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Michael R Blatt

Publications and source records attributed to Michael R Blatt.

12 recordsLinked to original sources
PubMed2006

Membrane trafficking and polar growth in root hairs and pollen tubes.

Root hairs and pollen tubes extend by rapid elongation that occurs exclusively at the tip. Fundamental for such local, tip-focused growth (so-called 'tip growth') is the polarization of the cytoplasm that directs secretory events to the tip, and the presence of internal gradients and transmembrane flux of ions, notably Ca2+, H+, K+, and Cl-. Electrophysiological and imaging studies using fluorescent markers have sought to link ion gradients with growth and membrane trafficking. Current models recognize membrane trafficking as fundamental to tip growth, notably its role in supplying lipid and protein to the new plasma membrane and cell wall that extend the apex of the cell, and a complementary role for endocytosis in retrieving excess membrane and in recycling various protein fractions. The current state of knowledge is reviewed here in order to highlight the major gaps in the present understanding of trafficking as it contributes to polar growth in these cells and recent results, that suggest a role for membrane trafficking in the active regulation of ion channel turnover and activity during polar tip growth, are discussed.

Animals↗
PubMed2006

Selective mobility and sensitivity to SNAREs is exhibited by the Arabidopsis KAT1 K+ channel at the plasma membrane.

Recent findings indicate that proteins in the SNARE superfamily are essential for cell signaling, in addition to facilitating vesicle traffic in plant cell homeostasis, growth, and development. We previously identified SNAREs SYP121/Syr1 from tobacco (Nicotiana tabacum) and the Arabidopsis thaliana homolog SYP121 associated with abscisic acid and drought stress. Disrupting tobacco SYP121 function by expressing a dominant-negative Sp2 fragment had severe effects on growth, development, and traffic to the plasma membrane, and it blocked K(+) and Cl(-) channel responses to abscisic acid in guard cells. These observations raise questions about SNARE control in exocytosis and endocytosis of ion channel proteins and their organization within the plane of the membrane. We have used a dual, in vivo tagging strategy with a photoactivatable green fluorescent protein and externally exposed hemagglutinin epitopes to monitor the distribution and trafficking dynamics of the KAT1 K(+) channel transiently expressed in tobacco leaves. KAT1 is localized to the plasma membrane within positionally stable microdomains of approximately 0.5 microm in diameter; delivery of the K(+) channel, but not of the PMA2 H(+)-ATPase, to the plasma membrane is suppressed by Sp2 fragments of tobacco and Arabidopsis SYP121, and Sp2 expression leads to profound changes in KAT1 distribution and mobility within the plane of the plasma membrane. These results offer direct evidence for SNARE-mediated traffic of the K(+) channel and a role in its distribution within subdomains of the plasma membrane, and they implicate a role for SNAREs in positional anchoring of the K(+) channel protein.

Abscisic Acid↗
PubMed2006

Interactive domains between pore loops of the yeast K+ channel TOK1 associate with extracellular K+ sensitivity.

Gating of the outward-rectifying K+ channel TOK1 of Saccharomyces cerevisiae is controlled by membrane voltage and extracellular K+ concentration. Previous studies identified two kinetically distinct effects of K+, and site-mutagenic analysis associated these K+-dependencies with domains of the extracellular turrets of the channel protein. We have mapped the TOK1 pore domains to extant K+ channel crystal structures to target additional residues contributing to TOK1 gating. Leu270, located in the first pore domain of TOK1, was found to be critical for gating and its K+ sensitivity. Analysis of amino acid substitutions indicated that spatial position of the polypeptide backbone is a primary factor determining gating sensitivity to K+. The strongest effects, with L270Y, L270F and L270W, led to more than a 30-fold decrease in apparent K+ affinity and an inversion in the apparent K+-dependence of voltage-dependent gating compared with the wild-type current. A partial rescue of wild-type gating was obtained on substitution in the second pore domain with the double mutant L270D/A428Y. These, and additional results, demarcate extracellular domains that are associated with the K+-sensitivity of TOK1 and they offer primary evidence for a synergy in gating between the two pore domains of TOK1, demonstrating an unexpected degree of long-distance interaction across the mouth of the K+ channel.

Amino Acid Sequence↗
PubMed2006

External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism.

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants.

Amino Acid Sequence↗
PubMed2006

Setting SNAREs in a different wood.

