Specific TCP transcription factors interact with and stabilize PRR2 within different nuclear sub-domains.

Plants possess a large set of transcription factors both involved in the control of plant development or in plant stress responses coordination. We previously identified PRR2, a Pseudo-Response Regulator, as a plant-specific CML-interacting partner. We reported that PRR2 acts as a positive actor of plant defense by regulating the production of antimicrobial compounds. Here, we report new data on the interaction between PRR2 and transcription factors belonging to the Teosinte branched Cycloidea and PCF (TCP) family. TCPs have been described to be involved in plant development and immunity. We evaluated the ability of PRR2 to interact with seven TCPs representative of the different subclades of the family. PRR2 is able to interact with TCP13, TCP15, TCP19 and TCP20 in yeast two-hybrid system and in planta interactions were validated for TCP19 and TCP20. Transient expression in tobacco highlighted that PRR2 protein is more easily detected when co-expressed with TCP19 or TC20. This stabilization is associated with a specific sub-nuclear localization of the complex in Cajal bodies or in nuclear speckles according to the interaction of PRR2 with TCP19 or TCP20 respectively. The interaction between PRR2 and TCP19 or TCP20 would contribute to the biological function in specific nuclear compartments.

PRR2, a pseudo-response regulator, promotes salicylic acid and camalexin accumulation during plant immunity.

Calcium signalling mediated by Calmodulin (CaM) and calmodulin-like (CML) proteins is critical to plant immunity. CaM and CML regulate a wide range of target proteins and cellular responses. While many CaM-binding proteins have been identified, few have been characterized for their specific role in plant immunity. Here, we report new data on the biological function of a CML-interacting partner, PRR2 (PSEUDO-RESPONSE REGULATOR 2), a plant specific transcription factor. Until now, the physiological relevance of PRR2 remained largely unknown. Using a reverse genetic strategy in A. thaliana, we identified PRR2 as a positive regulator of plant immunity. We propose that PRR2 contributes to salicylic acid (SA)-dependent responses when challenged with the phytopathogenic bacterium Pseudomonas syringae. PRR2 is transcriptionally upregulated by SA and P. syringae, enhances SA biosynthesis and SA signalling responses; e.g. in response to P. syringae, PRR2 induces the production of SA and the accumulation of the defence-related protein PR1. Moreover, PRR2 overexpressing lines exhibit an enhanced production of camalexin, a phytoalexin that confers enhanced resistance against pathogens. Together, these data reveal the importance of PRR2 in plant immune responses against P. syringae and suggest a novel function for this particular plant specific transcription factor in plant physiology.

The riddle of the plant vacuolar sorting receptors.

Proteins synthesized on membrane-bound ribosomes are sorted at the Golgi apparatus level for delivery to various cellular destinations: the plasma membrane or the extracellular space, and the lytic vacuole or lysosome. Sorting involves the assembly of vesicles, which preferentially package soluble proteins with a common destination. The selection of proteins for a particular vesicle type involves the recognition of proteins by specific receptors, such as the vacuolar sorting receptors for vacuolar targeting. Most eukaryotic organisms have one or two receptors to target proteins to the lytic vacuole. Surprisingly, plants have several members of the same family, seven in Arabidopsis thaliana. Why do plants have so many proteins to sort soluble proteins to their respective destinations? The presence of at least two types of vacuoles, lytic and storage, seems to be a partial answer. In this review we analyze the last experimental evidence supporting the presence of different subfamilies of plant vacuolar sorting receptors.

A bacterial artificial chromosome (BAC) library for sunflower, and identification of clones containing genes for putative transmembrane receptors.

Sunflower (Helianthus annuus L.) is an economically important oil seed crop with an estimated genome size of 3000 Mb. We have constructed a bacterial artificial chromosome (BAC) library for sunflower, which represents an estimated 4- to 5-fold coverage of the genome. Nuclei isolated from young leaves were used as a source of high-molecular-weight DNA and a partial restriction endonuclease digestion protocol was used to cleave the DNA. A random sample of 60 clones indicated an average insert size of 80 kb, implying a 95% probability of recovering any specific sequence of interest. The library was screened with chloroplast DNA probes. Only 0.1% of the clones were identified to be of chloroplast origin, indicating that contamination with organellar DNAs is very low. The utility of the library was evaluated by screening for the presence of genes for putative transmembrane receptors sharing epidermal growth factor (EGF) and integrin-like domains. First, a homologous sunflower EST (HaELP1) was obtained by degenerate RT-PCR cloning, using Arabidopsis thaliana genes (AtELP) as a source of consensus sequences. Three different BACs yielded positive hybridization signals when HaELP1 was used as a probe. BAC subcloning and sequencing demonstrated the presence of two different loci putatively homologous to genes for transmembrane proteins with EGF- and integrin-like domains from sunflower. This work demonstrates the suitability of the library for homology map-based cloning of sunflower genes and physical mapping of the sunflower genome.

Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high-efficiency expression library.

We constructed a high-efficiency expression library from Arabidopsis cDNA clones by introducing a poly (dC) stretch at the 5' end of the clones. This library enables the synthesis of proteins from all the cDNA clones present. We have screened the high-efficiency expression library with antibodies raised against total proteins from Arabidopsis plasmalemma and tonoplast. With the positive clones, we have constructed two cDNA ordered libraries enriched in genes encoding plasmalemma (522 clones) and tonoplast proteins (594 clones). Partial sequencing of both libraries shows that a high proportion (47%) of the clones encoded putative membrane proteins, or membrane-associated proteins. When sequenced, 55% of the cDNAs were new EST sequences for Arabidopsis, 26% were similar to genes present in other plants or organisms, and 29% were not referenced in any databank. Immunoscreening of the two cDNA ordered libraries with antibodies raised against proteins from Arabidopsis cells submitted to osmotic stress allows the selection of genes over- and under-expressed in stress conditions.

High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

Free-flow electrophoresis for fractionation of Arabidopsis thaliana membranes.

Highly purified tonoplast and plasma membrane vesicles were isolated from microsomes of Arabidopsis thaliana by preparative free-flow electrophoresis. The most electronegative fractions were identified as tonoplast using nitrate-inhibited Mg2+-ATPase as enzyme marker. The least electronegative fractions were identified as plasma membrane using glucan-synthase II, UDPG: sterol-glucosyl-transferase, and vanadate-inhibited Mg2+-ATPase as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (endoplasmic reticulum) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane. Immunoblot analysis of membrane fractions by antibodies directed against tonoplast and plasma membrane proteins confirmed the nature and the purity of the isolated membranes. The cytoskeletal protein actin, which was also identified by immunoblotting, was found to be specifically attached to the plasma membrane vesicles. The structural and functional integrity of the isolated membranes from Arabidopsis thaliana is discussed in the light of results obtained for the location of receptors and enzymes, or for the determination of ligand binding activity.

Osmotic stress activated expression of an Arabidopsis plasma membrane-associated protein: sequence and predicted secondary structure.

A cDNA clone At.MAMI (Arabidopsis thaliana membrane-associated mannitol-induced) was isolated from an Arabidopsis cDNA expression library by immunoselection. The cDNA was full-length (1.18 kb) with an open reading frame of 798 nucleotides encoding a 265 amino acid protein. The sequence of At.MAMI did not show any significant identity with other genes, as well as the deduced amino acid sequence with other proteins. However, prediction methods for the secondary structure of MAMI-30, together with homologous domains revealed some identity with VAP-33, a protein involved in membrane trafficking in neuronal tissues. In contrast to VAP-33, MAMI-30 did not exhibit a transmembrane domain, but positively charged loop regions could be involved in membrane anchoring. Indeed, MAMI-30 was immunodetected in purified plasma membrane from Arabidopsis cells. The gene was responsive to low turgor in Arabidopsis and its expression regulated developmentally. In addition, reduction of turgor caused a higher accumulation of mRNAs.

Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain.

We have characterized an Arabidopsis receptor-like serine/threonine kinase gene, Ath.lecRK1 (Arabidopsis thaliana lectin-receptor kinase), defining a new and putatively important class of plant receptor kinases. Structural features of the predicted polypeptide include an amino-terminal membrane-targeting signal sequence, a legume lectin-like extracellular domain, a single membrane-spanning domain, and a characteristic serine/threonine protein kinase domain. A recombinant protein containing the kinase domain can be autophosphorylated on a serine residue. Ath.lecRK1 is a member of a gene family of at least two closely related genes. Northern blot analysis indicates that the Ath.lecRK1 gene is weakly expressed in a variety of organs and is regulated in Arabidopsis cell suspension cultures according to the growth phase of cells. The role this new class of plant receptor kinase could play is discussed with regard to the transduction of oligosaccharide and plant hormone signals.

Isolation, sequencing and analysis of the expression of Bryonia calmodulin after mechanical perturbation.

A cDNA clone (Bc329) encoding calmodulin was isolated from a Bryonia cDNA library by screening with cloned Arabidopsis calmodulin cDNA. The cDNA Bc329 was 899 bp full-length clone. The predicted amino acid sequence consists of 149 residues and reveals a high homology with other known plant calmodulins (91 to 99% identity). Genomic southern blot suggests that Bryonia calmodulin is encoded by a single-copy gene. The Bc329 clone was used as a probe to study the expression of calmodulin mRNA after a mechanical stimulus applied on young Bryonia internodes. The steady-state of calmodulin mRNA reached a maximum 30 min after the treatment before it progressively decreased. The role of calcium and calmodulin as second messengers is discussed with regard to environmental changes.