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.

Increasing Phosphatidylinositol (4,5)-Bisphosphate Biosynthesis Affects Basal Signaling and Chloroplast Metabolism in Arabidopsis thaliana.

One challenge in studying the second messenger inositol(1,4,5)-trisphosphate (InsP3) is that it is present in very low amounts and increases only transiently in response to stimuli. To identify events downstream of InsP3, we generated transgenic plants constitutively expressing the high specific activity, human phosphatidylinositol 4-phosphate 5-kinase Iα (HsPIPKIα). PIP5K is the enzyme that synthesizes phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2); this reaction is flux limiting in InsP3 biosynthesis in plants. Plasma membranes from transgenic Arabidopsis expressing HsPIPKIα had 2-3 fold higher PIP5K specific activity, and basal InsP3 levels in seedlings and leaves were >2-fold higher than wild type. Although there was no significant difference in photosynthetic electron transport, HsPIPKIα plants had significantly higher starch (2-4 fold) and 20% higher anthocyanin compared to controls. Starch content was higher both during the day and at the end of dark period. In addition, transcripts of genes involved in starch metabolism such as SEX1 (glucan water dikinase) and SEX4 (phosphoglucan phosphatase), DBE (debranching enzyme), MEX1 (maltose transporter), APL3 (ADP-glucose pyrophosphorylase) and glucose-6-phosphate transporter (Glc6PT) were up-regulated in the HsPIPKIα plants. Our results reveal that increasing the phosphoinositide (PI) pathway affects chloroplast carbon metabolism and suggest that InsP3 is one component of an inter-organelle signaling network regulating chloroplast metabolism.

Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.

Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.

Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum.

A genetic approach was used to increase phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKIalpha in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P2 100-fold. In vivo studies revealed that the rate of 32Pi incorporation into whole-cell PtdIns(4,5)P2 increased >12-fold, and the ratio of [3H]PtdInsP2 to [3H]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [3H]inositol trisphosphate and [3H]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] binding assay showed that total cellular Ins(1,4,5)P3/g fresh weight was >40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKIalpha increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.

Ion chromatography of phytate in roots and tubers.

The ion chromatographic method for the quantification of phytate (InsP(6)) in foods was adapted for the analysis of roots and tubers. To maximize sensitivity, ultraviolet (UV) detection following postcolumn derivatization was compared with evaporative light-scattering detection (ELSD). Detection limits for phytate were 0.5 and 1 microg for UV and ELSD, respectively. Unidentified peaks eluting close to and after InsP(6) were removed by solid-phase extraction. Phytate was detected in 11 of 15 roots or tubers. The highest phytate levels were 0.169 and 0.133% of the fresh weight of taro (Colocasia esculenta) and yuca (Manihot esculenta), respectively. Potatoes (Solanum tuberosum) contained 0.035-0.073% phytate, whereas no phytate at a detection limit of 0.003% of fresh weight was observed in sweet potatoes (Ipomoea batatas).