CML10, a variant of calmodulin, modulates ascorbic acid synthesis.

Calmodulins (CaMs) regulate numerous Ca(2+) -mediated cellular processes in plants by interacting with their respective downstream effectors. Due to the limited number of CaMs, other calcium sensors modulate the regulation of Ca(2+) -mediated cellular processes that are not managed by CaMs. Of 50 CaM-like (CML) proteins identified in Arabidopsis thaliana, we characterized the function of CML10. Yeast two-hybrid screening revealed phosphomannomutase (PMM) as a putative interaction partner of CML10. In vitro and in vivo interaction assays were performed to analyze the interaction mechanisms of CML10 and PMM. PMM activity and the phenotypes of cml10 knock-down mutants were studied to elucidate the role(s) of the CML10-PMM interaction. PMM interacted specifically with CML10 in the presence of Ca(2+) through its multiple interaction motifs. This interaction promoted the activity of PMM. The phenotypes of cml10 knock-down mutants were more sensitive to stress conditions than wild-type plants, corresponding with the fact that PMM is an enzyme which modulates the biosynthesis of ascorbic acid, an antioxidant. The results of this research demonstrate that a calcium sensor, CML10, which is an evolutionary variant of CaM, modulates the stress responses in Arabidopsis by regulating ascorbic acid production.

Endoplasmic reticulum- and Golgi-localized phospholipase A2 plays critical roles in Arabidopsis pollen development and germination.

The phospholipase A(2) (PLA(2)) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA(2)s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA(2)s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA(2) paralogs (PLA(2)-ß, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA(2) using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA(2) inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA(2)s that are conserved in other organisms.

Single cystathionine ß-synthase domain-containing proteins modulate development by regulating the thioredoxin system in Arabidopsis.

Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine ß-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain-containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H(2)O(2) levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating photosynthesis-related [corrected] enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell.

An Arabidopsis cell growth defect factor-related protein, CRS, promotes plant senescence by increasing the production of hydrogen peroxide.

Arabidopsis thaliana Cell Growth Defect factor 1 (Cdf1) has been implicated in promotion of proapoptotic Bax-like cell death via the induction of reactive oxygen species (ROS). Here we report a conserved function of a chloroplast-targeting Cdf-related gene Responsive to Senescence (CRS) using CRS overexpression and loss of function in plants as well as CRS heterologous expression in yeast. CRS expression was strongly induced in senescent leaves, suggesting its main functions during plant senescence. CRS expression in yeast mitochondria increased the ROS level and led to cell death in a manner similar to Cdf1. In whole plants, overexpression of CRS caused the loss of chlorophylls (Chls) and the rapid onset of leaf senescence, while the lack of CRS led to the delay of leaf senescence in a loss-of-function mutant, crs. The higher and lower accumulation of H(2)O(2) was correlated with early and late senescence in CRS-overexpressing and crs mutant plants, respectively. Furthermore, expression of senescence-related marker genes and metacaspase genes was induced in CRS-overexpressing plants in response to dark. Our findings suggest that CRS plays a key role in the leaf senescence process that accompanies H(2)O(2) accumulation resulting in cell death promotion.

A cystathionine-ß-synthase domain-containing protein, CBSX2, regulates endothecial secondary cell wall thickening in anther development.

Anther formation and dehiscence are complex pivotal processes in reproductive development. The secondary wall thickening in endothecial cells of the anther is a known prerequisite for successful anther dehiscence. However, many gaps remain in our understanding of the regulatory mechanisms underlying anther dehiscence in planta, including a possible role for jasmonic acid (JA) and H(2)O(2) in secondary wall thickening of endothecial cells. Here, we report that the cystathionine ß-synthase domain-containing protein CBSX2 located in the chloroplast plays a critical role in thickening of the secondary cell walls of the endothecium during anther dehiscence in Arabidopsis. A T-DNA insertion mutant of CBSX2 (cbsx2) showed increased secondary wall thickening of endothecial cells and early anther dehiscence. Consistently, overexpression of CBSX2 resulted in anther indehiscence. Exogenous JA application induced secondary wall thickening and caused flower infertility in the cbsx2 mutant, whereas it partially restored fertility in the CBSX2-overexpressing lines lacking the wall thickening. CBSX2 directly modulated thioredoxin (Trx) in chloroplasts, which affected the level of H(2)O(2) and, consequently, expression of the genes involved in secondary cell wall thickening. Our findings have revealed that CBSX2 modulates the H(2)O(2) status, which is linked to the JA response and in turn controls secondary wall thickening of the endothecial cells in anthers for dehiscence to occur.

