Plant mechanosensing and Ca2+ transport

Review

Plant mechanosensing and Ca2+ transport Takamitsu Kurusu1,2,3, Kazuyuki Kuchitsu1,2, Masataka Nakano4, Yoshitaka Nakayama4, and Hidetoshi Iida4 1

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan 3 School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan 4 Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei, Tokyo 184-8501, Japan 2

Mechanical stimuli generate Ca2+ signals and influence growth and development in plants. Recently, candidates for Ca2+-permeable mechanosensitive (MS) channels have been identified. These channels are thought to be responsible for sensing osmotic shock, touch, and gravity. One candidate is the MscS-like (MSL) protein family, a homolog of the typical bacterial MS channels. Some of the MSL proteins are localized to plastids to maintain their shape and size. Another candidate is the mid1-complementing activity (MCA) protein family, which is structurally unique to the plant kingdom. MCA proteins are localized in the plasma membrane and are suggested to be involved in mechanosensing and to be functionally related to reactive oxygen species (ROS) signaling. Here, we review their structural features and role in planta. Plants sense mechanical stimuli Charles Darwin described how, when the petioles of some climbing plants were touched with sticks, the petioles clasped them and increased in thickness [1]. Rubbing the internode for approximately 10 s once or twice a day resulted in morphological changes and inhibition of growth [2]. Recently, this response, thigmomorphogenesis, was shown to play a role in protection against pests [3]. Touching twice a day was sufficient to retard elongation of inflorescences and delay flowering in Arabidopsis thaliana [4]. These reports are examples of how mechanical stimuli, such as touching and shaking, can affect morphogenesis, growth, and development in plants. The examples given above represent mechanical stimulus-induced gradual alterations, which are often hardly visible to the human eye. However, plants also show rapid responses. The two lobes of the leaf of the Venus flytrap Dionaea muscipula rapidly close to capture insects that contact the trigger-hairs at the center of the leaf [5]. The leaflet of Mimosa pudica folds after mechanical stimulation. In both cases, the responses occur within 1 s. Whether the responses are fast or slow, plants are able to sense and respond to mechanical stimuli. There has been a long history of studies on these processes, but the molecular mechanisms by which mechanical sensing leads to Corresponding authors: Iida, H. ([email protected]); Kuchitsu, K. ([email protected])

responses have only recently started to be elucidated (reviewed in [3,4,6–9]). Mechanosensing and Ca2+ transients How do plants sense mechanical stimuli? Some progress towards answering this question was made using Arabidopsis plants expressing a Ca2+-sensitive photoprotein, aequorin, from the jellyfish Aequorea victoria [10]. The recombinant plants emitted light immediately after touch, osmotic stress [6], and gravistimulation [11,12], suggesting that an immediate early event in the mechanoresponse is to increase cytosolic Ca2+ concentration ([Ca2+]cyt). Hypoosmotic shock, as well as trinitrophenol, an activator of MS channels in Escherichia coli [13], induced a rapid and transient rise in [Ca2+]cyt, predominantly due to plasma membrane Ca2+ influx in plant cells [14,15]. Although mechanical stimuli, such as touch, bending, and barriers to growth, all elicited rapid and transient increases in [Ca2+]cyt, their spatiotemporal patterns (Ca2+ signatures) were stimulus specific [16]. Consistent with these observations, cellular Ca2+-binding regulatory proteins, such as calmodulin and calmodulin-like proteins, were induced by mechanical stimuli [17]. These downstream events, as well as adaptive responses including the induction of transcription factors, could be suppressed when Ca2+ influx was compromised by Ca2+ chelators, such as ethylene glycol tetraacetic acid (EGTA) or 1,2-bis(o-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA), or by Ca2+-channel blockers [15,18,19]. Therefore, it is possible that plasma membrane Ca2+-permeable MS channels are key molecules that sense mechanical stimuli. In this review, we describe the properties and the molecular basis of Ca2+-permeable MS channels in various plant species. We also highlight the important roles of the MS channels in mechanical signaling and stress adaptation in plant cells. Plant MS channels and candidates MSL protein family Genome-wide screening to search for plant MS channels homologous to eukaryotic and prokaryotic MS channels revealed the MSL protein family [20,21]. MscS, one of three types of prokaryotic MS channel, has MS channel activity with small conductance, and functions as a safety valve to avoid cell rupture by releasing cytoplasmic solutes upon

1360-1385/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2012.12.002 Trends in Plant Science, April 2013, Vol. 18, No. 4

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Table 1. Presence or absence of MS channels in various kingdoms and a domain Kingdom/domain Plants Animals Fungi Bacteria

MscL – – – +

MscS + – + +

MCA + – – –

TRP – + + –

Piezo + + ? –

hypo-osmotic shock [22]. MSLs are found in land plants, algae, and fungi, but not in animals (Table 1). The overall amino acid sequences of MSLs are not homologous to those of prokaryotic MscS; only a pore-forming transmembrane segment and the secondary structure adjacent to this segment have been conserved. Conserved motifs of MSL2 have recently been characterized [23]. Based on the sequence of this conserved region, eukaryotic MSLs can be grouped into two main classes; Class I and Class II [24]. Several Class I MSLs contain organelletargeting sequences predicted to localize to mitochondria or plastids. By contrast, Class II MSLs do not have a targeting sequence and are found in fungi and protists, which are organisms that lack plastids. MSLs in fission yeast are localized at the endoplasmic reticulum (ER) membrane and regulate [Ca2+]cyt to support the survival of cells upon hypo-osmotic shock [25]. Arabidopsis has ten MSL genes (MSL1–10) in its genome. MSL1–3 and MSL4– 10 are assigned to Class I and Class II, respectively. Several lines of evidence indicate that some of the MSL family proteins have MS channel activity. MSL9 and MSL10, two of seven Class II Arabidopsis MSLs localized in the plasma membrane in roots, provide MS channel activity, although their physiological function is not yet known [26]. Phenotypically, the double (msl9/10) and quintuple (msl4/ 5/6/9/10) mutant plants grow normally. Electrophysiological studies suggest that MSL9 and MSL10 permeate Cl– rather than Ca2+ [26,27]. Recently, electrophysiological properties of MSL10 were clearly demonstrated [28]. Chlamydomonas reinhardtii MSC1, a chloroplast-localized Class I MSL, was shown electrophysiologically to have anionselective MS channel activity in E. coli giant spheroplasts [29]. Fission yeast Msy1, an ER-localized Class II MSL, has also been demonstrated electrophysiolologically to have MS channel activity [25]. Genetically, MSL2 and MSL3 are suggested to act as MS channels because both proteins can complement the lethality of an E. coli mutant lacking MscS under hypo-osmotic conditions [30]. It is still unknown whether these MSLs are involved in mechanical stimuliinduced generation of Ca2+ signals as Ca2+-permeable MS channels. The physiological evidence obtained so far suggests that MSL2 and MSL3 are plastid MS channels that participate in maintaining the shape and size of the plastid [30]. Both proteins are also required to protect plastids from hypoosmotic stress during normal plant growth [31]. The Arabidopsis msl2 msl3 double mutant has large, round plastids that lack dynamic tubular structures, known as stromules, on the surface. This phenotype can be rescued by increasing cytoplasmic osmolarity through exogenously provided osmolytes, or by withholding water. The enlarged plastids in the msl2 msl3 double mutant contain multiple 228

