Salt-dependent regulation of a CNG channel subfamily in Arabidopsis

Background  

In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies.     

CNGCs: prime targets of plant cyclic nucleotide signalling?

Cyclic nucleotide-gated channels (CNGCs) are a recently identified family of plant ion channels. They show a high degree of similarity to Shaker-type voltage-gated channels and contain a C-terminal cyclic nucleotide-binding domain with an overlapping calmodulin-binding domain. Heterologously expressed plant CNGCs show activation by cyclic nucleotides and permeability to monovalent and divalent cations. In plants, downstream effectors of cyclic nucleotide signals have so far remained obscure, and CNGCs might be their prime targets. The unique position of CNGCs as ligand-gated Ca(2+)-permeable channels suggests that they function at key sites where cyclic nucleotide and Ca(2+) signalling pathways interact. Such processes include plant defence responses, and two recently characterized Arabidopsis mutants in CNGC genes indeed show altered pathogen responses.     

Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels.

Cyclic nucleotide gated cation channels (CNGCs) are a large (20 genes in Arabidopsis thaliana) family of plant ligand gated (i.e. cyclic nucleotides activate currents) ion channels, however, little is known about their functional properties. One reason for this is the recalcitrance of plant CNGC expression in heterologous systems amenable to patch clamp studies. Here, we show results demonstrating the efficacy of using growth of a K+ uptake-deficient yeast (trk1,2) as a functional assay of CNGCs as inwardly-conducting cell membrane cation (K+) transporters. Prior work demonstrated that trk1,2 is hypersensitive to the antibiotic hygromycin (hyg) and that expression of an inwardly conducting K+ transporter suppresses hyg hypersensitivity. We find that increasing [hyg] in solid YPD medium inhibits trk1,2 growth around a filter disk saturated with 3 M K+. Northern analysis indicated that message is transcribed in trk1,2 transformed with the CNGC coding sequences. Confocal imaging of yeast expressing CNGC-fluorescent fusion proteins indicated channel targeting to the cell membrane. Trk1,2 expressing several plant CNGCs grown in the presence of hyg demonstrated (a) greater growth than trk1,2 transformed with empty plasmid, and (b) enhanced growth when cAMP was added to the medium. Alternatively, cAMP inhibited growth of yeast transformed with either the empty plasmid, or the plant K+ channel KAT1; this channel is not a CNGC. Growth of trk1,2 was dependent on filter disk [K+]; suggesting that complementation of hyg hypersensitivity due to presence of a functional plant CNGC was dependent on K+ movement into the cytosol. We conclude that plant CNGC functional characterization can be facilitated by this assay system.     

Cyclic Nucleotide Gated Channels 7 and 8 Are Essential for Male Reproductive Fertility

The Arabidopsis thaliana genome contains 20 CNGCs, which are proposed to encode cyclic nucleotide gated, non-selective, Ca²⁺-permeable ion channels. CNGC7 and CNGC8 are the two most similar with 74% protein sequence identity, and both genes are preferentially expressed in pollen. Two independent loss-of-function T-DNA insertions were identified for both genes and used to generate plant lines in which only one of the two alleles was segregating (e.g., cngc7-1+/−/cngc8-2−/− and cngc7-3−/−/cngc8-1+/−). While normal pollen transmission was observed for single gene mutations, pollen harboring mutations in both cngc7 and 8 were found to be male sterile (transmission efficiency reduced by more than 3000-fold). Pollen grains harboring T-DNA disruptions of both cngc7 and 8 displayed a high frequency of bursting when germinated in vitro. The male sterile defect could be rescued through pollen expression of a CNGC7 or 8 transgene including a CNGC7 with an N-terminal GFP-tag. However, rescue efficiencies were reduced ∼10-fold when the CNGC7 or 8 included an F to W substitution (F589W and F624W, respectively) at the junction between the putative cyclic nucleotide binding-site and the calmodulin binding-site, identifying this junction as important for proper functioning of a plant CNGC. Using confocal microscopy, GFP-CNGC7 was found to preferentially localize to the plasma membrane at the flanks of the growing tip. Together these results indicate that CNGC7 and 8 are at least partially redundant and provide an essential function at the initiation of pollen tube tip growth.     

