Patrick Doumas

A Role for Auxin Redistribution in the Responses of the Root System Architecture to Phosphate Starvation in Arabidopsis

The changes in root system architecture (RSA) triggered by phosphate (P) deprivation were studied in Arabidopsis (Arabidopsis thaliana) plants grown for 14 d on 1 mm or 3 μm P. Two different temporal phases were observed in the response of RSA to low P. First, lateral root (LR) development was promoted between days 7 and 11 after germination, but, after day 11, all root growth parameters were negatively affected, leading to a general reduction of primary root (PR) and LR lengths and of LR density. Low P availability had contrasting effects on various stages of LR development, with a marked inhibition of primordia initiation but a strong stimulation of activation of the initiated primordia. The involvement of auxin signaling in these morphological changes was investigated in wild-type plants treated with indole-3-acetic acid or 2,3,5-triiodobenzoic acid and in axr4-1, aux1-7, and eir1-1 mutants. Most effects of low P on RSA were dramatically modified in the mutants or hormone-treated wild-type plants. This shows that auxin plays a major role in the P starvation-induced changes of root development. From these data, we hypothesize that several aspects of the RSA response to low P are triggered by local modifications of auxin concentration. A model is proposed that postulates that P starvation results in (1) an overaccumulation of auxin in the apex of the PR and in young LRs, (2) an overaccumulation of auxin or a change in sensitivity to auxin in the lateral primordia, and (3) a decrease in auxin concentration in the lateral primordia initiation zone of the PR and in old laterals. Measurements of local changes in auxin concentrations induced by low P, either by direct quantification or by biosensor expression pattern (DR5::β-glucuronidase reporter gene), are in line with these hypotheses. Furthermore, the observation that low P availability mimicked the action of auxin in promoting LR development in the alf3 mutant confirmed that P starvation stimulates primordia emergence through increased accumulation of auxin or change in sensitivity to auxin in the primordia. Both the strong effect of 2,3,5-triiodobenzoic acid and the phenotype of the auxin-transport mutants (aux1, eir1) suggest that low P availability modifies local auxin concentrations within the root system through changes in auxin transport rather than auxin synthesis.

Towards the recovery of hydrophobic proteins on two‐dimensional electrophoresis gels

An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including hydrophobic proteins which are rarely detectable on two‐dimensional electrophoresis (2‐DE) gels. In this study, two methods were employed for the purification of hydrophobic proteins from Arabidopsis thaliana leaf plasma membrane (PM) model plants, prior to analysis on 2‐DE immobilized pH gradient (IPG) gels. Solubilization efficiency of two detergents, (3‐[(3‐cholomidopropyl)‐1‐propanesulfonic acid] (CHAPS)) and C8∅︁, were tested for the recovery of hydrophobic proteins. An immunological approach was used to determine the efficiency of the above methods. Fractionation of proteins by Triton X‐114 combined with solubilization with CHAPS resulted in the inability to detect hydrophobic proteins on 2‐DE gels. The use of C8∅︁ for protein solubilization did not improve this result. On the contrary, after treatment of membranes with alkaline buffer, the solubilization of PM proteins with detergent C8∅︁ permitted the recovery of such proteins on 2‐DE gels. The combination of membrane washing and the use of zwitterionic detergent resulted in the resolution of several integral proteins and the disappearance of peripheral proteins. In the resolution of expressed genome proteins, both large pH gradients in the first dimension and various acrylamide concentrations in the second dimension must be used. Notwithstanding, it is important to combine various sample treatments and different detergents in order to resolve soluble and hydrophobic proteins.