filamentous temperature-sensitive Z (FtsZ) rings [32]. MSL2 and MSL3 are suggested to function as components of the plastid division machinery in the pathway to regulate size and division of plastids, such as the Min system, serving to restrict FtsZ ring formation to the middle of plastids. The Piezo protein family Piezo proteins in mouse and Drosophila are pore-forming subunits of MS channels and respond to mechanical stimuli. Drosophila larvae lacking the DmPiezo gene showed a reduced behavioral response to noxious mechanical stimuli, compared with their wild type counterparts [33,34]. Many eukaryotic species, including plant species, have a single Piezo protein, but yeast and bacteria have no clear homologs [35]. Studies on plant Piezo homologs have not yet been reported. MCA protein family A genetic screen of Arabidopsis cDNAs, which complements the conditional lethality of a yeast mutant lacking the functional MID1 gene, resulted in the identification of MCA1 as a candidate MS channel [14]. The yeast Mid1 protein shows MS channel activity when expressed in mammalian cells [36]. Ca2+ uptake activity has been shown for MCA1 and its paralog MCA2, as well as rice (Oryza sativa) OsMCA1 and tobacco (Nicotiana tabacum) NtMCA1 and NtMCA2 [14,15,37–39]. MCA proteins are present in all land plants, including ferns and mosses, but not in algae, animals, protists, and fungi, suggesting that the function of MCA proteins is fundamental to land plants. Phylogenetic analyses indicate that genetic diversity of MCA genes occurred relatively recently, and only one MCA gene is present in various Poaceae [38] (Figure 1a). Interestingly, among the MCA proteins in dicots, those in Brassicaceae, including Arabidopsis and Brassica, are diverged from those in other dicots, and exist as two distinct paralogs (MCA1 and MCA2), suggesting their functional divergence. Functional diversity between Arabidopsis MCA1 and MCA2 has indeed been suggested [37]. Localization, ion permeability, and physiological functions of MSLs and MCAs characterized so far are summarized in Table 2. Structural features of MCA proteins The amino acid sequences of MCA proteins show relatively high similarity throughout the entire sequence, except for the serine-rich region in the middle, which shows substantial diversity among these proteins (Figure 1b). The N-terminal half has a region similar to the putative regulatory domain of protein kinase candidates found in grass species, and coiled-coil and EF-hand-like motifs [14,38,40]. Despite their activity in mediating Ca2+ uptake, MCA proteins have no homology to other known ion channels or transporters. Truncation analysis showed that the N-terminal half without the coiled-coil motif of MCA1 and MCA2 (MCA11-173 and MCA21-173) is necessary and sufficient for Ca2+ uptake activity [40]. This region has one putative transmembrane segment that contains a highly conserved Asp21. Replacement of Asp21 with Asn resulted in complete and partial loss of Ca2+ uptake activity in MCA1 and

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Table 2. Properties of MSLs and MCAs Channel MSL2

Species Arabidopsis thaliana

Localization Plastid

Mechanosensitivity Nd c

Conductance Nd

Permeability a Nd

MSL3

A. thaliana

Plastid

Nd

Nd

Nd

MSL9 MSL10 MSC1

Plasma membrane Plasma membrane Plastid

Yes Yes Yes

45 pS 103–137 pS 400 pS

Anions Anions Anions

Perinuclear ER

Yes

250 pS

Nd

Msy2

A. thaliana A. thaliana Chlamydomonas reinhardtii Schizosaccharomyces pombe S. pombe

Cortical ER

Nd

Nd

Nd

MCA1

A. thaliana

Plasma membrane

Yes

34 pS

Ca2+

MCA2 NtMCA1

A. thaliana Nicotiana tabacum

Yes Nd

Nd Nd

NtMCA2

N. tabacum

Nd

OsMCA1

Oryza sativa

Plasma membrane Plasma membrane and Hechtian strand Plasma membrane and Hechtian strand Plasma membrane

Nd

Msy1

Physiological function b Plastid morphology and division Plastid morphology and division Nd Nd Plastid morphology

Refs [30,32]

[25]

Ca2+ Ca2+

Hypo-osmotic response and Ca2+ sequestration Hypo- osmotic response and Ca2+ mobilization Touch and hypo-osmotic responses Hypo-osmotic response Hypo-osmotic response

Nd

Ca2+

Hypo-osmotic response

[38]

Nd

Ca2+

Hypo-osmotic response

[15]

[30,32] [26] [26,28] [29]

[25] [14,46] [37,46] [38]

a

Only permeable ions determined so far are listed.

b

Major physiological functions characterized so far are listed.

c

Not determined.

MCA2, respectively. Both MCA1 and MCA2 form a tetramer, suggesting that this region is the functional motif of the MCA protein, whereas the putative transmembrane segments of the MCA tetramer form a Ca2+-permeable pore (Figure 1b; see also Figure 1 of [40]). The EF-hand-like and coiled-coil motifs are suggested to have distinct effects on the Ca2+ influx activity of Arabidopsis MCA1 and MCA2 expressed in yeast cells, as assessed by truncation analysis [40]. A truncated form of MCA1 lacking the EF-hand-like motif (MCA11-135) did not show activity, whereas that of MCA2 (MCA21-135) did, suggesting that this motif positively regulates MCA1. Another truncated form of MCA1 with the coiled-coil motif (MCA11-237) did not have activity, whereas that of MCA2 (MCA21-237) did. As mentioned above, the truncated form of MCA1 lacking the coiled-coil motif but having the EFhand-like motif (MCA11-173) showed activity, suggesting that the coiled-coil motif negatively regulates MCA1. Thus, it is possible that the two motifs regulate MCA1 and MCA2 differently. Another explanation is that both proteins are not properly regulated in yeast cells, which do not contain intrinsic plant regulatory proteins. The C-terminal half of MCA proteins has a cysteine-rich domain called the Plac8 or DUF614 motif. The function of this motif is unknown [41]. In general, the functions of proteins containing this motif (the Plac8 super-family) are largely unknown, but some are suggested to mediate cadmium resistance [42,43] and are localized in plasma membrane microdomains [44,45]. All of the MCA proteins examined so far are localized at the plasma membrane [14,15,37,38]. Some are localized at Hechtian strands or the plasma membrane–cell wall interface and at punctuated structures on the cell surface [38], which may be related to plasma membrane microdomains. Plant MCA proteins may form clusters or complexes with other signaling molecules via the Plac8 motif.

