Calcium signalling in plants

Virtual Issue Articles: Unravelling response-specificity in Ca2+ signalling pathways in plant cells Jason J. Rudd, Vernonica E. Franklin-Tong Shaping the calcium signature Martin R. McAinsh and Jon K. Pittman Synergism between calcium ions and abscisic acid in preventing stomatal opening D. L. R. De Silva, A. M. Hetherington, T. A. Mansfield Signal transduction during fertilization in algae and vascular plants Colin Brownlee Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis Mireille Chabaud, Andrea Genre, Björn J. Sieberer, Antonella Faccio, Joëlle Fournier, Mara Novero, David G. Barker, Paola Bonfante Sesbania rostrata: a case study of natural variation in legume nodulation Ward Capoen, Giles Oldroyd, Sofie Goormachtig, Marcelle Holsters Flavonoid-induced calcium signalling in Rhizobium leguminosarum bv. viciae Roberto Moscatiello, Andrea Squartini, Paola Mariani, Lorella Navazio Ca2+ conduction by plant cyclic nucleotide gated channels and associated signaling components in pathogen defense signal transduction cascades Wei Ma, Gerald A. Berkowitz

A plastid protein crucial for Ca2+-regulated stomatal responses Stefan Weinl, Katrin Held, Kathrin Schlücking, Leonie Steinhorst, Sebastian Kuhlgert, Michael Hippler, Jörg Kudla The chloroplast as a regulator of Ca2+ signalling Alex A. R. Webb Mitochondrial morphology transition is an early indicator of subsequent cell death in Arabidopsis Iain Scott, David C. Logan Calcium homeostasis in plant cell nuclei Christian Mazars, Stéphane Bourque, Axel Mithöfer, Alain Pugin, Raoul Ranjeva Calcium channels in photosynthetic eukaryotes: implications for evolution of calcium -based signalling Frédéric Verret, Glen Wheeler, Alison R. Taylor, Garry Farnham, Colin Brownlee A cyclic nucleotide-gated channel is necessary for optimum fertility in high-calcium environments Suraphon Chaiwongsar, Allison K. Strohm, Joshua R. Roe, Roxana Y. Godiwalla, Catherine W. M. Chan Identification of hyperpolarization-activated calcium calcium channels in apical pollen tubes of Pyrus pyrifolia Hai-Yong Qu, Zhong-Lin Shang, Shao-Ling Zhang, Lian-Mei Liu, Ju-You Wu Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel Yansheng Wu, Xiaodong Xu, Sujuan Li, Ting Liu, Ligeng Ma, Zhonglin Shang

Virtual Issue Articles: Depolarization-activated calcium channels shape the calcium signatures induced by low-temperature stress Philip J. White Two voltage-dependent calcium channels co-exist in the apical plasma membrane of Arabidopsis thaliana root hairs Henk Miedema, Vadim Demidchik, Anne-Aliénor Véry, John H. F. Bothwell, Colin Brownlee, Julia M. Davies Physiological roles of nonselective cation channels in plants: from salt stress to signalling and development Vadim Demidchik, Frans J. M. Maathuis Annexins Anuphon Laohavisit, Julia M. Davies The CBL–CIPK Ca2+-decoding signaling network: function and perspectives Stefan Weinl, Jörg Kudla Nuclear protein kinases: still enigmatic components in plant cell signalling Jennifer Dahan, David Wendehenne, Raoul Ranjeva, Alain Pugin, Stéphane Bourque Nitric oxide modulates the influx of extracellular Ca2+ and actin filament organization during cell wall construction in Pinus bungeana pollen tubes Yuhua Wang, Tong Chen, Chunyang Zhang, Huaiqing Hao, Peng Liu, Maozhong Zheng, Frantisek Baluska, Jozef Samaj, Jinxing Lin Arabidopsis VILLIN4 is involved in root hair growth through regulating actin organization in a Ca2+-dependent manner Yi Zhang, Yingyu Xiao, Fei Du, Lijuan Cao, Huaijian Dong, Haiyun Ren

Phosphatidic acid formation is required for extracellular ATPmediated nitric oxide production in suspension-cultured tomato cells Daniela J. Sueldo, Noelia P. Foresi, Claudia A. Casalongué, Lorenzo Lamattina, Ana M. Laxalt Ethylene activates a plasma membrane Ca2+-permeable channel in tobacco suspension cells Min-Gui Zhao, Qiu-Ying Tian, Wen-Hao Zhang Calcium–calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants Xiuli Hu, Mingyi Jiang, Jianhua Zhang, Aying Zhang, Fan Lin, Mingpu Tan

Cover image: Localization of Ca2+-sensing receptor::Green Fluorescent Proteins in epidermal cells of N.benthamiana leaves using laser- scanning microscopy.

From Weinl et al., 2008. New Phytologist 179: 675-686.

Introduction The complex roles played by Ca2+ in the regulation of a multitude of processes in eukaryotes and algae have become widely accepted and better understood over the past two decades. A number of seminal studies extending back half a century or more laid the foundation for these more detailed investigations though our understanding of plant Ca2+ signalling progressed at a slower pace compared with the rapid advances in animal Ca2+ signalling research in the 1980s and 1990s.

