Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants.

Inositol pyrophosphates are eukaryotic signaling molecules that have been recently identified as key regulators of plant phosphate sensing and homeostasis. Given the importance of phosphate to current and future agronomic practices, we sought to design plants, which could be used to sequester phosphate, as a step in a phytoremediation strategy. To achieve this, we expressed diadenosine and diphosphoinositol polyphosphate phosphohydrolase (DDP1), a yeast (Saccharomyces cerevisiae) enzyme demonstrated to hydrolyze inositol pyrophosphates, in Arabidopsis thaliana and pennycress (Thlaspi arvense), a spring annual cover crop with emerging importance as a biofuel crop.

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

The IPK1 genes, which code for 2-kinases that can synthesize Ins(1,2,4,5,6)P from Ins(1,4,5,6)P, are expressed throughout cotton plants, resulting in the highest Ins(1,2,4,5,6)P concentrations in young leaves and flower buds. Cotton leaves contain large amounts of Ins(1,2,4,5,6)P and InsP compared to plants not in the Malvaceae family. The inositol polyphosphate pathway has been linked to stress tolerance in numerous plant species.

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

Phosphate is a major plant macronutrient and low phosphate availability severely limits global crop productivity. In , 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 ( and ) are responsible for the synthesis and turnover of InsP, the most implicated molecule.

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.

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.

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 (InsP) 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 InsP synthesis and its conversion into PP-InsPs, containing seven and eight phosphate groups (InsP and InsP).

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.

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.

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.

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.

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 ).

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.

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.

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.

Inositol polyphosphate phosphatidylinositol 5-phosphatase9 (At5ptase9) controls plant salt tolerance by regulating endocytosis.

Phosphatidylinositol 5-phosphatases (5PTases) that hydrolyze the 5' position of the inositol ring are key components of membrane trafficking system. Recently, we reported that mutation in At5PTase7 gene reduced production of reactive oxygen species (ROS) and decreased expression of stress-responsive genes, resulting in increased salt sensitivity. Here, we describe an even more salt-sensitive 5ptase mutant, At5ptase9, which also hydrolyzes the 5' phosphate groups specifically from membrane-bound phosphatidylinositides.

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.

myo-Inositol Oxygenase is Required for Responses to Low Energy Conditions in Arabidopsis thaliana.

myo-Inositol is a precursor for cell wall components, is used as a backbone of myo-inositol trisphosphate (Ins(1,4,5)P(3)) and phosphatidylinositol phosphate signaling molecules, and is debated about whether it is also a precursor in an alternate ascorbic acid synthesis pathway. Plants control inositol homeostasis by regulation of key enzymes involved in myo-inositol synthesis and catabolism. Recent transcriptional profiling data indicate up-regulation of the myo-inositol oxygenase (MIOX) genes under conditions in which energy or nutrients are limited.

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.

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.

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.

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.

Switches in nutrient and inositol signaling.

Studies of signal transduction networks such as the inositol signaling pathway can provide important insights for our understanding of the regulation of various biological events, including growth and development, disease and stress responses. Recently, we have identified a -inositol polyphosphate 5-phosphatase (5PTase13, At1g05630) that hydrolyzes the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P] and also interacts with the sucrose nonfermenting-1-related kinase (SnRK1.1) in the yeast two hybrid system and in vitro.

Inositol phosphate signaling and gibberellic acid.

To respond to physical signals and endogenous hormones, plants use specific signal transduction pathways. We and others have previously shown that second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] is used in abscisic acid (ABA) signaling, and that some mutants with altered Ins(1,4,5)P(3) have altered responses to ABA. Specifically, mutants defective in the myo-inositol polyphosphate 5-phosphatases (5PTases) 1 and 2 genes that hydrolyze 5-phosphates from Ins(1,4,5)P(3) and other PtdInsP and InsP substrates, have elevated Ins (1,4,5)P(3), and are ABA-hypersensitive.

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.