MCA proteins mediate mechanical signal-induced Ca2+ transport and regulation of cell growth and development Several lines of evidence suggest that the MCA protein functions as a Ca2+-permeable MS channel component. Firstly, ectopic overexpression of MCA proteins increased Ca2+ uptake in Arabidopsis seedlings as well as in cultured rice cells, and enhanced the hypo-osmotic shock-induced increase in [Ca2+]cyt [14,15,38]. Secondly, hypo-osmotic shock-induced changes in [Ca2+]cyt and Ca2+ influx were partially impaired in OsMCA1-suppressed cultured rice cells, whereas changes triggered by chitin fragments, a major microbe-associated molecular pattern, were not affected [15]. Thirdly, a cell stretching-induced increase in [Ca2+]cyt was enhanced in Chinese hamster ovary cells heterologously expressing MCA1 [14]. Fourthly, electrophysiological characterization of Xenopus oocytes expressing MCA1 suggested that MCA1 is a possible MS channel with a conductance of 34 pS [46]. MCA2 is also shown to generate membrane stretch-activated currents in oocytes [46]. Lastly, whole-cell patch-clamp recordings on Arabidopsis mesophyll protoplasts have shown that the anionic amphipath trinitrophenol, which penetrates the outer leaflet of the plasma membrane and thus causes membrane distortion, induces Ca2+ currents more in MCA1-overexpressing protoplasts than in their wild type counterparts [14]. In a knockout mutant of Arabidopsis MCA1 (but not MCA2), the roots were less able to penetrate a layer of hard agar, suggesting some defect in the mechanical response [14]. Rice OsMCA1-RNAi lines showed stunted growth and shortened rachises [15], which were reminiscent of morphological changes under drought stress [47]. OsMCA1 may play a role in the drought stress response through sensing changes in membrane distortion. Growth of the Arabidopsis mca1 mca2 double mutant showed enhanced sensitivity to Mg2+ [37]. Growth of OsMCA1-suppressed cultured rice cells was significantly restricted under Ca2+ 229

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(a) C. sinensis C. clemenna

N. tabacum (NtMCA1: AB622811) N. tabacum (NtMCA2: AB622812) S. lycopersicum

Most dicots

B. rapa T. halophila MCA2 MCA2

M. esculenta MCA 2 M. esculenta MCA 1 M. domesca P. persica

P. trichocarpa

Brassicaceae

C. papaya

A. coerulea C. savus

A. lyrata MCA1 C. rubella MCA1 A. thaliana (MCA1: At4G35920) B. rapa MCA1 T. halophila MCA1 B. napus

E. grandis G. max MCA4 G. max MCA3 P. vulgaris MCA2 M. truncatula MCA2 L. japonicus M. truncatula MCA1

C. rubella MCA2

A. thaliana (MCA2: At2G17780) A. lyrata MCA2

L. usitassimum

G. max MCA1 P. vulgaris G. max MCA2 MCA1

S. italica T. aesvum H. vulgare

S. bicolor Z. mays

O. sava (OsMCA1: AB601973) B. distachyon

Poaceae P. sitchensis

0.05

P. patens MCA1

S. moellendorffii P. patens MCA2

Mosses and/or lycophytes

(b)

TM

MCA1 MCA2 NtMCA1 OsMCA1 G. max L. usitatissimum M. esculenta P. trichocarpa E. grandis P. sitchensis S. moellendorffii P. patens

1 1 1 1 1 1 1 1 1 1 1 1

MSHSWDGLGEIASVAQLTGLDAVKLIGLIVKAANTAWMHKKNCRQFAQHLKLIGNLLEQLKISEMKKYPETREPLEGLEDALRRSYLLVNSCRDRSYLYLLAMGWNIVYQFRKHQDEIDR MANSWDQLGEIASVAQLTGIDALKLIGMIVNAANTARMHKKNCRQFAHHLKLIRNLLEQIKNSEMNQRSEILEPLQGLDDALRRSYILVKSCQEKSYLYLLAMGWNIVNQFEKAQNEIDL MA-TWEHFGEVANFAQLAGLDAVRLIGMIVKAASTARMHKKNCRQFAQHLKLIGNLLEQLKITELKKYPETREPLEYLEDALRRSYMLVHSCHDRSYLYLLAMGWNIVYQFRKAQNEIDQ MA-SWENLGDVATVVQLTGLDAVRLISMIVKAASTARLHKRNCRRFAQHLKLIGGLLEQLRVSELKKYPETREPLEQLEDALRRAYLLVHSCQDRSYLYLLAMGWNIVYQFRKAQNEIDN MA-SWDQMGELANVAQLTGVDAVRLIGMIVRAASTARMHKKNCRQFAQHLKLIGNLLEQLKISELKKYPETREPLEQLEDALRRSYILVNSCQDRSYLYLLAMGWNIVYQFRKAQNEIDR MA-AWGNFGEIANVAQITGVDAVRLIGLIVKAASTARMHKKNCRQFAQHLKLIGGLLEQLKISELKKYPETREPLEQLEDSLRKSYLLVNSCQDRSYLYLLAMGWNIVYQFRKAQNEIDR MS-SWEHFGEIANVAQLTGVDAVRLIGMIVKAASTARMHKKNCRQFAQHLKLIGNLLEQLKISELKKYPETREPLEQLEDALRRSYILVNSCQDRSYLYLLAMGWNIVYQFRKAQNEIDR MA-TWEHLGEVANVVQLTGIDAVRLIAMIGKAATTARMHKKNCRQFAQHLKLIGNLLEQLKISELKRYPETREPLEQLEDALRRSYLLVNSCQDRSYLYLLAMGWNIVYQFRKAQNEIDR MA-TWEAFGEVANVAQLTGLDATRLIGMIVQAANTARMHKRNCRQFAQHLKLIGNLLEQLKISELKRYPETREPIEQLEDALRRSYILVNSCQDRSYLYLLAMGWNIVYQFRRAQNEIDR ----WENVGDLANVTQLTGLNAVSLIALIVKAASNARMHKKNCRQFAQHLKLIGNLLEQLKATELKKYPETREPLEQLEDALRRSYVLVDSCQNRSYLYLLAMGWTIVNQFRQAHAEIDR MV-F--PVGDVATVAQIAGLDSLKLIAAVAAAAKNARMHKKNCRNFAQHLKLIGNLLEKLRLSELREHPETSEPLERLEEALRKAYILVNSCKNKSYLYLLAMGWNIVNQFKLRQAEIDR MP--WLALGDVASFGQLAGINAVQLIAMIVKAANNARMHKKNCRQFAQHLKLIANLLEQLNLTDLKERPECREPLEHLEQALRKAVVLVESCRDKSYLYLVAMGWMYVTKFRDYQDEIDK

