Regulation of inositol 1,2,4,5,6-pentakisphosphate and inositol hexakisphosphate levels in Gossypium hirsutum by IPK1.

MAIN CONCLUSION: The IPK1 genes, which code for 2-kinases that can synthesize Ins(1,2,4,5,6)P5 from Ins(1,4,5,6)P4, are expressed throughout cotton plants, resulting in the highest Ins(1,2,4,5,6)P5 concentrations in young leaves and flower buds. Cotton leaves contain large amounts of Ins(1,2,4,5,6)P5 and InsP6 compared to plants not in the Malvaceae family. The inositol polyphosphate pathway has been linked to stress tolerance in numerous plant species. Accordingly, we sought to determine why cotton and other Malvaceae have such high levels of these inositol phosphates. We have quantified the levels of InsP5 and InsP6 in different tissues of cotton plants and determined the expression of IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene) in vegetative and reproductive tissues. Gossypium hirsutum was found to contain four IPK1 genes that were grouped into two pair (AB, CD) where each pair consists of very similar sequences that were measured together. More IPK1AB is expressed in leaves than in roots, whereas more IPK1CD is expressed in roots than in leaves. Leaves and flower buds have more InsP5 and InsP6 than stems and roots. Leaves and roots contain more InsP5 than InsP6, whereas flower buds and stems contain more InsP6 than InsP5. Dark-grown seedlings contain more InsP5 and InsP6 than those grown under lights, and the ratio of InsP5 to InsP6 is greater in the light-grown seedlings. During 35 days of the life cycle of the third true leaf, InsP5 and InsP6 gradually decreased by more than 50%. Silencing IPK1AB and IPK1CD with Cotton Leaf Crumple Virus-induced gene silencing (VIGS) resulted in plants with an intense viral phenotype, reduced IPK1AB expression and lowered amounts of InsP5. The results are consistent with Ins(1,2,4,5,6)P5 synthesis from Ins(1,4,5,6)P4 by IPK1. This study detailed the central role of IPK1 in cotton inositol polyphosphate metabolism, which has potential to be harnessed to improve the resistance of plants to different kinds of stress.

Certain Malvaceae Plants Have a Unique Accumulation of myo-Inositol 1,2,4,5,6-Pentakisphosphate.

Methods used to quantify inositol phosphates in seeds lack the sensitivity and specificity necessary to accurately detect the lower concentrations of these compounds contained in the leaves of many plants. In order to measure inositol hexakisphosphate (InsP6) and inositol pentakisphosphate (InsP5) levels in leaves of different plants, a method was developed to concentrate and pre-purify these compounds prior to analysis. Inositol phosphates were extracted from leaves with diluted HCl and concentrated on small anion exchange columns. Reversed-phase solid phase extraction cartridges were used to remove compounds that give peaks that sometimes interfere during HPLC. The method permitted the determination of InsP6 and InsP5 concentrations in leaves as low as 10 µM and 2 µM, respectively. Most plants analyzed contained a high ratio of InsP6 to InsP5. In contrast, certain members of the Malvaceae family, such as cotton (Gossypium) and some hibiscus (Hibiscus) species, had a preponderance of InsP5. Radiolabeling of cotton seedlings also showed increased amounts of InsP5 relative to InsP6. Why some Malvaceae species exhibit a reversal of the typical ratios of these inositol phosphates is an intriguing question for future research.

Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants.

