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The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering., The Plant Cell, 35 (1), ISSN:1040-4651, ISSN:1531-298X, ISSN:1532-298X

SUMMARY Abscisic acid (ABA) is an important phytohormone controlling responses to abiotic stresses and is sensed by proteins from the PYR/PYL/RCAR family. To explore the genetic contribution of PYLs toward ABA-dependent and ABA-independent processes, we generated and characterized high-order Arabidopsis mutants with mutations in the PYL family. We obtained a pyl quattuordecuple mutant and found that it was severely impaired in growth and failed to produce seeds. Thus, we carried out a detailed characterization of a pyl duodecuple mutant, pyr1pyl1/2/3/4/5/7/8/9/10/11/12. The duo-decuple mutant was extremely insensitive to ABA effects on seed germination, seedling growth, stomatal closure, leaf senescence, and gene expression. The activation of SnRK2 protein kinases by ABA was blocked in the duodecuple mutant, but, unexpectedly, osmotic stress activation of SnRK2s was enhanced. Our results demonstrate an important role of basal ABA signaling in growth, senescence, and abscission and reveal that PYLs antagonize ABA-independent activation of SnRK2s by osmotic stress., In Brief Zhao et al. generated duodecuple and quattuordecuple Arabidopsis PYL ABA receptor mutants. Characterization of the mutants revealed that the ABA receptors are critical for plant growth and development and negatively regulate ABA-independent SnRK2 activity by interacting with and inhibiting osmotic stress-activated SnRK2 protein kinases.

The Arabidopsis-related model species (ARMS) Thellungiella salsuginea and Thellungiella parvula have generated broad interest in salt stress research. While general growth characteristics of these species are similar to Arabidopsis, some aspects of their life cycle require particular attention in order to obtain healthy plants, with a large production of seeds in a relatively short time. This chapter describes basic procedures for growth, maintenance, and Agrobacterium-mediated transformation of ARMS. Where appropriate, differences in requirements between Thellungiella spp. and Arabidopsis are highlighted, along with basic growth requirements of other less studied candidate model species. Current techniques for comparative genomics analysis between Arabidopsis and ARMS are also described in detail

A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na from the transpiration stream. However, TsHKT1;2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a Ktransporter in the presence of Na in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1;2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T. salsuginea and most other HKT1 sequences contain Asn (N) in this position. Wild-type TsHKT1;2 and altered AtHKT1 (AtHKT1) complemented K-uptake deficiency of yeast cells. Mutanthkt1-1 plants complemented with both AtHKT1 and TsHKT1;2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1. Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na and Kbased on the N/D variance in the pore region. This change also dictated inward-rectification for Na transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats.

The glutamate-induced excitotoxicity pathway has been reported in several neurodegenerative diseases. Molecules that inhibit the release of glutamate or cause the overactivation of glutamate receptors can minimize neuronal cell death in these diseases. Osmotin, a homolog of mammalian adiponectin, is a plant protein from Nicotiana tabacum that was examined for the first time in the present study to determine its protective effects against glutamate-induced synaptic dysfunction and neurodegeneration in the rat brain at postnatal day 7. The results indicated that glutamate treatment induced excitotoxicity by overactivating glutamate receptors, causing synaptic dysfunction and neuronal apoptosis after 4 h in the cortex and hippocampus of the postnatal brain. In contrast, post-treatment with osmotin significantly reversed glutamate receptor activation, synaptic deficit and neuronal apoptosis by stimulating the JNK/PI3K/Akt intracellular signaling pathway. Moreover, osmotin treatment abrogated glutamate-induced DNA damage and apoptotic cell death and restored the localization and distribution of p53, p-Akt and caspase-3 in the hippocampus of the postnatal brain. Finally, osmotin inhibited glutamate-induced PI3K-dependent ROS production in vitro and reversed the cell viability decrease, cytotoxicity and caspase-3/7 activation induced by glutamate. Taken together, these results suggest that osmotin might be a novel neuroprotective agent in excitotoxic diseases.