This has been due, at least in part, to the difficulties in measuring plant cytosolic Ca2+ concentrations. Notable early advances indicating roles for Ca2+ as a regulator of cellular function in plants came from studies of ionic currents associated with morphogenesis in fucoid algae, and phytochrome responses in giant algae such as Nitella. It was at least a decade later that the first measurements of cytosolic Ca2+ were made in plant and algal cells. Since then the field of Ca2+ signalling in plants has advanced rapidly on a number of fronts. The development of model systems for studying Ca2+ signalling, such as the stomatal guard cell and the application of cell physiology to model higher plant cells, along with advances in molecular genetic and genomics approaches are providing increasingly clearer pictures of both the similarities and substantial differences between the ways animals, algae and plants use Ca2+ to relay information within and between cells.

This Virtual Special Issue (VSI) presents a number of recent research articles and reviews that address some key features of signal transduction (stimulus perception, generation of and decoding information from Ca2+ signatures; and interactions with other signals and messengers) in plants and algae. The articles provide a snapshot of this rapidly advancing field and point to requirements for future research.

Tansley review Unravelling response-specificity in Ca2+ signalling pathways in plant cells

Author for correspondence: V. E. Franklin-Tong Fax: +44 121414 5925 Email: V.E.Franklin-Tong@bham.ac.uk

Jason J. Rudd, Vernonica E. Franklin-Tong

Summary New Phytologist (2001) 151: 7–33 doi: 10.1046/j.1469-8137.2001.00173.x

Keywords: Ca2+ signature; Ca2+ signalling in plants; signal-response coupling; response specificity

Considerable advances have been made, both in the technologies available to study changes in intracellular cytosolic free Ca2+ ([Ca2+]i), and in our understanding of Ca2+ signalling cascades in plant cells, but how specificity can be generated from such a ubiquitous component as Ca2+ is questionable. Recently the concept of ‘Ca2+ signatures’ has been formulated; tight control of the temporal and spatial characteristics of alterations in [Ca2+]i signals is thought to be responsible, at least in part, for the specificity of the response. However, the way in which Ca2+ signatures are decoded, which depends on the nature and location of the targets of the Ca2+ signals, has received little attention. In a few key systems, progress is being made on how diverse Ca2+ signatures might be transduced within cells in response to specific signals.

Valuable pieces of the signal-specificity puzzle are being put together and this is illustrated here using some key examples; these emphasize the global importance of Ca2+-mediated signal-transduction cascades in the responses of plants to a wide diversity of extracellular signals. However, the way in which signal specificity is encoded and transduced is still far from clear.

Tansley review Shaping the calcium signature

Author for correspondence: Tel: +44 161 275 5235 Fax: +44 161 275 5082 Email: jon.pittman@manchester.ac.uk

Martin R. McAinsh and Jon K. Pittman

Summary New Phytologist (2009) 181: 275–294 doi: 10.1111/j.1469-8137.2008.02682.x

Keywords: Ca2+-ATPase; Ca2+ channels; Ca2+ signature; H+/Ca2+ exchanger; modelling Ca2+ signals; signal transduction

In numerous plant signal transduction pathways, Ca2+ is a versatile second messenger which controls the activation of many downstream actions in response to various stimuli. There is strong evidence to indicate that information encoded within these stimulus-induced Ca2+ oscillations can provide signalling specificity. Such Ca2+ signals, or ‘Ca2+ signatures’, are generated in the cytosol, and in noncytosolic locations including the nucleus and chloroplast, through the coordinated action of Ca2+ influx and efflux pathways. An increased understanding of the functions and regulation of these various Ca2+ transporters has improved our appreciation of the role these transporters play in specifically shaping the Ca2+ signatures. Here we review the evidence which indicates that Ca2+ channel, Ca2+-ATPase and Ca2+ exchanger isoforms can indeed modulate specific Ca2+ signatures in response to an individual signal.

Synergism between calcium ions and abscisic acid in preventing stomatal opening D. L. R. De Silva, A. M. Hetherington, T. A. Mansfield

Summary Author for correspondence: Department of Biological Sciences, University of Lancaster, Bailrigg, Lancaster LAI 4YQ, UK

New Phytologist (1985) 100: 473-482 doi: 10.1111/j.1469-8137.1985.tb02795.x

KEYWORDS: Calcium; abscisic acid; stomata; Commelina communis

The inhibition of stomatal opening in Commelina communis L. by abscisic acid (ABA) appears to be dependent on the availability of calcium ions. Incubation of epidermal strips in 10−6 mol m−3 ABA had little effect in the absence of calcium, and a factorial experiment showed a highly significant calcium x ABA interaction. The effect of calcium appeared to be on the later stages of the opening process, and might have been the result of an inhibition of potassium uptake by the guard cells. There was no detectable effect of ABA when EGTA was used to chelate calcium from the apoplastic space.

The data are consistent with the hypothesis that ABA increases the permeability of the plasma membranes of the guard cells to calcium. Calcium might then operate as a second messenger to regulate the ionic fluxes that determine guard cell turgor.

Tansley review Signal transduction during fertilization in algae and vascular plants

Author for correspondence: Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK

Colin Brownlee

Summary New Phytologist (1994) 127: 399-423 doi: 10.1111/j.1469-8137.1994.tb03960.x

Keywords: Fertilization; Chlamydomonas; Fucus; gamete; signalling; angiosperms

Fertilization involves interaction between two gametes and exchange of genetic information. In addition, information is transferred which results in triggering of the particular cellular and developmental responses of the organism. This review considers the cellular signals involved during different stages of gamete interactions in plants, including gamete attraction, recognition, fusion, zygotic activation and development. A wide range of signalling mechanisms can be seen to operate during fertilization, reflecting both the diversity of systems bringing gametes together and the different cellular responses to fertilization throughout the plant kingdom. Moreover, signals involved in triggering early development following gamete fusion appear to differ significantly even between species showing at least superficially similar cellular responses. Much of our current understanding comes from studies of a few easily accessible systems though recent developments in gamete isolation, in vitro fertilization and embryo culture should increase the scope for studies of signalling mechanisms in vascular plants.

Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis Mireille Chabaud, Andrea Genre, Björn J. Sieberer, Antonella Faccio, Joëlle Fournier, Mara Novero, David G. Barker, Paola Bonfante

Summary Author for correspondence: Paola Bonfante Tel: +39 011 6705965 Email: p.bonfante@ipp.cnr.it

• The aim of this study was to investigate Ca2+ responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate.

New Phytologist (2011) 189: 347–355 doi: 10.1111/j.1469-8137.2010.03464.x

• Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca2+ responses in host root tissues.

KEYWORDS: arbuscular mycorrhiza; calcium spiking; common SYM pathway; fungal–plant signalling; Gigaspora spp.; Medicago truncatula; nuclear-localized Cameleon; spore exudates

• Ca2+ spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca2+ spiking within the AMresponsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. • These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca2+ response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleonexpressing root organ cultures represent a novel AM-specific bio-assay for such signals.

Minireview Sesbania rostrata: a case study of natural variation in legume nodulation

Author for correspondence: Marcelle Holsters Tel: +32 9 3313900 Email: mahol@psb.vib-ugent.be

Ward Capoen, Giles Oldroyd, Sofie Goormachtig, Marcelle Holsters

Summary New Phytologist (2010) 186: 340–345 doi: 10.1111/j.1469-8137.2009.03124.x

Keywords: calcium spiking; ethylene; intercellular invasion; Nod factor; Rhizobium

Legumes acquired the ability to engage in a symbiotic interaction with soil-borne bacteria and establish a nitrogen-fixing symbiosis in a novel root organ, the nodule.

Most legume crops and the model legumes Medicago truncatula and Lotus japonicus are infected intracellularly in root hairs via infection threads that lead the bacteria towards a nodule primordium in the root cortex. This infection process, however, does not reflect the great diversity of infection strategies that are used by leguminous plants. An alternative, intercellular invasion occurs in the semiaquatic legume Sesbania rostrata. Bacteria colonize epidermal fissures at lateral root bases and trigger cortical cell death for infection pocket formation and subsequent intercellular and intracellular infection thread progression towards the primordium. This infection mode evolved as an adaptation to waterlogged conditions that inhibit intracellular invasion. In this review, we discuss the molecular basis for this adaptation and how insights into this process contribute to general knowledge of the rhizobial infection process.

Flavonoid-induced calcium signalling in Rhizobium leguminosarum bv. viciae Roberto Moscatiello, Andrea Squartini, Paola Mariani, Lorella Navazio

Summary Author for correspondence: Lorella Navazio Tel: +39 049 8276295 Email: lorella.navazio@unipd.it

New Phytologist (2010) 188: 814–823 doi: 10.1111/j.1469-8137.2010.03411.x

• Legume–rhizobium symbiosis requires a complex dialogue based on the exchange of diffusible signals between the partners. Compatible rhizobia express key nodulation (nod) genes in response to plant signals – flavonoids – before infection. Host plants sense counterpart rhizobial signalling molecules – Nod factors – through transient changes in intracellular free-calcium. Here we investigate the potential involvement of Ca2+ in the symbiotic signalling pathway activated by flavonoids in Rhizobium leguminosarum bv. viciae.

• By using aequorin-expressing rhizobial strains, we monitored intracellular Ca2+ dynamics and the Ca2+ dependence of nod gene transcriptional activation.

KEYWORDS: aequorin; calcium signalling; flavonoids; legume– rhizobium symbiosis; NodD; Rhizobium leguminosarum bv. viciae

• Flavonoid inducers triggered, in R. leguminosarum, transient increases in the concentration of intracellular Ca2+ that were essential for the induction of nod genes.

Signalling molecules not specifically related to rhizobia, such as strigolactones, were not perceived by rhizobia through Ca2+ variations. A Rhizobium strain cured of the symbiotic plasmid responded to inducers with an unchanged Ca2+ signature, showing that the transcriptional regulator NodD is not directly involved in this stage of flavonoid perception and plays its role downstream of the Ca2+ signalling event.

• These findings demonstrate a key role played by Ca2+ in sensing and transducing plantspecific flavonoid signals in rhizobia and open up a new perspective in the flavonoid–NodD paradigm of nod gene regulation.

Minireview Ca2+ conduction by plant cyclic nucleotide gated channels and associated signaling components in pathogen defense signal transduction cascades Author for correspondence: Wei Ma Tel: +1 517 353 3205 Email: mawei@msu.edu

Wei Ma, Gerald A. Berkowitz

Summary New Phytologist (2011) 190: 566–572 doi: 10.1111/j.1469-8137.2010.03577.x

Keywords: Arabidopsis; calcium; cyclic nucleotide gated channel; hypersensitive response; ion channel; nitric oxide; plant innate immunity

Ca2+ elevation in the cytosol is an essential early event during pathogen response signaling cascades. However, the specific ion channels involved in Ca2+ influx into plant cells, and how Ca2+ signals are initiated and regulate downstream events during pathogen defense responses, are at present unclear. Plant cyclic nucleotide gated ion channels (CNGCs) provide a pathway for Ca2+ conductance across the plasma membrane (PM) and facilitate cytosolic Ca2+ elevation in response to pathogen signals.