MCA1 MCA2 NtMCA1 OsMCA1 G. max L. usitatissimum M. esculenta P. trichocarpa E. grandis P. sitchensis S. moellendorffii P. patens

121 121 120 120 120 120 120 120 120 117 118 119

FLKIIPLITLVDNARI--R-ERFEYIDRDQREYTLDEEDRHVQDVILKQESTRE-AASVLKKTLSCSYPNLRFCEALKTENEKLQIELQRSQEHYDVAQCEVIQRLIGVTQAAAAVEPDS FLKIVPLINMADNARI--R-ERLEAIERDQREYTLDEEDRKVQDVILKQESTREAATSVLKKTLSRSYPNMGFCEALKTEEEKLQLELQRSRARYDADQCEVIQRLIDVTQTAATVEPNL YLKIIPLITLVDNARV--R-ERFEIIEKDQCEYTLEAEDMKVQEVILKREPSKH-DTVVLKKTLSRSYPSMPINEAIQKENEKLQLELQRSQANLDVSHCEFIQHLLEVTEVVASESLSE YLRLVPLITLVDNARV--R-ERMEYIERDQCEYSFDDEDKEVQDALLNPDPSTN-PTVVLKKTLSCSYPNLPFNEALRKESEKLQVELQRSQSNMDMGQCEVIQHLLGVTKTVAS-SIPE YLRLVPLITLVDNARV--R-ERLEVIEMDQREYTLDDEDQKAQTVIFKPEPDKD-DTAVLKKTLSCSYPNCSFTEALKKENEKLKLELQRSQANLDMNQCEVIQRLLDVTEVAA-YSVPA YLRLIPLITLVDNTRI--R-DRLEYIERDQCEYTLDEEDRKVQDLILKSESVKD-QTVVLKKTLSCSYPNMCFNEALKKESEKLQIELQRSQSNMDVNPSEVIQHLLEVTEVAAASVVPE YLRLVPLITLVDNARV--R-ERLEDIQKDQHEYTLDEEDRKVQDVILKPEPLKD-QTIVLKKTLSSSYPNLGFNEALQKESQKLQLELQRSQANLDVKQCEVIQHLIDVTEAAAANSLPQ YLRLVPLITLVDNSRV--R-ERLEDIERDQREYTLDDEDRRVQDVILKPDCSGE-HTTMLKKTLSCSYPNMCFNEALRKENEKLQLELQRSQAHLDVNQCEVIQHLIEVTEVAAASSLPE YLKLVPLITLVDNARVRVR-ERLEVIERDQREYTLDDEDQKVQTVILTHEPSKN-DAMMLKKTLSCSYPNLCFNEALKKENEKLRMELQRSQTNMDMNQCEVIQRLLEVTETVASNSLPD YLKIVPLINLLDNHRV--K-DRLQAIEKDQREYTLDEEEEKVHDAILHPESSVL-NSSMLRKSLSRRYPYLGFEEALQKENEKLQVELHRSRANMDVDQCDVIRHLIEVTENVANI-PPE LLQIIPLISLVDNNRV--RWQQLREIQRDQKEYTLDEDELRLQETVLKPDLSVN-ESRELRRNLSRNYPGLALEEALRKENKKLKKELQKMQSLMEEDQCDVIRRLIDITETDVKGTYEK YLKLIPLISLVENS----R-ERIRAIVKDKRSYTMERSDVKVQETLLKPEHTRR-DSMRLSRQLSRRYPGMPLDIALREENAKLQKELEHMRACMELEKCGVIEHLIDFTEAAAADPVIL

MCA1 MCA2 NtMCA1 OsMCA1 G. max L. usitatissimum M. esculenta P. trichocarpa E. grandis P. sitchensis S. moellendorffii P. patens

237 238 236 235 235 236 236 236 238 232 235 233

EKELTKKASKKS-ERSSSMK-T---EYSYDEDSPK----KSSTRAASRSTSNVSSGHDLLSRR--ASQAQHHEEWHTDLLACCSEPSLCFKTFFFPCGTLAKIATAASNRHISSAEACNE EKVLTKK----E-ELTSSKK-R---DDLYDTDS-------SSIRADSRSTSYVSSGHELLSGR--SLQ--HRGNWHADLLDCCSEPCLCLKTLFFPCGTLAKISTVATSRQISSTEVCKN KSSPTKPTKKLE-HSHSDVN-SD--KEHNDRSYTK----SDEKQSTSRITSSVSSQRELLSSK--GSD--RYDEWHSDLLGCCSEPLLCIKTCFFPCGTFSKVASAAADRHISSADACNE KCATPKVSEKAD-SNHTKVS-EDSAKTYHDDSPKK----QKDACTAPRSSPPSSYGHDLVSSR--GSY--S-DEWHADLLGCCSEPSLCLRTFFFPCGTFSKIASIAKNRPMSSSEACND KCSPEKSHKKEE-YNYSDANSDK--DHSSDEKYHA----KIDKHSPSRYSV---AQKDLASTG--GSY--QQEDWHTDLLACCSEPSLCMKTFFYPCGTFSKIASVARNRPISSGEACND KRSSKTLSKKLE-PNYSDMN-VK--EHTYDDSYSR----KSDSRTISRDTSSVSSGQDLLSRK--GSS--HDEEWQTDLLGCCSEPLLCVKTLFFPCGTFSKIATVTSNRHMASAEACNE KTSPMKASKKLE-SNNSDVS-EK--NDSSDENYPK----KSDSRTTSRNTSSFSSGDNLLSHR--NSY--QHEEWHSDLLGCCSEPSLCLKTFFYPCGTFSKIATVATNKHMSPAEACNE KSSSTKSSKKLE-PAYSDAS-EN--KHSFDDSYST----KSDSHKTSRNTSSVSSRDDLLSSR--GSH--QQEEWHADLLGCCSEPYLCIKTLFYPCGTFAKIATVAKNRHISSAEACNE YGSPVKGSKKKE-KYTSDADSDR--EQSTGETYP-------------NNSS---LDNDLLPSK--GSY--GHYEWHTDLLGCCSEPLLCIKTFFCPCDTFSKIATVATNRHMSKGEACND RKHFQHSNCEEN-HPCADCCEEQ--PSPMDGSYLR----KQD----GRQDSSISSGRDLISQR--GSH--RYEEWKTDLFGCCMEPYLCFKTCIYPCGTFSNIAAVASNGKISPEQACND DPDYMKEAKKYEQSNYEQSN---------SDAYTY----Q-E--------S---HKSNETAS---KSW--QLQDWHHDLYGCCGSPLLCVGTFLCPCCTFATVAATATNGIMPKHAACTN AEAMAEIPEE--------DN-EE--EERHRKPHTRTEVVESSKRHMKHLKPSFST-LSLSSQKVVQCR--HEDNWRYDLFDCCVDPCLCIETFCYPCGTFTLVASSVTDGGTSEDSACSQ

