Vegetative Tissues In Response Research Articles

A Type I and a Type II Metacaspase Are Differentially Regulated during Corolla Development and in Response to Abiotic and Biotic Stresses in Petunia × hybrida

Metacaspases are structural homologs of the metazoan caspases that are found in plants, fungi, and protozoans. They are cysteine proteases that function during programmed cell death, stress, and cell proliferation. A putative metacaspase designated PhMC2 was cloned from Petunia × hybrida, and sequence alignment and phylogenetic analysis revealed that it encodes a type II metacaspase. PhMC2 cleaved protease substrates with an arginine residue at the P1 site and cysteine (iodoacetamide) and arginal (leupeptin) protease inhibitors nearly abolished this activity. The activity of PhMC2 was highest at pH 8, and the putative catalytic site cysteine residue was required for optimal activity. Quantitative PCR showed that PhMC2 transcripts were detectable in petunia corollas, styles, and ovaries. Expression patterns were not upregulated during petal senescence but were higher at the middle stages of development when flower corollas were fully open but not yet starting to wilt. PhMC1, a type I metacaspase previously identified in petunia, and PhMC2 were differentially regulated in vegetative tissues in response to biotic and abiotic stresses. PhMC2 expression was upregulated to a greater extent than PhMC1 following Botrytis cinerea infection, while PhMC1 was upregulated more by drought, salinity, and low nutrient stress. These results suggest that petunia metacaspases are involved in flower development, senescence, and stress responses.

Lipid Droplet Isolation from Arabidopsis thaliana Leaves.

Lipid droplets (LDs) are neutral lipid aggregates surrounded by a phospholipid monolayer and specific proteins. In plants, they play a key role as energy source after seed germination, but are also formed in vegetative tissues in response to developmental or environmental conditions, where their functions are poorly understood. To elucidate these, it is essential to isolate LDs with good yields, while retaining their protein components. LD isolation protocols are based on their capacity to float after centrifugation in sucrose gradients. Early strategies using stringent conditions and LD-abundant plant tissues produced pure LDs where core proteins were identified. To identify more weakly bound LD proteins, recent protocols have used low stringency buffers, but carryover contaminants and low yields were often a problem. We have developed a sucrose gradient-based protocol to isolate LDs from Arabidopsis leaves, using Tween-20 and fresh tissue to increase yield. In both healthy and bacterially-infected Arabidopsis leaves, this protocol allowed to identify LD proteins that were later confirmed by microscopy analysis.

Desiccation tolerance in plants: Structural characterization of the cell wall hemicellulosic polysaccharides in three Selaginella species

Drought-induced dehydration of vegetative tissues in lycopods affects growth and survival. Different species of Selaginella have evolved a series of specialized mechanisms to tolerate desiccation in vegetative tissues in response to water stress. In the present study, we report on the structural characterization of the leaf cell wall of the desiccation-tolerant species S. involvens and two desiccation-sensitive species, namely S. kraussiana and S. moellendorffii. Isolated cell walls from hydrated and desiccated leaves of each species were fractionated and the resulting oligosaccharide fragments were analyzed to determine their structural features. Our results demonstrate that desiccation induces substantial modifications in the cell wall composition and structure. Altogether, these data highlight the fact that structural remodeling of cell wall hemicellulosic polysaccharides including XXXG-rich xyloglucan, arabinoxylan and acetylated galactomannan is an important process in order to mitigate desiccation stress in Selaginella.

Changes in gene expression in Camelina sativa roots and vegetative tissues in response to salinity stress

The response of Camelina sativa to salt stress was examined. Salt reduced shoot, but not root length. Root and shoot weight were affected by salt, as was photosynthetic capacity. Salt did not alter micro-element concentration in shoots, but increased macro-element (Ca and Mg) levels. Gene expression patterns in shoots indicated that salt stress may have led to shuttling of Na+ from the cytoplasm to the tonoplast and to an increase in K+ and Ca+2 import into the cytoplasm. In roots, gene expression patterns indicated that Na+ was exported from the cytoplasm by the SOS pathway and that K+ was imported in response to salt. Genes involved in chelation and storage were up-regulated in shoots, while metal detoxification appeared to involve various export mechanisms in roots. In shoots, genes involved in secondary metabolism leading to lignin, anthocyanin and wax production were up-regulated. Partial genome partitioning was observed in roots and shoots based on the expression of homeologous genes from the three C. sativa sub-genomes. Sub-genome I and II were involved in the response to salinity stress to about the same degree, while about 10% more differentially-expressed genes were associated with sub-genome III.