Recent studies indicate that the recognition of pathogens results in cyclic nucleotide production and the activation of CNGCs, which leads to downstream generation of pivotal signaling molecules (such as nitric oxide (NO)). Calmodulins (CaMs) and CaMlike proteins (CMLs) are also involved in this signaling, functioning as Ca2+ sensors and mediating the synthesis of NO during the plant pathogen response signaling cascade. In this article, these and other pivotal signaling components downstream from the Ca2+ signal, such as Ca2+-dependent protein kinases (CDPKs) and CaM-binding transcription activators (CAMTAs), are discussed in terms of their involvement in the pathogen response signal transduction cascade.

A plastid protein crucial for Ca2+-regulated stomatal responses

Stefan Weinl, Katrin Held, Kathrin Schlücking, Leonie Steinhorst, Sebastian Kuhlgert, Michael Hippler, Jörg Kudla

Summary Author for correspondence: Stefan Weinl Tel: +49 251/83 24811 Email: Stefan.Weinl@unimuenster.de

New Phytologist (2008) 179: 675–686 doi: 10.1111/j.1469-8137.2008.02492.x

KEYWORDS: calcium; chloroplast; Ca2+-sensing receptor (CAS); guard cells; signal transduction; stomatal movement

• Guard cell movements are regulated by environmental cues including, for example, elevations in extracellular Ca2+ concentration. Here, the subcellular localization and physiological function of the Ca2+-sensing receptor (CAS) protein was investigated.

• CAS protein localization was ascertained by microscopic analyses of green fluorescent protein (GFP) fusion proteins and biochemical fractionation assays. Comparative guard cell movement investigations were performed in wild-type and cas loss-of-function mutant lines of Arabidopsis thaliana. Cytoplasmic Ca2+ dynamics were addressed in plants expressing the yellow cameleon reporter protein YC3.6.

• This study identified CAS as a chloroplast-localized protein that is crucial for proper stomatal regulation in response to elevations of external Ca2+. CAS fulfils this role through modulation of the cytoplasmic Ca2+ concentration.

• This work reveals a novel role of the chloroplast in cellular Ca2+ signal transduction.

The chloroplast as a regulator of Ca2+ signalling

Alex A. R. Webb

Summary Author for correspondence: Alex A. R. Webb tel +44 1223 333948 Alex.webb@plantsci.cam.ac.uk

New Phytologist (2008) 179: 568-570 doi: 10.1111/j.1469-8137.2008.02550.x

KEYWORDS: Arabidopsis; calcium; CAS; chloroplast; guard cell; signalling; stroma; thylaroid

This is a commentary on Weinl S, Held K, Schlßcking K, Steinhorst L, Kuhlgert S, Hippler M, Kudla J. 2008. A plastid protein crucial for Ca2+-regulated stomatal responses. New Phytologist 179: 675–686.

Mitochondrial morphology transition is an early indicator of subsequent cell death in Arabidopsis Iain Scott, David C. Logan

Summary Author for correspondence: David C. Logan Tel:+44 (0) 1334 463367 Email: david.logan@standrews.ac.uk

New Phytologist (2008) 177: 90–101 doi: 10.1111/j.1469-8137.2007.02255.x

KEYWORDS: Arabidopsis; cell death; mitochondria; mitochondrial dynamics; mitochondrial permeability transition; morphology

• Mitochondrial morphology and dynamics were investigated during the onset of cell death in Arabidopsis thaliana. Cell death was induced by either chemical (reactive oxygen species (ROS)) or physical (heat) shock.

• Changes in mitochondrial morphology in leaf tissue, or isolated protoplasts, each expressing mitochondrial-targeted green fluorescent protein (GFP), were observed by epifluorescence microscopy, and quantified.

• Chemical induction of ROS production, or a mild heat shock, caused a rapid and consistent change in mitochondrial morphology (termed the mitochondrial morphology transition) that preceded cell death. Treatment of protoplasts with a cell-permeable superoxide dismutase analogue, TEMPOL, blocked this morphology change. Incubation of protoplasts in micromolar concentrations of the calcium channel-blocker lanthanum chloride, or the permeability transition pore inhibitor cyclosporin A, prevented both the mitochondrial morphology transition and subsequent cell death.

• It is concluded that the observed mitochondrial morphology transition is an early and specific indicator of cell death and is a necessary component of the cell death process.

Tansley review Calcium homeostasis in plant cell nuclei

Author for correspondence: Raoul Ranjeva Tel: +33 5 62 19 35 17 Email: ranjeva@scsv.ups-tlse.fr

Christian Mazars, Stéphane Bourque, Axel Mithöfer, Alain Pugin, Raoul Ranjeva

Summary New Phytologist (2009) 181: 261–274 doi: 10.1111/j.1469-8137.2008.02680.x

Keywords: aequorin; autonomy; calcium; cell nucleus; homeostasis

In plant cells, calcium-based signaling pathways are involved in a large array of biological processes, including cell division, polarity, growth, development and adaptation to changing biotic and abiotic environmental conditions. Free calcium changes are known to proceed in a nonstereotypical manner and produce a specific signature, which mirrors the nature, strength and frequency of a stimulus. The temporal aspects of calcium signatures are well documented, but their vectorial aspects also have a profound influence on biological output. Here, we will focus on the regulation of calcium homeostasis in the nucleus. We will discuss data and present hypotheses suggesting that, while interacting with other organelles, the nucleus has the potential to generate and regulate calcium signals on its own.

