Silvia M. Velasquez

O-Glycosylated Cell Wall Proteins Are Essential in Root Hair Growth

Sequential protein posttranslational modifications facilitate cell wall self-assembly and root hair elongation in Arabidopsis. Root hairs are single cells that develop by tip growth and are specialized in the absorption of nutrients. Their cell walls are composed of polysaccharides and hydroxyproline-rich glycoproteins (HRGPs) that include extensins (EXTs) and arabinogalactan-proteins (AGPs). Proline hydroxylation, an early posttranslational modification of HRGPs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs (which are mainly arabinosylated) and AGPs (which are mainly arabinogalactosylated). We explored the biological function of P4Hs, arabinosyltransferases, and EXTs in root hair cell growth. Biochemical inhibition or genetic disruption resulted in the blockage of polarized growth in root hairs and reduced arabinosylation of EXTs. Our results demonstrate that correct O-glycosylation on EXTs is essential for cell-wall self-assembly and, hence, root hair elongation in Arabidopsis thaliana.

An update on post-translational modifications of hydroxyproline-rich glycoproteins: toward a model highlighting their contribution to plant cell wall architecture

Plant cell walls are composite structures mainly composed of polysaccharides, also containing a large set of proteins involved in diverse functions such as growth, environmental sensing, signaling, and defense. Research on cell wall proteins (CWPs) is a challenging field since present knowledge of their role into the structure and function of cell walls is very incomplete. Among CWPs, hydroxyproline (Hyp)-rich O-glycoproteins (HRGPs) were classified into three categories: (i) moderately glycosylated extensins (EXTs) able to form covalent scaffolds; (ii) hyperglycosylated arabinogalactan proteins (AGPs); and (iii) Hyp/proline (Pro)-Rich proteins (H/PRPs) that may be non-, weakly- or highly-glycosylated. In this review, we provide a description of the main features of their post-translational modifications (PTMs), biosynthesis, structure, and function. We propose a new model integrating HRGPs and their partners in cell walls. Altogether, they could form a continuous glyco-network with non-cellulosic polysaccharides via covalent bonds or non-covalent interactions, thus strongly contributing to cell wall architecture.

Auxin and Cellular Elongation

Multiple modes of auxin action are integrated as a crucial growth regulator in the single cell model of the root hair. Auxin is a crucial growth regulator in plants. However, a comprehensive understanding of how auxin induces cell expansion is perplexing, because auxin acts in a concentration- and cell type-dependent manner. Consequently, it is desirable to focus on certain cell types to exemplify the underlying growth mechanisms. On the other hand, plant tissues display supracellular growth (beyond the level of single cells); hence, other cell types might compromise the growth of a certain tissue. Tip-growing cells do not display neighbor-induced growth constraints and, therefore, are a valuable source of information for growth-controlling mechanisms. Here, we focus on auxin-induced cellular elongation in root hairs, exposing a mechanistic view of plant growth regulation. We highlight a complex interplay between auxin metabolism and transport, steering root hair development in response to internal and external triggers. Auxin signaling modules and downstream cascades of transcription factors define a developmental program that appears rate limiting for cellular growth. With this knowledge in mind, the root hair cell is a very suitable model system in which to dissect cellular effectors required for cellular expansion.

Molecular link between auxin and ROS-mediated polar growth

Significance Tip-growing root hairs are an excellent model system for deciphering the molecular mechanism underlying reactive oxygen species (ROS)-mediated cell elongation. Root hairs are able to expand in response to external signals, increasing several hundred-fold their original size, which is important for survival of the plant. Although their final cell size is of fundamental importance, the molecular mechanisms that control it remain largely unknown. In this study, we propose a molecular mechanism that links the auxin–auxin response factors module to activation of RSL4, which directly targets genes encoding ROS-producing enzymes such as NADPH oxidases (or RESPIRATORY BURST OXIDASE HOMOLOG proteins) and secreted type III peroxidases. Activation of these genes impacts on apoplastic ROS homeostasis, thereby stimulating root hair cell elongation. Root hair polar growth is endogenously controlled by auxin and sustained by oscillating levels of reactive oxygen species (ROS). These cells extend several hundred-fold their original size toward signals important for plant survival. Although their final cell size is of fundamental importance, the molecular mechanisms that control it remain largely unknown. Here we show that ROS production is controlled by the transcription factor RSL4, which in turn is transcriptionally regulated by auxin through several auxin response factors (ARFs). In this manner, auxin controls ROS-mediated polar growth by activating RSL4, which then up-regulates the expression of genes encoding NADPH oxidases (also known as RESPIRATORY BURST OXIDASE HOMOLOG proteins) and class III peroxidases, which catalyze ROS production. Chemical or genetic interference with ROS balance or peroxidase activity affects root hair final cell size. Overall, our findings establish a molecular link between auxin and ROS-mediated polar root hair growth.

