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.

Populus trichocarpa clade A PP2C protein phosphatases: their stress-induced expression patterns, interactions in core abscisic acid signaling, and potential for regulation of growth and development.

KEY MESSAGE: Overexpression of the poplar PP2C protein phosphatase gene PtrHAB2 resulted in increased tree height and altered leaf morphology and phyllotaxy, implicating PP2C phosphatases as growth regulators functioning under favorable conditions. We identified and studied Populus trichocarpa genes, PtrHAB1 through PtrHAB15, belonging to the clade A PP2C family of protein phosphatases known to regulate abscisic acid (ABA) signaling. PtrHAB1 through PtrHAB3 and PtrHAB12 through PtrHAB15 were the most highly expressed genes under non-stress conditions. The poplar PP2C genes were differentially regulated by drought treatments. Expression of PtrHAB1 through PtrHAB3 was unchanged or downregulated in response to drought, while all other PtrHAB genes were weakly to strongly upregulated in response to drought stress treatments. Yeast two-hybrid assays involving seven ABA receptor proteins (PtrRCAR) against 12 PtrHAB proteins detected 51 interactions involving eight PP2Cs and all PtrRCAR proteins with 22 interactions requiring the addition of ABA. PtrHAB2, PtrHAB12, PtrHAB13 and PtrHAB14 also interacted with the sucrose non-fermenting related kinase 2 proteins PtrSnRK2.10 and PtrSnRK2.11, supporting conservation of a SnRK2 signaling cascade regulated by PP2C in poplar. Additionally, PtrHAB2, PtrHAB12, PtrHAB13 and PtrHAB14 interacted with the mitogen-activated protein kinase protein PtrMPK7. Due to its interactions with PtrSnRK2 and PtrMPK7 proteins, and its reduced expression during drought stress, PtrHAB2 was overexpressed in poplar to test its potential as a growth regulator under non-stress conditions. 35S::PtrHAB2 transgenics exhibited increased growth rate for a majority of transgenic events and alterations in leaf phyllotaxy and morphology. These results indicate that PP2Cs have additional roles which extend beyond canonical ABA signaling, possibly coordinating plant growth and development in response to environmental conditions.

Biosynthesis and possible functions of inositol pyrophosphates in plants.

Inositol phosphates (InsPs) are intricately tied to lipid signaling, as at least one portion of the inositol phosphate signaling pool is derived from hydrolysis of the lipid precursor, phosphatidyl inositol (4,5) bisphosphate. The focus of this review is on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e., PPx) attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6)-hexakisphosphate (InsP6), which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals (referred to as phytic acid). Even though plants produce huge amounts of precursor InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. Recent work has delineated that Arabidopsis has two genes capable of PP-InsP5 synthesis, and PPx-InsPs have been detected across the plant kingdom. This review will detail the known roles of PPx-InsPs in yeast and animal systems, and provide a description of recent data on the synthesis and accumulation of these novel molecules in plants, and potential roles in signaling.

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.