Fetal alcohol syndrome is a neurological and developmental disorder caused by exposure of developing brain to ethanol. Administration of osmotin to rat pups reduced ethanol-induced apoptosis in cortical and hippocampal neurons. Osmotin, a plant protein, mitigated the ethanol-induced increases in cytochrome c, cleaved caspase-3, and PARP-1. Osmotin and ethanol reduced ethanol neurotoxicity both in vivo and in vitro by reducing the protein levels of cleaved caspase-3, intracellular [Ca(2+)]cyt, and mitochondrial transmembrane potential collapse, and also upregulated antiapoptotic Bcl-2 protein. Osmotin is a homolog of adiponectin, and it controls energy metabolism via phosphorylation. Adiponectin can protect hippocampal neurons against ethanol-induced apoptosis. Abrogation of signaling via receptors AdipoR1 or AdipoR2, by transfection with siRNAs, reduced the ability of osmotin and adiponectin to protect neurons against ethanol-induced neurodegeneration. Metformin, an activator of AMPK (adenosine monophosphate-activated protein kinase), increased whereas Compound C, an inhibitor of AMPK pathway, reduced the ability of osmotin and adiponectin to protect against ethanol-induced apoptosis. Osmotin exerted its neuroprotection via Bcl-2 family proteins and activation of AMPK signaling pathway. Modulation of AMPK pathways by osmotin, adiponectin, and metformin hold promise as a preventive therapy for fetal alcohol syndrome.

Molecular genetics has confirmed older research and generated new insights into the ways how plants deal with adverse conditions. This body of research is now being used to interpret stress behavior of plants in new ways, and to add results from most recent genomics- based studies. The new knowledge now includes genome sequences of species that show extreme abiotic stress tol- erances, which enables new strategies for applications through either molecular breeding or transgenic engineer- ing. We will highlight some physiological features of the extremophile lifestyle, outline emerging features about halophytism based on genomics, and discuss conclusions about underlying mechanisms.

Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.

In this work, pollen mother cells (PMCs) isolated in Arabidopsis thaliana by laser-capture microdissection (LCM) were subjected to transcription profiling by microarray (LMM). PMCs covering all meiotic stages, from leptotene to tetrad, were collected in an Atmcc1 characterized by overexpression of a GCN5-like histone acetylase (AtMCC1). A total of 150 genes showed differential expression in Atmcc1 PMCs as compared to the wild type. Histone hyperacetylation affected the transcription of genes belonging to categories such as the meiotic and mitotic cycle, the ubiquitinproteasome-system, and the chromatin structure. We also discuss the putative role of ASK1 and RAD51C upregulation in the meiotic defects observed in Atmcc1. PMCLMM experiments allowed identification of candidate AtMCC1 targets with known and potential function in meiosis, providing data for further investigation on plant meiosis. © 2012 Korean Society of Plant Biologists and Springer-Verlag Berlin Heidelberg.

Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance.

Thellungiella parvula1 is related to Arabidopsis thaliana and is endemic to saline, resource-poor habitats2, making it a model for the evolution of plant adaptation to extreme environments. Here we present the draft genome for this extremophile species. Exclusively by next generation sequencing, we obtained the de novo assembled genome in 1,496 gap-free contigs, closely approximating the estimated genome size of 140 Mb. We anchored these contigs to seven pseudo chromosomes without the use of maps. We show that short reads can be assembled to a near-complete chromosome level for a eukaryotic species lacking prior genetic information. The sequence identifies a number of tandem duplications that, by the nature of the duplicated genes, suggest a possible basis for T. parvula’s extremophile lifestyle. Our results provide essential background for developing genomically influenced testable hypotheses for the evolution of environmental stress tolerance.

We report the identification and characterization of an Arabidopsis mutant, hos10-1 (for high expression of osmotically responsive genes), in which the expression of RD29A and other stress-responsive genes is activated to higher levels or more rapidly activated than in wild-type by low temperature, exogenous abscisic acid (ABA), or salt stress (NaCl). The hos10-1 plants are extremely sensitive to freezing temperatures, completely unable to acclimate to the cold, and are hypersensitive to NaCl. Induction of NCED3 (the gene that encodes the rate-limiting enzyme in ABA biosynthesis) by polyethylene glycol-mediated dehydration and ABA accumulation are reduced by this mutation. Detached shoots from the mutant plants display an increased transpiration rate compared with wild-type plants. The hos10-1 plants exhibit several developmental alterations, such as reduced size, early flowering, and reduced fertility. The HOS10 gene encodes a putative R2R3-type MYB transcription factor that is localized to the nucleus. Together, these results indicate that HOS10 is an important coordinating factor for responses to abiotic stress and for growth and development.

