Gene Expression In Barley Research Articles

Expression analysis of barley (Hordeum vulgare L.) during salinity stress

Barley (Hordeum vulgare L.) is a salt-tolerant crop species with considerable economic importance in salinity-affected arid and semiarid regions of the world. In this work, barley cultivar Morex was used for transcriptional profiling during salinity stress using a microarray containing approximately 22,750 probe sets. The experiment was designed to target the early responses of genes to a salinity stress at seedling stage. We found a comparable number of probe sets up-regulated and down-regulated in response to salinity. The differentially expressed genes were broadly characterized using gene ontology and through expression-based hierarchical clustering to identify interesting features in the data. A prominent feature of the response to salinity was the induction of genes involved in jasmonic acid biosynthesis and genes known to respond to jasmonic acid treatment. A large number of abiotic stress (heat, drought, and low temperature) related genes were also found to be responsive to salinity stress. Our results also indicate osmoprotection to be an early response of barley under salinity stress. Additionally, we compared the results of our studies with two other reports characterizing gene expression of barley under salinity stress and found very few genes in common.

Transcriptional regulation by abscisic acid in barley (Hordeum vulgare L.) seeds involves autoregulation of the transcription factor HvABI5

The barley bZIP transcription factor HvABI5 mediates abscisic acid (ABA)-upregulated gene expression in barley (Hordeum vulgare L.) seeds. HvABI5 specifically recognizes cis-elements of the ABA response complexes present in the promoters of the ABA-induced genes HVA1 and HVA22. HvABI5 together with another transcription factor, HvVP1, are required for the transactivation of these promoters, and this transactivation process is insensitive to the negative regulator abi1-1. The expression of HvABI5 itself appeared to be induced by ABA and can be suppressed by abi1-1. Gain- and loss-of-function studies in barley aleurone cells show that HvABI5 expression is positively regulated by a feed-forward circuit that involves HvABI5 itself and HvVP1. Mutation of the Ser residue in HvABI5, which has been shown to be phosphorylated in an ABA-dependent manner in the rice orthologue of HvABI5, reduces the transactivation activity of the factor by 50%. Although levels of HvABI5 and its transcript are enhanced by ABA treatment, the nuclear localization of HvABI5 is not affected by ABA. A model based on these observations is presented to explain the ABA upregulation of gene expression.

Photoperiod and Temperature Interactions Regulate Low-Temperature-Induced Gene Expression in Barley

Vernalization and photoperiod (PP) responses are developmental mechanisms that allow plants to synchronize their growth and reproductive cycles with the seasonal weather changes. Vernalization requirement has been shown to influence the length of time that low-temperature (LT)-induced genes are up-regulated when cereal species are exposed to acclimating temperatures. The objective of the present study was to determine whether expression of LT-inducedWcs and Wcor gene families is also developmentally regulated by PP response. The LT-tolerant, highly short-day (SD)-sensitive barley (Hordeum vulgare L. cv Dicktoo) was subjected to 8-h SD and 20-h long-day PPs at cold-acclimating temperatures over a period of 70 d. A delay in transition from the vegetative to the reproductive stage under SD resulted in an increased level and longer retention of LT tolerance. Similar WCS and WCOR protein homologs were expressed, but levels of expression were much higher in plants acclimated under SD, indicating that the poor LT tolerance of long-day plants was the result of an inability to maintain LT-induced genes in an up-regulated state. These observations indicate that the PP and vernalization genes influence the expression of LT-induced genes in cereals through separate pathways that eventually converge to activate genes controlling plant development. In both instances, the delay in the transition from the vegetative to the reproductive stage produces increased LT tolerance that is sustained for a longer period of time, indicating that the developmental genes determine the duration of expression of LT-induced structural genes.

