KATANIN-dependent mechanical properties of the stigmatic cell wall mediate the pollen tube path in Arabidopsis.

Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules. Pollen tubes first grow within the cell wall of the papilla cells, applying pressure to the cell. Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules (CMTs), which in turn mediate deposition of cellulose microfibrils (CMFs). Here, by combining imaging, genetic and chemical approaches, we show that isotropic reorientation of CMTs and CMFs in aged Col-0 and katanin1-5 (ktn1-5) papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae. We show that this coiled phenotype is associated with specific mechanical properties of the cell walls that provide less resistance to pollen tube growth. Our results reveal an unexpected role for KTN1 in pollen tube guidance on the stigma by ensuring mechanical anisotropy of the papilla cell wall.

Flowering plants produce small particles known as pollen that ­ with the help of the wind, bees and other animals ­ carry male sex cells (sperm) to female sex cells (eggs) contained within flowers. When a grain of pollen lands on the female organ of a flower, called the pistil, it gives rise to a tube that grows through the pistil towards the egg cells at the base. The surface of the pistil is covered in a layer of long cells named papillae. Like most plant cells, the papillae are surrounded by a rigid structure known as the cell wall, which is mainly composed of strands known as microfibrils. The pollen tube exerts pressure on a papilla to allow it to grow through the cell wall towards the base of the pistil. Previous studies have shown that the pistil produces signals that guide pollen tubes to the eggs. However, it remains unclear how pollen tubes orient themselves on the surface of papillae to grow in the right direction through the pistil. Riglet et al. combined microscopy, genetic and chemical approaches to study how pollen tubes grow through the surface of the pistils of a small weed known as Arabidopsis thaliana. The experiments showed that an enzyme called KATANIN conferred mechanical properties to the cell walls of papillae that allowed pollen tubes to grow towards the egg cells, and also altered the orientation of the microfibrils in these cell walls. In A. thaliana plants that were genetically modified to lack KATANIN the pollen tubes coiled around the papillae and sometimes grew in the opposite direction to where the eggs were. KATANIN is known to cut structural filaments inside the cells of plants, animals and most other living things. By revealing an additional role for KATANIN in regulating the mechanical properties of the papilla cell wall, these findings indicate this enzyme may also regulate the mechanical properties of cells involved in other biological processes.

SYNERGISTIC ON AUXIN AND CYTOKININ 1 positively regulates growth and attenuates soil pathogen resistance.

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.

CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) Mediates Plant Resistance against Pseudomonas syringae.

Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na+:K+:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.

Xyloglucans and Microtubules Synergistically Maintain Meristem Geometry and Phyllotaxis.

The shoot apical meristem (SAM) gives rise to all aerial plant organs. Cell walls are thought to play a central role in this process, translating molecular regulation into dynamic changes in growth rate and direction, although their precise role in morphogenesis during organ formation is poorly understood. Here, we investigated the role of xyloglucans (XyGs), a major, yet functionally poorly characterized, wall component in the SAM of Arabidopsis (Arabidopsis thaliana). Using immunolabeling, biochemical analysis, genetic approaches, microindentation, laser ablation, and live imaging, we showed that XyGs are important for meristem shape and phyllotaxis. No difference in the Young's modulus (i.e. an indicator of wall stiffness) of the cell walls was observed when XyGs were perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and pectin methylation status. Interestingly, control of cortical microtubule dynamics by the severing enzyme KATANIN became vital when XyGs were perturbed or absent. This suggests that the cytoskeleton plays an active role in compensating for altered cell wall composition.

Xyloglucan endotransglucosylase-hydrolase30 negatively affects salt tolerance in Arabidopsis.

Plants have evolved various strategies to sense and respond to saline environments, which severely reduce plant growth and limit agricultural productivity. Alteration to the cell wall is one strategy that helps plants adapt to salt stress. However, the physiological mechanism of how the cell wall components respond to salt stress is not fully understood. Here, we show that expression of XTH30, encoding xyloglucan endotransglucosylase-hydrolase30, is strongly up-regulated in response to salt stress in Arabidopsis. Loss-of-function of XTH30 leads to increased salt tolerance and overexpression of XTH30 results in salt hypersensitivity. XTH30 is located in the plasma membrane and is highly expressed in the root, flower, stem, and etiolated hypocotyl. The NaCl-induced increase in xyloglucan (XyG)-derived oligosaccharide (XLFG) of the wild type is partly blocked in xth30 mutants. Loss-of-function of XTH30 slows down the decrease of crystalline cellulose content and the depolymerization of microtubules caused by salt stress. Moreover, lower Na+ accumulation in shoot and lower H2O2 content are found in xth30 mutants in response to salt stress. Taken together, these results indicate that XTH30 modulates XyG side chains, altered abundance of XLFG, cellulose synthesis, and cortical microtubule stability, and negatively affecting salt tolerance.