Natural Variation of Root Hydraulics in Arabidopsis Grown in Normal and Salt-Stressed Conditions

To gain insights into the natural variation of root hydraulics and its molecular components, genotypic differences related to root water transport and plasma membrane intrinsic protein (PIP) aquaporin expression were investigated in 13 natural accessions of Arabidopsis (Arabidopsis thaliana). The hydraulic conductivity of excised root systems (Lpr) showed a 2-fold variation among accessions. The contribution of aquaporins to water uptake was characterized using as inhibitors mercury, propionic acid, and azide. The aquaporin-dependent and -independent paths of water transport made variable contributions to the total hydraulic conductivity in the different accessions. The distinct suberization patterns observed among accessions were not correlated with their root hydraulic properties. Real-time reverse transcription-polymerase chain reaction revealed, by contrast, a positive overall correlation between Lpr and certain highly expressed PIP transcripts. Root hydraulic responses to salt stress were characterized in a subset of five accessions (Bulhary-1, Catania-1, Columbia-0, Dijon-M, and Monte-Tosso-0 [Mr-0]). Lpr was down-regulated in all accessions except Mr-0. In Mr-0 and Catania-1, cortical cell hydraulic conductivity was unresponsive to salt, whereas it was down-regulated in the three other accessions. By contrast, the five accessions showed qualitatively similar aquaporin transcriptional profiles in response to salt. The overall work provides clues on how hydraulic regulation allows plant adaptation to salt stress. It also shows that a wide range of root hydraulic profiles, as previously reported in various species, can be observed in a single model species. This work paves the way for a quantitative genetics analysis of root hydraulics.

Large scale characterization of plant plasma membrane proteins

After a brief review of the strategies used to date to identify systematically plasma membrane (PM) proteins, emphasis was given to the proteomic approach of PM proteins from the model plant Arabidopsis thaliana. Comparative analysis of two-dimensional gels from PM and cytosolic fractions was used to assess the cellular origin of proteins found in PM fraction. The classification obtained was confirmed by protein sequencing that showed, in addition, that most analyzed proteins were peripheral proteins. A large proportion of these appeared to correspond to PM-constitutive proteins that were present in the PM from different plant organs, but were not uniquely located at the PM depending on the organ. In addition, the presence of organ-specific sets of PM-specific proteins was also demonstrated. Additional procedures were developed to identify integral PM proteins. The combined use of PM washes with alkaline carbonate buffer or Triton X-100/KBr, and of a new detergent to solubilize protein, resulted in improved recovery of hydrophobic proteins on gels. Results are discussed in terms of construction of comprehensive proteomes for PM and other membranes and organelles.

Cloning of the V-ATPase subunit G in plant: functional expression and sub-cellular localization

A 13‐kDa tobacco plasma membrane protein was isolated from two‐dimensional electrophoresis gels. After microsequencing, RT‐PCR techniques and cDNA library screening allowed for the cloning of two cDNAs. These cDNAs encoded for the subunit G of the vacuolar H+‐ATPase, the first one identified in plants. Analysis of mRNA distribution showed a maximum level in the leaves and in the stem of the apical part of the tobacco plant. Heterologous functional complementation of the yeast mutant (Δvma10::URA3) was achieved with the two cDNAs. After fractionation of microsomal membranes on linear sucrose gradient, Western blots were performed using antibodies against recombinant protein and three peaks were identified: one which comigrated with the tonoplast marker and the others at slightly higher density corresponding to endoplasmic reticulum and to plasma membrane fractions.

Reconstitution of an electrogenic auxin transport activity mediated by Arabidopsis thaliana plasma membrane proteins

Plasma membrane proteins from Arabidopsis thaliana leaves were reconstituted into proteoliposomes and a K+ diffusion potential was generated. The resulting ionic fluxes, determined in the presence of the plant hormone auxin (indole‐3 acetic acid), showed an additional electrogenic and saturable component, with a K M of 6 μM. This flux was neither detected in liposomes in the presence of indole‐3 acetic acid, nor in proteoliposomes in the presence of an inactive auxin analog and was completely inhibited by 3 μM naphtylphthalamic acid, a specific inhibitor of the auxin efflux carrier. The efficiency of the reconstituted carrier and the mechanism of its regulation by naphtylphthalamic acid are discussed.