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Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species. [ABSTRACT FROM AUTHOR]

Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterolπ8,π7-isomerases and sterol-π14-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H+-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H+-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H+- pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H+-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H+-pyrophosphatases are major determinants of plant tolerance to amine fungicides. [ABSTRACT FROM AUTHOR]

Flowering is a crucial process that demands substantial resources. Carbon metabolism must be coordinated with development through a control mechanism that optimizes fitness for any physiological need and growth stage of the plant. However, how sugar allocation is controlled during the floral transition is unknown. Recently, the role of a CONSTANS (CO) ortholog (Cr -CO) in the control of the photoperiod response in the green alga Chlamydomonas reinhardtii and its influence on starch metabolism was demonstrated. In this work, we show that transitory starch accumulation and glycan composition during the floral transition in Arabidopsis thaliana are regulated by photoperiod. Employing a multidisciplinary approach, we demonstrate a role for CO in regulating the level and timing of expression of the GRANULE BOUND STARCH SYNTHASE (GBSS) gene. Furthermore, we provide a detailed characterization of a GBSS mutant involved in transitory starch synthesis and analyze its flowering time phenotype in relation to its altered capacity to synthesize amylose and to modify the plant free sugar content. Photoperiod modification of starch homeostasis by CO may be crucial for increasing the sugar mobilization demanded by the floral transition. This finding contributes to our understanding of the flowering process. [ABSTRACT FROM AUTHOR]