SUMMARYInositol 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 (InsP 6 ). Here, we report that Arabidopsis and maize InsP 6 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 InsP 7 and InsP 8 . Many tissues, including, seed, seedlings, roots and leaves accumulate InsP 7 and InsP 8 , thus synthesis is not confined to tissues with high InsP 6 . 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 InsP 7 synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP 6 kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting nonredundant 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 InsP 7 and InsP 8 in plants.

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, with suggested roles in bioenergetic homeostasis and inorganic phosphate (Pi) sensing. Information regarding the biosynthesis of these unique class of signaling molecules in plants is scarce, however the enzymes responsible for their biosynthesis in other eukaryotes have been well described. Here we report the characterization of the two Arabidopsis VIP kinase domains, a newly discovered activity of the Arabidopsis ITPK1 and ITPK2 enzymes, and the subcellular localization of the enzymes involved in the synthesis of InsP6 and PP-InsPs. Our data indicate that AtVIP1-KD and AtVIP2-KD act primarily as 1PP-specific Diphosphoinositol Pentakisphosphate Kinases (PPIP5) Kinases. The AtITPK enzymes, in contrast, can function as InsP6 kinases, and thus are the missing enzyme in the plant PP-InsP synthesis pathway.Together, these enzyme classes can function in plants to produce PP-InsPs, which have been implicated in signal transduction and Pi sensing pathways. We measured a higher InsP7 level (increased InsP7/InsP8 ratio) in vip1/vip2 double loss-of-function mutants, and an accumulation of InsP8 (decreased InsP7/InsP8 ratio) in the 35S:VIP2 overexpression line relative to wild-type plants. We also report that enzymes involved in the synthesis of InsPs and PP-InsPs accumulate within the nucleus and cytoplasm of plant cells. Our work defines a molecular basis for understanding how plants synthesize PP-InsPs which is crucial for determining the roles of these signaling molecules in processes such as Pi sensing. 3 SIGNIFICANCE STATEMENTInositol pyrophosphate signaling molecules are of agronomic importance as they can control complex responses to the limited nutrient, phosphate. This work fills in the missing steps in the inositol pyrophosphate synthesis pathway and points to a role for these molecules in the plant cell nucleus. This is an important advance that can help us design future strategies to increase phosphate efficiency in plants.

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.

Although many reports characterize the transcriptional response of Arabidopsis seedlings to microgravity, few investigate the effect of partial or fractional gravity on gene expression. Understanding plant responses to fractional gravity is relevant for plant growth on lunar and Martian surfaces. The plant signaling flight experiment utilized the European Modular Cultivation System (EMCS) onboard the International Space Station (ISS). The EMCS consisted of two rotors within a controlled chamber allowing for two experimental conditions, microgravity (stationary rotor) and simulated gravity in space. Seedlings were grown for 5 days under continuous light in seed cassettes. The arrangement of the seed cassettes within each experimental container results in a gradient of fractional g (in the spinning rotor). To investigate whether gene expression patterns are sensitive to fractional g, we carried out transcriptional profiling of root samples exposed to microgravity or partial g (ranging from 0.53 to 0.88 g). Data were analyzed using DESeq2 with fractional g as a continuous variable in the design model in order to query gene expression across the gravity continuum. We identified a subset of genes whose expression correlates with changes in fractional g. Interestingly, the most responsive genes include those encoding transcription factors, defense, and cell wall-related proteins and heat shock proteins.

Gravitropism is a universal plant response. It is initiated by the sensing of the primary signal (mass or pressure), which is then converted into chemical signals that are transduced and propagated in a precise spatial and temporal fashion, resulting in a differential growth response. Our thesis is that membrane lipids and lipid-mediated signaling pathways play critical roles in the initial signaling and in the establishment of polarity. In this review, we highlight results from recent literature and discuss the major questions that remain unanswered.