An Apoplastic β-Glucosidase is Essential for the Degradation of Flavonol 3-O-β-Glucoside-7-O-α-Rhamnosides in Arabidopsis.

Flavonol bisglycosides accumulate in plant vegetative tissues in response to abiotic stress, including simultaneous environmental perturbations (i.e. nitrogen deficiency and low temperature, NDLT), but disappear with recovery from NDLT. Previously, we determined that a recombinant Arabidopsis β-glucosidase (BGLU), BGLU15, hydrolyzes flavonol 3-O-β-glucoside-7-O-α-rhamnosides and flavonol 3-O-β-glucosides, forming flavonol 7-O-α-rhamnosides and flavonol aglycones, respectively. In this study, the transient expression of a BGLU15-Cherry fusion protein in onion epidermal cells demonstrated that BGLU15 was localized to the apoplast. Analysis of BGLU15 T-DNA insertional inactivation lines (bglu15-1 and bglu15-2) revealed negligible levels of BGLU15 transcripts, whereas its paralogs BGLU12 and BGLU16 were expressed in wild-type and bglu15 plants. The recombinant BGLU16 did not hydrolyze quercetin 3-O-β-glucoside-7-O-α-rhamnoside or rhamnosylated flavonols, but was active with the synthetic substrate, p-nitrophenyl-β-d-glucoside. In addition, shoots of both bglu15 mutants contained negligible flavonol 3-O-β-glucoside-7-O-α-rhamnoside hydrolase activity, whereas this activity increased by 223% within 2 d of NDLT recovery in wild-type plants. The levels of flavonol 3-O-β-glucoside-7-O-α-rhamnosides and quercetin 3-O-β-glucoside were high and relatively unchanged in shoots of bglu15 mutants during recovery from NDLT, whereas rapid losses were apparent in wild-type shoots. Moreover, losses of two flavonol 3-O-β-neohesperidoside-7-O-α-rhamnosides and kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside were evident during recovery from NDLT, regardless of whether BGLU15 was present. A spike in a kaempferol 7-O-α-rhamnoside occurred with stress recovery, regardless of germplasm, suggesting a contribution from hydrolysis of kaempferol 3-O-β-neohesperidoside-7-O-α-rhamnosides and/or kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside by hitherto unknown mechanisms. Thus, BGLU15 is essential for catabolism of flavonol 3-O-β-glucoside-7-O-α-rhamnosides and flavonol 3-O-β-glucosides in Arabidopsis.

Molecular cloning and characterization of salt inducible dehydrin gene from the C4 plant Pennisetum glaucum

Dehydrins (DHNs) or group 2 LEA (late embryogenesis abundant) proteins play a protective role in plants under different abiotic stress conditions like drought, salinity, cold and heat stress. DHNs are expressed in late embryogenesis and accumulate in vegetative tissues in response to desiccation stress in all photosynthetic organisms. Here we report the cloning and characterization of a PgDHN gene from the C4 plant Pennisetum glaucum. The PgDHN cDNA encoded for a polypeptide of 133 amino acids with an estimated molecular weight of 13.87kDa and isoelectric point of 6.81. The protein sequence analysis of PgDHN classified it into the YnSKn subgroup of dehydrins. Phylogenetic analysis revealed that PgDHN is evolutionarily related to a Setaria italica DHN. In silico sequence analysis of the PgDHN promoter identified a distinct set of cis-elements and transcription factor binding sites. PgDHN mRNA accumulated in leaves of P. glaucum upon treatment with NaCl stress. Recombinant PgDHN transformed E. coli cells showed improved tolerance and exhibited better growth rate under high salt concentration (750mM) and heat stress in comparison to their respective controls. Heterologous expression of PgDHN in transgenic yeast showed increased tolerance to multiple abiotic stresses. This study provides a possible role of PgDHN in stress adaptation and stress tolerance in pearl millet.

Significance of galactinol and raffinose family oligosaccharide synthesis in plants.

Abiotic stress induces differential expression of genes responsible for the synthesis of raffinose family of oligosaccharides (RFOs) in plants. RFOs are described as the most widespread D-galactose containing oligosaccharides in higher plants. Biosynthesis of RFOs begin with the activity of galactinol synthase (GolS; EC 2.4.1.123), a GT8 family glycosyltransferase that galactosylates myo-inositol to produce galactinol. Raffinose and the subsequent higher molecular weight RFOs (Stachyose, Verbascose, and Ajugose) are synthesized from sucrose by the subsequent addition of activated galactose moieties donated by Galactinol. Interestingly, GolS, the key enzyme of this pathway is functional only in the flowering plants. It is thus assumed that RFO synthesis is a specialized metabolic event in higher plants; although it is not known whether lower plant groups synthesize any galactinol or RFOs. In higher plants, several functional importance of RFOs have been reported, e.g., RFOs protect the embryo from maturation associated desiccation, are predominant transport carbohydrates in some plant families, act as signaling molecule following pathogen attack and wounding and accumulate in vegetative tissues in response to a range of abiotic stresses. However, the loss-of-function mutants reported so far fail to show any perturbation in those biological functions. The role of RFOs in biotic and abiotic stress is therefore still in debate and their specificity and related components remains to be demonstrated. The present review discusses the biology and stress-linked regulation of this less studied extension of inositol metabolic pathway.

