In animal cells, phosphoinositides are key components of the inositol 1,4,5-trisphosphate/diacylglycerolbased signaling pathway, but also have many other cellular functions. These lipids are also believed to fulfill similar functions in plant cells, although many details concerning the components of a plant phosphoinositide system, and their regulation are still missing. Only recently have the different phosphoinositide isomers been unambiguously identified in plant cells. Another problem that hinders the study of the function of phosphoinositides and their derivatives, as well as the regulation of their metabolism, in plant cells is the need for a homogenous, easily obtainable material, from which the extraction and purification of phospholipids is relatively easy and quantitatively reproducible. We present here a thorough characterization of the phospholipids purified from [ 32 P]orthophosphate-and myo-[2-3 H]inositol-radiolabeled Arabidopsis thaliana suspension-cultured cells. We then show that NaCl treatment induces dramatic increases in the levels of phosphatidylinositol 4,5-bisphosphate and diacylglycerol pyrophosphate and also affects the turnover of phosphatidylcholine. The increase in phosphatidylinositol 4,5-bisphosphate was also observed with a non-ionic hyperosmotic shock. In contrast, the increase in diacylglycerol pyrophosphate and the turnover of phosphatidylcholine were relatively specific to salt treatments as only minor changes in the metabolism of these two phospholipids were detected when the cells were treated with sorbitol instead of NaCl.Phosphoinositides are quantitatively minor phospholipids that play an important role in the transduction of physiological signals, such as hormones, growth factors, and neurotransmitters in animal cells (1). One of the key early events triggered by these physiological stimuli is the activation of phosphoinositide-specific phospholipase C (PI-PLC), 1 resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) to the two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) and diacylglycerol, which induce Ca 2ϩ release from internal stores and stimulate protein kinase C, respectively (1, 2). During the last decade, it has become evident that in addition to serving as precursors to Ins(1,4,5)P 3 and diacylglycerol, phosphoinositides actively participate in other cellular functions: they have been shown to regulate the dynamics of the actin cytoskeleton through the interaction with actin-binding proteins (3, 4), and to potentiate the activation of protein kinase C (5) and PI-PLC (6, 7). In addition, phosphoinositides phosphorylated at the D 3 -hydroxy group of the inositol headgroup are required for specific vesicle trafficking steps (8, 9) and are able to activate the recently identified novel protein kinases Akt/PKB and phosphoinositide-dependent kinases (10). Recently, a new 3-phosphorylated phosphoinositide, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ), was identified and shown to accumulate in yeast cells ...

CHL27, the Arabidopsis homologue to Chlamydomonas Crd1, a plastid-localized putative diiron protein, is required for the synthesis of protochlorophyllide and therefore is a candidate subunit of the aerobic cyclase in chlorophyll biosynthesis. ␦-Aminolevulinic acid-fed antisense Arabidopsis plants with reduced amounts of Crd1͞CHL27 accumulate Mg-protoporphyrin IX monomethyl ester, the substrate of the cyclase reaction. Mutant plants have chlorotic leaves with reduced abundance of all chlorophyll proteins. Fractionation of Arabidopsis chloroplast membranes shows that Crd1͞ CHL27 is equally distributed on a membrane-weight basis in the thylakoid and inner-envelope membranes.T he chlorophyll (Chl) biosynthetic pathway, occurring in all photosynthetic organisms, has been described through genetic analysis of bacterial mutants and in vitro reconstitution of individual reactions (1-3). Chl production begins with the condensation of eight molecules of ␦-aminolevulinic acid (ALA) to uroporphyrinogen III, the first cyclic tetrapyrrole. Uroporphyrinogen III is then converted to protoporphyrin IX, which is the branch-point intermediate to hemes and Chls. The chelation of magnesium into protoporphyrin IX results in the formation of Mg-protoporphyrin IX (MgP), which is converted to MgP monomethyl ester (MgPMME) by a methyl transferase (4). MgPMME is the substrate for the so-called cyclase reaction, which results in the formation of divinyl protochlorophyllide (Pchlide) containing the fifth ring (ring E) characteristic of all Chls (Fig. 1). In angiosperms, the subsequent steps include the extensively studied, light-dependent conversion of Pchlide to chlorophyllide a via NADPH-Pchlide oxidoreductase and the addition of a polyisoprene tail to complete Chl a production.Labeling experiments (5) suggested two different mechanisms for the cyclase reaction and presumably two different enzymes. The bchE gene product is implicated in the anaerobic reaction because bchE mutants in Rhodobacter sphaeroides accumulate pigments corresponding to MgP and MgPMME (6). The aerobic enzyme is clearly distinct. Although its genetic identity remained elusive for a long time, biochemical studies did define a reaction path and several key features of the enzyme. The aerobic cyclase reaction, an overall six-electron oxidation, is proposed to occur in three sequential steps (7) (Fig. 1). The first step is the stereospecific hydroxylation of the methyl-esterified ring C propionate followed by oxidation of the alcohol to the corresponding ketone. The now-activated methylene group reacts with the ␥-meso carbon of the porphyrin nucleus in an oxidative reaction involving removal of two H to yield ring E. Both the hydroxylated and the keto compounds were suggested to be genuine intermediates, because they could function as substrates for the enzyme (8, 9). Molecular oxygen is required at the step of hydroxylation and also for the conversion of the keto intermediate to divinyl Pchlide (8).The enzyme is iron-dependent: iron chelators inhibit the cyclase enzyme ...