Emerging Functions for Cell Wall Polysaccharides Accumulated during Eudicot Seed Development.

The formation of seeds is a reproductive strategy in higher plants that enables the dispersal of offspring through time and space. Eudicot seeds comprise three main components, the embryo, the endosperm and the seed coat, where the coordinated development of each is important for the correct formation of the mature seed. In addition, the seed coat protects the quiescent progeny and can provide transport mechanisms. A key underlying process in the production of seed tissues is the formation of an extracellular matrix termed the cell wall, which is well known for its essential function in cytokinesis, directional growth and morphogenesis. The cell wall is composed of a macromolecular network of polymers where the major component is polysaccharides. The attributes of polysaccharides differ with their composition and charge, which enables dynamic remodeling of the mechanical and physical properties of the matrix by adjusting their production, modification or turnover. Accordingly, the importance of specific polysaccharides or modifications is increasingly being associated with specialized functions within seed tissues, often through the spatio-temporal accumulation or remodeling of particular polymers. Here, we review the evolution and accumulation of polysaccharides during eudicot seed development, what is known of their impact on wall architecture and the diverse roles associated with these in different seed tissues.

Suppression of Arabidopsis GGLT1 affects growth by reducing the L-galactose content and borate cross-linking of rhamnogalacturonan-II.

Boron is a micronutrient that is required for the normal growth and development of vascular plants, but its precise functions remain a subject of debate. One established role for boron is in the cell wall where it forms a diester cross-link between two monomers of the low-abundance pectic polysaccharide rhamnogalacturonan-II (RG-II). The inability of RG-II to properly assemble into a dimer results in the formation of cell walls with abnormal biochemical and biomechanical properties and has a severe impact on plant productivity. Here we describe the effects on RG-II structure and cross-linking and on the growth of plants in which the expression of a GDP-sugar transporter (GONST3/GGLT1) has been reduced. In the GGLT1-silenced plants the amount of L-galactose in side-chain A of RG-II is reduced by up to 50%. This leads to a reduction in the extent of RG-II cross-linking in the cell walls as well as a reduction in the stability of the dimer in the presence of calcium chelators. The silenced plants have a dwarf phenotype, which is rescued by growth in the presence of increased amounts of boric acid. Similar to the mur1 mutant, which also disrupts RG-II cross-linking, GGLT1-silenced plants display a loss of cell wall integrity under salt stress. We conclude that GGLT1 is probably the primary Golgi GDP-L-galactose transporter, and provides GDP-L-galactose for RG-II biosynthesis. We propose that the L-galactose residue is critical for RG-II dimerization and for the stability of the borate cross-link.

GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) Is a GlcNAc-Containing Glycosylinositol Phosphorylceramide Glycosyltransferase.

Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structures, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis (Arabidopsis thaliana) and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain GlcNAc and GlcN [collectively GlcN(Ac)]. In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, "rice-like" GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.

Xyloglucans fucosylation defects do not alter plant boundary domain definition.

The CUP-SHAPED COTYLEDON (CUC) transcription factors play a fundamental role in plant morphogenesis by defining boundary domains throughout plant development. Despite their central roles in plant development, little is known about the CUC molecular network. In a recent work, we identified a role for MUR1, a protein involved in the production of GDP-L-Fucose, in this network and showed that fucose per se is required for proper boundary definition in various developmental contexts. Which pathway involving fucose is required to determine boundary is not yet known. Here, we use a previously described mutant and transgenic line with reduced fucosylated xyloglucans (XyG) to explore one such pathway. By quantitatively comparing leaf shape, we show that defects in XyG fucosylation do not impact leaf serrations development suggesting that fucose absence in XyG does not impact boundary development in mur1-1 mutant. Thus another - not yet identified - pathway or fucosylated compound contribute to boundary domain definition.