Group II late embryogenesis abundant (LEA) proteins: structural and functional aspects in plant abiotic stress

Group II late embryogenesis abundant (LEA) proteins are crucial phytomolecules which accumulate mainly in the late phases of seed development and also in the vegetative tissues in response to exogenous stress. In spite of considerable research, their mechanism of action to generate plant tolerance against abiotic stresses still remains obscure. The present review focuses on the varied structural aspects which ultimately dictate the multifarious functions of the Group II LEA proteins when the plants are exposed to intense desiccation. Currently, several reports have been documented regarding newer in silico approaches in predicting LEA protein structure and the corresponding cis-elements. Coupled to recent transgenic approaches, these reports need to be properly structured to further characterize the physico-chemical and functional importance of LEA proteins in regulating tolerance against multiple abiotic stresses.

Galactinol synthase transcriptional profile in two genotypes of Coffea canephora with contrasting tolerance to drought.

Increased synthesis of galactinol and raffinose family oligosaccharides (RFOs) has been reported in vegetative tissues in response to a range of abiotic stresses. In this work, we evaluated the transcriptional profile of a Coffea canephora galactinol synthase gene (CcGolS1) in two clones that differed in tolerance to water deficit in order to assess the contribution of this gene to drought tolerance. The expression of CcGolS1 in leaves was differentially regulated by water deficit, depending on the intensity of stress and the genotype. In clone 109A (drought-susceptible), the abundance of CcGolS1 transcripts decreased upon exposure to drought, reaching minimum values during recovery from severe water deficit and stress. In contrast, CcGolS1 gene expression in clone 14 (drought-tolerant) was stimulated by water deficit. Changes in galactinol and RFO content did not correlate with variation in the steady-state transcript level. However, the magnitude of increase in RFO accumulation was higher in the tolerant cultivar, mainly under severe water deficit. The finding that the drought-tolerant coffee clone showed enhanced accumulation of CcGolS1 transcripts and RFOs under water deficit suggests the possibility of using this gene to improve drought tolerance in this important crop.

Characterisation of an SKn-type Dehydrin Promoter from Wheat and Its Responsiveness to Various Abiotic and Biotic Stresses

Dehydrins (DHNs; late embryogenesis abundant D11 family) are a family of intrinsically unstructured plant proteins that accumulate in seed embryos or in vegetative tissues in response to environmental stresses. To elucidate the regulatory mechanisms of wheat-derived DHNs under these stresses, we isolated and characterised the full-length cDNA, genomic and promoter sequences of dehydrin wzy1-2 (SK3-type) from the wheat (Triticum aestivum L.) Zhengyin 1. We then analysed the responsiveness of the wzy1-2 promoter to various abiotic and biotic stresses. The sequence analysis indicated that the gene is 1,255 bp long and contains one 103-bp intron, which is inserted in the nucleotide sequence encoding the S-motif and characterised by a GT–AG border. The promoter contains several potential stress-related cis-acting regulatory elements, including ABA-, dehydration-, low temperature-, methyl jasmonate (MeJA)-, gibberellin (GA)-, salicylic acid (SA)-, and defence and stress-responsive elements. The quantitative PCR analysis demonstrated that the transcript accumulation occurred in response to osmotic stress, cold, MeJA, abscisic acid (ABA), GA and SA treatments. The histochemical analysis of GUS expression in wheat transgenic calli demonstrated that wzy1-2 promoter activity could be upregulated by osmotic stress, ABA, GA or SA treatment. Quantitative fluorometric GUS assays further demonstrated that one SA-responsive element was located between −59 and −39 bp and that one GA-responsive element was situated between −39 and +107 bp. Combined, these results indicate that multiple cis-acting elements are involved in the induction of the wzy1-2 gene, which implies that different cis-acting elements interact in stress response cross-talk.