Vesicle traffic is essential for cell homeostasis, growth and development in plants, as it is in other eukaryotes, and is facilitated by a superfamily of proteins known as soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs). Although SNAREs are well-conserved across phylla, genomic analysis for two model angiosperm species available to date, rice and Arabidopsis, highlights common patterns of divergence from other eukaryotes. These patterns are associated with the expansion of some gene subfamilies of SNAREs, the absence of others and the appearance of new proteins that show no significant homologies to SNAREs of mammals, yeast or Drosophila. Recent findings indicate that the functions of these plant SNAREs also extend beyond the conventional 'housekeeping' activities associated with vesicle trafficking. A number of SNAREs have been implicated in environmental responses as diverse as stomata movements and gravisensing as well as sensitivity to salt and drought. These proteins are essential for signal transduction and response and, in most cases, appear also to maintain additional roles in membrane trafficking. One common theme to this added functionality lies in control of non-SNARE proteins, notably ion channels. Other examples include interactions between the SNAREs and scaffolding or other structural components within the plant cell.

Abscisic Acid↗
PubMed2005

Protein phosphorylation is a prerequisite for intracellular Ca2+ release and ion channel control by nitric oxide and abscisic acid in guard cells.

Recent work has indicated that nitric oxide (NO) and its synthesis are important elements of signal cascades in plant-pathogen defence, and are a prerequisite for drought and abscisic acid (ABA) responses in Arabidopsis thaliana and Vicia faba guard cells. NO regulates inward-rectifying K+ channels and Cl- channels of Vicia guard cells via intracellular Ca2+ release. However, its integration with related signals, including the actions of serine-threonine protein kinases, is less well defined. We report here that the elevation of cytosolic-free [Ca2+] ([Ca2+]i) mediated by NO in guard cells is reversibly inhibited by the broad-range protein kinase antagonists staurosporine and K252A, but not by the tyrosine kinase antagonist genistein. The effects of kinase antagonism translate directly to a loss of NO-sensitivity of the inward-rectifying K+ channels and background (Cl- channel) current, and to a parallel loss in sensitivity of the K+ channels to ABA. These results demonstrate that NO-dependent signals can be modulated through protein phosphorylation upstream of intracellular Ca2+ release, and they implicate a target for protein kinase control in ABA signalling that feeds into NO-dependent Ca2+ release.

Abscisic Acid↗
PubMed2004

Nitric oxide block of outward-rectifying K+ channels indicates direct control by protein nitrosylation in guard cells.

Recent work has indicated that nitric oxide (NO) and its synthesis are important elements of signal cascades in plant pathogen defense and are a prerequisite for drought and abscisic acid responses in Arabidopsis (Arabidopsis thaliana) and Vicia faba guard cells. Nonetheless, its mechanism(s) of action has not been well defined. NO regulates inward-rectifying K+ channels of Vicia guard cells through its action on Ca2+ release from intercellular Ca2+ stores, but alternative pathways are indicated for its action on the outward-rectifying K+ channels (I(K,out)), which are Ca2+ insensitive. We report here that NO affects I(K,out) when NO is elevated above approximately 10 to 20 nm. NO action on I(K,out) was consistent with oxidative stress and was suppressed by several reducing agents, the most effective being British anti-Lewisite (2,3-dimercapto-1-propanol). The effect of NO on the K+ channel was mimicked by phenylarsine oxide, an oxidizing agent that cross-links vicinal thiols. Neither intracellular pH buffering nor the phosphotyrosine kinase antagonist genistein affected NO action on I(K,out), indicating that changes in cytosolic pH and tyrosine phosphorylation are unlikely to contribute to NO or phenylarsine oxide action in this instance. Instead, our results strongly suggest that NO directly modifies the K+ channel or a closely associated regulatory protein, probably by nitrosylation of cysteine sulfhydryl groups.

Arsenicals↗
PubMed2004

A new catch in the SNARE.

Vesicle traffic underpins cell homeostasis, growth and development in plants. Traffic is facilitated by a superfamily of proteins known as SNAREs ( soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) that interact to draw vesicle and target membrane surfaces together for fusion of the bilayers. Several recent findings now indicate that plant SNAREs might not be limited to the conventional 'housekeeping' activities commonly attributed to vesicle trafficking. In the past five years, six different SNAREs have been implicated in stomatal movements, gravisensing and pathogen resistance. These proteins almost certainly do contribute to specific membrane fusion events but they are also essential for signal transduction and response. Some SNAREs can modulate the activity of non-SNARE proteins, notably ion channels. Other examples might reflect SNARE interactions with different scaffolding and structural components of the cell.

