Slocum RD, Wang H, Tomasevich A, Clark G, Roux SJ. Role of apyrase in salvaging of phosphate from extracellular nucleotides and in regulating phosphate uptake in Arabidopsis. . Preprint of article submitted.Abstract

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.

McWhite C, Papoulas O, Drew K, Cox R, June V, Dong R, Kwon T, Wan C, Salmi M, Roux SJ, et al. A pan-protein compex map reveals deep conservation and novel assemblies. Cell [Internet]. 181 :460-474. Publisher's VersionAbstract
Plants are foundational for global ecological and economic systems, but most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scientific and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. By using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified previously unknown interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical for agriculture. We also observed plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers a cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.
Veerappa R, Slocum RD, Siegenthaler A, Wang J, Roux SJ. Ectopic expression of a pea apyrase enhances root system architecture and drought survival in Arabidopsis and soybean. Plant Cell and Environment [Internet]. 42 :337-353. Publisher's VersionAbstract
Ectoapyrases (ecto-NTPDases) function to decrease levels of extracellular ATP and ADP in animals and plants. Prior studies showed that ectopic expression of a pea ectoapyrase, psNTP9, enhanced growth in Arabidopsis seedlings and that the overexpression of the two Arabidopsis apyrases most closely related to psNTP9 enhanced auxin transport and growth in Arabidopsis. These results predicted that ectopic expression of psNTP9 could promote a more extensive root system architecture (RSA) in Arabidopsis. We confirmed that transgenic Arabidopsis seedlings had longer primary roots, more lateral roots, and more and longer root hairs than wild-type plants. Because RSA influences water uptake, we tested whether the transgenic plants could tolerate osmotic stress and water deprivation better than wild-type plants, and we confirmed these properties. Transcriptomic analyses revealed gene expression changes in the transgenic plants that helped account for their enhanced RSA and improved drought tolerance. The effects of psNTP9 were not restricted to Arabidopsis, because its expression in soybeans improved the RSA, growth, and seed yield of this crop and supported higher survival in response to drought. Our results indicate that in both Arabidopsis and soybeans, the constitutive expression of psNTP9 results in a more extensive RSA and improved survival in drought stress conditions.
Clark G, Roux SJ. Role of Ca2+ in mediating plant responses to extracellular ATP and ADP. International Journal of Molecular Sciences [Internet]. 19. Publisher's VersionAbstract
Among the most recently discovered chemical regulators of plant growth and development are extracellular nucleotides, especially extracellular ATP (eATP) and extracellular ADP (eADP). Plant cells release ATP into their extracellular matrix under a variety of different circumstances, and this eATP can then function as an agonist that binds to a specific receptor and induces signaling changes, the earliest of which is an increase in the concentration of cytosolic calcium ([Ca2+]cyt). This initial change is then amplified into downstream-signaling changes that include increased levels of reactive oxygen species and nitric oxide, which ultimately lead to major changes in the growth rate, defense responses, and leaf stomatal apertures of plants. This review presents and discusses the evidence that links receptor activation to increased [Ca2+]cyt and, ultimately, to growth and diverse adaptive changes in plant development. It also discusses the evidence that increased [Ca2+]cyt also enhances the activity of apyrase (nucleoside triphosphate diphosphohydrolase) enzymes that function in multiple subcellular locales to hydrolyze ATP and ADP, and thus limit or terminate the effects of these potent regulators.
Wang J, Song J, Clark G, Roux SJ. ANN1 and ANN2 function in post-phloem sugar transport in root tips to affect primary root growth. Plant Physiology. 178 :390-401.Abstract

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.

Cannon AE, Salmi ML, Cantero A, Roux SJ. Generation of transgenic spores of the fern Ceratopteris richardii to analyze Ca2+ dynamics during gravity-directed polarization. In: Current Advances in Fern Research. Springer International Publishing AG ; pp. 285-303. Publisher's VersionAbstract

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.

Park J, Salmi ML, Wan Salim WWA, Rademacher A, Wickizer B, Schooley A, Benton J, Cantero A, Argote PF, Ren M, et al. An autonomous lab on a chip for space flight claibration of gravity-induced transcellular calcium polarization in signle-cell fern spores. Lab on a Chip. 17 :1095-1103.Abstract

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

Tripathy M, Weeraratne G, Clark G, Roux SJ. Apyrase inhibitors enhance the ability of diverse fungicides to inhibit the growth of different plant pathogenic fungi. Molecular Plant Pathology [Internet]. :1-12. Publisher's VersionAbstract


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.


Clark G, Russell J, Enveart P, Gracia B, Wessel A, Jarmoskaite I, Polioudakis D, Stuart Y, Gonzalez T, MacKrell A, et al. Science Educational Outreach Programs That Benefit Students and Scientists. PLoS Biology [Internet]. 14 (2). Publisher's VersionAbstract

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.