Expression of plant cyclic nucleotide-gated cation channels in yeast

The functional properties of inwardly conducting plant cyclic nucleotide-gated cation channels (CNGCs) have not been thoroughly characterized due in part to the recalcitrance of their functional expression in heterologous systems. Here, K+ uptake-deficient mutants of yeast (trk1,2) and Escherichia coli (LB650), as well as the Ca2+-uptake yeast mutant mid1,cch1, were used for functional characterization of Arabidopsis thaliana CNGCs, with the aim of identifying some of the cultural and physiological conditions that impact on plant CNGC function in heterologous systems. Use of the Ca2+-uptake yeast mutant provided the first evidence consistent with Ca2+ conduction by the A. thaliana CNGC AtCNGC1. Expression of AtCNGC1 in LB650 demonstrated that mutants of Escherichia coli (which has no endogenous calmodulin) can also be used to study functional properties of CNGCs. Expression of AtCNGC2 and AtCNGC4 enhanced growth of trk1,2 in the presence of hygromycin; AtCNGC1 has less of an effect. Deletion of the AtCNGC1 calmodulin-binding domain enhanced growth of trk1,2 at low external K+ but not of LB650, suggesting that yeast calmodulin may bind to, and down-regulate this plant channel. In vitro binding studies confirmed this physical interaction. Northern analysis, green fluorescent protein:AtCNGC1 fusion protein expression, as well as an antibody raised against a portion of AtCNGC1, were used to monitor expression of AtCNGC1 and deletion constructs of the channel in the heterologous systems. In the presence of the activating ligand cAMP, expression of the AtCNGC1 channel with the calmodulin-binding domain deleted increased intracellular [K+] of trk1,2. Trk1,2 is hypersensitive to the toxic cations spermine, tetramethylamine, and NH4+. These compounds, as well as amiloride, inhibited trk1,2 growth and thereby improved the efficacy of this yeast mutant as a heterologous expression system for CNGCs. In addition to characterizing mutants of yeast and E. coli as assay systems for plant CNGCs, work presented in this report demonstrates, for the first time, that a plant CNGC can retain ion channel function despite (partial) deletion of its calmodulin-binding domain and that yeast calmodulin can bind to and possibly down-regulate a plant CNGC.     

Molecular characterization and expression of cyclic nucleotide gated ion channels 19 and 20 in Arabidopsis thaliana for their potential role in salt stress

Cyclic nucleotide gated ion channels (CNGCs) in plants have very important role in signaling and development. The study reports role of CNGC19 and CNGC20 in salt stress in A. thaliana. In-silico, genome wide analysis showed that CNGC19 and CNGC20 are related to salt stress with maximum expression after 6 h in A. thaliana. The position of inserted T-DNA was determined (in-vivo) through TAIL-PCR for activation tagged mutants of CNGC19 and CNGC20 under salt stress. The expression of AtCNGC19 and AtCNGC20 after cloning under 35S promoter of expression vectors pBCH1 and pEarleyGate100 was determined in A. thaliana by real-time PCR analysis. Genome wide analysis showed that AtCNGC11 had lowest and AtCNGC20 highest molecular weight as well as number of amino acid residues. In-vivo expression of AtCNGC19 and AtCNGC20 was enhanced through T-DNA insertion and 35S promoter in over-expressed plants under high salt concentration. AtCNGC19 was activated twice in control and about five times under 150 mM NaCl stress level, and expression value was also higher than AtCNGC20. Phenotypically, over-expressed plants and calli were healthier while knock-out plants and calli showed retarded growth under salinity stress. The study provides new insight for the role of AtCNGC19 and AtCNGC20 under salt stress regulation in A. thaliana and will be helpful for improvement of crop plants for salt stress to combat food shortage and security.     

CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling

Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca²⁺-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca²⁺ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca²⁺ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca²⁺ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS–YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca²⁺ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

One-sentence summary: The immunity-related Ca²⁺ channel CYCLIC NUCLEOTIDE-GATED CHANNEL 2 modulates auxin homeostasis and balances cellular auxin perception by influencing auxin biosynthesis.     

Arabidopsis thaliana cyclic nucleotide gated channel 3 forms a non-selective ion transporter involved in germination and cation transport

The Arabidopsis thaliana genome contains 20 cyclic nucleotide gated channel (CNGC) genes encoding putative non-selective ion channels. Classical and reverse genetic approaches have revealed that two members of this family (CNGC2 and CNGC4) play a role in plant defence responses whereas CNGC1 and CNGC10 may participate in heavy metal and cation transport. Yet, it remains to be resolved how the ion transport attributes of CNGCs are integrated into their physiological function. In this study, CNGC3 is characterized through heterologous expression, GUS- and GFP-reporter gene fusions, and by adopting a reverse genetics approach. A CNGC3-GFP fusion protein shows that it is mainly targeted to the plasma membrane. Promoter GUS studies demonstrate CNGC3 expression predominantly in the cortical and epidermal root cells, but also a ubiquitous presence in shoot tissues. Expression of CNGC3 in yeast indicates it can function as a Na(+) uptake and a K(+) uptake mechanism. cngc3 null mutations decreased seed germination in the presence of NaCl but not KCl. Relative to the wild type, mutant seedling growth is more resistant to the presence of toxic concentrations of NaCl and KCl. The ionic composition and ion uptake characteristics of wild-type and mutant seedlings suggests that the growth advantage in these conditions may be due to restricted ion influx in mutant plants, and that CNGC3 functions in the non-selective uptake of monovalent cations in Arabidopsis root tissue.     

Cyclic nucleotide-gated ion channel gene family in rice,identification, characterization and experimental analysis of expression response to plant hormones,biotic and abiotic stresses

Background

Cyclic nucleotide-gated channels (CNGCs) are Ca²⁺-permeable cation transport channels, which are present in both animal and plant systems. They have been implicated in the uptake of both essential and toxic cations, Ca²⁺ signaling, pathogen defense, and thermotolerance in plants. To date there has not been a genome-wide overview of the CNGC gene family in any economically important crop, including rice (Oryza sativa L.). There is an urgent need for a thorough genome-wide analysis and experimental verification of this gene family in rice.

Results

In this study, a total of 16 full length rice CNGC genes distributed on chromosomes 1–6, 9 and 12, were identified by employing comprehensive bioinformatics analyses. Based on phylogeny, the family of OsCNGCs was classified into four major groups (I-IV) and two sub-groups (IV-A and IV- B). Likewise, the CNGCs from all plant lineages clustered into four groups (I-IV), where group II was conserved in all land plants. Gene duplication analysis revealed that both chromosomal segmentation (OsCNGC1 and 2, 10 and 11, 15 and 16) and tandem duplications (OsCNGC1 and 2) significantly contributed to the expansion of this gene family. Motif composition and protein sequence analysis revealed that the CNGC specific domain “cyclic nucleotide-binding domain (CNBD)” comprises a “phosphate binding cassette” (PBC) and a “hinge” region that is highly conserved among the OsCNGCs. In addition, OsCNGC proteins also contain various other functional motifs and post-translational modification sites. We successively built a stringent motif: (LI-X(2)-[GS]-X-[FV]-X-G-[1]-ELL-X-W-X(12,22)-SA-X(2)-T-X(7)-[EQ]-AF-X-L) that recognizes the rice CNGCs specifically. Prediction of cis-acting regulatory elements in 5′ upstream sequences and expression analyses through quantitative qPCR demonstrated that OsCNGC genes were highly responsive to multiple stimuli including hormonal (abscisic acid, indoleacetic acid, kinetin and ethylene), biotic (Pseudomonas fuscovaginae and Xanthomonas oryzae pv. oryzae) and abiotic (cold) stress.