Inositol pyrophosphates are unique cellular signaling molecules with recently discovered roles in energy sensing and metabolism. Studies in eukaryotes have revealed that these compounds have a rapid turnover, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of investigation in plants even though seeds produce large amounts of their precursor, myo-inositol hexakisphosphate (InsP6 ). Here, we report that Arabidopsis and maize InsP6 transporter mutants have elevated levels of inositol pyrophosphates in their seed, providing unequivocal identification of their presence in plant tissues. We also show that plant seeds store a little over 1% of their inositol phosphate pool as InsP7 and InsP8 . Many tissues, including, seed, seedlings, roots and leaves accumulate InsP7 and InsP8 , thus synthesis is not confined to tissues with high InsP6 . We have identified two highly similar Arabidopsis genes, AtVip1 and AtVip2, which are orthologous to the yeast and mammalian VIP kinases. Both AtVip1 and AtVip2 encode proteins capable of restoring InsP7 synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP6 kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting non-redundant or non-overlapping functions in plants. These results contribute to our knowledge of inositol phosphate metabolism and will lay a foundation for understanding the role of InsP7 and InsP8 in plants.

Regulation of Sucrose non-Fermenting Related Kinase 1 genes in Arabidopsis thaliana.

The Sucrose non-Fermenting Related Kinase 1 (SnRK1) proteins have been linked to regulation of energy and stress signaling in eukaryotes. In plants, there is a small SnRK1 gene family. While the SnRK1.1 gene has been well studied, the role other SnRK1 isoforms play in energy or stress signaling is less well understood. We used promoter:GUS analysis and found SnRK1.1 is broadly expressed, while SnRK1.2 is spatially restricted. SnRK1.2 is expressed most abundantly in hydathodes, at the base of leaf primordia, and in vascular tissues within both shoots and roots. We examined the impact that sugars have on SnRK1 gene expression and found that trehalose induces SnRK1.2 expression. Given that the SnRK1.1 and SnRK1.2 proteins are very similar at the amino acid level, we sought to address whether SnRK1.2 is capable of re-programming growth and development as has been seen previously with SnRK1.1 overexpression. While gain-of-function transgenic plants overexpressing two different isoforms of SnRK1.1 flower late as seen previously in other SnRK1.1 overexpressors, SnRK1.2 overexpressors flower early. In addition, SnRK1.2 overexpressors have increased leaf size and rosette diameter during early development, which is the opposite of SnRK1.1 overexpressors. We also investigated whether SnRK1.2 was localized to similar subcellular compartments as SnRK1.1, and found that both accumulate in the nucleus and cytoplasm in transient expression assays. In addition, we found SnRK1.1 accumulates in small puncta that appear after a mechanical wounding stress. Together, these data suggest key differences in regulation of the SnRK1.1 and SnRK1.2 genes in plants, and highlights differences overexpression of each gene has on the development of Arabidopsis.

Assaying inositol and phosphoinositide phosphatase enzymes.

One critical aspect of phosphoinositide signaling is the turnover of signaling molecules in the pathway. These signaling molecules include the phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates (InsPs). The enzymes that catalyze the breakdown of these molecules are thus important potential regulators of signaling, and in many cases the activity of such enzymes needs to be measured and compared to other enzymes. PtdInsPs and InsPs are broken down by sequential dephosphorylation reactions which are catalyzed by a set of specific phosphatases. Many of the phosphatases can act on both PtdInsP and InsP substrates. The protocols described in this chapter detail activity assays that allow for the measurement of PtdInsP and InsP phosphatase activities in vitro starting with native or recombinant enzymes. Three different assays are described that have different equipment requirements and allow one to test a range of PtdInsP and InsP phosphatases that act on different substrates.

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli.

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.

The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppression of cell death.