MCA1 MCA2 NtMCA1 OsMCA1 G. max L. usitatissimum M. esculenta P. trichocarpa E. grandis P. sitchensis S. moellendorffii P. patens

346 338 344 344 341 344 344 344 335 337 325 339

LMAYSLILSCCCYTCCVRRKLRKTLNITGGFIDDFLSHVMCCCCALVQELREVEIRGAY--GTEK--TKISP-PSSQFMEH LIVYSLILSCCCYTCCIRKKLRKTLNITGGCIDDFLSHLMCCCCALVQELREVEIHRASYAGTEKSNKEMSP-PTPQFMEE LMAYSLILSCCCYTCCIRKKLRKKLNITGGIIDDFLSHLMCCCCALVQELREVEIRGAH--GTGK--TKTSP-PPSQFMES IMAYSLILSCCCYTCCVRRKLRQKLNIAGGCIDDFLSHLMCCCCALVQEWREVEIRGAY--G-ER--TKISP-PSFQYMEH LMAYSLILSCCCYTCCVRRKLRKMLNITGGFIDDFLSHLMCCCCALVQEWREVEIRGLS--G-SK--TKTSP-PPSQYMES LMAYSLILSCCCYTCCIRRKLRNMFNISGGFIDDFLSHLMCCCCALVQEWREVEIRGAY--G-QK--TKTSP-PVSQSMELMAYSLILSCCCYTCCIRRKLRKTLNISGGFVDDFLSHLMCCCCALVQEWREVETRGVY--GPEK--TKTSP-PPSQFMES LMAYSMMLSCCCYTCCVRRELRKTLNITGGFIDDFLSHLMCCCCALVQEWREVEIRGVY--GPEK--TKTSP-PPSQFMES LLAYSLILSCCCYTCCVRRKLRKMLNI-GGWFDDFLSHFMCCCCALVQEWREVEIRGLS--G------------------K LMTYSLVFGCCCYTCCMRRKLRKLLNIAGGMCDDFLTHMTCCCCALVQEWREIECRGLD--DSHM--KKMSP-PPSQQMECLIYTMVLSCCFYTCCFRRKLRKLYNIEGGSCDDCWAHFLCFCCALVQEAREIKARERD--GGNC--H------TLLLFIT LAFHSLYGGCCCYTSCIRRKVRRRFDIPGDCFSDYWAHVCCCCCAVLQELHELRFREKQ--GKYT--TQFFLYSLSGMQNV

12 0 12 0 11 9 11 9 11 9 11 9 11 9 11 9 11 9 11 6 11 7 11 8

Putaitve funconal domain of MCA

EF-hand-like

23 6 23 7 23 5 23 4 23 4 23 5 23 5 23 5 23 7 23 1 23 4 23 2

Coiled-coil 34 5 33 7 34 3 34 3 34 0 34 3 34 3 34 3 33 4 33 6 32 4 33 8

42 1 41 7 41 9 41 8 41 5 41 7 41 9 41 9 39 4 41 1 39 5 41 5

Plac8 TRENDS in Plant Science

Figure 1. Phylogenetic study and domain structure of the mid1-complementing activity (MCA) protein family. (a) A phylogenetic tree of the MCA protein family based on amino acid sequences of the MCA functional domain constructed using the MEGA5.1 software by the Neighbor-Joining method. The alignment was performed by MTRAP [67]. The scale bar (0.05) represents the relative branch length. (b) Alignment of the MCA protein family from Arabidopsis thaliana, tobacco (Nicotiana tabacum), rice (Oryza sativa), soybean (Glycine max), flax (Linum usitatissimum), cassava (Manihot esculenta), poplar (Populus trichocarpa), eucalyptus (Eucalyptus grandis), spruce (Picea sitchensis), lycophyte (Selaginella moellendorffii), and moss (Physcomitrella patens). The black or gray boxes indicate identical or conserved residues in the whole

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Cell wall metabolism Mechanical stress Cl

–

H2O2 Ca2+

MSL9 and MSL10

O2

O2–

•

MCAs EFL CC

PM ?

Rbohs

EFL CC

N

EF

EF

FAD NADPH

C

?

CH2–SH

MSL2 and MSL3 Ca2+ sensors(?)

Redox signaling

?

TCH3(= CML12), NtERF4 Plasd

TF (?)

Gene TRENDS in Plant Science

Figure 2. Intracellular localization and possible roles of MscS-like (MSL) and mid1-complementing activity (MCA) proteins. How the mechanical stress is sensed by MSLs and MCAs is unknown. Question marks represent uncertainties to be clarified by future studies. For MSL9 and MSL10, Cl– permeability was shown by patch-clamp studies [26,28], Cl– efflux-induced membrane depolarization is speculated to activate voltage-dependent channels, such as Shaker-type K+ channels and Ca2+ channels, leading to the release of osmolytes and the generation of a Ca2+ signal, respectively [28]. MCAs mediate Ca2+ influx to induce Ca2+-dependent cellular responses, including expression of mechanical stimulus-inducible genes [e.g., TOUCH 3 (TCH3/CML12) and tobacco ethylene response factor 4 (NtERF4)] and activation of respiratory burst oxidase homologs (Rbohs) through the Ca2+-binding EF-hand motifs, leading to deliberate reactive oxygen species (ROS) production. ROS generated in the apoplast provide substrates for peroxidases to affect cell wall metabolism, and could also be transported to the cytosol by plasma membrane (PM) aquaporins to modify cysteine residues of target proteins to promote redox signaling. Ca2+-binding regulatory proteins (Ca2+-sensors) and transcription factors (TFs) have not yet been identified, except for TCH3/CML12 [14]. EFL and CC represent EF-hand-like and coiled-coil motifs, respectively.