Yang X, Wang B, Farris B, Clark G, Roux SJ. Modulation of Root Skewing in Arabidopsis by Apyrases and Extracellular ATP. Plant & Cell Physiology. 56 :2197-2206.Abstract

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.

Wang X, Ma X, Wang H, Li B, Clark G, Guo Y, Roux S, Sun D, Tang W. Proteomic study of microsomal proteins reveals a key role for Arabidopsis Annexin 1 in mediating heat stress-induced increase in intracellular calcium levels. . Molecular & Cellular Proteomics. 14 :686-694.Abstract

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.

Chiu T-Y, Lao J, Manalansan B, Loqué D, Roux SJ, Heazlewood JL. Biochemical characterization of Arabidopsis APYRASE family reveals their roles in regulating endomembrane NDP/NMP homeostasis. Biochemical J. 472 :43-54.Abstract


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.


Lee H-S, Kim Y, Pham G, Kim JW, Song J-H, Lee Y, Hwang Y-S, Roux SJ, Kim S-H. Brassinazole resistant 1 (BZR1)-dependent brassinosteroid signalling pathway leads to ectopic activation of quiescent cell division and suppresses columella stem cell differentiation. J Exp Bot. 66 (15) :4835-49.Abstract
Previous publications have shown that BRI1 EMS suppressor 1 (BES1), a positive regulator of the brassinosteroid (BR) signalling pathway, enhances cell divisions in the quiescent centre (QC) and stimulates columella stem cell differentiation. Here, it is demonstrated that BZR1, a BES1 homologue, also promotes cell divisions in the QC, but it suppresses columella stem cell differentiation, opposite to the action of BES1. In addition, BR and its BZR1-mediated signalling pathway are shown to alter the expression/subcellular distribution of pin-formed (PINs), which may result in changes in auxin movement. BR promotes intense nuclear accumulation of BZR1 in the root tip area, and the binding of BZR1 to the promoters of several root development-regulating genes, modulating their expression in the root stem cell niche area. These BZR1-mediated signalling cascades may account for both the ectopic activation of QC cell divisions as well as the suppression of the columella stem cell differentiation. They could also inhibit auxin-dependent distal stem cell differentiation by antagonizing the auxin/WOX5-dependent pathway. In conclusion, BZR1-/BES1-mediated BR signalling pathways show differential effects on the maintenance of root apical meristem activities: they stimulate ectopic QC division while they show opposite effects on the differentiation of distal columella stem cells in a BR concentration- and BZR1-/BES1-dependent manner.
Vanegas DC, Clark G, Cannon AE, Roux S, Chaturvedi P, McLamore ES. A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems. Biosens Bioelectron. 74 :37-44.Abstract
The objective of this study was to develop a self-referencing electrochemical biosensor for the direct measurement of ATP flux into the extracellular matrix by living cells/organisms. The working mechanism of the developed biosensor is based on the activity of glycerol kinase and glycerol-3-phosphate oxidase. A stratified bi-enzyme nanocomposite was created using a protein-templated silica sol gel encapsulation technique on top of graphene-modified platinum electrodes. The biosensor exhibited excellent electrochemical performance with a sensitivity of 2.4±1.8nA/µM, a response time of 20±13s and a lower detection limit of 1.3±0.7nM. The self-referencing biosensor was used to measure exogenous ATP efflux by (i) germinating Ceratopteris spores and (ii) growing Zea mays L. roots. This manuscript demonstrates the first development of a non-invasive ATP micro-biosensor for the direct measurement of eATP transport in living tissues. Before this work, assays of eATP have not been able to record the temporally transient movement of ATP at physiological levels (nM and sub-nM). The method demonstrated here accurately measured [eATP] flux in the immediate vicinity of plant cells. Although these proof of concept experiments focus on plant tissues, the technique developed herein is applicable to any living tissue, where nanomolar concentrations of ATP play a critical role in signaling and development. This tool will be invaluable for conducting hypothesis-driven life science research aimed at understanding the role of ATP in the extracellular environment.
Cannon AE, Salmi ML, Bushart TJ, Roux SJ. Studying molecular changes during gravity perception and response in a single cell. Methods Mol Biol. 1309 :199-207.Abstract
Early studies revealed a highly predictable pattern of gravity-directed growth and development in Ceratopteris richardii spores. This makes the spores a valuable model system for the study of how a single cell senses and responds to the force of gravity. Gravity regulates both the direction and magnitude of a trans-cell calcium current in germinating spores, and the orientation of this current predicts the polarization of spore development. Molecular techniques have been developed to evaluate the transcriptomic and proteomic profiles of spores before and after gravity establishes the polarity of their development. Here we describe these techniques, along with protocols for sterilizing the spores, sowing them in a solid or liquid growth media, and evaluating germination.
Sessa EB, Banks JA, Barker MS, Der JP, Duffy AM, Graham SW, Hasebe M, Langdale J, Li F-W, Marchant BD, et al. Between two fern genomes. Gigascience. 3 :15.Abstract
Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves.