Salinity is an abiotic stress that limits both yield and the expansion of agricultural crops to new areas. In the last 20 years our basic understanding of the mechanisms underlying plant tolerance and adaptation to saline environments has greatly improved owing to active development of advanced tools in molecular, genomics, and bioinformatics analyses. However, the full potential of investigative power has not been fully exploited, because the use of halophytes as model systems in plant salt tolerance research is largely neglected. The recent introduction of halophytic Arabidopsis-Relative Model Species (ARMS) has begun to compare and relate several unique genetic resources to the well-developed Arabidopsis model. In a search for candidates to begin to understand, through genetic analyses, the biological bases of salt tolerance, 11 wild relatives of Arabidopsis thaliana were compared: Barbarea verna, Capsella bursa-pastoris, Hirschfeldia incana, Lepidium densiflorum, Malcolmia triloba, Lepidium virginicum, Descurainia pinnata, Sisymbrium officinale, Thellungiella parvula, Thellungiella salsuginea (previously T. halophila), and Thlaspi arvense. Among these species, highly salt-tolerant (L. densiflorum and L. virginicum) and moderately salt-tolerant (M. triloba and H. incana) species were identified. Only T. parvula revealed a true halophytic habitus, comparable to the better studied Thellungiella salsuginea. Major differences in growth, water transport properties, and ion accumulation are observed and discussed to describe the distinctive traits and physiological responses that can now be studied genetically in salt stress research.

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.

The kinase-associated protein phosphatase (KAPP) is a regulator of the receptor-like kinase (RLK) signaling pathway. Loss-of-function mutations rag1-1 (root attenuated growth1-1) and rag1-2, in the locus encoding KAPP, cause NaCl hypersensitivity in Arabidopsis thaliana. The NaCl hypersensitive phenotype exhibited by rag1 seedlings includes reduced shoot and primary root growth, root tip swelling, and increased lateral root formation. The phenotype exhibited by rag1-1 seedlings is associated with a specific response to Na(+) toxicity. The sensitivity to Na(+) is Ca(2+) independent and is not due to altered intracellular K(+)/Na(+). Analysis of the genetic interaction between rag1-1 and salt overly sensitive1 (sos1-14) revealed that KAPP is not a component of the SOS signal transduction pathway, the only Na(+) homeostasis signaling pathway identified so far in plants. All together, these results implicate KAPP as a functional component of the RLK signaling pathway, which also mediates adaptation to Na(+) stress. RLK pathway components, known to be modulated by NaCl at the messenger RNA level, are constitutively down-regulated in rag1-1 mutant plants. The effect of NaCl on their expression is not altered by the rag1-1 mutation.

The historical beginning of understanding the importance of water to life started with the Ionic Philosophers (from the Ionic Sea), who began to think that physiology (referring to nature, from the ancient Greek physis = nature ; logos = talk ) could explain life better than theology (referring to

We report the identification and characterization of an Arabidopsis mutant, hos10-1 (for high expression of osmotically responsive genes), in which the expression of RD29A and other stress-responsive genes is activated to higher levels or more rapidly activated than in wild-type by low temperature, exogenous abscisic acid (ABA), or salt stress (NaCl). The hos10-1 plants are extremely sensitive to freezing temperatures, completely unable to acclimate to the cold, and are hypersensitive to NaCl. Induction of NCED3 (the gene that encodes the rate-limiting enzyme in ABA biosynthesis) by polyethylene glycol-mediated dehydration and ABA accumulation are reduced by this mutation. Detached shoots from the mutant plants display an increased transpiration rate compared with wild-type plants. The hos10-1 plants exhibit several developmental alterations, such as reduced size, early flowering, and reduced fertility. The HOS10 gene encodes a putative R2R3-type MYB transcription factor that is localized to the nucleus. Together, these results indicate that HOS10 is an important coordinating factor for responses to abiotic stress and for growth and development.