limitation [15]. The growth defects in mutants with reduced levels of MCA proteins may have been due to reduced Ca2+ uptake, resulting in a low Ca2+:Mg2+ ratio. MCA-overexpressing plants showed more obvious defects in development, with short stems, small rosettes, no petals, and shrunken seed pods [14,37]. Cultured plant cells overexpressing MCA proteins also showed restricted cell proliferation [15,38]. Similar severe growth retardation has also been reported for plants and cells overexpressing other putative Ca2+-permeable channels [48,49]. [Ca2+]cyt plays critical roles in developmental and morphogenetic processes, such as cell division and elongation, formation of polarity, and cell differentiation [50]. Overproduction of MCA proteins may provoke excessive stressinduced [Ca2+]cyt increases or disturb Ca2+ homeostasis, leading to impairment of growth or differentiation. The level of tobacco NtMCA2 transcripts fluctuated throughout the cell cycle in synchronous-cultured tobacco

BY-2 cells, reaching its maximum in the G1 phase, where cell elongation and expansion occurred, and its minimum in the S phase [38]. Arabidopsis MCA1 also showed a peak in expression in the G1 phase [51]. Cell elongation and expansion are associated with ion uptake and mechanical stimulation at the plasma membrane. Ca2+–ROS signaling network in the osmotic and mechanical responses In various plant cells, hypo-osmotic shock as a mechanical stimulus triggered NADPH oxidase-mediated production of ROS following Ca2+ influx [15,52–54]. In Arabidopsis and rice, respiratory burst oxidase homologs (Rbohs) showed ROS-producing activity that was synergistically activated by binding of Ca2+ to the EF-hand motifs in the N-terminal cytosolic domain and phosphorylation [55–58]. ROS-activated Ca2+-permeable channels and Rbohs have been suggested to constitute a positive feedback mechanism

sequence in the alignment, respectively. The double line indicates a putative functional region of MCA proteins. Bars indicate the position of a potential transmembrane (TM) segment, an EF-hand-like motif and a coiled-coil region. A cysteine-rich region of the C terminus is similar to the Plac8 motif found in plant and animal proteins. These motifs were predicted by the Network Protein Sequence Analysis (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html) and the online program SOSUI (http:// bp.nuap.nagoya-u.ac.jp/sosui/). The amino acid sequences of the MCA protein family were collected using the following databases: Phytozome (http://www.phytozome.net/), the Gene Index Project (http://compbio.dfci.harvard.edu/tgi/plant.html), Lotus japonicus (http://www.kazusa.or.jp/lotus/blast.html) from the Kazusa DNA Research Institute, and NCBI (http://www.ncbi.nlm.nih.gov/). Full binominal names are as follows: Aquilegia coerulea (AcoGoldSmith_v1.005671m.g), Arabidopsis lyrata (MCA1; Al491044, MCA2; Al480672), Arabidopsis thaliana (MCA1; At4G35920, MCA2; At2G17780), Brachypodium distachyon (Bradi1g74650), Brassica napus (EV190704), Brassica rapa (MCA1; Bra011649, MCA2; Bra037250), Capsella rubella (MCA1; Carubv10004901m.g, MCA2; Carubv10013809m.g), Carica papaya (evm.TU.supercontig_6.294), Citrus clementina (clementine0.9_011316m.g), Citrus sinensis (orange1.1g014734m.g), Glycine max (MCA1; Glyma01g36860, MCA2; Glyma11g08430, MCA3; Glyma02g05130, MCA4; Glyma16g23240), Cucumis sativus (Cucsa.322780), Eucalyptus grandis (Eucgr.I02323), Hordeum vulgare (FLbaf94d24), Linum usitatissimum (Lus10025955.g), Lotus japonicus (chr2.CM0272.840.r2.m), Malus domestica (MDP0000258953), Manihot esculenta (MCA1; cassava4.1_008447m.g, MCA2; cassava4.1_008443m.g), Medicago truncatula (MCA1; Medtr5g022570, MCA2; Medtr8g089340), Nicotiana tabacum (MCA1; AB622811, MCA2; AB622812), Oryza sativa (AB601973), Phaseolus vulgaris (MCA1; Phvul.002G106200, MCA2; Phvul.003G265800), Physcomitrella patens (MCA1; XP_001768983, MCA2; XP_001785848), Picea sitchensis (DR536368), Populus trichocarpa (POPTR_0005s11200), Prunus persica (ppa006351m.g), Selaginella moellendorffii (Smo93428), Setaria italica (SiPROV010555m.g), Solanum lycopersicum (C02SLm0020N19.1_0000003), Sorghum bicolor (Sb01g046630), Thellungiella halophila (MCA1; Thhalv10025310m.g, MCA2; Thhalv10023167m.g), Triticum aestivum (CJ663875), and Zea mays (GRMZM2G027821).

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Review to enhance both Ca2+ and ROS signals [56,57]. A functional NADPH oxidase, AtRbohC/RHD2, affected mechanical stress-induced ROS generation, and regulated root hair elongation in a Ca2+-dependent manner [16,56]. It is plausible that MCAs play a role in the regulation of mechanical responses via signal transduction pathways dependent on Ca2+ and ROS. Overexpression of rice OsMCA1 enhanced NADPH oxidase-mediated ROS production in cultured cells [15]. Both Arabidopsis MCA1 and ROS generated by AtRbohD and/or AtRbohF were suggested to be involved in regulating osmosensitive metabolic changes [59]. Apoplastic ROS production plays critical roles in the modification of cell wall metabolism, including crosslinking to regulate its rigidity [60,61]. MCA1 was required for cell wall damage-induced deposition of lignin, which confers mechanical strength to the cell wall [62]. Expression of the Arabidopsis touch-inducible gene TOUCH 3 (TCH3/CML12), which encodes a calmodulinlike protein, and tobacco ethylene response factor 4 (NtERF4), which is responsive to mechanical stress, are upregulated in the overexpressors of MCAs in Arabidopsis plants and tobacco BY-2 cells [14,38]. These findings suggest the following initial plasma membrane signaling in response to osmotic and mechanical stimulation: activation of the plasma membrane MS Ca2+-permeable channels, such as the MCA protein family, induces the influx of Ca2+, leading to activation of Rboh(s) through the Ca2+-binding EF-hand motifs to generate ROS (Figure 2). The signaling network dependent on Ca2+ and ROS may play a crucial role in regulating downstream events, such as regulation of the mechanical properties of the cell wall, metabolic changes, and gene expression. ROS generated in the apoplast provide substrates for peroxidases to affect cell wall metabolism, such as crosslinking, and are also transported to the cytosol by plasma membrane aquaporins [63] to modify cysteine residues of target proteins to promote redox signaling [64]. Concluding remarks and future perspectives Mechanical stresses greatly affect plant growth and development. To better understand such general phenomena of life, it is crucial to understand the molecular mechanisms of mechanosensing and mechanotransduction through elucidating the structure and function of MS channels and their interacting molecules [9,65,66]. Recent molecular and genetic studies represent a new era for studies on MS channels and their candidates, including MSLs and MCAs. For example, molecular characterization of MSLs and MCAs would facilitate determination of the structural basis of mechanosensory machineries that might comprise molecules in the cell wall and/or extracellular matrix, the plasma membrane, intracellular regulatory proteins, cytoskeletons, and MS channel itself. Importantly, such studies would help elucidate asyet-unknown mechanisms of how plant MS channels sense a variety of mechanical stresses. MS channels have been suggested to be mechanosensors that transduce the shear forces in the cell wall–membrane–cytoskeleton system [66]. We anticipate that the study of MSLs and MCAs will help decisively to unravel the mechanisms involved. 232