Rice Sucrose Partitioning Mediated by a Putative Pectin Methyltransferase and Homogalacturonan Methylesterification.

Homogalacturonan (HG) is the main component of pectins. HG methylesterification has recently emerged as a key determinant controlling cell attachment, organ formation, and phyllotaxy. However, whether and how HG methylesterification affects intercellular metabolite transport has rarely been reported. Here, we identified and characterized knockout mutants of the rice (Oryza sativa) OsQUA2 gene encoding a putative pectin methyltransferase. Osqua2 mutants exhibit a remarkable decrease in the degree of methylesterification of HG in the culm-sieve element cell wall and a markedly reduced grain yield. The culm of Osqua2 mutant plants contains excessive sucrose (Suc), and a 13CO2 feeding experiment showed that the Suc overaccumulation in the culm was caused by blocked Suc translocation. These and other findings demonstrate that OsQUA2 is essential for maintaining a high degree of methylesterification of HG in the rice culm-sieve element cell wall, which may be critical for efficient Suc partitioning and grain filling. In addition, our results suggest that the apoplastic pathway is involved in long-distance Suc transport in rice. The identification and characterization of the OsQUA2 gene and its functionality revealed a previously unknown contribution of HG methylesterification and provided insight into how modification of the cell wall regulates intercellular transport in plants.

Xyloglucan Metabolism Differentially Impacts the Cell Wall Characteristics of the Endosperm and Embryo during Arabidopsis Seed Germination.

Cell wall remodeling is an essential mechanism for the regulation of plant growth and architecture, and xyloglucans (XyGs), the major hemicellulose, are often considered as spacers of cellulose microfibrils during growth. In the seed, the activity of cell wall enzymes plays a critical role in germination by enabling embryo cell expansion leading to radicle protrusion, as well as endosperm weakening prior to its rupture. A screen for Arabidopsis (Arabidopsis thaliana) mutants affected in the hormonal control of germination identified a mutant, xyl1, able to germinate on paclobutrazol, an inhibitor of gibberellin biosynthesis. This mutant also exhibited reduced dormancy and increased resistance to high temperature. The XYL1 locus encodes an α-xylosidase required for XyG maturation through the trimming of Xyl. The xyl1 mutant phenotypes were associated with modifications to endosperm cell wall composition that likely impact on its resistance, as further demonstrated by the restoration of normal germination characteristics by endosperm-specific XYL1 expression. The absence of phenotypes in mutants defective for other glycosidases, which trim Gal or Fuc, suggests that XYL1 plays the major role in this process. Finally, the decreased XyG abundance in hypocotyl longitudinal cell walls of germinating embryos indicates a potential role in cell wall loosening and anisotropic growth together with pectin de-methylesterification.

The ABA-deficiency suppressor locus HAS2 encodes the PPR protein LOI1/MEF11 involved in mitochondrial RNA editing.

The hot ABA-deficiency suppressor2 (has2) mutation increases drought tolerance and the ABA sensitivity of stomata closure and seed germination. Here we report that the HAS2 locus encodes the mitochondrial editing factor11 (MEF11), also known as lovastatin insensitive1. has2/mef11 mutants exhibited phenotypes very similar to the ABA-hypersensitive mutant, hai1-1 pp2ca-1 hab1-1 abi1-2, which is impaired in four genes encoding type 2C protein phosphatases (PP2C) that act as upstream negative regulators of the ABA signaling cascade. Like pp2c, mef11 plants were more resistant to progressive water stress and seed germination was more sensitive to paclobutrazol (a gibberellin biosynthesis inhibitor) as well as mannitol and NaCl, compared with the wild-type plants. Phenotypic alterations in mef11 were associated with the lack of editing of transcripts for the mitochondrial cytochrome c maturation FN2 (ccmFN2) gene, which encodes a cytochrome c-heme lyase subunit involved in cytochrome c biogenesis. Although the abundance of electron transfer chain complexes was not affected, their dysfunction could be deduced from increased respiration and altered production of hydrogen peroxide and nitric oxide in mef11 seeds. As minor defects in mitochondrial respiration affect ABA signaling, this suggests an essential role for ABA in mitochondrial retrograde regulation.