Tansley review Calcium channels in photosynthetic eukaryotes: implications for evolution of calcium-based signalling Author for correspondence: Colin Brownlee Tel: +44 1752633331 Email: cbr@mba.ac.uk

Frédéric Verret, Glen Wheeler, Alison R. Taylor, Garry Farnham, Colin Brownlee

Summary New Phytologist (2010) 187: 23–43 doi: 10.1111/j.1469-8137.2010.03271.x

Keywords: algae; Ca2+ signalling; channels; embryophytes; evolution; photosynthetic eukaryotes; plants

Much of our current knowledge on the mechanisms by which Ca2+ signals are generated in photosynthetic eukaryotes comes from studies of a relatively small number of model species, particularly green plants and algae, revealing some common features and notable differences between ‘plant’ and ‘animal’ systems. Physiological studies from a broad range of algal cell types have revealed the occurrence of animallike signalling properties, including fast action potentials and fast propagating cytosolic Ca2+ waves. Genomic studies are beginning to reveal the widespread occurrence of conserved channel types likely to be involved in Ca2+ signalling. However, certain widespread ‘ancient’ channel types appear to have been lost by certain groups, such as the embryophytes. More recent channel gene loss is also evident from comparisons of more closely related algal species. The underlying processes that have given rise to the current distributions of Ca2+ channel types include widespread retention of ancient Ca2+ channel genes, horizontal gene transfer (including symbiotic gene transfer and acquisition of bacterial genes), gene loss and gene expansion within taxa. The assessment of the roles of Ca2+ channel genes in diverse physiological, developmental and life history processes represents a major challenge for future studies.

A cyclic nucleotide-gated channel is necessary for optimum fertility in high-calcium environments Suraphon Chaiwongsar, Allison K. Strohm, Joshua R. Roe, Roxana Y. Godiwalla, Catherine W. M. Chan

Summary Author for correspondence: Catherine Chan Tel: +1 262 472 5133 Email: chanc@uww.edu

• Arabidopsis cngc2 plants are hypersensitive to external calcium and exhibit reduced plant size and fertility, especially when they are treated with elevated but physiologically relevant levels of calcium. This report focuses on the role of cyclic nucleotide-gated channel 2 (CNGC2) in plant fertility.

New Phytologist (2009) 183: 76–87 doi: 10.1111/j.1469-8137.2009.02833.x

• To determine the cause of the reduced fertility, we investigated the flower structure and growth potential of both male and female reproductive organs in cngc2 plants grown in high-calcium conditions.

KEYWORDS: Arabidopsis thaliana, calcium, channel 2 (CNGC2), cyclic nucleotide, cyclic nucleotide-gated fertility, ion channel.

• cngc2 mutants had short stamens that may limit pollen deposition and pistils that were not conducive to pollen tube growth.

• Our data indicate that sporophytic, but not gametophytic, defects are the main cause of the observed reduction in seed yield in cngc2 plants, and suggest that correct cyclic nucleotide and calcium signaling are important for cell elongation and pollen tube guidance.

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia Hai-Yong Qu, Zhong-Lin Shang, Shao-Ling Zhang, Lian-Mei Liu, Ju-You Wu

Summary Author for correspondence: Shao-ling Zhang Tel: +86 025 84396580 Fax: +86 025 84395262 Email: nnzsl@sina.comc.cn

• The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca2+]cyt) is essential for pollen-tube growth. Local Ca2+ influx mediated by Ca2+-permeable channels plays a key role in maintaining this [Ca2+]cyt gradient.

New Phytologist (2007) 174: 524–536 doi: 10.1111/j.1469-8137.2007.02069.x

• Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique.

KEYWORDS: channels, patch clamp, pollen tube spheroplasts, Pyrus pyrifolia.

Ca2+-permeable

• We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba2+) ≈ Ca2+ > magnesium (Mg2+) > strontium (Sr2+) > manganese (Mn2+). This channel conductance was selective for Ca2+ over chlorine (Cl−) (relative permeability PCa/PCl = 14 in 10 mm extracellular Ca2+). We also showed that the channel was inhibited by the Ca2+ channel blockers lanthanum (La3+) and gadolinium (Gd3+). Furthermore, channel activity depended on extracellular pH and pollen viability.

• We propose that the Ca2+-permeable channel is likely to play a role in mediating Ca2+ influx into the growing pollen tubes to maintain the [Ca2+]cyt gradient.

Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel Yansheng Wu, Xiaodong Xu, Sujuan Li, Ting Liu, Ligeng Ma, Zhonglin Shang

Summary Author for correspondence: Zhonglin Shang Tel:+86 31186269814 Email: zls22@cam.ac.uk; zhonglinshang@hotmail.com

New Phytologist (2007) 176: 550–559 doi: 10.1111/j.1469-8137.2007.02214.x

KEYWORDS: Arabidopsis thaliana; heterotrimeric G protein; hyperpolarization-activated calcium channel; patch clamp; pollen

• The role of heterotrimeric G proteins in pollen germination and tube growth was investigated using Arabidopsis thaliana plants in which the gene (GPA) encoding the Gprotein a subunit (Ga) was null or overexpressed.

• Pollen germination, free cytosolic calcium concentration ([Ca2+]cyt) and Ca2+ channel activity in the plasma membrane (PM) of pollen cells were investigated.

