Positional cloning of the barley tillering gene uniculme4

Manipulation of plant architectural traits such as the number of tillers can effectively increase grain yield in cereals. Within the frame of the TriticeaeGenome project (www.triticeaegenome.eu), the objective of our group was the fine mapping and positional cloning of uniculme4 (cul4), a gene required for tillering in barley. Based on initial medium resolution mapping of the locus, a segregating population including 4900 F3 plants was developed and genotyped with three tightly linked SNP markers (Tavakol et al., abstract P321, PAG XIX). The locus was further resolved through mapping of 8 synteny-derived markers allowing the identification of a candidate gene that co-segregates with the cul4 phenotype. The two genes that flank the candidate gene in Brachypodium and rice were positioned 0.11 cM and 0.12 cM from cul4, respectively: development of new markers is underway using sequence information from two BACs anchored to the physical map and spanning this region. The intron-exon structure of the candidate gene was determined from a cDNA isolated from wild-type plants. Resequencing of independent cul4 stocks identified three distinct mutations within the candidate gene, including a deletion of the 5\u2019 region. Comparison of expression levels and patterns in mutant and wild-type plants is underway

The genetic basis of composite spike form in barley and "Miracle-Wheat"

Inflorescences of the tribe Triticeae, which includes wheat (Triticum sp. L.) and barley (Hordeum vulgare L.) are characterized by sessile spikelets directly borne on the main axis, thus forming a branchless spike. "Compositum-Barley" and tetraploid "Miracle-Wheat" (T. turgidum convar. compositum (L.f.) Filat.) display non-canonical spike-branching in which spikelets are replaced by lateral branch-like structures resembling small-sized secondary spikes. As a result of this branch formation "Miracle-Wheat" produces significantly more grains per spike, leading to higher spike yield. In this study, we first isolated the gene underlying spike-branching in "Compositum-Barley", i.e. compositum 2 (com2). Moreover, we found that COM2 is orthologous to the branched head(t) (bh(t)) locus regulating spike-branching in tetraploid "Miracle-Wheat". Both genes possess orthologs with similar functions in maize BRANCHED SILKLESS 1 (BD1) and rice FRIZZY PANICLE/BRANCHED FLORETLESS 1 (FZP/BFL1) encoding AP2/ERF transcription factors. Sequence analysis of the bh(t) locus in a collection of mutant and wild type tetraploid wheat accessions revealed that a single amino acid substitution in the DNA-binding domain gave rise to the domestication of "Miracle-Wheat". mRNA in situ hybridization, microarray experiments, and independent qRT-PCR validation analyses revealed that the branch repression pathway in barley is governed through the spike architecture gene Six-rowed spike 4 regulating COM2 expression, while HvIDS1 (barley ortholog of maize INDETERMINATE SPIKELET 1) is a putative down-stream target of COM2. These findings presented here provide new insights into the genetic basis of spike architecture in Triticeae, and have disclosed new targets for genetic manipulations aiming at boosting wheat's yield potential

Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids : evidence for a phospholipid binding site which overlaps the calmodulin-binding site

The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate>phosphatidylserine>phosphatidylcholine=phosphatidylethanolamine=0. Acidic phospholipids increased Vmax-Ca2+ and lowered the value of K0.5-Ca2+ below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K0.5-Ca2+ value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1-116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant D74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca2+-ATPase provides new evidence that type 2B Ca2+-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein

Intracellular localisation of PPI1 (proton pump interactor, isoform 1), a regulatory protein of the plasma membrane H plus -ATPase of Arabidopsis thaliana

PPI1 (proton pump interactor isoform 1) is a novel protein able to interact with the C-terminal autoinhibitory domain of the Arabidopsis thaliana plasma membrane (PM) H+-ATPase. In vitro, PPI1 binds the PM H+-ATPase in a site different from the known 14-3-3 binding site and stimulates its activity. In this study, we analysed the intracellular localisation of PPI1. The intracellular distribution was monitored in A. thaliana cultured cells by immunolocalisation using an antiserum against the PPI1 N-terminus and in Vicia faba guard cells and epidermal cells by transient expression of a GFP::PPI1 fusion. The results indicate that the bulk of PPI1 is localised at the endoplasmic reticulum, from which it might be recruited to the PM for interaction with the H+-ATPase in response to as yet unidentified signals

Mutations in the actuator of ACA8, a Ca-ATPase of A. thaliana, generate partially deregulated pumps

ACA8, a type 2B Ca ATPase, has a regulatory N-terminus with autoinhibitory action suppressed by binding of calmodulin (CaM). ACA8 N-terminus binds a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To define the role of this interaction in autoinhibition we have analysed a number of single point mutants of ACA8 E252-N345 sequence. Mutation to Ala of any of 6 acidic residues (E252, D273, D291, D303, E302, D332) originates an enzyme with normal activity in presence of CaM, but less CaM-stimulated. These results highlight the relevance in autoinhibition of a negative charge in the small cytoplasmic loop of ACA8. The most deregulated mutant is D291A, which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the D291A mutant is stronger than that of D291N suggesting a more direct involvement of this residue in autoinhibition. Of the other mutants (I284A, N286A, P289A, P322A, V344A, N345A), only P322A has a basal activity higher than that of the WT. These results provide the first evidence that the small cytoplasmic loop of a type 2B Ca ATPase plays a role in the attainment of the autoinhibited stat

Intracellular localisation of PPI1 (proton pump interactor, isoform 1), a regulatory protein of the plasma membrane H(+)-ATPase of Arabidopsis thaliana.

PPI1 (proton pump interactor isoform 1) is a novel protein able to interact with the C-terminal autoinhibitory domain of the Arabidopsis thaliana plasma membrane (PM) H(+)-ATPase. In vitro, PPI1 binds the PM H(+)-ATPase in a site different from the known 14-3-3 binding site and stimulates its activity. In this study, we analysed the intracellular localisation of PPI1. The intracellular distribution was monitored in A. thaliana cultured cells by immunolocalisation using an antiserum against the PPI1 N-terminus and in Vicia faba guard cells and epidermal cells by transient expression of a GFP::PPI1 fusion. The results indicate that the bulk of PPI1 is localised at the endoplasmic reticulum, from which it might be recruited to the PM for interaction with the H(+)-ATPase in response to as yet unidentified signals