Inhibition of germination of dormant barley (Hordeum vulgare L.) grains by blue light as related to oxygen and hormonal regulation.

Germination of primary dormant barley grains is promoted by darkness and temperatures below 20 °C, but is strongly inhibited by blue light. Exposure under blue light at 10 °C for periods longer than five days, results in a progressive inability to germinate in the dark, considered as secondary dormancy. We demonstrate that the inhibitory effect of blue light is reinforced in hypoxia. The inhibitory effect of blue light is associated with an increase in embryo abscisic acid (ABA) content (by 3.5- to 3.8-fold) and embryo sensitivity to both ABA and hypoxia. Analysis of expression of ABA metabolism genes shows that increase in ABA mainly results in a strong increase in HvNCED1 and HvNCED2 expression, and a slight decrease in HvABA8'OH-1. Among the gibberellins (GA) metabolism genes examined, blue light decreases the expression of HvGA3ox2, involved in GA synthesis, increases that of GA2ox3 and GA2ox5, involved in GA catabolism, and reduces the GA signalling evaluated by the HvExpA11 expression. Expression of secondary dormancy is associated with maintenance of high embryo ABA content and a low HvExpA11 expression. The partial reversion of the inhibitory effect of blue light by green light also suggests that cryptochrome might be involved in this hormonal regulation.

ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination.

Dormancy is an adaptive trait that enables seed germination to coincide with favorable environmental conditions. It has been clearly demonstrated that dormancy is induced by abscisic acid (ABA) during seed development on the mother plant. After seed dispersal, germination is preceded by a decline in ABA in imbibed seeds, which results from ABA catabolism through 8'-hydroxylation. The hormonal balance between ABA and gibberellins (GAs) has been shown to act as an integrator of environmental cues to maintain dormancy or activate germination. The interplay of ABA with other endogenous signals is however less documented. In numerous species, ethylene counteracts ABA signaling pathways and induces germination. In Brassicaceae seeds, ethylene prevents the inhibitory effects of ABA on endosperm cap weakening, thereby facilitating endosperm rupture and radicle emergence. Moreover, enhanced seed dormancy in Arabidopsis ethylene-insensitive mutants results from greater ABA sensitivity. Conversely, ABA limits ethylene action by down-regulating its biosynthesis. Nitric oxide (NO) has been proposed as a common actor in the ABA and ethylene crosstalk in seed. Indeed, convergent evidence indicates that NO is produced rapidly after seed imbibition and promotes germination by inducing the expression of the ABA 8'-hydroxylase gene, CYP707A2, and stimulating ethylene production. The role of NO and other nitrogen-containing compounds, such as nitrate, in seed dormancy breakage and germination stimulation has been reported in several species. This review will describe our current knowledge of ABA crosstalk with ethylene and NO, both volatile compounds that have been shown to counteract ABA action in seeds and to improve dormancy release and germination.

Epoxycarotenoid cleavage by NCED5 fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with other NCED family members.

Carotenoid cleavage, catalyzed by the 9-cis-epoxycarotenoid dioxygenase (NCED) constitutes a key step in the regulation of ABA biosynthesis. In Arabidopsis, this enzyme is encoded by five genes. NCED3 has been shown to play a major role in the regulation of ABA synthesis in response to water deficit, whereas NCED6 and NCED9 have been shown to be essential for the ABA production in the embryo and endosperm that imposes dormancy. Reporter gene analysis was carried out to determine the spatiotemporal pattern of NCED5 and NCED9 gene expression. GUS activity from the NCED5 promoter was detected in both the embryo and endosperm of developing seeds with maximal staining after mid-development. NCED9 expression was found at early stages in the testa outer integument layer 1, and after mid-development in epidermal cells of the embryo, but not in the endosperm. In accordance with its temporal- and tissue-specific expression, the phenotypic analysis of nced5 nced6 nced9 triple mutant showed the involvement of the NCED5 gene, together with NCED6 and NCED9, in the induction of seed dormancy. In contrast to nced6 and nced9, however, nced5 mutation did not affect the gibberellin required for germination. In vegetative tissues, combining nced5 and nced3 mutations reduced vegetative growth, increased water loss upon dehydration, and decreased ABA levels under both normal and stressed conditions, as compared with nced3. NCED5 thus contributes, together with NCED3, to ABA production affecting plant growth and water stress tolerance.