• Results showed that, compared with pollen grains of the wild type (ecotype Wassilewskija, ws), in vitro germinated pollen of Ga null mutants (gpa1-1 and gpa1-2) had lower germination percentages and shorter pollen tubes, while pollen from Ga overexpression lines (wGa and cGa) had higher germination percentages and longer pollen tubes. Compared with ws pollen cells, [Ca2+]cyt was lower in gpa1-1 and gpa1-2 and higher in wGa and cGa. In whole-cell patch clamp recordings, a hyperpolarization-activated Ca2+permeable conductance was identified in the PM of pollen protoplasts. The conductance was suppressed by trivalent cations but insensitive to organic blockers; its permeability to divalent cations was Ba2+ > Ca2+ > Mg2+ > Sr2+ > Mn2+. The activity of the Ca2+-permeable channel conductance was down-regulated in pollen protoplasts of gpa1-1 and gpa1-2, and up-regulated in wGa and cGa. • The results suggest that Ga may participate in pollen germination through modulation of the hyperpolarization-activated Ca2+ channel in the PM of pollen cells.

Depolarization-activated calcium channels shape the calcium signatures induced by low-temperature stress Philip J. White

Summary Author for correspondence: Philip J. White Tel +441382560043 philip.white@scri.ac.uk

New Phytologist (2009) 183: 6-8 doi: 10.1111/j.1469-8137.2009.02857.x

KEYWORDS: calcium (Ca); Ca2+ signature; cation channels; low temperature stress; modelling Ca2+ signals; plasma membrane; signal transduction

This is a commentary on McAinsh MR, Pittman JK. 2009. Shaping the calcium signature.

New Phytologist 181: 275-294.

Two voltage-dependent calcium channels co-exist in the apical plasma membrane of Arabidopsis thaliana root hairs Henk Miedema, Vadim Demidchik, Anne-Aliénor Véry, John H. F. Bothwell, Colin Brownlee, Julia M. Davies

Summary Author for correspondence: J. M. Davies Tel: +44 1223 333 939 Fax: +44 1223 333 953 Email: jmd32@cam.ac.uk

• Calcium (Ca2+)-permeable plasma membrane ion channels are critical to root hair elongation and signalling. Arabidopsis thaliana root hair plasma membrane contains a hyperpolarization-activated Ca2+ channel (HACC) conductance. Here, the co-residence of HACC with a depolarization-activated Ca2+ channel (DACC) conductance has been investigated.

New Phytologist (2008) 179: 378–385 doi: 10.1111/j.1469-8137.2008.02465.x

• Whole-cell patch-clamping of apical plasma membrane has been used to study Ca2+ conductances and reveal the negative slope conductance typical of DACCs. Specific voltage protocols, Ba2+-permeation and inhibition by the cation channel blocker Gd3+ have been used to identify the DACC conductance.

KEYWORDS: anion; Arabidopsis thaliana; calcium; channel; depolarization; plasma membrane; root hair

• The Gd3+ sensitive DACC conductance was identified in only a minority of cells. DACC activity was quickly masked by the development of the HACC conductance. However, in the period between the disappearance of the negative slope conductance and the predominance of HACC, DACC activity could still be detected.

• A DACC conductance coexists with HACC in the root hair apical plasma membrane and could provide Ca2+ influx over a wide voltage range, consistent with a role in signalling.

Tansley review Physiological roles of nonselective cation channels in plants: from salt stress to signalling and development Author for correspondence: Vadim Demidchik Tel: +44 1206873322 Email: vdemid@essex.ac.uk

Vadim Demidchik, Frans J. M. Maathuis

Summary New Phytologist (2010) 175: 387-404 doi: 10.1111/j.1469-8137.2007.02128.x

Keywords: cytosolic free calcium; development; mineral nutrition; nonselective cation channels (NSCCs); plant neurotransmitter receptors; reactive oxygen species; salinity; signalling.

Nonselective cation channels (NSCCs) catalyse passive fluxes of cations through plant membranes. NSCCs do not, or only to a small extent, select between monovalent cations, and several are also permeable to divalent cations. Although a number of NSCC genes has been identified in plant genomes, a direct correlation between gene products and in vivo observed currents is still largely absent for most NSCCs. In this review, physiological functions and molecular properties of NSCCs are critically discussed. Recent studies have demonstrated that NSCCs are directly involved in a multitude of stress responses, growth and development, uptake of nutrients and calcium signalling. NSCCs can also function in the perception of external stimuli and as signal transducers for reactive oxygen species, pathogen elicitors, cyclic nucleotides, membrane stretch, amino acids and purines.

Tansley review Annexins

Author for correspondence: Julia Davies Tel: +44 1223 333939 Email: jmd32@cam.ac.uk

Anuphon Laohavisit, Julia M. Davies

Summary New Phytologist (2011) 189: 40–53 doi: 10.1111/j.1469-8137.2010.03533.x

Keywords: annexin; calcium; death; pathogen; reactive oxygen; stress

Annexins are multifunctional lipid-binding proteins. Plant annexins are expressed throughout the life cycle and are under environmental control. Their association or insertion into membranes may be governed by a range of local conditions (Ca2+, pH, voltage or lipid identity) and nonclassical sorting motifs. Protein functions include exocytosis, actin binding, peroxidase activity, callose synthase regulation and ion transport. As such, annexins appear capable of linking Ca2+, redox and lipid signalling to coordinate development with responses to the biotic and abiotic environment.

Significant advances in plant annexin research have been made in the past 2 yr. Here, we review the basis of annexin multifunctionality and suggest how these proteins may operate in the life and death of a plant cell.