Conclusions

There are 16 CNGC genes in rice, which were probably expanded through chromosomal segmentation and tandem duplications and comprise a PBC and a “hinge” region in the CNBD domain, featured by a stringent motif. The various cis-acting regulatory elements in the upstream sequences may be responsible for responding to multiple stimuli, including hormonal, biotic and abiotic stresses.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-853) contains supplementary material, which is available to authorized users.     

The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells

Background  

The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots.     

A Suppressor Screen of the Chimeric AtCNGC11/12 Reveals Residues Important for Intersubunit Interactions of Cyclic Nucleotide-Gated Ion Channels

To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22 (cpr22). Based on computational modeling, two new alleles, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), were identified as counterparts of human CNGA3 (a human CNGC) mutants. Both mutants lost all cpr22-mediated phenotypes. Transient expression in Nicotiana benthamiana as well as functional complementation in yeast (Saccharomyces cerevisiae) showed that both AtCNGC11/12:G459R and AtCNGC11/12:R381H have alterations in their channel function. Site-directed mutagenesis coupled with fast-protein liquid chromatography using recombinantly expressed C-terminal peptides indicated that both mutations significantly influence subunit stoichiometry to form multimeric channels. This observation was confirmed by bimolecular fluorescence complementation in planta. Taken together, we have identified two residues that are likely important for subunit interaction for plant CNGCs and likely for animal CNGCs as well.Cyclic nucleotide-gated ion channels (CNGCs) were first discovered in retinal photoreceptors and olfactory sensory neurons (Zagotta and Siegelbaum, 1996; Kaupp and Seifert, 2002). CNGCs play crucial roles for the signal transduction in these neurons that are excited by photons and odorants, respectively. In mammalian genomes, six CNGC genes have been found and named CNGA1 to CNGA4, CNGB1, and CNGB3 (Kaupp and Seifert, 2002). It has been reported that in mammalian cells, CNGCs function as heterotetramers that are composed of A and B subunits with cell-specific stoichiometry (Kaupp and Seifert, 2002; Cukkemane et al., 2011). For example, CNGCs in rod photoreceptors are composed of three A1 subunits and one B1a subunit, whereas in cone photoreceptors, they are believed to be composed of two A3 and two B3 subunits (Zhong et al., 2002; Peng et al., 2004). The structure of each subunit is similar to that of the voltage-gated K⁺-selective ion channel (Shaker) proteins, including a cytoplasmic N terminus, six membrane-spanning regions (S1–S6), a pore domain located between S5 and S6, and a cytoplasmic C terminus (Zagotta and Siegelbaum, 1996). However, CNGCs are only weakly voltage dependent and are opened by the direct binding of cyclic nucleotides (cAMP and cGMP), which are universally important secondary messengers that control diverse cellular responses (Fesenko et al., 1985). The cytoplasmic C terminus contains a cyclic nucleotide-binding domain (CNBD) and a C-linker region that connects the CNBD to the S6 domain. CNGC activity is also regulated by feedback inhibitory mechanisms involving the Ca²⁺ sensor protein, calmodulin (CaM). CaM-binding sites in animal CNGCs have been found in various regions of both the C- and N-terminal domains (Ungerer et al., 2011). It has been reported that the subunit composition has significant influence on the mode of CaM-mediated regulation (Kramer and Siegelbaum, 1992; Bradley et al., 2004; Song et al., 2008).On the other hand, plant CNGCs have only been investigated much more recently. The first plant CNGC, HvCBT1, was identified as a CaM-binding transporter protein in barley (Hordeum vulgare; Schuurink et al., 1998). Subsequently, several CNGCs were identified from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum; Arazi et al., 1999; Köhler and Neuhaus, 1998; Köhler et al., 1999). Interestingly, the Arabidopsis genome sequencing project identified a large family comprising 20 members (AtCNGC1–AtCNGC20), indicating a significant expansion of Arabidopsis CNGCs that suggests a higher level of diversity and functional importance in plants (Mäser et al., 2001). To date, possible biological functions of Arabidopsis CNGCs in development, ion homeostasis, thermal sensing, as well as pathogen resistance have been reported (Kaplan et al., 2007; Chin et al., 2009; Dietrich et al., 2010; Moeder et al., 2011; Finka et al., 2012). With respect to structure, plant CNGCs are believed to have a similar architecture to their animal counterparts (Chin et al., 2009). However, only a handful of studies on the structure-function analysis of plant CNGCs have been published so far, and this field is still very much in its infancy (Hua et al., 2003; Bridges et al., 2005; Kaplan et al., 2007; Baxter et al., 2008; Chin et al., 2010).Previously, we have reported two functionally important residues in plant CNGCs (Baxter et al., 2008; Chin et al., 2010). These residues were discovered using a suppressor screen of the rare gain-of-function Arabidopsis mutant constitutive expresser of PR genes22 (cpr22; Yoshioka et al., 2006). The cpr22 mutant, which has a deletion between AtCNGC11 and AtCNGC12 resulting in a novel but functional chimeric CNGC (AtCNGC11/12), exhibits multiple resistance responses without pathogen infection in the hemizygous state and conditional lethality in the homozygous state (Yoshioka et al., 2001, 2006; Moeder et al., 2011). It has been reported that the cpr22 phenotype is attributable to the expression of AtCNGC11/12 and its channel activity (Yoshioka et al., 2006; Baxter et al., 2008), thereby making the suppressor screen an invaluable tool for identifying intragenic mutants to further elucidate the structure-function relationship of plant CNGCs (Baxter et al., 2008; Chin et al., 2010).In this study, we describe a total of 29 mutant alleles of AtCNGC11/12, including the three previously published alleles (Baxter et al., 2008; Chin at al., 2010), and compare their predicted three-dimensional structural positions with equivalent mutations of a human CNGC, CNGA3. In this analysis, two AtCNGC11/12 mutations emerged as counterparts of human mutations (Wissinger et al., 2001). Both the AtCNGC11/12 as well as the human CNGA3 mutations were computationally predicted to affect intersubunit interactions. This prediction was experimentally validated by size-exclusion chromatography (FPLC) as well as bimolecular fluorescence complementation (BiFC) in combination with site-direct mutagenesis using recombinant C-terminal peptides.     