Novel bifunctional nucleases, OmBBD and AtBBD1, are involved in abscisic acid-mediated callose deposition in Arabidopsis.

Screening of the expressed sequence tag library of the wild rice species Oryza minuta revealed an unknown gene that was rapidly and strongly induced in response to attack by a rice fungal pathogen (Magnaporthe oryzae) and an insect (Nilaparvata lugens) and by wounding, abscisic acid (ABA), and methyl jasmonate treatments. Its recombinant protein was identified as a bifunctional nuclease with both RNase and DNase activities in vitro. This gene was designated OmBBD (for O. minuta bifunctional nuclease in basal defense response). Overexpression of OmBBD in an Arabidopsis (Arabidopsis thaliana) model system caused the constitutive expression of the PDF1.2, ABA1, and AtSAC1 genes, which are involved in priming ABA-mediated callose deposition. This activation of defense responses led to an increased resistance against Botrytis cinerea. atbbd1, the knockout mutant of the Arabidopsis ortholog AtBBD1, was susceptible to attack by B. cinerea and had deficient callose deposition. Overexpression of either OmBBD or AtBBD1 in atbbd1 plants complemented the susceptible phenotype of atbbd1 against B. cinerea as well as the deficiency of callose deposition. We suggest that OmBBD and AtBBD1 have a novel regulatory role in ABA-mediated callose deposition.

An actinoporin plays a key role in water stress in the moss Physcomitrella patens.

* Modern land plants arose from a green algae-like ancestor c. 480 million years ago. While several novel morphological features were critical for survival in the aerial environment, physiological innovation undoubtedly played a key role in the colonization of terrestrial habitats. Recently, actinoporin genes, a small group of pore-forming toxins from sea anemones, have been found in the bryophyte and lycophyte lineages of land plants where they are upregulated in water-stressed tissues. * The bryoporin gene in the moss Physcomitrella patens (PpBP) was functionally characterized by RNA blot analyses and overexpression in P. patens. In order to examine functional homology between PpBP and sea anemone actinoporins, the recombinant PpBP was subjected to hemolytic analysis of pig blood cells, which is one of the specific activities of actinoporins. * PpBP was upregulated by various abiotic stresses, in particular most strongly by dehydration stress. Overexpression of the bryoporin gene heightens drought tolerance in P. patens significantly. In addition, PpBP shared the highest structural homology with actinoporins in a three-dimensional structural database and showed hemolytic activity. * These results suggest that this phylogenetic distribution may have resulted from an ancient horizontal gene transfer and actinoporins may have played an important role in early land plants.

Overexpression of lipid transfer protein (LTP) genes enhances resistance to plant pathogens and LTP functions in long-distance systemic signaling in tobacco.

The lipid signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. To investigate the biological functions of pepper lipid transfer protein (LTP), we carried out virus-induced gene silencing (VIGS) in pepper, constitutive expression of CALTPs and grafting experiments in the tobacco plant. Suppression of endogenous CALTPI and CALTPII by VIGS, respectively, resulted in enhanced susceptibility to Xanthomonas campestris pv. vescatoria and pepper mosaic mottle virus in pepper. On the other hand, the constitutive expression of CALTPI and CALTPII genes in tobacco plants showed enhanced resistance to oomycete pathogen, Phytophthora nicotianae and bacterial pathogen, Pseudomonas syringae pv. tabaci. Enhanced resistance is found to be associated with the enhanced CALTP transcript levels in the independent transgenic CALTPI or II tobacco lines. Induced resistance responses in grafted scion leaves revealed that LTP plays a role in long-distance systemic signaling in plants.

