NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase.

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.

Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme.

In plants, a proposed ascorbate (vitamin C) biosynthesis pathway occurs via GDP-D-mannose (GDP-D-Man), GDP-L-galactose (GDP-L-Gal), and L-galactose. However, the steps involved in the synthesis of L-Gal from GDP-L-Gal in planta are not fully characterized. Here we present evidence for an in vivo role for L-Gal-1-P phosphatase in plant ascorbate biosynthesis. We have characterized a low ascorbate mutant (vtc4-1) of Arabidopsis thaliana, which exhibits decreased ascorbate biosynthesis. Genetic mapping and sequencing of the VTC4 locus identified a mutation (P92L) in a gene with predicted L-Gal-1-P phosphatase activity (At3g02870). Pro-92 is within a beta-bulge that is conserved in related myo-inositol monophosphatases. The mutation is predicted to disrupt the positioning of catalytic amino acid residues within the active site. Accordingly, L-Gal-1-P phosphatase activity in vtc4-1 was approximately 50% of wild-type plants. In addition, vtc4-1 plants incorporate significantly more radiolabel from [2-(3)H]Man into L-galactosyl residues suggesting that the mutation increases the availability of GDP-L-Gal for polysaccharide synthesis. Finally, a homozygous T-DNA insertion line, which lacks a functional At3g02870 gene product, is also ascorbate-deficient (50% of wild type) and deficient in L-Gal-1-P phosphatase activity. Genetic complementation tests revealed that the insertion mutant and VTC4-1 are alleles of the same genetic locus. The significantly lower ascorbate and perturbed L-Gal metabolism in vtc4-1 and the T-DNA insertion mutant indicate that L-Gal-1-P phosphatase plays a role in plant ascorbate biosynthesis. The presence of ascorbate in the T-DNA insertion mutant suggests there is a bypass to this enzyme or that other pathways also contribute to ascorbate biosynthesis.

Analysis of the root-hair morphogenesis transcriptome reveals the molecular identity of six genes with roles in root-hair development in Arabidopsis.

Root-hair morphogenesis is a model for studying the genetic regulation of plant cell development, and double-mutant analyses have revealed a complex genetic network underlying the development of this type of cell. Therefore, to increase knowledge of gene expression in root hairs and to identify new genes involved in root-hair morphogenesis, the transcriptomes of the root-hair differentiation zone of wild-type (WT) plants and a tip-growth defective root-hair mutant, rhd2-1, were compared using Affymetrix ATH1 GeneChips. A set of 606 genes with significantly greater expression in WT plants defines the 'root-hair morphogenesis transcriptome'. Compared with the whole genome, this set is highly enriched in genes known to be involved in root-hair morphogenesis. The additional gene families and functional groups enriched in the root-hair morphogenesis transcriptome are cell wall enzymes, hydroxyproline-rich glycoproteins (extensins) and arabinogalactan proteins, peroxidases, receptor-like kinases and proteins with predicted glycosylphosphatidylinositol (GPI) anchors. To discover new root-hair genes, 159 T-DNA insertion lines identified from the root-hair morphogenesis transcriptome were screened for defects in root-hair morphogenesis. This identified knockout mutations in six genes (RHM1-RHM6) that affected root-hair morphogenesis and that had not previously been identified at the molecular level: At2g03720 (similar to Escherichia coli universal stress protein); At3g54870 (armadillo-repeat containing kinesin-related protein); At4g18640 (leucine-rich repeat receptor-like kinase subfamily VI); At4g26690 (glycerophosphoryl diester phosphodiesterase-like GPI-anchored protein); At5g49270 (COBL9 GPI-anchored protein) and At5g65090 (inositol-1,4,5 triphosphate 5-phosphatase-like protein). The mutants were transcript null, their root-hair phenotypes were characterized and complementation testing with uncloned root-hair genes was performed. The results suggest a role for GPI-anchored proteins and lipid rafts in root-hair tip growth because two of these genes (At4g26690 and At5g49270) encode predicted GPI-anchored proteins likely to be associated with lipid rafts, and several other genes previously shown to be required for root-hair development also encode proteins associated with sterol-rich lipid rafts.

Proline metabolism and transport in maize seedlings at low water potential.

The growing zone of maize seedling primary roots accumulates proline at low water potential. Endosperm removal and excision of root tips rapidly decreased the proline pool and greatly reduced proline accumulation in root tips at low water potential. Proline accumulation was not restored by exogenous amino acids. Labelling root lips with [14C]glutamate and [14C]proline showed that the rate of proline utilization (oxidation and protein synthesis) exceeded the rate of biosynthesis by five-fold at high and low water potentials. This explains the reduction in the proline pool following root and endosperm excision and the inability to accumulate proline at low water potential. The endosperm is therefore the source of the proline that accumulates in the root tips of intact seedlings. Proline constituted 10% of the amino acids released from the endosperm. [14C]Proline was transported from the scutellum to other parts of the seedling and reached the highest concentration in the root tip. Less [14C]proline was transported at low water potential but because of the lower rate of protein synthesis and oxidation, more accumulated as proline in the root tip. Despite the low biosynthesis capacity of the roots, the extent of proline accumulation in relation to water potential is precisely controlled by transport and utilization rate.