Synthesis of new pyrazoles and pyrozolo [3,4-b] pyridines as anti-inflammatory agents by inhibition of COX-2 enzyme

New pyrazoles and pyrazolo[3,4-b] pyridines were synthesized and their structure was confirmed by elemental analyses as well as IR, ¹H NMR, ¹³C NMR, and mass spectral data. All the newly synthesized derivatives were evaluated in vitro for inhibitory activity against COX-1 and COX-2 enzymes and their IC₅₀ values were calculated, most of the derivatives showed good inhibitory activity with derivatives IVb, IVh and IVJ showing inhibitory activity better than celecoxib. Moreover, the eight most potent derivatives IVa, IVb, IVc, IVd, IVe, IVh, IVJ, and IVL were selected for in vivo assay to measure their effect on paw edema in rates and their ulcerogenic effect. Compounds IVa, IVb and IVc were found to be the most active and selective as COX-2 inhibitors and most effective in protection from edema, they were also found to have lowest ulcerogenic effect among all derivatives.     

Cyclic Nucleotide Gated Channels and Ca2+-Mediated Signal Transduction During Plant Innate Immune Response to Pathogens

Transitory perturbations in the level of cytosolic Ca²⁺ are well known to be involved in numerous cell signaling pathways in both plant and animal systems. However, not much is known at present about the molecular identity of plant plasma membrane Ca²⁺ conducting ion channels or their specific roles in signal transduction cascades. A recent study employing genetic approaches as well as patch clamp electrophysiological analysis of channel currents has provided the first such direct evidence linking a specific gene product with inward Ca²⁺ currents across the plant cell membrane. This work identified Ca²⁺ permeation through (Arabidopsis) cyclic nucleotide gated channel isoform 2 (CNGC2) as contributing to the plant innate immunity signaling cascade initiated upon perception of a pathogen. Here, we expand on the implications of CNGC2 mediated cytosolic Ca²⁺ elevations associated with plant cell response to pathogen recognition, and propose some additional steps that may be involved in the innate immunity signal cascade.Key Words: calcium, CNGC, hypersensitive response, nitric oxide, plant innate immunity, plant ion channel, reactive oxygen species     

植物环核苷酸门控离子通道及其功能研究进展

环核苷酸门控离子通道(CNGC)是非选择性的阳离子通道, 可以直接被细胞内信使小分子——环核苷酸(cAMP和cGMP)活化。该通道蛋白包含6个跨膜α-螺旋, C端各具一个交叠的环核苷酸与钙调蛋白结合区。CNGC广泛存在于各种植物细胞中。研究表明, 模式植物拟南芥(Arabidopsis thaliana)的CNGC家族有20个成员, 分为4个亚群, 它们在抗病、花粉管生长、对Ca²⁺响应、抵抗重金属离子毒害和抗盐等多种信号途径中发挥重要作用, 协助植物细胞应对各种生物与非生物胁迫。该文简要介绍了CNGC的结构、表达谱及其调控因子, 并着重总结了近年来CNGC生物学功能的研究进展, 以期为今后系统开展其功能研究提供理论依据。     

Sequence and expression analysis of the Arabidopsis IQM family

Four major families have been found so far to possess the calmodulin binding IQ motif/s in plants: the IQD, the myosin, the CAMTA and the CNGC family. We have systematically identified and characterized a novel IQ motif-containing protein family, IQM, in Arabidopsis (Arabidopsis thaliana) using bioinformatics methods. IQM family contains six-member proteins (IQM1-6) which share sequence homology with a pea heavy metal-induced protein 6 and a ribosome-inactivating protein, trichosanthin, as well as IQ motif. IQM family can be divided into two groups, IQM3 and the other member proteins, based on sequence similarity and phylogenetic analysis of sequences. Though almost constitutive expression patterns were found in various plant organs of 6-week-old plants for IQM1 and 2, the other genes exhibited distinct organ-specific expression patterns. Light irradiation and treatment with heavy metals such as CdCl₂ or Pb(NO₃)₂ and high concentrations of mannitol or NaCl also changed expression of each IQM gene in a distinct manner in 7-day-old seedlings. However, treatment with various hormones, such as auxin, abscisic acid, gibberellin, methyl jasmonate and ethylene precursor, did not affect gene expression significantly. These results suggest that each IQM family gene plays a different role in plant development and responses to environmental cues.     

Trypsin Inhibitor Polymorphism: Multigene Family Expression and Posttranslational Modification

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Based on amino acid composition, molecular mass, and N-terminal sequence, the six inhibitors are closely related to one another and belong to the Bowman–Birk family of inhibitors. To define the relations among them, molecular mass and amino acid composition of peptides obtained from digestion with trypsin were determined. The sequence and the biosynthetic mechanism of the isoform formation have been partially resolved for four major isoforms. Two isoinhibitor forms (PSTI IVa, IVb) in pea seeds are due to expression of two distinct genes; PSTI IVa has four amino acid replacements when its sequence is compared with the sequence of PSTI IVb. Two others (PSTI I, II) result from posttranslational proteolytic cleavage of nine C-terminal residues of forms PSTI IVa and IVb, respectively.     