Identification of putative MAPK kinases in Oryza minuta and O. sativa responsive to biotic stresses.

The mitogen-activated protein kinase (MAPK) signaling cascade is critical for regulating plant defense systems against various kinds of pathogen and environmental stresses. One component of this cascade, the MAP kinase kinases (MAPKK), has not yet been shown to be induced in plants following biotic attacks, such as those by insects and fungi. We describe here a gene coding for a blast (Magnaporthe grisea)- and insect (Nilaparvata lugens)-responsive putative MAPK kinase, OmMKK1 (Oryza minuta MAPKK 1), which was identified in a library of O. minuta expressed sequence tags (ESTs). Two copies of OmMKK1 are present in the O. minuta genome. They encode a predicted protein with molecular mass 39 kDa and pI of 6.2. Transcript patterns following imbibition of plant hormones such as methyl jasmonic acid (MeJA), ethephone, salicylic acid (SA) and abscisic acid (ABA), as well as exposure to methyl viologen (MV), revealed that the expression of OmMKK1 is related to defense response signaling pathways. A comparative analysis of OmMKK1 and its O. sativa ortholog OsMKK1 showed that both were induced by stress-related hormones and biotic stresses, but that the kinetics of their responses differed despite their high amino acid sequence identity (96%).

Calcineurin B-like 3 calcium sensor associates with and inhibits 5'-methylthioadenosine nucleosidase 2 in Arabidopsis.

Calcineurin B-like (CBL) proteins constitute a unique family of calcium sensor relays in plants. It is well known that CBLs detect the calcium signals elicited by a variety of abiotic stresses and relay the information to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs). In this study, we found that a few CBL members can also target another group of enzymes 5'-methylthioadenosine nucleosidases (MTANs), which are encoded by two genes in Arabidopsis, AtMTAN1 and AtMTAN2. In the yeast two-hybrid system, AtMTAN1 interacted with multiple CBL members such as CBL2, CBL3 and CBL6, whereas AtMTAN2 associated exclusively with CBL3. We further demonstrated that the CBL3-AtMTAN2 association occurs in a calcium-dependent manner, which results in a significant decrease in the enzyme activity of the AtMTAN2 protein. Taken together, these results clearly indicate that the CBL family can target at least two distinct groups of enzymes (CIPKs and MTANs), conferring an additional level of complexity on the CBL-mediated signaling networks. In addition, our finding also provides a novel molecular mechanism by which calcium signals are transduced to alter metabolite profiles in plants.

WITHDRAWN: A novel calmodulin-interacting protein (CUGT1) is rapidly induced by stress signals in Arabidopsis.

The manuscript has been withdrawn on request of the authors and will not be published in that version in the print edition of Planta.

Identification of genes induced in wound-treated wild rice (Oryza minuta).

A subtracted library was constructed from wound-treated wild rice (Oryza minuta) by suppression subtractive hybridization (SSH) in combination with mirror orientation selection (MOS). To distinguish between differentially expressed transcripts and false positive clones, DNA chips containing 960 random clones were applied as a form of reverse Northern screening. Based on the signal intensities and expression ratios obtained from experiments performed in triplicate, 371 clones were selected. ESTs produced from the subtracted library showed 63.2% redundancy, and 72% of all clones could be matched to the GenBank nonredundant database. Functional categorization placed the identified enriched genes in categories of subcellular localization, metabolism, cell rescue and defense, and transcription. These EST-related resistance mechanisms could be used in investigations into the defense mechanisms of wild species, and to provide new routes to improving the germplasm of cultivated rice.

An Arabidopsis R2R3-MYB transcription factor, AtMYB20, negatively regulates type 2C serine/threonine protein phosphatases to enhance salt tolerance.

