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.

Inositol Pyrophosphate Pathways and Mechanisms: What Can We Learn from Plants?

The ability of an organism to maintain homeostasis in changing conditions is crucial for growth and survival. Eukaryotes have developed complex signaling pathways to adapt to a readily changing environment, including the inositol phosphate (InsP) signaling pathway. In plants and humans the pyrophosphorylated inositol molecules, inositol pyrophosphates (PP-InsPs), have been implicated in phosphate and energy sensing. PP-InsPs are synthesized from the phosphorylation of InsP6, the most abundant InsP. The plant PP-InsP synthesis pathway is similar but distinct from that of the human, which may reflect differences in how molecules such as Ins(1,4,5)P3 and InsP6 function in plants vs. animals. In addition, PP-InsPs can potentially interact with several major signaling proteins in plants, suggesting PP-InsPs play unique signaling roles via binding to protein partners. In this review, we will compare the biosynthesis and role of PP-InsPs in animals and plants, focusing on three central themes: InsP6 synthesis pathways, synthesis and regulation of the PP-InsPs, and function of a specific protein domain called the Syg1, Pho1, Xpr1 (SPX ) domain in binding PP-InsPs and regulating inorganic phosphate (Pi) sensing. This review will provide novel insights into the biosynthetic pathway and bioactivity of these key signaling molecules in plant and human systems.

Arabidopsis Phospholipase C3 is Involved in Lateral Root Initiation and ABA Responses in Seed Germination and Stomatal Closure.

Phospholipase C (PLC) is well known for its role in animal signaling, where it generates the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), by hydrolyzing the minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), upon receptor stimulation. In plants, PLC's role is still unclear, especially because the primary targets of both second messengers are lacking, i.e. the ligand-gated Ca2+ channel and protein kinase C, and because PIP2 levels are extremely low. Nonetheless, the Arabidopsis genome encodes nine PLCs. We used a reversed-genetic approach to explore PLC's function in Arabidopsis, and report here that PLC3 is required for proper root development, seed germination and stomatal opening. Two independent knock-down mutants, plc3-2 and plc3-3, were found to exhibit reduced lateral root densities by 10-20%. Mutant seeds germinated more slowly but were less sensitive to ABA to prevent germination. Guard cells of plc3 were also compromised in ABA-dependent stomatal closure. Promoter-ß-glucuronidase (GUS) analyses confirmed PLC3 expression in guard cells and germinating seeds, and revealed that the majority is expressed in vascular tissue, most probably phloem companion cells, in roots, leaves and flowers. In vivo 32Pi labeling revealed that ABA stimulated the formation of PIP2 in germinating seeds and guard cell-enriched leaf peels, which was significantly reduced in plc3 mutants. Overexpression of PLC3 had no effect on root system architecture or seed germination, but increased the plant's tolerance to drought. Our results provide genetic evidence for PLC's involvement in plant development and ABA signaling, and confirm earlier observations that overexpression increases drought tolerance. Potential molecular mechanisms for the above observations are discussed.

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.

Biochemical evaluation of the decarboxylation and decarboxylation-deamination activities of plant aromatic amino acid decarboxylases.

Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.

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.

The role of phosphoinositides and inositol phosphates in plant cell signaling.

Work over the recent years has greatly expanded our understanding of the specific molecules involved in plant phosphoinositide signaling. Physiological approaches, combined with analytical techniques and genetic mutants have provided tools to understand how individual genes function in this pathway. Several key differences between plants and animals have become apparent. This chapter will highlight the key areas where major differences between plants and animals occur. In particular, phospholipase C and levels of phosphatidylinositol phosphates differ between plants and animals, and may influence how inositol second messengers form and function in plants. Whether inositol 1,4,5-trisphosphate and/or inositol hexakisphosphate (InsP6) function as second messengers in plants is discussed. Recent data on potential, novel roles of InsP6 in plants is considered, along with the existence of a unique InsP6 synthesis pathway. Lastly, the complexity of myo-inositol synthesis in plants is discussed in reference to synthesis of phosphoinositides and impact on plant growth and development.

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine.

Inositol polyphosphate 5-phosphatase7 regulates the production of reactive oxygen species and salt tolerance in Arabidopsis.

Plants possess remarkable ability to adapt to adverse environmental conditions. The adaptation process involves the removal of many molecules from organelles, especially membranes, and replacing them with new ones. The process is mediated by an intracellular vesicle-trafficking system regulated by phosphatidylinositol (PtdIns) kinases and phosphatases. Although PtdIns comprise a fraction of membrane lipids, they function as major regulators of stress signaling. We analyzed the role of PtdIns 5-phosphatases (5PTases) in plant salt tolerance. The Arabidopsis (Arabidopsis thaliana) genome contains 15 At5PTases. We analyzed salt sensitivity in nine At5ptase mutants and identified one (At5ptase7) that showed increased sensitivity, which was improved by overexpression. At5ptase7 mutants demonstrated reduced production of reactive oxygen species (ROS). Supplementation of mutants with exogenous PtdIns dephosphorylated at the D5' position restored ROS production, while PtdIns(4,5)P(2), PtdIns(3,5)P(2), or PtdIns(3,4,5)P(3) were ineffective. Compromised salt tolerance was also observed in mutant NADPH Oxidase, in agreement with the low ROS production and salt sensitivity of PtdIns 3-kinase mutants and with the inhibition of NADPH oxidase activity in wild-type plants. Localization of green fluorescent protein-labeled At5PTase7 occurred in the plasma membrane and nucleus, places that coincided with ROS production. Analysis of salt-responsive gene expression showed that mutants failed to induce the RD29A and RD22 genes, which contain several ROS-dependent elements in their promoters. Inhibition of ROS production by diphenylene iodonium suppressed gene induction. In summary, our results show a nonredundant function of At5PTase7 in salt stress response by regulating ROS production and gene expression.

