Apyrases are NTPDases that remove the terminal phosphate from NTPs and NDPs, but not from NMPs. They have conserved structures and functions in yeast, plants and animals. Among the most studied APYs in plants are those in Arabidopsis (AtAPYs) and peas (PsAPYs), both of which have been shown to play major roles in regulating plant growth and development. Valuable insights on their functional roles have been gained by transgenically altering their transcript abundance, either by constitutively expressing them or by their suppression. This review focuses on studies of transgenic lines of yeast and multiple different plants that revealed insights on the growth-altering functions of plant apyrases in different organisms. APY expression can also be inhibited post-translationally by chemically blocking its enzymatic activity, so this review also briefly covers studies that used inhibitors to suppress APY activity in plants and fungi.

Annexins are a multigene family of calcium-dependent membrane-binding proteins that play important roles in plant cell signaling. Annexins are multifunctional proteins, and their function in plants is not comprehensively understood. Arabidopsis (Arabidopsis thaliana) annexins ANN1 and ANN2 are 64% identical in their primary structure, and both are highly expressed in

seedlings. Here, we showed that ann-mutant seedlings grown in the absence of sugar show decreased primary root growth and altered columella cells in root caps; however, these mutant defects are rescued by Suc, Glc, or Fru. In seedlings grown without sugar, significant up-regulation of photosynthetic gene expression and chlorophyll accumulation was found in ann-mutant cotyledons compared to that in wild type, which indicates potential sugar starvation in the roots of ann-mutant seedlings. Unexpectedly, the overall sugar content of ann-mutant primary roots was significantly higher than that of wild-type roots when grown without sugar. To examine the diffusion of sugar along the entire root to the root tip, we examined the unloading pattern of carboxyfluorescein dye and found that post-phloem sugar transport was impaired in ann-mutant root tips compared to that in wild type. Increased levels of ROS and callose were detected in the root tips of ann-mutant seedlings grown without Suc, the latter of which would restrict plasmodesmal sugar transport to root tips. Our results indicate that ANN1 and ANN2 play an important role in post-phloem sugar transport to the root tip, which in turn indirectly influences photosynthetic rates in cotyledons. This study expands our understanding of the function of annexins in plants.

Spores from the fern, Ceratopteris richardii, have been used to study gravity-directed cell polarization for over three decades. This system is ideal for these studies because it has a highly-predictable growth and developmental pattern and primarily responds to the mechanical force of gravity during polarization. Early studies on the development in this system showed that during the first 24 h of germination, Ceratopteris spores establish a Ca²⁺ concentration differential along the outer periphery of the cell that is defined by the uptake of Ca²⁺ through channels at the bottom of the spore and an efflux of Ca²⁺ through pumps at the top. This 100-fold [Ca²⁺] differential is sensitive to the direction and magnitude of the gravitational force. In a low-gravity environment or when the uptake of Ca²⁺ is blocked, spore polarization becomes random. These results support the hypothesis that the uptake of Ca²⁺ is necessary for gravity-directed polarization in Ceratopteris spores. For many years, studies of Ceratopteris were limited by the inability to produce stable transformants. However, recent protocols have led to the first stably transformed lines in Ceratopteris richardii. This work reviews and discusses the latest studies of gravity-induced changes in Ca²⁺ transport dynamics in Ceratopteris spores, the use of transformation protocols to overexpress or knock-out genes relevant to the transport of Ca²⁺ in Ceratopteris, and the potential value of mutants to more thoroughly understand the role of Ca²⁺ transport dynamics in gravity-directed polarization of spores.

This report describes the development of lab-on-a-chip device designed to measure changes in cellular ion gradients that are induced by changes in gravitational (g) forces. The bioCD presented here detects differential calcium ion concentrations outside of individual cells. The device includes sufficient replicates for statistical analysis of the gradients around multiple single cells and around control wells that are empty or include dead cells. In the data presented, the degree of the cellular response correlates with the magnitude of the g-force applied via rotation of the bioCD. The experiments recorded the longest continuous observation of a cellular response to hypergravity made to date, and they demonstrate the potential utility of this device for assaying the threshold of cells' g-force responses in spaceflight conditions