Transcription and mRNA processing are regulated by phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II, which consists of tandem repeats of a Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptapeptide. Previous studies showed that members of the plant CTD phosphatase-like (CPL) protein family differentially regulate osmotic stress-responsive and abscisic acid-responsive transcription in Arabidopsis thaliana . Here we report that AtCPL1 and AtCPL2 specifically dephosphorylate Ser-5 of the CTD heptad in Arabidopsis RNA polymerase II, but not Ser-2. An N-terminal catalytic domain of CPL1, which suffices for CTD Ser-5 phosphatase activity in vitro , includes a signature DXDXT acylphosphatase motif, but lacks a breast cancer 1 CTD, which is an essential component of the fungal and metazoan Fcp1 CTD phosphatase enzymes. The CTD of CPL1, which contains two putative double-stranded RNA binding motifs, is essential for the in vivo function of CPL1 and includes a C-terminal 23-aa signal responsible for its nuclear targeting. CPL2 has a similar domain structure but contains only one double-stranded RNA binding motif. Combining mutant alleles of CPL1 and CPL2 causes synthetic lethality of the male but not the female gametes. These results indicate that CPL1 and CPL2 exemplify a unique family of CTD Ser-5-specific phosphatases with an essential role in plant growth and development.

Arabidopsis stt3a-1 and stt3a-2 mutations cause NaCl/osmotic sensitivity that is characterized by reduced cell division in the root meristem. Sequence comparison of the STT3a gene identified a yeast ortholog, STT3, which encodes an essential subunit of the oligosaccharyltransferase complex that is involved in protein N-glycosylation. NaCl induces the unfolded protein response in the endoplasmic reticulum (ER) and cell cycle arrest in root tip cells of stt3a seedlings, as determined by expression profiling of ER stress–responsive chaperone (BiP-GUS) and cell division (CycB1;1-GUS) genes, respectively. Together, these results indicate that plant salt stress adaptation involves ER stress signal regulation of cell cycle progression. Interestingly, a mutation (stt3b-1) in another Arabidopsis STT3 isogene (STT3b) does not cause NaCl sensitivity. However, the stt3a-1 stt3b-1 double mutation is gametophytic lethal. Apparently, STT3a and STT3b have overlapping and essential functions in plant growth and developmental processes, but the pivotal and specific protein glycosylation that is a necessary for recovery from the unfolded protein response and for cell cycle progression during salt/osmotic stress recovery is associated uniquely with the function of the STT3a isoform.

Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3–1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1 sos3–1 . Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3–1 hkt1–1 and sos3–1 hkt1–2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1–1 and hkt1–2 mutations can suppress to an equivalent extent the Na + sensitivity of sos3–1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3–1 hkt1–1 and sos3–1 hkt1–2 seedlings are able to maintain [K + ] int in medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K + /Na + than the sos3–1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3–1 mutant that is caused by K + deficiency on culture medium with low Ca 2+ (0.15 mM) and + . Interestingly, the capacity of hkt1 mutations to suppress the Na + hypersensitivity of the sos3–1 mutant is reduced substantially when seedlings are grown in medium with low Ca 2+ (0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na + entry and high affinity K + uptake. The hkt1 mutations have revealed the existence of another Na + influx system(s) whose activity is reduced by high [Ca 2+ ] ext .

3 pages, 1 figure, 24 references., Recent reports from Jian Kang Zhu et al. have provided clear evidence for a signal transduction pathway that mediates salt tolerance of plants by controlling ion homeostasis. Earlier research had identified several salt overly sensitive (sos) mutants of Arabidopsis thaliana ecotype Columbia, based on Na+/Li+ hypersensitivity. Genetic complementation studies allowed the mutants to be placed into five allelic groups, sos 1–sos 5. Phenotypic additivity analyses indicate that SOS1, SOS2 and SOS3 function in a common pathway, with SOS1 being epistatic to the other loci. Recently, mapped-based cloning and gene complementation has identified the genes at these three loci. SOS3 is a Ca2+-binding protein that contains EF-hand structures and a myristoylation site in the N terminus. It has greatest sequence homology with yeast calcineurin subunit B and with animal neuronal Ca2+ sensors. SOS2 encodes a serine/threonine kinase with a catalytic domain similar to the yeast sucrose nonfermenting (SNF1) kinase and the mammalian AMP-activated protein kinase (AMPK), but is regulated differently through a distinctive C-terminal regulatory domain. SOS1 is a putative plasma membrane Na+/H+ antiporter resembling the mammalian NHE and bacterial NhaP exchangers.