Photoperiod and Temperature Interactions Regulate Low-Temperature-Induced Gene Expression in Barley

Vernalization and photoperiod (PP) responses are developmental mechanisms that allow plants to synchronize their growth and reproductive cycles with the seasonal weather changes. Vernalization requirement has been shown to influence the length of time that low-temperature (LT)-induced genes are up-regulated when cereal species are exposed to acclimating temperatures. The objective of the present study was to determine whether expression of LT-induced Wcs and Wcor gene families is also developmentally regulated by PP response. The LT-tolerant, highly short-day (SD)-sensitive barley (Hordeum vulgare L. cv Dicktoo) was subjected to 8-h SD and 20-h long-day PPs at cold-acclimating temperatures over a period of 70 d. A delay in transition from the vegetative to the reproductive stage under SD resulted in an increased level and longer retention of LT tolerance. Similar WCS and WCOR protein homologs were expressed, but levels of expression were much higher in plants acclimated under SD, indicating that the poor LT tolerance of long-day plants was the result of an inability to maintain LT-induced genes in an up-regulated state. These observations indicate that the PP and vernalization genes influence the expression of LT-induced genes in cereals through separate pathways that eventually converge to activate genes controlling plant development. In both instances, the delay in the transition from the vegetative to the reproductive stage produces increased LT tolerance that is sustained for a longer period of time, indicating that the developmental genes determine the duration of expression of LT-induced structural genes.

Octadecanoid-derived alteration of gene expression and the "oxylipin signature" in stressed barley leaves. Implications for different signaling pathways.

Stress-induced gene expression in barley (Hordeum vulgare cv Salome) leaves has been correlated with temporally changing levels of octadecanoids and jasmonates, quantified by means of gas chromatography/mass spectrometry-single ion monitoring. Application of sorbitol-induced stress led to a low and transient rise of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA), and the methyl esters JAME and OPDAME, respectively, followed by a large increase in their levels. JA and JAME peaked between 12 and 16 h, about 4 h before OPDA and OPDAME. However, OPDA accumulated up to a 2.5-fold higher level than the other compounds. Dihomo-JA and 9, 13-didehydro-OPDA were identified as minor components. Kinetic analyses revealed that a transient threshold of jasmonates or octadecanoids is necessary and sufficient to initiate JA-responsive gene expression. Although OPDA and OPDAME applied exogenously were metabolized to JA in considerable amounts, both of them can induce gene expression, as evidenced by those genes that did not respond to endogenously formed JA. Also, coronatine induces JA-responsive genes independently from endogenous JA. Application of deuterated JA showed that endogenous synthesis of JA is not induced by JA treatment. The data are discussed in terms of distinct signaling pathways.

Plastid photodamage and Cab gene expression in barley leaves

The effects of norflurazon (NF) and amitrole (AM), two bleaching herbicides which inhibit carotenogenesis, were compared in leaves of 7‐day‐old barley (Hordeum vulgare L. cv Express) plants grown in damaging light. The herbicide effects were analysed with respect to chloroplast organization, photosynthetic functionality and nuclear photodependent expression of the Lhcb1 gene, which codes for the Lhcb1 light‐harvesting chlorophyll a/b binding protein of photosystem II. Both herbicides caused dramatic photooxidation of organelles, which were photosynthetically unfunctional. Plastids of NF‐treated plants lacked thylakoids and pigments. Plastids of AM‐treated plants had some strikingly altered membranes and contained only very small quantities of chlorophylls. Despite the presence of severely photodamaged plastids, cells of AM‐treated leaves contained high levels of Lhcb1 transcript. This transcript, on the contrary, was completely absent in the cells of NF‐treated plants. These findings suggest that in order to block expression of nuclear genes coding for plastid‐resident proteins, photodamage leading to the complete dismantling of thylakoids and to the total absence of any form of photosynthetic pigment is required.

Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B1- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B1- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T0 leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T1 progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T1 progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T5 progeny in one line, T4 progeny in one line, T3 progeny in three lines and T2 or T1 progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B1- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T1 progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T2 or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T2 or later generations. We conclude that the B1- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering.