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Both extracellular and intracellular mechanical stresses can be sensed via various types of MS channel. Indeed, some MSLs are specific to organellar membranes, whereas others are localized in the plasma membrane. So far, plant MSLs have been studied only in Arabidopsis. It will be of great interest to investigate the MSLs of other plant species, including monocots. By contrast, all the MCA proteins examined to date are localized in the plasma membrane and are involved in the hypo-osmotic response and probably also in the touch response. Other physical and chemical stresses, including gravity, might also activate MS channels. Therefore, not only MSLs and MCAs, but also the plant Piezo proteins should provide further clues for understanding how plants perceive and respond to such a variety of stresses. Acknowledgments We would like to thank Kazuo Shinozaki (RIKEN Plant Science Center, Yokohama, Japan) for his critical reading of the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Area No. 21026009 (to H.I.), 21117516 (to K.K.), 23117718 (to K.K.), and No. 23120509 (to H.I.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, a Grant-in-Aid for Scientific Research B No. 19370023 (to K.K.), 21370017 (to H.I.) and 23380027 (to K.K.) from the Japan Society for the Promotion of Science (JSPS), and a Grant-in-Aid for JSPS Fellows No. 10J02008 (to Y.N.).

References 1 Darwin, C. (1865) On the movements and habits of climbing plants. J. Linn. Soc. Lond. (Bot.) 9, 1–118 2 Jaffe, M.J. (1973) Thigmomorphogenesis: the response of plant growth and development to mechanical stimulation. Planta 114, 143–157 3 Chehab, E.W. et al. (2009) Thigmomorphogenesis: a complex plant response to mechano-stimulation. J. Exp. Bot. 60, 43–56 4 Braam, J. (2005) In touch: plant responses to mechanical stimuli. New Phytol. 165, 373–389 5 Escalante-Perez, M. et al. (2011) A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap. Proc. Natl. Acad. Sci. U.S.A. 108, 15492–15497 6 Trewavas, A. and Knight, M. (1994) Mechanical signalling, calcium and plant form. Plant Mol. Biol. 26, 1329–1341 7 Jaffe, M.J. et al. (2002) Thigmo responses in plants and fungi. Am. J. Bot. 89, 375–382 8 Telewski, F.W. (2006) A unified hypothesis of mechanoperception in plants. Am. J. Bot. 93, 1466–1476 9 Monshausen, G.B. and Gilroy, S. (2009) Feeling green: mechanosensing in plants. Trends Cell Biol. 19, 228–235 10 Knight, M.R. et al. (1991) Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352, 524–526 11 Plieth, C. and Trewavas, A.J. (2002) Reorientation of seedlings in the earth’s gravitational field induces cytosolic calcium transients. Plant Physiol. 129, 786–796 12 Toyota, M. et al. (2008) Cytoplasmic calcium increases in response to changes in the gravity vector in hypocotyls and petioles of Arabidopsis seedlings. Plant Physiol. 146, 505–514 13 Martinac, B. et al. (1990) Mechanosensitive ion channels of E. coli activated by amphipaths. Nature 348, 261–263 14 Nakagawa, Y. et al. (2007) Arabidopsis plasma membrane protein crucial for Ca2+ influx and touch sensing in roots. Proc. Natl. Acad. Sci. U.S.A. 104, 3639–3644 15 Kurusu, T. et al. (2012) Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+ influx and modulates generation of reactive oxygen species in cultured rice cells. BMC Plant Biol. http://dx.doi.org/10.1186/1471-2229-12-11 16 Monshausen, G.B. et al. (2009) Ca2+ regulates reactive oxygen species production and pH during mechanosensing in Arabidopsis roots. Plant Cell 21, 2341–2356 17 Braam, J. and Davis, R.W. (1990) Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes in Arabidopsis. Cell 60, 357–364