We have characterized the function of a plant R2R3-MYB transcription factor, Arabidopsis thaliana MYB20 (AtMYB20). Transgenic plants overexpressing AtMYB20 (AtMYB20-OX) enhanced salt stress tolerance while repression lines (AtMYB20-SRDX) were more vulnerable to NaCl than wild-type plants. Following NaCl treatment, the expressions of ABI1, ABI2 and AtPP2CA, which encode type 2C serine/threonine protein phosphatases (PP2Cs) that act as negative regulators in abscisic acid (ABA) signaling, were suppressed in AtMYB20-OX but induced in AtMYB20-SRDX. The electrophoretic mobility shift assay results revealed that AtMYB20 binds to the promoter regions containing the MYB recognition sequence (TAACTG) and an ACGT core element of ABI1 and AtPP2CA. These findings suggest that AtMYB20 down-regulates the expression of PP2Cs, the negative regulator of ABA signaling, and enhances salt tolerance.

CBSXs are sensor relay proteins sensing adenosine-containing ligands in Arabidopsis.

We recently determined that CBSX proteins, which have only one pair of cystathionine ß-synthase (CBS) domains, directly regulate the activation of thioredoxins and thereby control cellular H2O2 levels and modulate both plant development and growth. The Arabidopsis genome contains six CBSXs, and these are localized to different subcellular compartments­ CBSX1 and CBSX2 in the chloroplast, CBSX3 in the mitochondria, CBSX4 in the cytosol, and CBSX5 and CBSX6 in the endoplasmic reticulum. The CBSXs have been identified in prokaryotes and plants, but not in animals. The considerable differences in length and amino acid sequence between CBSX members may result in variations in protein structure and in their specificity to interact with ligands and/or target proteins. Here, we discuss the possibility that the CBSXs are novel sensor relay proteins that use adenosine-containing molecules as a ligand.

Barley DNA-binding methionine aminopeptidase, which changes the localization from the nucleus to the cytoplasm by low temperature, is involved in freezing tolerance.

The polymerase chain reaction-based Mirror Orientation Selection (MOS) method was used to isolate low temperature-induced genes from cold-treated winter barley (Hordeum vulgare L. cv. Dongbori). MOS screening identified a novel methionine (Met) aminopeptidase (MAP) designated as HvMAP. The deduced HvMAP protein was determined to possess an aminopeptidase domain and a nuclear localization signal. An in vitro enzyme assay using recombinant HvMAP protein demonstrated MAP activity. The expression of this gene was induced by low temperature and abscisic acid treatment, and overexpression of this gene conferred stronger freezing tolerance to Arabidopsis transgenic plants as compared to wild-type plants. Interestingly, low temperature treatment changed the localization of HvMAP from the nucleus to the cytoplasm. These findings suggest that HvMAP is a novel MAP that functions in freezing tolerance by facilitating protein maturation.

The Arabidopsis calcium sensor calcineurin B-like 3 inhibits the 5'-methylthioadenosine nucleosidase in a calcium-dependent manner.

Calcineurin B-like (CBL) proteins represent a unique family of calcium sensors in plant cells. Sensing the calcium signals elicited by a variety of abiotic stresses, CBLs transmit the information to a group of serine/threonine protein kinases (CBL-interacting protein kinases [CIPKs]), which are currently known as the sole targets of the CBL family. Here, we report that the CBL3 member of this family has a novel interaction partner in addition to the CIPK proteins. Extensive yeast two-hybrid screenings with CBL3 as bait identified an interesting Arabidopsis (Arabidopsis thaliana) cDNA clone (named AtMTAN, for 5'-methylthioadenosine nucleosidase), which encodes a polypeptide similar to EcMTAN from Escherichia coli. Deletion analyses showed that CBL3 utilizes the different structural modules to interact with its distinct target proteins, CIPKs and AtMTAN. In vitro and in vivo analyses verified that CBL3 and AtMTAN physically associate only in the presence of Ca(2+). In addition, we empirically demonstrated that the AtMTAN protein indeed possesses the MTAN activity, which can be inhibited specifically by Ca(2+)-bound CBL3. Overall, these findings suggest that the CBL family members can relay the calcium signals in more diverse ways than previously thought. We also discuss a possible mechanism by which the CBL3-mediated calcium signaling regulates the biosynthesis of ethylene and polyamines, which are involved in plant growth and development as well as various stress responses.