2 pages, 1 figure, 19 references., Long ago plants ventured forth from the relatively stable world of the oceans to the land, where mineral nutrients and water were often scarce commodities. In their quest to cope with the changing conditions of life on land, many plant species appear to have lost much of their ability to deal with higher sodium concentrations. With the exception of the halophytes, which occupy the sodium-rich terrestrial habitats of estuaries, marshes and other niche environments, land plants have evolved a requirement for fresh water. However, in recent years, we have learned that the cells of most higher plants are capable of adjusting to high levels of NaCl. Indeed, if exposed in a gradual manner, plants can grow and reproduce during exposure to very high concentrations of sodium[1]. It is this ability to adjust that has led many to believe that most of the cellular machinery for dealing with excessive sodium still exists in the majority of plant species. What distinguishes many salt tolerant species is the ability to engage that machinery when needed. By understanding the signaling system that allows a plant to sense excess sodium in the environment and to make appropriate adjustments, plant biologists hope to be able to influence the growth behavior of crop plants in arid and inhospitable conditions. When plants are challenged with salinity stress, an increase in the concentration of Ca2+ often can ameliorate the inhibitory effects on growth[2]. Although the underlying mechanism has remained largely unexplained, prevailing models for Ca2+ function include both membrane stabilization and signaling roles. Numerous studies indicate that a variety of stress conditions, including salinity, induce cytosolic Ca2+ accumulation[3]. The role of Ca2+ as a second messenger in many biological systems, coupled with these observations, indicates that plants are able to adjust to high salt environments by activating a signal transduction system involving Ca2+.

We isolated from a tomato cDNA library the tomPRO1locus, which encodes γ-glutamyl kinase (GK) and γ-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. InEscherichia coli, tomPRO1directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified fromE. colirecognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.

Three cysteine proteinase inhibitor cDNA clones (pL1, pR1, and pN2) have been isolated from a soybean (Glycine max L. Merr.) embryo library. The proteins encoded by the clones are between 60 and 70% identical and contain the consensus QxVxG motif and W residue in the appropriate spatial context for interaction with the cysteine proteinase papain. L1, R1, and N2 mRNAs were differentially expressed in different organs of plants (juvenile and mature) and seedlings, although N2 mRNA was constitutive only in flowers. R1 and N2 transcripts were induced by wounding or methyl jasmonate (M-JA) treatment in local and systemic leaves coincident with increased papain inhibitory activity, indicating a role for R1 and N2 in plant defense. The L1 transcript was constitutively expressed in leaves and was induced slightly by M-JA treatment in roots. Unlike the chymotrypsin/trypsin proteinase inhibitor II gene (H. Pena-Cortes, J. Fisahn, L. Willmitzer [1995] Proc Natl Acad Sci USA 92: 4106–4113), expression of the soybean genes was only marginally induced by abscisic acid and only in certain tissues. Norbornadiene, a competitive inhibitor of ethylene binding, abolished the wounding or M-JA induction of R1 and N2 mRNAs but not the accumulation of the wound-inducible vspA transcript. Presumably, ethylene binding to its receptor is involved in the wound inducibility of R1 and N2 but not vspA mRNAs. Bacterial recombinant L1 and R1 proteins, expressed as glutathione S-transferase fusion proteins, exhibited substantial inhibitory activities against vicilin peptidohydrolase, the major thiol endopeptidase in mung bean seedlings. Recombinant R1 protein had much greater cysteine proteinase inhibitor activity than recombinant L1 protein, consistent with the wound inducibility of the R1 gene and its presumed role in plant defense.

6 pages, 2 figuress, 3 tables, 35 references., We describe the isolation of salt-sensitive Chlamydomonas reinhardtii mutants by insertional mutagenesis using the nitrate reductase (Nit1)gene. The plasmid pMN24, containing Nitl, was used for transformation of 305CW15 (nit1 cw75 mt+), and transformants were selected for complementation of the nit- phenotype. From 6875 nit+ colonies, four transformants (S4, S18, S46, and S66) were isolated that exhibited both Na+ and Li+ sensitivity (sod-), and another transformant (S33) was selected that exhibited sensitivity to Li+ but not Na+ (lit-) based on relative growth comparisons with the wild-type strain. S33, S46, and S66 were no more growth inhibited by sorbitol than was 305CW15. In comparison, S4 and S18 exhibited substantial growth inhibition in medium supplemented with sorbitol. Genetic analyses indicated that the salt-sensitive mutants were each defective in a single recessive gene. The mutant genes in S4 (sodl), S33 (lit1), and S66 (sod3) are linked to a functional copy of Nit1 and are presumably tagged with a pMN24 insertion.