Low Sugar Is Not Always Good: Impact of Specific O-Glycan Defects on Tip Growth in Arabidopsis

Mutants of the O-glycosylation pathway of extensins as well as molecular dynamics simulations uncover the effects of the O-glycosylation machinery on root hair tip growth.

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1–5 β- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-β1,4Araf-β1,2Araf-β1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its’ product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.

Response of the fungus Pseudocercospora griseola f. mesoamericana to Tricyclazole

Pseudocercospora griseola, an anamorph of Mycosphaerella, causes Angular Leaf Spot (ALS). The mycelia and conidia from P. griseola are coloured due to the synthesis of 1,8 dihydroxynaphthalene (DHN)-melanin. The aim of this work was to identify in P. griseola f. mesoamericana isolate T4, a highly pigmented fungus, intermediary compounds as a result of the inhibition of melanin synthesis by tricyclazole, and to analyze at the structural level the localization of these dark pigments. The main metabolites were analyzed using ultraviolet matrix-assisted laser desorption-ionization mass spectrometry (UV–MALDI MS). Tricyclazole affected P. griseola f. mesoamericana in several different ways. The most evident effect was the reduction of melanin synthesis, and therefore diffusible shunt products were found and identified. Flaviolin was the main intermediate metabolite found in cultures supplemented with tricyclazole. This inhibitor, which affected pigmentation and the cell wall structure of mycelium, revealed macroscopically by the reduction in growth, decreased the stratification and deposition of melanin in the hyphal wall. These results suggest a possible role of tricyclazole to control ALS.

Root hair sweet growth.

Root hairs are single cells specialized in the absorption of water and nutrients from the soil. Growing root hairs require intensive cell-wall changes to accommodate cell expansion at the apical end by a process known as tip or polarized growth. We have recently shown that cell wall glycoproteins such as extensions (EXTs) are essential components of the cell wall during polarized growth. Proline hydroxylation, an early posttranslational modification of cell wall EXTs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs. Biochemical inhibition or genetic disruption of specific P4Hs resulted in the blockage of polarized growth in root hairs. Our results demonstrate that correct hydroxylation and also further O-glycosylation on EXTs are essential for cell-wall self-assembly and, hence, root hair elongation. The changes that O-glycosylated cell-wall proteins like EXTs undergo during cell growth represent a starting point to unravel the entire biochemical pathway involved in plant development.

Corrigendum: Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD

Scientific Reports 7: Article number: 45341; published online: 30 March 2017; updated: 23 May 2017. The original version of this Article contained an error in the spelling of the author Zhang Yang, which was incorrectly given as Yang Zhang. The Author Contributions Statement, S.R.M., X.Y., S.M.V., S.G.Scientific Reports 7: Article number: 45341; published online: 30 March 2017; updated: 23 May 2017. The original version of this Article contained an error in the spelling of the author Zhang Yang, which was incorrectly given as Yang Zhang. The Author Contributions Statement, S.R.M., X.Y., S.M.V., S.G.

Cell biology: Zipping the Casparian strip

Roots must sort the good from the bad and distinguish the inside from the outside. In endodermal cells, a ring-like apoplastic diffusion barrier called the Casparian strip is established, splitting the cells down the middle into inner and outer lateral halves. Its integrity and polarity depends on a novel protein kinase called SCHENGEN1.