ChemInform Abstract: Structure—Activity Relations of Substituted, Deleted or Stereospecifically Altered Jasmonic Acid in Gene Expression of Barley Leaves.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Structure–activity relations of substituted, deleted or stereospecifically altered jasmonic acid in gene expression of barley leaves

Abstract Jasmonic acid and 66 structurally related compounds were tested to find the structural requirements which induce the expression of jasmonate-responsive genes in barley. An intact cyclopentanone ring as well as a pentenyl side chain exhibiting only minor alterations are necessary for this activity. The (−)-enantiomeric and the (+)-7- iso -enantiomeric structure increase activity of jasmonoyl compounds.

Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves -- the link between jasmonate and abscisic acid

In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.

Amino acid conjugates of jasmonic acid induce jasmonate-responsive gene expression in barley ( Hordeum vulgare L.) leaves

Leaves of barley ( Hordeum vulgare L. cv. Salome) treated with jasmonic acid (JA), its methyl ester (JM), or its amino acid conjugates exhibit up-regulation of specific genes and down-regulation of house-keeping genes. This transcriptional regulation exhibits several specificities. (i) The (−)-enantiomers are more active, and conjugates are mainly active if they carry an l-amino acid moiety. (ii) The various JA-responsive genes respond differentially to enantiomeric and chiralic forms. (iii) Both JA and its amino acid conjugates exhibiting no or negligible interconversion induce/repress genes.

Temporal pattern of jasmonate-induced alterations in gene expression of barley leaves.

Leaf tissues of barley (Hordeum vulgare L. cv. Salome) respond to methyl jasmonate (JaMe) treatment with a characteristic pattern of gene expression. Jasmonate-induced proteins (JIPs), such as leaf thionins (jip15 gene product) and ribosome-inactivating proteins (jip60 gene product), rapidly accumulate. Their genes are transiently transcriptionally activated, as shown here by the determination of in-vitro transcription rates in run-off assays. In contrast to jip genes, expression of photosynthetic genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS gene product) and a type III light-harvesting chlorophyll-a/b-binding protein (LHCP; lhbC1 gene product), for example, was rapidly down-regulated in JaMe-treated barley leaves. Despite decreasing rates of rbcS and lhbC1 gene transcription, their transcripts were maintained in JaMe-treated leaf tissues for at least 36 h. Only at a later stage, was there a decline in the levels of rbcS and lhbC1, but not jip, transcripts, suggesting a selective destabilization of photosynthetic mRNAs in JaMe-treated leaf tissues.

Modular Nature of Abscisic Acid (ABA) Response Complexes: Composite Promoter Units That Are Necessary and Sufficient for ABA Induction of Gene Expression in Barley

Modular Nature of Abscisic Acid (ABA) Response Complexes: Composite Promoter Units That Are Necessary and Sufficient for ABA Induction of Gene Expression in Barley

Effect of Salt Stress on Germin Gene Expression in Barley Roots.

Germin gene expression in barley (Hordeum vulgare L.) seedlings responds to developmental and environmental cues. During seed germination, germin mRNA levels were maximal 2 d after the start of imbibition in control seedlings and declined to low levels by 6 d. When seeds were sown in the presence of 200 mM NaCl, germin mRNA levels were also maximal after 2 d, but NaCl treatment, which slowed seedling growth, prolonged germin gene expression for an additional 1 d. In 4-d-old seedlings, germin mRNA levels were highest in roots and higher in the vascular transition region than in shoots. In roots of 6-d-old seedlings, germin gene expression was regulated by salt shock and plant growth regulators. Induced germin mRNA levels were maximal 8 h after treatment with NaCl, salicylate, methyl salicylate, or methyl jasmonate and 4 h after treatment with abscisic acid and indoleacetic acid. Like germin mRNA, dehydrin mRNA levels were maximal 8 h after NaCl treatment. In contrast, peroxidase mRNA levels declined to less than control levels within 30 min of treatment. Germin gene expression is regulated developmentally by salt stress and by treatments with plant hormones. Since germin is an oxalate oxidase, these result imply that oxalate has important roles in plant development and homeostasis.

Isolation and transformation of rice aleurone protoplasts

Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.

Isolation and transformation of rice aleurone protoplasts

Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.