A previous study has demonstrated that the treatment of Arabi-

dopsis plants with chemical inhibitors of apyrase enzymes

increases their sensitivity to herbicides. In this study, we found

that the addition of the same or related apyrase inhibitors could

potentiate the ability of different fungicides to inhibit the growth

of five different pathogenic fungi in plate growth assays. The

growth of all five fungi was partially inhibited by three commonly

used fungicides: copper octanoate, myclobutanil and propicona-

zole. However, when these fungicides were individually tested in

combination with any one of four different apyrase inhibitors

(AI.1, AI.10, AI.13 or AI.15), their potency to inhibit the growth

of five fungal pathogens was increased significantly relative to

their application alone. The apyrase inhibitors were most effec-

tive in potentiating the ability of copper octanoate to inhibit fun-

gal growth, and least effective in combination with

propiconazole. Among the five pathogens assayed, that most

sensitive to the fungicide-potentiating effects of the inhibitors

was Sclerotinia sclerotiorum. Overall, among the 60 treatment

combinations tested (five pathogens, four apyrase inhibitors,

three fungicides), the addition of apyrase inhibitors increased sig-

nificantly the sensitivity of fungi to the fungicide treatments in

53 of the combinations. Consistent with their predicted mode of

action, inhibitors AI.1, AI.10 and AI.13 each increased the level

of propiconazole retained in one of the fungi, suggesting that

they could partially block the ability of efflux transporters to

remove propiconazole from these fungi.

Both scientists and the public would benefit from improved communication of basic scientific research and from integrating scientists into education outreach, but opportunities to support these efforts are limited. We have developed two low-cost programs—"Present Your PhD Thesis to a 12-Year-Old" and "Shadow a Scientist”—that combine training in science communication with outreach to area middle schools. We assessed the outcomes of these programs and found a 2-fold benefit: scientists improve their communication skills by explaining basic science research to a general audience, and students' enthusiasm for science and their scientific knowledge are increased. Here we present details about both programs, along with our assessment of them, and discuss the feasibility of exporting these programs to other universities.

When plant primary roots grow along a tilted surface that is impenetrable, they can undergo a slanted deviation from the direction of gravity called skewing. Skewing is induced by touch stimuli which the roots experience as they grow along the surface. Touch stimuli also induce the release of extracellular ATP (eATP) into the plant's extracellular matrix, and two apyrases (NTPDases) in Arabidopsis, APY1 and APY2, can help regulate the concentration of eATP. The primary roots of seedlings overexpressing APY1 show less skewing than wild-type plants. Plants suppressed in their expression of APY1 show more skewing than wild-type plants. Correspondingly, chemical inhibition of apyrase activity increased skewing in mutants and wild-type roots. Exogenous application of ATP or ATPγS also increased skewing in wild-type roots, which could be blocked by co-incubation with a purinergic receptor antagonist. These results suggest a model in which gradients of eATP set up by differential touch stimuli along roots help direct skewing in roots growing along an impenetrable surface.

To understand the early signaling steps in the response of plant cells to increased environmental temperature, 2-D difference gel electrophoresis was used to study the proteins in microsomes of Arabidopsis seedlings that are regulated early during heat stress. Using mass spectrometry, 19 microsomal proteins that showed an altered expression level within 5 min after heat treatment were identified. Among these proteins, annexin 1 (AtANN1) was one of those up-regulated rapidly after heat-shock treatment. Functional studies show loss-of-function mutants for AtANN1 and its close homolog AtANN2 were more sensitive to heat-shock treatment, whereas plants overexpressing AtANN1 showed more resistance to this treatment. Correspondingly, the heat-induced expression of heat-shock proteins and heat-shock factors is inhibited in ann1/ann2 double mutant, and the heat-activated increase in cytoplasmic calcium concentration ([Ca(2+)]cyt) is greatly impaired in the ann1 mutant and almost undetectable in ann1/ann2 double mutant. Taken together these results suggest that AtANN1 is important in regulating the heat-induced increase in [Ca(2+)]cyt and in the response of Arabidopsis seedlings to heat stress.

Plant apyrases are nucleoside triphosphate diphosphohydolases and have been implicated in an array of functions within the plant including the regulation of extracellular ATP. Arabidopsis encodes a family of seven membrane bound apyrases (AtAPY1 to 7) comprised of three distinct clades all of which contain the five conserved apyrase domains. Subcellular localization of all seven Arabidopsis apyrases indicated that they all localize to internal membranes comprising the cis-Golgi, ER and endosome, indicating an endo-apyrase classification for the entire family. The complementation of a yeast double mutant (Δynd1Δgda1) which lacks apyrase activity indicated that all members could function as endo-apyrases except AtAPY7 which was unable to complement the yeast mutant. Complementation of the mutant yeast using characterized human apyrases demonstrated that functional complementation of the mutant could only be accomplished using a functional endo-apyrase (NTPDase6), with no complementation occurring when using a characterized human ecto-apyrase (NTPDase1). An analysis of substrate specificity for the Arabidopsis apyrases AtAPY1 to 6 indicated that each member has a distinct set of preferred substrates covering various NDPs and NTPs. The Arabidopsis apyrase AtAPY3 was the only member to display a strong preference for NTPs, with other members mainly exhibiting NDP substrate preferences.