by their abilityto shelter colonies of some ant species in hollow structures,or ant-domatia, that are often formed by hypertrophy of theinternal tissue at specific locations (i.e. trunk, branches,thorns and leaf pouches). In Hirtella physophora (Chrysobalanaceae),the focal species of this study, the ant-domatia consist ofleaf pouches formed when the leaf rolls over onto itself tocreate two spheres at the base of the blade. Methods: The morphological and anatomical changes through which foliarant-domatia developed from the laminas are studied for the firsttime by using fresh and fixed mature leaves from the same H.physophora individuals. Key results: Ant-domatia were characterized by larger extra-floral nectaries,longer stomatal apertures and lower stomatal density. The anatomicalstructure of the domatia differed in the parenchymatous tissuewhere palisade and spongy parenchyma were indistinct; chloroplastdensity was lower and lignified sclerenchymal fibres were morenumerous compared with the lamina. In addition, the domatiawere thicker than the lamina, largely because the parenchymatousand epidermal cells were enlarged. Conclusions: Herein, the morphological and anatomical changes that permitfoliar ant-domatia to be defined as a specialized leaf structureare highlighted. Similarities as well as structural modificationsin the foliar ant-domatia compared with the lamina are discussedfrom botanical, functional and mutualistic points of view. Theseresults are also important to understanding the reciprocal evolutionarychanges in traits and, thus, the coevolutionary processes occurringin insect–plant mutualisms.     

The actin cytoskeleton is involved in signalling protoplast embryogenesis induced by agarose embedding

The organization of actin microfilaments was studied by immunofluorescence in protoplasts isolated from sunflower hypocotyls and cultured in an agarose matrix. Removal of the cell wall completely disrupted the actin cytoskeleton, which became progressively reorganized into cortical microfilament arrays and actin cables during protoplast culture. Treatment of protoplasts with arginine-glycine-aspartic acid (Arg-Gly-Asp) motif-containing peptides, to inhibit putative cell contacts with the agarose matrix, strongly affected this repair process: microfilament elongation and cable formation were inhibited and the connectivity between the cortical network and the perinuclear basket was lost. Furthermore, embryoid formation induced by agarose embedding was reduced. Similar effects were observed with a short treatment with latrunculin B, known to disrupt actin microfilaments. These results indicate that the actin network is involved in the signalling process that leads to polarity acquisition and embryoid determination in agarose-embedded protoplasts.     

Pectin immunolocalization and calcium visualization in differentiating derivatives from poplar cambium

Summary Calcium distribution and pectin esterification patterns in the cambial zone of poplar branches were studied with ionic microscopy and immunological tools respectively. Dynamic changes correlating with cell growth and cell differentiation were observed both on the xylem and on the phloem sides. In expanding cell walls of xylem derivatives, unesterified pectins were restricted to cell junctions and middle lamellae, occasionally accompanied by calcium ions. In contrast, in differentiating and mature phloem cells, acidic pectins and Ca²⁺ were present all over the walls leading to early stiffening of the polysaccharide network. Significant labelling was detected with JIM5 antibodies in some dictyosomes suggesting exocytosis of low methylated polymers towards the cell walls. At cell junctions, unesterified pectins might originate from the activity of pectinmethylesterases localized in these areas. Thus un- and deesterified pectins might be located in different cell wall domains whose distribution, varying with cell type, will confer specific extensibility to the wall matrix.Abbreviations BSA bovine serum albumin - DM degree of methylation - FITC fluorescein isothiocyanate - HM highly methylated pectins - LM low methylated pectins - PME pectin methylesterase - SIMS secondary ion mass spectrometry - TBS tris-buffered saline     

Dissection of Bacterial Wilt on Medicago truncatula Revealed Two Type III Secretion System Effectors Acting on Root Infection Process and Disease Development

Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease, which colonizes susceptible Medicago truncatula via the intact root tip. Infection involves four steps: appearance of root tip symptoms, root tip cortical cell invasion, vessel colonization, and foliar wilting. We examined this pathosystem by in vitro inoculation of intact roots of susceptible or resistant M. truncatula with the pathogenic strain GMI1000. The infection process was type III secretion system dependent and required two type III effectors, Gala7 and AvrA, which were shown to be involved at different stages of infection. Both effectors were involved in development of root tip symptoms, and Gala7 was the main determinant for bacterial invasion of cortical cells. Vessel invasion depended on the host genetic background and was never observed in the resistant line. The invasion of the root tip vasculature in the susceptible line caused foliar wilting. The avrA mutant showed reduced aggressiveness in all steps of the infection process, suggesting a global role in R. solanacearum pathogenicity. The roles of these two effectors in subsequent stages were studied using an assay that bypassed the penetration step; with this assay, the avrA mutant showed no effect compared with the GMI1000 strain, indicating that AvrA is important in early stages of infection. However, later disease symptoms were reduced in the gala7 mutant, indicating a key role in later stages of infection.