The cellular language of myo-inositol signaling.

The simple polyol, myo-inositol, is used as a building block of a cellular language that plays various roles in signal transduction. This review describes the terminology used to denote myo-inositol-containing molecules, with an emphasis on how phosphate and fatty acids are added to create second messengers used in signaling. Work in model systems has delineated the genes and enzymes required for synthesis and metabolism of many myo-inositol-containing molecules, with genetic mutants and measurement of second messengers playing key roles in developing our understanding. There is increasing evidence that molecules such as myo- inositol(1,4,5)trisphosphate and phosphatidylinositol(4,5)bisphosphate are synthesized in response to various signals plants encounter. In particular, the controversial role of myo-inositol(1,4,5)trisphosphate is addressed, accompanied by a discussion of the multiple enzymes that act to regulate this molecule. We are also beginning to understand new connections of myo-inositol signaling in plants. These recent discoveries include the novel roles of inositol phosphates in binding to plant hormone receptors and that of phosphatidylinositol(3)phosphate binding to pathogen effectors.

Ten best practices for taking experiential learning online.

Like many institutions around the world, the COVID-19 pandemic prompted us to shift our summer 2020 in-person undergraduate experiential learning program to a remote, virtual format. Here, we present our observations, summarized in 10 best practices, for moving a STEM-focused research experience for undergraduates, experiential learning program or research-based course online. We will also discuss how our program was originally designed and implemented, and how we adapted our activities to deliver an at-home research experience that maintained student engagement, mentorship, and a shared sense of community.

A Role for Inositol Pyrophosphates in the Metabolic Adaptations to Low Phosphate in Arabidopsis.

Phosphate is a major plant macronutrient and low phosphate availability severely limits global crop productivity. In Arabidopsis, a key regulator of the transcriptional response to low phosphate, phosphate starvation response 1 (PHR1), is modulated by a class of signaling molecules called inositol pyrophosphates (PP-InsPs). Two closely related diphosphoinositol pentakisphosphate enzymes (AtVIP1 and AtVIP2) are responsible for the synthesis and turnover of InsP8, the most implicated molecule. This study is focused on characterizing Arabidopsis vip1/vip2 double mutants and their response to low phosphate. We present evidence that both local and systemic responses to phosphate limitation are dampened in the vip1/vip2 mutants as compared to wild-type plants. Specifically, we demonstrate that under Pi-limiting conditions, the vip1/vip2 mutants have shorter root hairs and lateral roots, less accumulation of anthocyanin and less accumulation of sulfolipids and galactolipids. However, phosphate starvation response (PSR) gene expression is unaffected. Interestingly, many of these phenotypes are opposite to those exhibited by other mutants with defects in the PP-InsP synthesis pathway. Our results provide insight on the nexus between inositol phosphates and pyrophosphates involved in complex regulatory mechanisms underpinning phosphate homeostasis in plants.

Can Inositol Pyrophosphates Inform Strategies for Developing Low Phytate Crops?

Inositol pyrophosphates (PP-InsPs) are an emerging class of "high-energy" intracellular signaling molecules, containing one or two diphosphate groups attached to an inositol ring, that are connected with phosphate sensing, jasmonate signaling, and inositol hexakisphosphate (InsP6) storage in plants. While information regarding this new class of signaling molecules in plants is scarce, the enzymes responsible for their synthesis have recently been elucidated. This review focuses on InsP6 synthesis and its conversion into PP-InsPs, containing seven and eight phosphate groups (InsP7 and InsP8). These steps involve two types of enzymes: the ITPKs that phosphorylate InsP6 to InsP7, and the PPIP5Ks that phosphorylate InsP7 to InsP8. This review also considers the potential roles of PP-InsPs in plant hormone and inorganic phosphate (Pi) signaling, along with an emerging role in bioenergetic homeostasis. PP-InsP synthesis and signaling are important for plant breeders to consider when developing strategies that reduce InsP6 in plants, as this will likely also reduce PP-InsPs. Thus, this review is primarily intended to bridge the gap between the basic science aspects of PP-InsP synthesis/signaling and breeding/engineering strategies to fortify foods by reducing InsP6.

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.

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.

Genomic organization and regulation of the LeIMP-1 and LeIMP-2 genes encoding myo-inositol monophosphatase in tomato.

Myo-inositol (inositol) monophosphatase (IMP), an enzyme which catalyzes the synthesis of free inositol from various inositol monophosphates, is encoded by a small multigene family in many organisms. The tomato IMP gene family encodes three IMP isoforms with identical in vitro biochemical properties. To determine the role of each tomato LeIMP gene in plant growth, we isolated the genomic DNA copies of the LeIMP-1 and LeIMP-2 genes. The LeIMP-1 gene spans approximately 5.8 kb and consists of 12 exons, whereas the LeIMP-2 gene consists of an uninterrupted, single open reading frame (ORF). We have previously shown that steady-state levels of LeIMP-2 mRNA were very low in comparison to LeIMP-1 and LeIMP-3 mRNA levels. To determine whether LeIMP-2 gene expression was spatially restricted to a discreet domain within the plant we constructed transgenic plants containing an LeIMP-2 promoter::uidA gene fusion. Analysis of transgenic seedlings revealed that the LeIMP-2 promoter directed gene expression within epidermal and cortex cells of specific stem/leaf junctions in an abaxial-specific pattern and in the shoot apical meristem. Further, inositol, the product of IMP catalysis, and Li+, an inhibitor of IMP catalysis, decreased expression of the LeIMP-2 promoter as measured by a decrease in beta-glucuronidase activity after treatment.

Characterization of the inositol monophosphatase gene family in Arabidopsis.

Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca(2+) signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.

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.