Group 1 LEA proteins, an ancestral plant protein group, are also present in other eukaryotes, and in the archeae and bacteria domains

Water is an essential element for living organisms, such that various responses have evolved to withstand water deficit in all living species. The study of these responses in plants has had particular relevance given the negative impact of water scarcity on agriculture. Among the molecules highly associated with plant responses to water limitation are the so-called late embryogenesis abundant (LEA) proteins. These proteins are ubiquitous in the plant kingdom and accumulate during the late phase of embryogenesis and in vegetative tissues in response to water deficit. To know about the evolution of these proteins, we have studied the distribution of group 1 LEA proteins, a set that has also been found beyond the plant kingdom, in Bacillus subtilis and Artemia franciscana. Here, we report the presence of group 1 LEA proteins in green algae (Chlorophyita and Streptophyta), suggesting that these group of proteins emerged before plant land colonization. By sequence analysis of public genomic databases, we also show that 34 prokaryote genomes encode group 1 LEA-like proteins; two of them belong to Archaea domain and 32 to bacterial phyla. Most of these microbes live in soil-associated habitats suggesting horizontal transfer from plants to bacteria; however, our phylogenetic analysis points to convergent evolution. Furthermore, we present data showing that bacterial group 1 LEA proteins are able to prevent enzyme inactivation upon freeze-thaw treatments in vitro, suggesting that they have analogous functions to plant LEA proteins. Overall, data in this work indicate that LEA1 proteins' properties might be relevant to cope with water deficit in different organisms.

Isolation and expression analysis of LEA genes in peanut (Arachis hypogaea L.)

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides. A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.

Molecular mechanism of dehydrin in response to environmental stress in plant

Abstract Dehydrins, known as the D-11 subgroup of late embryogenesis abundant (LEA) protein, are an immunologically distinct family of proteins, which typically accumulate in desiccation-tolerant seed embryo or in vegetative tissues in response to various environmental stresses such as drought, salinity and freezing. The existence of conservative sequences designated as K, 5, and Y segments is a structural feature of dehydrins, and the K segment found in all dehydrins represents a highly conserved 15 amino acid motif (EKKGIMDKIKEKLPG) and forms an amphiphilic α-helix. According to the arrangement of these domains and clustering analysis, dehydrins are subdivided into 5 subtypes: YnSK, Kn, KnS, SKn and YnK. Different types of dehydrins are induced by different environmental stress in plants. Study results showed that dehydrins might play important protective roles under abiotic stress via a number of different mechanisms, including improving or protecting enzyme activities by the cryoprotective activity in responding to freez/thaw or dehydration; stabilizing vesicles or other endomembrane structures by function as the membrane stabilizer during freeze induced dehydration, and preventing the membrane system from the oxidative damage induced by reactive oxygen radicals as the radical scavenger. Here, the gene expression and molecular mechanisms of dehydrin in response to stress in plants are discussed.

Isolation and Characterization of cDNA Encoding Three Dehydrins Expressed During Coffea canephora (Robusta) Grain Development

Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized. The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT-PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2). The CcDH1 and CcDH2 genes encode Y(3)SK(2) dehydrins and the CcDH3 gene encodes an SK(3) dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized. cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.

The molecular characterization of a cDNA encoding the putative integral membrane protein, HvSec61α, expressed during early stage of barley kernel development

The Sec61 complex is essential for the translocation of both soluble proteins and integral membrane proteins into the membrane of the endoplasmic reticulum. To study the molecular events that occur during kernel development, the suppression subtractive hybridization (SSH) method was performed using grains of barley ( Hordeum vulgare L.) cv. Karl at different developmental stages (14 DAF and 5 DAF). One of the SSH clones showed high homology to cDNA encoding, a Sec61α subunit in wheat, but not to any clones previously registered in barley. The full-length clone was screened and then designated as HvSec61α ( Hordeum vulgare Sec61 α subunit). The transcription level of HvSec61α was low in grains at 5 DAF, increased at 8 DAF and was highest at 11 DAF. The transgenic Arabidopsis over expressing HvSec61α grew more vigorously than wild-type plants during drought stress. The expression of HvSec61α was high in grains but low in other tissues, such as the pericarp, stem, and leaf. The expression of HvSec61α increased in vegetative tissues in response to abiotic stresses such as PEG, cold, or salt as well as to hormones such as ABA, MeJA, and cytokinin. We speculate that the physiological function of the Sec61 complex might not only be to translocate secretory proteins, but as well integral membrane proteins in the ER during early kernel development in barley.

The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences.