Tansley review The CBL–CIPK Ca2+-decoding signaling network: function and perspectives

Author for correspondence: Jörg Kudla Tel: +49 (0) 251 83 24813 Email: jkudla@uni-muenster.de

Stefan Weinl, Jörg Kudla

Summary New Phytologist (2009) 184: 517–528 doi: 10.1111/j.1469-8137.2009.02938.x

Keywords: calcineurin B-like (CBL), calcium, CBL-interacting protein kinases (CIPKs), protein kinases, signaling.

Calcium serves as a versatile messenger in many adaptation and developmental processes in plants. Cellular calcium signals are detected and transmitted by calciumbinding proteins functioning as sensor molecules. The family of calcineurin B-like (CBL) proteins represents a unique group of calcium sensors and contributes to the decoding of calcium transients by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). In higher plants, CBL proteins and CIPKs form a complex signaling network that allows for flexible but specific signal–response coupling during environmental adaptation reactions. This review presents novel findings concerning the evolution of this signaling network and key insights into the physiological function of CBL–CIPK complexes. These aspects will be presented and discussed in the context of emerging functional principles governing efficient and specific information processing in this signaling system.

Tansley review Nuclear protein kinases: still enigmatic components in plant cell signalling

Author for correspondence: Stéphane Bourque Tel: +33 3 80 69 34 76 Email: bourque@dijon.inra.fr

Jennifer Dahan, David Wendehenne, Raoul Ranjeva, Alain Pugin, Stéphane Bourque

Summary New Phytologist (2010) 185: 355–368 doi: 10.1111/j.1469-8137.2009.03085.x

Keywords: nuclear translocation; nucleus; protein (de)phosphorylation; protein kinase; signal transduction

Plants constantly face changing conditions in their environment. Unravelling the transduction mechanisms from signal perception at the plasma membrane level down to gene expression in the nucleus is a fascinating challenge. Protein phosphorylation, catalysed by protein kinases, is one of the major posttranslational modifications involved in the specificity, kinetic(s) and intensity of a signal transduction pathway.

Although commonly assumed, the involvement of nuclear protein kinases in signal transduction is often poorly characterized. In particular, both their regulation and mode of action remain to be elucidated and may lead to the unveiling of new original mechanisms. For example, unlike animal cells, plant cells contain only a few strictly nucleus-localized protein kinases, which calls into question the role of this cellular distribution between the cytosol and the nucleus in their activation and functions. The control of their nucleocytoplasmic trafficking appears to play a major role in their regulation, probably through promoting interactions with their substrates under specific cellular conditions. However, recent findings showing that the nucleus can generate complex networks of second messengers (e.g. Ca2+or diacyglycerol) suggest that nuclear protein kinases could play an active role in the decoding of such signals.

Nitric oxide modulates the influx of extracellular Ca2+ and actin filament organization during cell wall construction in Pinus bungeana pollen tubes Yuhua Wang, Tong Chen, Chunyang Zhang, Huaiqing Hao, Peng Liu, Maozhong Zheng, Frantisek Baluska, Jozef Samaj, Jinxing Lin

Summary Author for correspondence: Jinxing Lin Tel: 0086 10 62836211 Email: linjx@ibcas.ac.cn

• Nitric oxide (NO) plays a key role in many physiological processes in plants, including pollen tube growth. Here, effects of NO on extracellular Ca2+ flux and microfilaments during cell wall construction in Pinus bungeana pollen tubes were investigated.

New Phytologist (2009) 182: 851–862 doi: 10.1111/j.1469-8137.2009.02820.x

• Extracellular Ca2+ influx, the intracellular Ca2+ gradient, patterns of actin organization, vesicle trafficking and cell wall deposition upon treatment with the NO donor S-nitroso-Nacetylpenicillamine (SNAP), the NO synthase (NOS) inhibitor Nω-nitro-L-arginine (L-NNA) or the NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were analyzed.

KEYWORDS: actin filaments; calcium influx,cell wall; nitric oxide (NO); pollen tube

• SNAP enhanced pollen tube growth in a dose-dependent manner, while L-NNA and cPTIO inhibited NO production and arrested pollen tube growth. Noninvasive detection and microinjection of a Ca2+ indicator revealed that SNAP promoted extracellular Ca2+ influx and increased the steepness of the tip-focused Ca2+ gradient, while cPTIO and L-NNA had the opposite effect. Fluorescence labeling indicated that SNAP, cPTIO and L-NNA altered actin organization, which subsequently affected vesicle trafficking. Finally, the configuration and/or distribution of cell wall components such as pectins and callose were significantly altered in response to L-NNA. Fourier transform infrared (FTIR) microspectroscopy confirmed the changes in the chemical composition of walls.

• Our results indicate that NO affects the configuration and distribution of cell wall components in pollen tubes by altering extracellular Ca2+ influx and F-actin organization.

Arabidopsis VILLIN4 is involved in root hair growth through regulating actin organization in a Ca2+-dependent manner Yi Zhang, Yingyu Xiao, Fei Du, Lijuan Cao, Huaijian Dong, Haiyun Ren

Summary Author for correspondence: Haiyun Ren Tel: +86 10 58806090 Email: hren@bnu.edu.cn

New Phytologist (2011) 190: 667–682 doi: 10.1111/j.1469-8137.2010.03632.x

KEYWORDS: actin-binding protein; actin bundle; actin cytoskeleton; cytoplasmic streaming; villin

• Villin is one of the major actin filament bundling proteins in plants. The function of Arabidopsis VILLINs (AtVLNs) is still poorly understood in living cells. In this report, the biochemical activity and cellular function of AtVLN4 were examined.

