Biswa R. Acharya

Hormone interactions in stomatal function

Research in recent years on the biology of guard cells has shown that these specialized cells integrate both extra- and intra-cellular signals in the control of stomatal apertures. Among the phytohormones, abscisic acid (ABA) is one of the key players regulating stomatal function. In addition, auxin, cytokinin, ethylene, brassinosteroids, jasmonates, and salicylic acid also contribute to stomatal aperture regulation. The interaction of multiple hormones can serve to determine the size of stomatal apertures in a condition-specific manner. Here, we discuss the roles of different phytohormones and the effects of their interactions on guard cell physiology and function.

A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein–protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r2 = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Border control--a membrane-linked interactome of Arabidopsis.

Degrees of Separation Proteins embedded in membranes represent an interesting point of communication between the cell and its environment, but their localization to membranes can make them difficult to study. Jones et al. (p. 711) found an approach to catalog thousands of interactions involving membrane proteins and membrane-associated signaling machinery—including many previously uncharacterized proteins. With a focus on the model plant Arabidopsis, several of the identified interactions fill gaps in important signal transduction chains, while others point to functions for enigmatic unknown proteins. Amembrane and signaling protein interaction network for gene discovery and hypothesis generation is identified in Arabidopsis. Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 106 pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split–green fluorescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis guard cells.

Open Stomata 1 (OST1) (SnRK2.6 or SRK2E), a serine/threonine protein kinase, is a positive regulator in abscisic acid (ABA)-mediated stomatal response, but OST1-regulation of K(+) and Ca(2+) currents has not been studied directly in guard cells and it is unknown whether OST1 activity is limiting in ABA-mediated stomatal responses. We employed loss-of-function and gain-of-function approaches to study native ABA responses of Arabidopsis guard cells. We performed stomatal aperture bioassays, patch clamp analyses and reactive oxygen species (ROS) measurements. ABA inhibition of inward K(+) channels and light-induced stomatal opening are reduced in ost1 mutants while transgenic plants overexpressing OST1 show ABA hypersensitivity in these responses. ost1 mutants are insensitive to ABA-induced stomatal closure, regulation of slow anion currents, Ca(2+) -permeable channel activation and ROS production while OST1 overexpressing lines are hypersensitive for these responses, resulting in accelerated stomatal closure in response to ABA. Overexpression of OST1 in planta in the absence of ABA application does not affect basal apertures or ion currents. Moreover, we demonstrate the physical interaction of OST1 with the inward K(+) channel KAT1, the anion channel SLAC1, and the NADPH oxidases AtrbohD and AtrbohF. Our findings support OST1 as a critical limiting component in ABA regulation of stomatal apertures, ion channels and NADPH oxidases in Arabidopsis guard cells.

The guard cell metabolome: functions in stomatal movement and global food security.

Guard cells represent a unique single cell-type system for the study of cellular responses to abiotic and biotic perturbations that affect stomatal movement. Decades of effort through both classical physiological and functional genomics approaches have generated an enormous amount of information on the roles of individual metabolites in stomatal guard cell function and physiology. Recent application of metabolomics methods has produced a substantial amount of new information on metabolome control of stomatal movement. In conjunction with other “omics” approaches, the knowledge-base is growing to reach a systems-level description of this single cell-type. Here we summarize current knowledge of the guard cell metabolome and highlight critical metabolites that bear significant impact on future engineering and breeding efforts to generate plants/crops that are resistant to environmental challenges and produce high yield and quality products for food and energy security.

Arabidopsis thaliana CML25 mediates the Ca2+ regulation of K+ transmembrane trafficking during pollen germination and tube elongation

The concentration alteration of cytosolic-free calcium ([Ca(2+) ]cyt ) is a well-known secondary messenger in plants and plays important roles during pollen grain germination and tube elongation. Here we demonstrate that CML25, a member of calmodulin-like proteins, has Ca(2+) -binding activity and plays a role in pollen grain germination, tube elongation and seed setting. CML25 transcript was abundant in mature pollen grains and pollen tubes, and its product CML25 protein was primarily directed to the cytoplasm. Two independent CML25 loss-of-function T-DNA insertion mutants suffered a major reduction in both the rate of pollen germination and the elongation of the pollen tube. Also, pollen grains of cml25 mutants were less sensitive to the external K(+) and Ca(2+) concentration than wild-type pollen. The disruption of CML25 increased the [Ca(2+) ]cyt in both the pollen grain and the pollen tube, which in turn impaired the Ca(2+) -dependent inhibition of whole-cell inward K(+) currents in protoplasts prepared from these materials (pollen grain and pollen tube). Complementation of cml25-1 mutant resulted in the recovery of wild-type phenotype. Our findings indicate that CML25 is an important transducer in the Ca(2+) -mediated regulation of K(+) influx during pollen germination and tube elongation.