l-myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the rate-limiting step in the synthesis of myo-inositol, a critical compound in the cell. Plants contain multiple MIPS genes, which encode highly similar enzymes. We characterized the expression patterns of the three MIPS genes in Arabidopsis thaliana and found that MIPS1 is expressed in most cell types and developmental stages, while MIPS2 and MIPS3 are mainly restricted to vascular or related tissues. MIPS1, but not MIPS2 or MIPS3, is required for seed development, for physiological responses to salt and abscisic acid, and to suppress cell death. Specifically, a loss in MIPS1 resulted in smaller plants with curly leaves and spontaneous production of lesions. The mips1 mutants have lower myo-inositol, ascorbic acid, and phosphatidylinositol levels, while basal levels of inositol (1,4,5)P(3) are not altered in mips1 mutants. Furthermore, mips1 mutants exhibited elevated levels of ceramides, sphingolipid precursors associated with cell death, and were complemented by a MIPS1-green fluorescent protein (GFP) fusion construct. MIPS1-, MIPS2-, and MIPS3-GFP each localized to the cytoplasm. Thus, MIPS1 has a significant impact on myo-inositol levels that is critical for maintaining levels of ascorbic acid, phosphatidylinositol, and ceramides that regulate growth, development, and cell death.

VTC4 is a bifunctional enzyme that affects myoinositol and ascorbate biosynthesis in plants.

Myoinositol synthesis and catabolism are crucial in many multiceullar eukaryotes for the production of phosphatidylinositol signaling molecules, glycerophosphoinositide membrane anchors, cell wall pectic noncellulosic polysaccharides, and several other molecules including ascorbate. Myoinositol monophosphatase (IMP) is a major enzyme required for the synthesis of myoinositol and the breakdown of myoinositol (1,4,5)trisphosphate, a potent second messenger involved in many biological activities. It has been shown that the VTC4 enzyme from kiwifruit (Actinidia deliciosa) has similarity to IMP and can hydrolyze l-galactose 1-phosphate (l-Gal 1-P), suggesting that this enzyme may be bifunctional and linked with two potential pathways of plant ascorbate synthesis. We describe here the kinetic comparison of the Arabidopsis (Arabidopsis thaliana) recombinant VTC4 with d-myoinositol 3-phosphate (d-Ins 3-P) and l-Gal 1-P. Purified VTC4 has only a small difference in the V(max)/K(m) for l-Gal 1-P as compared with d-Ins 3-P and can utilize other related substrates. Inhibition by either Ca(2+) or Li(+), known to disrupt cell signaling, was the same with both l-Gal 1-P and d-Ins 3-P. To determine whether the VTC4 gene impacts myoinositol synthesis in Arabidopsis, we isolated T-DNA knockout lines of VTC4 that exhibit small perturbations in abscisic acid, salt, and cold responses. Analysis of metabolite levels in vtc4 mutants showed that less myoinositol and ascorbate accumulate in these mutants. Therefore, VTC4 is a bifunctional enzyme that impacts both myoinositol and ascorbate synthesis pathways.

Biochemical and Genetic Characterization of PspE and GlpE, Two Single-domain Sulfurtransferases of Escherichia coli.

The pspE and glpE genes of Escherichia coli encode periplasmic and cytoplasmic single-domain rhodaneses, respectively, that catalyzes sulfur transfer from thiosulfate to thiophilic acceptors. Strains deficient in either or both genes were constructed. Comparison of rhodanese activity in these strains revealed that PspE provides 85% of total rhodanese activity, with GlpE contributing most of the remainder. PspE activity was four times higher during growth on glycerol versus glucose, and was not induced by conditions that induce expression of the psp regulon. The glpE/pspE mutants displayed no apparent growth phenotypes, indicating that neither gene is required for biosynthesis of essential sulfur-containing molecules. PspE was purified by using cation exchange chromatography. Two distinct active peaks were eluted and differed in the degree of stable covalent modification, as assessed by mass spectrometry. The peak eluting earliest contained the equivalent mass of two additional sulfur atoms, whereas the second peak contained mainly one additional sulfur. Kinetic properties of purified PspE were consistent with catalysis occurring via a double-displacement mechanism via an enzyme-sulfur intermediate involving the active site cysteine. K(m)s for SSO(3) (2-) and CN(-) were 2.7 mM and 32 mM, respectively, and k(cat) was 64(s-1). The enzyme also catalyzed transfer of sulfur from thiosulfate to dithiothreitol, ultimately releasing sulfide.