SUMMARYStimulus-specific calcium (Ca 2+ ) signals have crucial functions in developmental processes in many organisms, and are deciphered by various Ca 2+ -binding proteins. In Arabidopsis thaliana, a signaling network consisting of calcineurin B-like (CBL) protein calcium sensors and CBL-interacting protein kinases (CIPKs) has been shown to fulfil pivotal functions at the plasma membrane in regulating ion fluxes and abiotic stress responses. However, the role of tonoplast-localized CBL proteins and especially their function in regulating developmental programs remains largely unknown. In this study, we analyzed single and double mutants of the closely related tonoplast-localized calcium sensors CBL2 and CBL3, which show either reduction of function (rf) or complete loss of function (lf). While single cbl2 or cbl3 mutants did not display discernable phenotypes, cbl2/cbl3 mutants exhibited defects in vegetative growth and were severely impaired in seed development and morphology. Seeds of the cbl2/3rf mutant were smaller in size and exhibited reduced weight and fatty acid content compared to wild-type, but accumulation of sucrose was not altered. Moreover, accumulation of inositol hexakisphosphate (InsP 6 ), the major storage form of phosphorus in seeds, was significantly reduced in mutant seeds. In addition, complete loss of CBL2 and CBL3 function in cbl2/3lf resulted in a high frequency of severe defects in embryonic development. Together, our findings reveal a crucial function of Ca 2+ -controlled processes at the vacuolar membrane as determinants of seed yield and size, and demonstrate the importance of vacuolar CBL calcium sensors for plant embryogenesis.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase implicated in cellular proliferation and survival. In animal cells, loss of PTEN leads to increased levels of phosphatidylinositol (3,4,5)-trisphosphate, stimulation of glucose and lipid metabolism, cellular growth, and morphological changes (related to adaptation and survival). Intriguingly, in plants, phosphatidylinositol (3,4,5)-trisphosphate has not been detected, and the enzymes that synthesize it were never reported. In this study we performed a genetic, biochemical, and functional characterization of the moss Physcomitrella patens PTEN gene family. P. patens has four PTENs, which are ubiquitously expressed during the entire moss life cycle. Using a knock-in approach, we show that all four genes are expressed in growing tissues, namely caulonemal and rhizoid cells. At the subcellular level, PpPTEN-green fluorescent protein fusions localized to the cytosol and the nucleus. Analysis of single and double knockouts revealed no significant phenotypes at different developmental stages, indicative of functional redundancy. However, compared with wild-type triple and quadruple pten knockouts, caulonemal cells grew faster, switched from the juvenile protonemal stage to adult gametophores earlier, and produced more rhizoids. Furthermore, analysis of lipid content and quantitative real-time polymerase chain reaction data performed in quadruple mutants revealed altered phosphoinositide levels [increase in phosphatidylinositol (3,5)-bisphosphate and decrease in phosphatidylinositol 3-phosphate] and up-regulation of marker genes from the synthesis phase of the cell cycle (e.g. P. patens proliferating cell nuclear antigen, ribonucleotide reductase, and minichromosome maintenance) and of the retinoblastoma-related protein gene P. patens retinoblastomarelated protein1. Together, these results suggest that PpPTEN is a suppressor of cell growth and morphogenic development in plants.