Transcriptional and metabolic signatures of Arabidopsis responses to chewing damage by an insect herbivore and bacterial infection and the consequences of their interaction

Plants use multiple interacting signaling systems to identify and respond to biotic stresses. Although it is often assumed that there is specificity in signaling responses to specific pests, this is rarely examined outside of the gene-for-gene relationships of plant-pathogen interactions. In this study, we first compared early events in gene expression and later events in metabolite profiles of Arabidopsis thaliana following attack by either the caterpillar Spodoptera exigua or avirulent (DC3000 avrRpm1) Pseudomonas syringae pv. tomato at three time points. Transcriptional responses of the plant to caterpillar feeding were rapid, occurring within 1 h of feeding, and then decreased at 6 and 24 h. In contrast, plant response to the pathogen was undetectable at 1 h but grew larger and more significant at 6 and 24 h. There was a surprisingly large amount of overlap in jasmonate and salicylate signaling in responses to the insect and pathogen, including levels of gene expression and individual hormones. The caterpillar and pathogen treatments induced different patterns of expression of glucosinolate biosynthesis genes and levels of glucosinolates. This suggests that when specific responses develop, their regulation is complex and best understood by characterizing expression of many genes and metabolites. We then examined the effect of feeding by the caterpillar Spodoptera exigua on Arabidopsis susceptibility to virulent (DC3000) and avirulent (DC3000 avrRpm1) P. syringae pv. tomato, and found that caterpillar feeding enhanced Arabidopsis resistance to the avirulent pathogen and lowered resistance to the virulent strain. We conclude that efforts to improve plant resistance to bacterial pathogens are likely to influence resistance to insects and vice versa. Studies explicitly comparing plant responses to multiple stresses, including the role of elicitors at early time points, are critical to understanding how plants organize responses in natural settings.

A new discrete dynamic model of ABA-induced stomatal closure predicts key feedback loops

Stomata, microscopic pores in leaf surfaces through which water loss and carbon dioxide uptake occur, are closed in response to drought by the phytohormone abscisic acid (ABA). This process is vital for drought tolerance and has been the topic of extensive experimental investigation in the last decades. Although a core signaling chain has been elucidated consisting of ABA binding to receptors, which alleviates negative regulation by protein phosphatases 2C (PP2Cs) of the protein kinase OPEN STOMATA 1 (OST1) and ultimately results in activation of anion channels, osmotic water loss, and stomatal closure, over 70 additional components have been identified, yet their relationships with each other and the core components are poorly elucidated. We integrated and processed hundreds of disparate observations regarding ABA signal transduction responses underlying stomatal closure into a network of 84 nodes and 156 edges and, as a result, established those relationships, including identification of a 36-node, strongly connected (feedback-rich) component as well as its in- and out-components. The network’s domination by a feedback-rich component may reflect a general feature of rapid signaling events. We developed a discrete dynamic model of this network and elucidated the effects of ABA plus knockout or constitutive activity of 79 nodes on both the outcome of the system (closure) and the status of all internal nodes. The model, with more than 1024 system states, is far from fully determined by the available data, yet model results agree with existing experiments in 82 cases and disagree in only 17 cases, a validation rate of 75%. Our results reveal nodes that could be engineered to impact stomatal closure in a controlled fashion and also provide over 140 novel predictions for which experimental data are currently lacking. Noting the paucity of wet-bench data regarding combinatorial effects of ABA and internal node activation, we experimentally confirmed several predictions of the model with regard to reactive oxygen species, cytosolic Ca2+ (Ca2+c), and heterotrimeric G-protein signaling. We analyzed dynamics-determining positive and negative feedback loops, thereby elucidating the attractor (dynamic behavior) repertoire of the system and the groups of nodes that determine each attractor. Based on this analysis, we predict the likely presence of a previously unrecognized feedback mechanism dependent on Ca2+c. This mechanism would provide model agreement with 10 additional experimental observations, for a validation rate of 85%. Our research underscores the importance of feedback regulation in generating robust and adaptable biological responses. The high validation rate of our model illustrates the advantages of discrete dynamic modeling for complex, nonlinear systems common in biology.

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab–GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1(Y94A) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H+-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.

Interaction between Calcium and Actin in Guard Cell and Pollen Signaling Networks

Calcium (Ca2+) plays important roles in plant growth, development, and signal transduction. It is a vital nutrient for plant physical design, such as cell wall and membrane, and also serves as a counter-cation for biochemical, inorganic, and organic anions, and more particularly, its concentration change in cytosol is a ubiquitous second messenger in plant physiological signaling in responses to developmental and environmental stimuli. Actin cytoskeleton is well known for its importance in cellular architecture maintenance and its significance in cytoplasmic streaming and cell division. In plant cell system, the actin dynamics is a process of polymerization and de-polymerization of globular actin and filamentous actin and that acts as an active regulator for calcium signaling by controlling calcium evoked physiological responses. The elucidation of the interaction between calcium and actin dynamics will be helpful for further investigation of plant cell signaling networks at molecular level. This review mainly focuses on the recent advances in understanding the interaction between the two aforementioned signaling components in two well-established model systems of plant, guard cell, and pollen.