Combinations of ethylene and methyl jasmonate (E/MeJA) synergistically induced members of both groups 1 and 5 of the pathogenesis-related (PR) superfamily of defense genes. E/MeJA caused a synergistic induction of PR-1b and osmotin (PR-5) mRNA accumulation in tobacco seedlings. E/MeJA also synergistically activated the osmotin promoter fused to a [beta]-glucuronidase marker gene in a tissue-specific manner. The E/MeJA responsiveness of the osmotin promoter was localized on a -248 to +45 fragment that exhibited responsiveness to several other inducers. E/MeJA induction also resulted in osmotin protein accumulation to levels similar to those induced by osmotic stress. Of the several known inducers of the osmotin gene, including salicylic acid (SA), fungal infection is the only other condition known to cause substantial osmotin protein accumulation in Wisconsin 38, a tobacco cultivar that does not respond hypersensitively to tobacco mosaic virus. Based on the ability of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine to block ethylene induction of PR-1b mRNA accumulation and its inability to block osmotin mRNA induction by ethylene, these two PR gene groups appeared to have at least partially separate signal transduction pathways. Stimulation of osmotin mRNA accumulation by okadaic acid indicated that another protein kinase system is involved in regulation of the osmotin gene. SA, which is known to induce pathogen resistance in tobacco, could not induce the osmotin gene as much as E/MeJA and neither could it induce PR-1b as much as SA and MeJA combined.

Higher plant proteins immunologically related to the animal substrate adhesion molecule vitronectin have recently been observed and implicated in a variety of biological processes, such as plasma membrane-cell wall adhesion, pollen tube extension, and bacterium-plant interaction. We provide evidence that, similar to vitronectin, one of these proteins, PVN1 (plant vitronectin-like 1), isolated from 428 mM NaCl-adapted tobacco cells binds to glass surfaces an heparin. PVN1 was isolated by glass bead affinity chromatography. Isolated PVN1 has adhesive activity based on results from a baby hamster kidney cell-spreading assay. This plant adhesion protein was detected in all tissues examined but was most abundant in roots and salt-adapted cultured cells. Immunogold labeling indicated that PVN1 is localized in the cell wall of cortical and transmitting tissue cells of pollinated mature styles. A partial amino acid sequence of PVN1 revealed no similarity with vitronectin but, instead, was nearly identical to the translational elongation factor-1 alpha (EF-1 alpha). A clone isolated by screening a tobacco cDNA expression library with anti-PVN1 encoded a protein with greater than 93% identity to sequences of EF-1 alpha from plants of numerous species. Immunological cross-reactivity between tobacco PVN1 and EF-1 alpha as well as the reaction between the EF-1 alpha antibody and the 65- and 75-kD vitronectin-like proteins of a fucoidal alga supported the conclusion that the plant extracellular adhesion protein PVN1 is related to EF-1 alpha.

The cell wall is a fundamental component in the response of plants to environmental changes. To directly assess the role of the cell wall we have increased the expression and activity of a cell wall associated peroxidase (TPX2), an enzyme involved in modifying cell wall architecture. Overexpression of TPX2 had no effect on wild-type development, but greatly increased the germination rate under high salt or osmotic stress. Differential scanning calorimetry showed that transgenic seeds were able to retain more water available for germination than wild-type seeds. Thermoporometry calculations indicated that this could be due to a lower mean pore size in the walls of transgenic seeds. Therefore, the higher capacity of transgenic seeds in retaining water could result in higher germination rates in conditions where the availability of water is restricted.

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp88, Glu108, Tyr134, Asn136, Leu226 and Gln227) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp88, Tyr134 or Asn136 eliminated GlcNAc binding activity by GS-II, while those at Glut108, Leu226 or Gln227 did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr134 eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu226 and Gln227 did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.