Novel calcium-binding GTPase (AtCBG) involved in ABA-mediated salt stress signaling in Arabidopsis.

We have identified a novel Ca(2+)-signal sensing GTPase (643 amino acid residues with an estimated molecular mass of 79 kDa) from the Arabidopsis genome database. This protein contains a RHO-like GTPase domain at the N-terminus (15-184 amino acids) and two calcium-binding EF-hand motifs (199-227 and 319-347 amino acids, respectively). It has the capability to bind calcium and hydrolyze GTP; in addition, its GTPase activity is regulated by changes in Ca(2+) concentration. The expression of this gene was induced by ABA and salt stresses, and specific knock-out mutants were highly sensitive to ABA and salt treatments. These findings suggest that this protein is a novel ABA- and salt stress-related Ca(2+) signal transducer.

Proteome analysis of gametophores identified a metallothionein involved in various abiotic stress responses in Physcomitrella patens.

Physcomitrella patens is a model plant for studying gene function using a knockout strategy. To establish a proteome database for P. patens, we resolved over 1,500 soluble proteins from gametophore and protonema tissues by two-dimensional electrophoresis (2-DE) and obtained peptide mass fingerprints (PMFs) by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Using expressed sequence tags (ESTs), we were able to predict the identities of 90 protein spots. Most of these were related to energy or primary metabolism. Comparative proteome analysis was used to identify proteins specific for each of the tissue types. One of these was a metallothionein type-2 (PpMT2) protein that was highly upregulated in gametophore tissue. PpMT2 was induced in both the gametophore and protonema following culture on solid media and in response to various abiotic stresses such as copper, cadmium, cold, indole-3-acetic acid, and ethylene. We suggest that PpMT2 is not only involved in metal binding and detoxification, but also in many biological aspects as a metal messenger or a protein with additional functions.

Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth.

The pollen-specific receptor kinases LePRK1 and LePRK2 have localization and expression profiles that strongly suggest they play roles in pollen germination and tube growth. To identify downstream components of LePRK signaling, we used their cytoplasmic domains (CDs) as baits in yeast two-hybrid screens of a tomato pollen cDNA library. A pollen-specific protein we named kinase partner protein (KPP) interacted with the CDs of both LePRK1 and LePRK2 in yeast and in an in vitro pull-down assay, and with LePRK2 in a co-immunoprecipitation assay. KPP is a peripheral membrane protein and is phosphorylated in pollen. Pollen tubes over-expressing KPP developed balloon-like tips with abnormal cytoplasmic streaming and F-actin arrangements and plants over-expressing KPP exhibited impaired transmission of the transgene through the male. KPP-like genes are found only in plants; the 14 family members in Arabidopsis thaliana exhibit diverse expression patterns and potentially play roles in signaling pathways in other tissues.

Novel CIPK1-associated proteins in Arabidopsis contain an evolutionarily conserved C-terminal region that mediates nuclear localization.

Environmental stimuli, including light, pathogens, hormones, and abiotic stresses, elicit changes in the cytosolic Ca(2+) signatures of plant cells. However, little is known about the molecular mechanisms by which plants sense and transmit the specific cytoplasmic Ca(2+) signal into the nucleus, where gene regulation occurs to respond appropriately to the stress. In this study, we have identified two novel Arabidopsis (Arabidopsis thaliana) proteins specifically associated with Calcineurin B-Like-Interacting Protein Kinase1 (CIPK1), a member of Ser/Thr protein kinases that interact with the calcineurin B-like Ca(2+)-binding proteins. These two proteins contain a very similar C-terminal region (180 amino acids in length, 81% similarity), which is required and sufficient for both interaction with CIPK1 and translocation to the nucleus. Interestingly, the conserved C-terminal region was also found in many proteins from various eukaryotic organisms, including humans. However, none of them have been characterized so far. Taken together, these findings suggest that the two proteins containing the evolutionarily conserved C-terminal region (ECT1 and ECT2) may play a critical role in relaying the cytosolic Ca(2+) signals to the nucleus, thereby regulating gene expression.