Ion channels in plants: From bioelectricity,via signaling,to behavioral actions

In his recent opus magnum review paper published in the October issue of Physiology Reviews, Rainer Hedrich summarized the field of plant ion channels.¹ He started from the earliest electric recordings initiated by Charles Darwin of carnivorous Dionaea muscipula,^1,2 known as Venus flytrap, and covered the topic extensively up to the most recent discoveries on Shaker-type potassium channels, anion channels of SLAC/SLAH families, and ligand-activated channels of glutamate receptor-like type (GLR) and cyclic nucleotide-gated channels (CNGC).¹     

Heterologous Expression of Wheat VERNALIZATION 2 (TaVRN2) Gene in Arabidopsis Delays Flowering and Enhances Freezing Tolerance

The vernalization gene 2 (VRN2), is a major flowering repressor in temperate cereals that is regulated by low temperature and photoperiod. Here we show that the gene from Triticum aestivum (TaVRN2) is also regulated by salt, heat shock, dehydration, wounding and abscissic acid. Promoter analysis indicates that TaVRN2 regulatory region possesses all the specific responsive elements to these stresses. This suggests pleiotropic effects of TaVRN2 in wheat development and adaptability to the environment. To test if TaVRN2 can act as a flowering repressor in species different from the temperate cereals, the gene was ectopically expressed in the model plant Arabidopsis. Transgenic plants showed no alteration in morphology, but their flowering time was significantly delayed compared to controls plants, indicating that TaVRN2, although having no ortholog in Brassicaceae, can act as a flowering repressor in these species. To identify the possible mechanism by which TaVRN2 gene delays flowering in Arabidopsis, the expression level of several genes involved in flowering time regulation was determined. The analysis indicates that the late flowering of the 35S::TaVRN2 plants was associated with a complex pattern of expression of the major flowering control genes, FCA, FLC, FT, FVE and SOC1. This suggests that heterologous expression of TaVRN2 in Arabidopsis can delay flowering by modulating several floral inductive pathways. Furthermore, transgenic plants showed higher freezing tolerance, likely due to the accumulation of CBF2, CBF3 and the COR genes. Overall, our data suggests that TaVRN2 gene could modulate a common regulator of the two interacting pathways that regulate flowering time and the induction of cold tolerance. The results also demonstrate that TaVRN2 could be used to manipulate flowering time and improve cold tolerance in other species.     

Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

The kingdom Stramenopile includes diatoms, brown algae, and oomycetes. Plant pathogenic oomycetes, including Phytophthora, Pythium and downy mildew species, cause devastating diseases on a wide range of host species and have a significant impact on agriculture. Here, we report comparative analyses on the genomes of thirteen straminipilous species, including eleven plant pathogenic oomycetes, to explore common features linked to their pathogenic lifestyle. We report the sequencing, assembly, and annotation of six Pythium genomes and comparison with other stramenopiles including photosynthetic diatoms, and other plant pathogenic oomycetes such as Phytophthora species, Hyaloperonospora arabidopsidis, and Pythium ultimum var. ultimum. Novel features of the oomycete genomes include an expansion of genes encoding secreted effectors and plant cell wall degrading enzymes in Phytophthora species and an over-representation of genes involved in proteolytic degradation and signal transduction in Pythium species. A complete lack of classical RxLR effectors was observed in the seven surveyed Pythium genomes along with an overall reduction of pathogenesis-related gene families in H. arabidopsidis. Comparative analyses revealed fewer genes encoding enzymes involved in carbohydrate metabolism in Pythium species and H. arabidopsidis as compared to Phytophthora species, suggesting variation in virulence mechanisms within plant pathogenic oomycete species. Shared features between the oomycetes and diatoms revealed common mechanisms of intracellular signaling and transportation. Our analyses demonstrate the value of comparative genome analyses for exploring the evolution of pathogenesis and survival mechanisms in the oomycetes. The comparative analyses of seven Pythium species with the closely related oomycetes, Phytophthora species and H. arabidopsidis, and distantly related diatoms provide insight into genes that underlie virulence.