Bushart TJ, Cannon A, Clark G, Roux SJ. Structure and function of CrACA1, the major PM-type Ca2+-ATPase, expressed at the peak of the gravity-directed trans-cell calcium current in spores of the fern Ceratopteris richardii. Plant Biol (Stuttg). 16 Suppl 1 :151-7.Abstract
Spores of the fern Ceratopteris richardii have proven to be a valuable single-cell system for studying gravity responses. The earliest cellular change directed by gravity in these cells is a trans-cell calcium current, which peaks near 10 h after the spores are induced to germinate. This current is needed for gravity-directed axis alignment, and its peak is coincident with the time period when gravity polarises the direction of subsequent nuclear migration and rhizoid growth. Transcriptomic analysis of genes expressed at the 10-h time point revealed several that encode proteins likely to be key components that either drive the current or regulate it. Notable among these is a plasma membrane (PM)-type Ca(2+) ATPase, CrACA1, whose activity pumping Ca(2+) out of cells is regulated by gravity. This report provides an initial characterisation of the structure and expression of this protein, and demonstrates its heterologous function complementing the K616 mutant of yeast, which is deficient in PM-type Ca(2+) pump activity. Gravity-induced changes in the trans-cell Ca(2+) current occur within seconds, a result consistent with the hypothesis that the force of gravity can rapidly alter the post-translational state of the channels and pumps that drive this current across spore cells. This report identifies a transporter likely to be a key driver of the current, CrACA1, and characterises the role of this protein in early germination and gravity-driven polarity fixation through analysis of expression levels, functional complementation and pharmacological treatments. These data, along with newly available transcriptomic data obtained at the 10-h time point, indicate that CrACA1 is present, functional and likely a major contributing component of the trans-cell Ca(2+) efflux. CrACA1 is not necessary for polar axis alignment, but pharmacological perturbations of it disrupt rhizoid development. These data support and help refine the post-translational modification model for gravity responses.
Clark GB, Morgan RO, Fernandez M-P, Salmi ML, Roux SJ. Breakthroughs spotlighting roles for extracellular nucleotides and apyrases in stress responses and growth and development. Plant Sci. 225 :107-16.Abstract
Animal and plant cells release nucleotides into their extracellular matrix when touched, wounded, and when their plasma membranes are stretched during delivery of secretory vesicles and growth. These released nucleotides then function as signaling agents that induce rapid increases in the concentration of cytosolic calcium, nitric oxide and superoxide. These, in turn, are transduced into downstream physiological changes. These changes in plants include changes in the growth of diverse tissues, in gravitropism, and in the opening and closing of stomates. The concentration of extracellular nucleotides is controlled by various phosphatases, prominent among which are apyrases EC (nucleoside triphosphate diphosphohydrolases, NTPDases). This review provides phylogenetic and pHMM analyses of plant apyrases as well as analysis of predicted post-translational modifications for Arabidopsis apyrases. This review also summarizes and discusses recent advances in research on the roles of apyrases and extracellular nucleotides in controlling plant growth and development. These include new findings that document how apyrases and extracellular nucleotides control auxin transport, modulate stomatal aperture, and mediate biotic and abiotic stress responses, and on how apyrase suppression leads to growth inhibition.
Lim MH, Wu J, Yao J, Gallardo IF, Dugger JW, Webb LJ, Huang J, Salmi ML, Song J, Clark G, et al. Apyrase suppression raises extracellular ATP levels and induces gene expression and cell wall changes characteristic of stress responses. Plant Physiol. 164 (4) :2054-67.Abstract
Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP (eATP) can modulate the rate of cell growth in diverse tissues. Two closely related apyrases (APYs) in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, function, in part, to control the concentration of eATP. The expression of APY1/APY2 can be inhibited by RNA interference, and this suppression leads to an increase in the concentration of eATP in the extracellular medium and severely reduces growth. To clarify how the suppression of APY1 and APY2 is linked to growth inhibition, the gene expression changes that occur in seedlings when apyrase expression is suppressed were assayed by microarray and quantitative real-time-PCR analyses. The most significant gene expression changes induced by APY suppression were in genes involved in biotic stress responses, which include those genes regulating wall composition and extensibility. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and two of these changes, wall lignification and decreased methyl ester bonds, were verified by direct analyses. Taken together, the results are consistent with the hypothesis that APY1, APY2, and eATP play important roles in the signaling steps that link biotic stresses to plant defense responses and growth changes.
Roux SJ. A start point for extracellular nucleotide signaling. Mol Plant. 7 (6) :937-8.