Animals↗
PubMed2003

Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways.

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca2+-sensitive ion channels of Vicia guard cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free [Ca2+]. NO-sensitive Ca2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca2+ channels and Ca2+-insensitive K+ channels, and NO scavengers failed to block the activation of these K+ channels evoked by ABA. These results place NO action firmly within one branch of the Ca2+-signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.

Abscisic Acid↗
PubMed2002

Protein phosphorylation activates the guard cell Ca2+ channel and is a prerequisite for gating by abscisic acid.

Protein phosphorylation and cytosolic-free [Ca2+] ([Ca2+]i) contribute to signalling cascades evoked by the water-stress hormone abscisic acid (ABA) that lead to stomatal closure in higher-plant leaves. ABA activates an inward-rectifying Ca2+ channel at the plasma membrane of stomatal guard cells, promoting Ca2+ entry by shifting the voltage-sensitivity of the channels. Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de-)phosphorylation in plasma membrane patches from Vicia guard cells. Ca2+ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K1/2, 1.3 +/- 0.7 mm) but not the non-hydrolyzable analog ATPgammaS. ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A-type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca2+ channel activity by increasing the open probability and number of active channels. Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage-sensitivities of the Ca2+ current and [Ca2+]i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca2+]i rise evoked by hyperpolarization and delayed its recovery. These results demonstrate a membrane-delimited interaction between 1/2 A-type protein phosphatase(s) and the Ca2+ channel or associated proteins, and they are consistent with a role for protein (de-)phosphorylation in ABA signalling mediated directly through Ca2+ channel gating that leads to [Ca2+]i increases in the guard cells.

Abscisic Acid↗
PubMed2002

The abscisic acid-related SNARE homolog NtSyr1 contributes to secretion and growth: evidence from competition with its cytosolic domain.

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion, and secretion. Previously, we isolated the syntaxin-related protein NtSyr1 (NtSyp121) from tobacco in a screen for abscisic acid-related signaling elements, demonstrating its role in determining the abscisic acid sensitivity of K(+) and Cl(-) channels in stomatal guard cells. NtSyr1 is localized to the plasma membrane and is expressed normally throughout the plant, especially in root tissues, suggesting that it might contribute to cellular homeostasis as well as to signaling. To explore its functions in vivo further, we examined stably transformed lines of tobacco that expressed various constructs of NtSyr1, including the full-length protein and a truncated fragment, Sp2, corresponding to the cytosolic domain shown previously to be active in suppressing ion channel response to abscisic acid. Constitutively overexpressing NtSyr1 yielded uniformly high levels of protein (>10 times the wild-type levels) and was associated with a significant enhancement of root growth in seedlings but not with any obvious phenotype in mature, well-watered plants. Similar transformations with constructs encoding the Sp2 fragment of NtSyr1 showed altered leaf morphology but gave only low levels of Sp2 fragment, suggesting a strong selective pressure against plants expressing this protein. High expression of the Sp2 fragment was achieved in stable transformants under the control of a dexamethasone-inducible promoter. Sp2 expression was correlated positively with altered cellular and tissue morphology in leaves and roots and with a cessation of growth in seedlings. Overexpression of the full-length NtSyr1 protein rescued the wild-type phenotype, even in plants expressing high levels of the Sp2 fragment, supporting the idea that the Sp2 fragment interfered specifically with NtSyr1 function by competing with NtSyr1 for its binding partners. To explore NtSyr1 function in secretion, we used a green fluorescent protein (GFP)-based section assay. When a secreted GFP marker was coexpressed with Sp2 in tobacco leaves, GFP fluorescence was retained in cytosolic reticulate and punctate structures. In contrast, in plants coexpressing secreted GFP and NtSyr1 or secreted GFP alone, no GFP fluorescence accumulated within the cells. A new yellow fluorescent protein-based secretion marker was used to show that the punctate structures labeled in the presence of Sp2 colocalized with a Golgi marker. These structures were not labeled in the presence of a dominant Rab1 mutant that inhibited transport from the endoplasmic reticulum to the Golgi. We propose that NtSyr1 functions as an element in SNARE-mediated vesicle trafficking to the plasma membrane and is required for cellular growth and homeostasis.

Abscisic Acid↗