• The biochemical property of AtVLN4 was characterized by co-sedimentation assays, fluorescence microscopy and spectroscopy of pyrene fluorescence. The in vivo function of AtVLN4 was analysed by ectopically expressing it in tobacco pollen and examining the phenotypes of its T-DNA insertional plants.

• Recombinant AtVLN4 protein exhibited multiple activities on actin, including actin filament bundling, calcium (Ca2+)-dependent filament severing and barbed end capping.

Expression of AtVLN4 in tobacco pollen induced the formation of supernumerary actin cables and reduced pollen tube growth. Loss of function of AtVLN4 resulted in slowing of root hair growth, alteration in cytoplasmic streaming routes and rate, and reduction of both axial and apical actin bundles.

• Our results demonstrated that AtVLN4 is involved in root hair growth through regulating actin organization in a Ca2+-dependent manner.

Rapid report Phosphatidic acid formation is required for extracellular ATP-mediated nitric oxide production in suspension-cultured tomato cells Author for correspondence: Ana M. Laxalt Tel: +54 223 4753030 Email: amlaxalt@mdp.edu.ar

Daniela J. Sueldo, Noelia P. Foresi, Claudia A. Casalongué, Lorenzo Lamattina, Ana M. Laxalt

Summary New Phytologist (2010) 185: 909–916 doi: 10.1111/j.1469-8137.2009.03165.x

Keywords: calcium; diacylglycerol kinase; extracellular ATP; nitric oxide; phosphatidic acid; phospholipase C; phospholipase D; tomato cells

• In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca2+, nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway.

The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown.

• In vivo phospholipid signaling upon extracellular ATP treatment was studied in 32Pilabeled suspension-cultured tomato (Solanum lycopersicum) cells.

• Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mm induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase.

• Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase activation.

Rapid report Ethylene activates a plasma membrane Ca2+permeable channel in tobacco suspension cells

Author for correspondence: Wen-Hao Zhang Tel: +86 10 62836697 Email: whzhang@ibcas.ac.cn

Min-Gui Zhao, Qiu-Ying Tian, Wen-Hao Zhang.

Summary New Phytologist (2007) 174: 507–515 doi: 10.1111/j.1469-8137.2007.02037.x

Keywords: cytosolic Ca2+ activity; ethylene; patch-clamp; plasma membrane Ca2+-permeable channel; tobacco (Nicotiana tabacum) suspension cells

• Here, the effects of the ethylene-releasing compound, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currents across plasma membranes and on the cytosolic Ca2+ activity ([Ca2+]c) of tobacco (Nicotiana tabacum) suspension cells were characterized using a patch-clamp technique and confocal laser scanning microscopy.

• Exposure of tobacco protoplasts to ethephon and ACC led to activation of a plasma membrane cation channel that was permeable to Ba2+, Mg2+ and Ca2+, and inhibited by La3+, Gd3+ and Al3+.

• The ethephon- and ACC-induced Ca2+-permeable channel was abolished by the antagonist of ethylene perception (1-metycyclopropene) and by the inhibitor of ACC synthase (aminovinylglycin), indicating that activation of the Ca2+-permeable channels results from ethylene. Ethephon elicited an increase in the [Ca2+]c of tobacco suspension cells, as visualized by the Ca2+-sensitive probe Fluo-3 and confocal microscopy. The ethephon-induced elevation of [Ca2+]c was markedly inhibited by Gd3+ and BAPTA, suggesting that an influx of Ca2+ underlies the elevation of [Ca2+]c.

• These results indicate that an elevation of [Ca2+]c, resulting from activation of the plasma membrane Ca2+-permeable channels by ethylene, is an essential component in ethylene signaling in plants.

Calcium–calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants Xiuli Hu, Mingyi Jiang, Jianhua Zhang, Aying Zhang, Fan Lin, Mingpu Tan

Summary Author for correspondence: Mingyi Jiang Tel: +86 25 84396372 Fax: +86 25 84396673 Email: myjiang@njau.edu.cn

New Phytologist (2007) 173: 27–38 doi: 10.1111/j.1469-8137.2006.01888.x

KEYWORDS: abscisic acid; antioxidant enzymes; calcium/calmodulin; hydrogen peroxide (H2O2); signal transduction; Zea mays

• Using pharmacological and biochemical approaches, the role of calmodulin (CaM) and the relationship between CaM and hydrogen peroxide (H2O2) in abscisic acid (ABA)induced antioxidant defense in leaves of maize (Zea mays) plants were investigated.

• Treatment with ABA or H2O2 led to significant increases in the concentration of cytosolic Ca2+ in the protoplasts of mesophyll cells and in the expression of the calmodulin 1 (CaM1) gene and the content of CaM in leaves of maize plants, and enhanced the expression of the antioxidant genes superoxide dismutase 4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes. The up-regulation of the antioxidant enzymes was almost completely blocked by pretreatments with two CaM antagonists.

• Pretreatments with CaM antagonists almost completely inhibited ABA-induced H2O2 production throughout ABA treatment, but pretreatment with an inhibitor or scavenger of reactive oxygen species (ROS) did not affect the initial increase in the contents of CaM induced by ABA. • Our results suggest that Ca2+–CaM is involved in ABA-induced antioxidant defense, and that cross-talk between Ca2+–CaM and H2O2 plays a pivotal role in ABA signaling.

Useful Links