Early induction of new mRNAs accompanies the resistance reaction of barley to the wheat pathogen, Erysiphe graminis f.sp. tritici

The results presented here show that inoculation of barley with wheat powdery mildew ( Erysiphe graminis f.sp. tritici), which is non-pathogenic on barley, is followed by an early and rapid activation of a specific set of genes as indicated by the accumulation of specific mRNAs. Two-dimensional polyacrylamide gel electrophoresis of in vitro translation products made from RNA isolated from inoculated and uninoculated barley leaves showed several new polypeptide products at 4 h after inoculation. These products were not apparent at 2 h and they are most probably of plant origin. Induction of new mRNA species correlated with interactions between the primary germ tubes of mildew spores and the epidermis. Analysis of RNA preparations from epidermal strips indicates that at least some of the induced mRNAs are also localized in the epidermis. The results are discussed in relation to other reports of gene expression in barley accompanying resistance responses, and in relation to induced resistance in barley against powdery mildew.

Overlap of gametophytic and sporophytic gene expression in barley

The overlap of gametophytic and sporophytic gene expression in barley was studied by means of enzyme electrophoresis. Of the isozymes found, 60% were expressed in both gametophyte and sporophyte, 30% were sporophyte specific, and 10% were gametophyte specific. A considerable amount of the barley genome is thus potentially amenable to gametophytic selection. The estimated sizes of the common, sporophytic and gametophytic domains in barley gene expression correspond with the estimates obtained in other plant species.

Differential mRNA transcription during salinity stress in barley

The molecular and genetic bases of salinity tolerance in plants are not understood. Gene expression at the mRNA level was investigated in a salt-tolerant and a salt-sensitive genotype of barley. Seedlings were exposed to NaCl stress and translatable mRNAs were isolated from root and shoot tissues. A reticulocyte cell-free system was programed with barley mRNAs and the in vitro products were resolved on two-dimensional polyacrylamide gels following isoelectric focusing or nonequilibrium pH gradient gel electrophoresis in the first dimension. The functional mRNAs in unstressed seedlings were qualitatively almost indistinguishable in the two genotypes. However, salinity stress triggered differential transcription of specific mRNAs depending upon genotype and tissue. In roots, 12 new mRNAs were induced that encoded proteins of 21-34 kDa, with a pI range of 6.1-7.7. In shoots, the 9 new mRNAs coded for proteins of 18-50.5 kDa, with a pI range of 5.4-7.8. These new stress mRNAs represented one of two main classes. Class I consisted of mRNAs shared by both genotypes. Class II represented mRNAs specific to each genotype; unique mRNAs of roots accumulated preferentially in the salt-tolerant genotype, whereas those of shoots accumulated in the salt-sensitive genotype. The findings suggest that transcriptional as well as posttranscriptional mechanisms regulate gene expression in barley during salinity stress.

Hormonal control of alpha-amylase gene expression in barley. Studies using a cloned CDNA probe.

Starting with an enriched alpha-amylase mRNA preparation, cDNA was synthesized by reverse transcription and was cloned at the Pst I site of plasmid vector pBR322 by the "G-C tailing" procedure. Clones containing alpha-amylase cDNA sequences were identified among the recombinant clones by in vitro translation of hybrid-selected mRNA followed by immunoprecipitation of translation products with antiserum to alpha-amylase. The longest alpha-amylase cDNA clone isolated was 630 base pairs long and was characterized by digestion with restriction enzymes. Using this clone as a labeled nucleic acid hybridization probe, we observed that multiple bands of DNA with alpha-amylase sequences were present in restriction digests of barley embryo DNA. This indicates the presence of a family of barley alpha-amylase or alpha-amylase-like genes. Analysis of RNA blots with the cloned alpha-amylase hybridization probe indicated that alpha-amylase mRNA is synthesized de novo in barley aleurones after addition of gibberellic acid. The gibberellic acid-induced accumulation of alpha-amylase mRNA is blocked by cycloheximide which suggests the requirement of a newly synthesized protein factor for efficient expression of alpha-amylase gene.