The abscisic acid (ABA)-induced protein kinase PKABA1 is present in dormant seeds and is a component of the signal transduction pathway leading to ABA-suppressed gene expression in cereal grains. We have identified a member of the ABA response element-binding factor (ABF) family of basic leucine zipper transcription factors from wheat (Triticum aestivum) that is specifically bound by PKABA1. This protein (TaABF) has highest sequence similarity to the Arabidopsis ABA response protein ABI5. In two-hybrid assays TaABF bound only to PKABA1, but not to a mutant version of PKABA1 lacking the nucleotide binding domain, suggesting that binding of TaABF requires prior binding of ATP as would be expected for binding of a protein substrate by a protein kinase. TaABF mRNA accumulated together with PKABA1 mRNA during wheat grain maturation and dormancy acquisition and TaABF transcripts increased transiently during imbibition of dormant grains. In contrast to PKABA1 mRNA, TaABF mRNA is seed specific and did not accumulate in vegetative tissues in response to stress or ABA application. PKABA1 produced in transformed cell lines was able to phosphorylate synthetic peptides representing three specific regions of TaABF. These data suggest that TaABF may serve as a physiological substrate for PKABA1 in the ABA signal transduction pathway during grain maturation, dormancy expression, and ABA-suppressed gene expression.

CpR18, a novel SAP-domain plant transcription factor, binds to a promoter region necessary for ABA mediated expression of the CDeT27-45 gene from the resurrection plant Craterostigma plantagineum Hochst.

CDeT27-45 is a lea-like gene from the resurrection plant Craterostigma plantagineum (Scrophulariaceae) which is strongly expressed in vegetative tissues in response to dehydration or treatment with abscisic acid (ABA). Expression of the gene is correlated with the acquisition of desiccation tolerance. Nuclear proteins bind to a 29-bp cis-regulatory region of the promoter which is essential for transcriptional activation of the CDeT27-45 gene by ABA. Using a yeast one-hybrid screen, the cDNA clone CpR18 was isolated, which encodes a protein with specific binding activity for the cis-regulatory element in the CDeT27-45 promoter. The protein contains an acidic region, a SAP-domain, a zinc finger of the C3H-type, and two motifs which are conserved in proteins from several plant species. One of the conserved regions is rich in basic residues and is predicted to form a helix-loop-helix structure. The R18 gene shows high similarities to genomic sequences and ESTs from other plant species. The tissue-specific expression pattern of the rare R18 mRNA and the distribution of nuclear protein binding activity for the CDeT27-45 promoter fragment are compared. The R18 protein is indeed localized in the nucleus, and activates transcription of CDeT27-45 promoter-GUS fusion constructs in tobacco protoplasts. DNA blot analysis and isolation of genomic clones reveal that two copies of R18 are present in the C. plantagineum genome.

Aldose reductase in rice ( Oryza sativa L.): stress response and developmental specificity

Aldose reductase (AR) protein and enzyme (alditol: NAD (P) + 1-oxidoreductase, EC 1.1.1.21) activity have been identified in mature seeds of indica rice cultivars. The protein begins to accumulate 15 days after pollination, reaches a peak at seed maturity and disappears upon imbibition. Furthermore, AR is induced in vegetative tissues in response to exogenous ABA application and other stress conditions, such as PEG mediated water stress and salinity. Increase in AR protein levels upon stress are in close agreement with a similar increase in enzyme activity. Varietal differences in AR levels have been demonstrated. Interestingly, all tested tolerant cultivars (as denoted by breeders) accumulate AR in vegetative tisssue in response to ABA application, while the sensitive line, Hamsa, does not do this under similar stress conditions, suggesting that AR may be associated with stress tolerance. Furthermore, AR protein has been identified in mature seeds of some selected cereals indicating the conserved nature of AR across grasses.

Novel Plant Ca2+-binding Protein Expressed in Response to Abscisic Acid and Osmotic Stress

A cDNA corresponding to an mRNA which accumulates in germinating rice seeds in response to the phytohormone abscisic acid was isolated by differential hybridization. Northern blotting indicated that the mRNA also accumulates in vegetative tissues in response to treatment with abscisic acid and to osmotic stress. Sequencing identified a major open reading frame encoding a novel protein of 27.4 kDa. The identity of the open reading frame was confirmed by comparing the translation products of cellular, hybrid-selected, and in vitro transcribed RNAs and by immunoprecipitation. Western blotting of cellular extracts indicated that the protein is associated with microsomal or membrane fractions. Data base searches indicated that it contains a conserved Ca(2+)-binding, EF-hand motif and that related proteins are similarly expressed in Arabidopsis thaliana. A fusion protein purified from Escherichia coli containing the putative EF-hand region was shown to bind Ca2+ in blot binding assays. These data identify a novel gene family encoding proteins involved in the response of plants to abscisic acid and osmotic stress.