extraction-quantification-soluble-radiolabeled-inositol

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

University of Bonn

SEC14 lipid transfer proteins are important regulators of phospholipid metabolism. Structural, genetic and cell biological studies in yeast suggest that they help phosphatidylinositol (PtdIns)/phosphoinositide (PIP) kinases to overcome their intrinsic inefficiency to recognize membrane-embedded substrate, thereby playing a key role in PIP homeostasis. Genomes of higher plants encode a high number and diversity of SEC14 proteins, often in combination with other domains. The Arabidopsis SEC14-Nlj16 protein AtSFH1, an important regulator of root hair development, plays an important role in the establishment of PIP microdomains. Key to this mechanism is a highly specific interaction of the Nlj16 domain with PtdIns(4,5)P and an interaction-triggered oligomerization of the protein. Nlj16/PtdIns(4,5)P interaction depends on a polybasic motif similar to those identified in other regulatory proteins.

The regulation of cell polarity by lipid transfer proteins of the SEC14 family.

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen's benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP to InsP 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana.

A 1-phytase type III effector interferes with plant hormone signaling.

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-β-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.

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

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains that are receptors for inositol pyrophosphates (PP-InsP), suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that deletion of two diphosphoinositol pentakisphosphate kinases VIH1/2 impairs plant growth and leads to constitutive Pi starvation responses. Deletion of phosphate starvation response transcription factors partially rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases in plant Pi signal transduction cascades. VIH1/2 are bifunctional enzymes able to generate and break-down PP-InsPs. Mutations in the kinase active site lead to increased Pi levels and constitutive Pi starvation responses. ATP levels change significantly in different Pi growth conditions. ATP-Mg2+ concentrations shift the relative kinase and phosphatase activities of diphosphoinositol pentakisphosphate kinases in vitro. Pi inhibits the phosphatase activity of the enzyme. Thus, VIH1 and VIH2 relay changes in cellular ATP and Pi concentrations to changes in PP-InsP levels, allowing plants to maintain sufficient Pi levels.

Article	Year
The regulation of cell polarity by lipid transfer proteins of the SEC14 family. Current opinion in plant biology\| PubMed ID: 29017091	2017
A 1-phytase type III effector interferes with plant hormone signaling. Nature communications\| PubMed ID: 29255246	2017
Arabidopsis Phospholipase C3 is Involved in Lateral Root Initiation and ABA Responses in Seed Germination and Stomatal Closure. Plant & cell physiology\| PubMed ID: 29309666	2018
Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis. eLife\| PubMed ID: 31436531	2019

Gabriel Schaaf

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