Review 18 Lee, J.S. et al. (1983) Reversible loss of gravitropic sensitivity in maize roots after tip application of calcium chelators. Science 220, 1375–1376 19 Fujita, Y. et al. (2011) ABA-mediated transcriptional regulation in response to osmotic stress in plants. J. Plant Res. 124, 509–525 20 Pivetti, C.D. et al. (2003) Two families of mechanosensitive channel proteins. Microbiol. Mol. Biol. Rev. 67, 66–85 21 Haswell, E.S. et al. (2011) Mechanosensitive channels: what can they do and how do they do it? Structure 19, 1356–1369 22 Hurst, A.C. et al. (2008) MscS, the bacterial mechanosensitive channel of small conductance. Int. J. Biochem. Cell Biol. 40, 581–585 23 Jensen, G.S. and Haswell, E.S. (2012) Functional analysis of conserved motifs in the mechanosensitive channel homolog MscS-like2 from Arabidopsis thaliana. PLoS ONE 7, e40336 24 Haswell, E.S. (2007) MscS-like proteins in plants. Curr. Top. Membr. 58, 329–359 25 Nakayama, Y. et al. (2012) Organellar mechanosensitive channels in fission yeast regulate the hypo–osmotic shock response. Nat. Commun. http://dx.doi.org/10.1038/ncomms2014 26 Haswell, E.S. et al. (2008) Two MscS homologs provide mechanosensitive channel activities in the Arabidopsis root. Curr. Biol. 18, 730–734 27 Peyronnet, R. et al. (2008) AtMSL9 and AtMSL10: sensors of plasma membrane tension in Arabidopsis roots. Plant Signal. Behav. 3, 726–729 28 Maksaev, G. and Haswell, E.S. (2012) MscS-Like10 is a stretchactivated ion channel from Arabidopsis thaliana with a preference for anions. Proc. Natl. Acad. Sci. U.S.A. 109, 19015–19020 29 Nakayama, Y. et al. (2007) Molecular and electrophysiological characterization of a mechanosensitive channel expressed in the chloroplasts of Chlamydomonas. Proc. Natl. Acad. Sci. U.S.A. 104, 5883–5888 30 Haswell, E.S. and Meyerowitz, E.M. (2006) MscS-like proteins control plastid size and shape in Arabidopsis thaliana. Curr. Biol. 16, 1–11 31 Veley, K.M. et al. (2012) Mechanosensitive channels protect plastids from hypoosmotic stress during normal plant growth. Curr. Biol. 22, 408–413 32 Wilson, M.E. et al. (2011) Two mechanosensitive channel homologs influence division ring placement in Arabidopsis chloroplasts. Plant Cell 23, 2939–2949 33 Coste, B. et al. (2012) Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483, 176–181 34 Kim, S.E. et al. (2012) The role of Drosophila Piezo in mechanical nociception. Nature 483, 209–212 35 Coste, B. et al. (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330, 55–60 36 Kanzaki, M. et al. (1999) Molecular identification of a eukaryotic, stretch-activated nonselective cation channel. Science 285, 882–886 [report clarification in Science (2000) 288, 1374] 37 Yamanaka, T. et al. (2010) MCA1 and MCA2 that mediate Ca2+ uptake have distinct and overlapping roles in Arabidopsis. Plant Physiol. 152, 1284–1296 38 Kurusu, T. et al. (2012) Involvement of the putative Ca2+-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca2+ uptake, Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells. J. Plant Res. 125, 555–568 39 Kurusu, T. et al. (2012) Roles of a putative mechanosensitive plasma membrane Ca2+-permeable channel OsMCA1 in generation of reactive oxygen species and hypo-osmotic signaling in rice. Plant Signal. Behav. 7, 796–798 40 Nakano, M. et al. (2011) Determination of structural regions important for Ca2+ uptake activity in Arabidopsis MCA1 and MCA2 expressed in yeast. Plant Cell Physiol. 52, 1915–1930 41 Galaviz-Hernandez, C. et al. (2003) Plac8 and Plac9, novel placentalenriched genes identified through microarray analysis. Gene 309, 81–89 42 Song, W.Y. et al. (2004) A novel family of cys-rich membrane proteins mediates cadmium resistance in Arabidopsis. Plant Physiol. 135, 1027–1039

Trends in Plant Science April 2013, Vol. 18, No. 4

43 Tanaka, S. et al. (2007) A novel gene with antisalt and anticadmium stress activities from a halotolerant marine green alga Chlamydomonas sp W80. FEMS Microbiol. Lett. 271, 48–52 44 Iomini, C. et al. (2006) Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas. Curr. Biol. 16, 1147–1153 45 Lefebvre, B. et al. (2007) Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system. Plant Physiol. 144, 402–418 46 Furuichi, T. et al. (2012) Expression of Arabidopsis MCA1 enhanced mechanosensitive channel activity in the Xenopus laevis oocyte plasma membrane. Plant Signal. Behav. 7, 1022–1026 47 Ji, X.M. et al. (2005) Tissue-specific expression and drought responsiveness of cell-wall invertase genes of rice at flowering. Plant Mol. Biol. 59, 945–964 48 Kim, S.A. et al. (2001) Overexpression of the AtGluR2 gene encoding an Arabidopsis homolog of mammalian glutamate receptors impairs calcium utilization and sensitivity to ionic stress in transgenic plants. Plant Cell Physiol. 42, 74–84 49 Kurusu, T. et al. (2004) Identification of a putative voltage-gated Ca2+permeable channel (OsTPC1) involved in Ca2+ influx and regulation of growth and development in rice. Plant Cell Physiol. 45, 693–702 50 White, P.J. and Broadley, M.R. (2003) Calcium in plants. Ann. Bot. 92, 487–511 51 Menges, M. et al. (2002) Cell cycle-regulated gene expression in Arabidopsis. J. Biol. Chem. 277, 41987–42002 52 Beffagna, N. et al. (2005) Modulation of reactive oxygen species production during osmotic stress in Arabidopsis thaliana cultured cells: involvement of the plasma membrane Ca2+-ATPase and H+ATPase. Plant Cell Physiol. 46, 1326–1339 53 Hayashi, T. et al. (2006) Ca2+ transient induced by extracellular changes in osmotic pressure in Arabidopsis leaves: differential involvement of cell wall-plasma membrane adhesion. Plant Cell Environ. 29, 661–672 54 Cazale, A.C. et al. (1998) Oxidative burst and hypoosmotic stress in tobacco cell suspensions. Plant Physiol. 116, 659–669 55 Ogasawara, Y. et al. (2008) Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation. J. Biol. Chem. 283, 8885–8892 56 Takeda, S. et al. (2008) Local positive feedback regulation determines cell shape in root hair cells. Science 319, 1241–1244 57 Kimura, S. et al. (2012) Protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca2+ and reactive oxygen species. Biochim. Biophys. Acta 1823, 398–405 58 Takahashi, S. et al. (2012) Reactive oxygen species production and activation mechanism of the rice NADPH oxidase OsRbohB. J. Biochem. 152, 37–43 59 Wormit, A. et al. (2012) Osmosensitive changes of carbohydrate metabolism in response to cellulose biosynthesis inhibition. Plant Physiol. 159, 105–117 60 Brady, J.D. and Fry, S.C. (1997) Formation of di-isodityrosine and loss of isodityrosine in the cell walls of tomato cell-suspension cultures treated with fungal elicitors or H2O2. Plant Physiol. 115, 87–92 61 O’Brien, J.A. et al. (2012) Reactive oxygen species and their role in plant defence and cell wall metabolism. Planta 236, 765–779 62 Denness, L. et al. (2011) Cell wall damage-induced lignin biosynthesis is regulated by a reactive oxygen species- and jasmonic acid-dependent process in Arabidopsis. Plant Physiol. 156, 1364–1374 63 Dynowski, M. et al. (2008) Plant plasma membrane water channels conduct the signalling molecule H2O2. Biochem. J. 414, 53–61 64 Spoel, S.H. and Loake, G.J. (2011) Redox-based protein modifications: the missing link in plant immune signalling. Curr. Opin. Plant Biol. 14, 358–364 65 Perbal, G. and Driss-Ecole, D. (2003) Mechanotransduction in gravisensing cells. Trends Plant Sci. 8, 498–504 66 Ding, J.P. and Pickard, B.G. (1993) Mechanosensory calcium-selective cation channels in epidermal cells. Plant J. 3, 83–110 67 Hara, T. et al. (2010) MTRAP: pairwise sequence alignment algorithm by a new measure based on transition probability between two consecutive pairs of residues. BMC Bioinformatics http://dx.doi.org/ 10.1186/1471-2105-11-235

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