Characterization of Expression of Drought- and Abscisic Acid-Regulated Tomato Genes in the Drought-Resistant Species Lycopersicon pennellii.

A number of genes are induced by drought stress, and some of these genes are regulated by the plant hormone abscisic acid (ABA). In tomato (Lycopersicon esculentum), four genes have been identified and isolated that require elevated levels of endogenous ABA for expression: le4, le16, le20, and le25. To gain a better understanding of the role of these genes during stress, their expression has been studied in the drought-resistant relative of tomato, Lycopersicon pennellii. It was determined that homologous genes to all four of the L. esculentum genes were present in the L. pennellii genome. Studies were undertaken to compare the expression characteristics of these genes in L. esculentum, L. pennellii, and their F1. Using two methods of water-deficit imposition, whole plants to which water was withheld and detached leaves that were wilted to 88% of their original fresh weight, it was demonstrated that transcripts of these genes accumulated in L. pennellii in response to water deficit. In general, the increase occurred after a longer period of water deficit in L. pennellii than in tomato. As in drought-sensitive species, ABA levels were elevated by drought stress in L. pennellii, although the levels were reduced compared with those in tomato. All four tomato genes were responsive to ABA in L. esculentum and the F1, but only three of the four genes (le16, le20, and le25) were induced in response to exogenous application of ABA in L. pennellii. The patterns of expression of these genes in L. pennellii are generally similar to that of L esculentum; therefore, it is suggested that these genes play a similar, yet undefined, role in both genotypes rather than being genes that are responsible for the greater drought resistance of L. pennellii.

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae.

During periods of water deficit, plants accumulate late embryogenesis-abundant (LEA) proteins which are thought to protect cells from stresses associated with dehydration. One of these genes, le25, is expressed in tomato leaves and roots in response to water deficit and abscisic acid accumulation. To study the function of this protein and to test the effect of overproduction of the LE25 protein in Saccharomyces cerevisiae (Sc), a recombinant plasmid in which le25 is expressed under the control of the GAL1 promoter was constructed. The content of LE25 was high in Sc cells transformed with the recombinant plasmid. The transformant exhibited several stress-tolerant phenotypes. Growth of the transformant in a medium with 1.2 M NaCl was improved, as compared to a control strain. While the control strain showed a long lag phase of 40 h, le25-expressing cells showed a shortened lag phase of 10 h. However, no growth improvement was observed in a medium with 2 M sorbitol. In addition, the transformant had an increased survival rate after freezing stress, but not after high-temperature stress. These results, together with its predicted secondary structure, may indicate that LE25 functions as an ion scavenger.

Abscisic acid regulation of gene expression during water-deficit stress in the era of the Arabidopsis genome.

Changes in gene expression may lead to cellular adaptation of water-deficit stress, yet all of the induced mRNAs may not play this role. Changes in gene expression must be signalled by transduction mechanisms that first sense a water deficit. This first step triggers changes in gene expression that function to synthesize additional signals such as abscisic acid (ABA). The enzymes involved in ABA biosynthesis have been cloned and their regulation during water-deficit stress is being characterized. Once ABA levels are increased, further signalling mechanisms are initiated to signal new gene expression patterns that are proposed to play a role in cellular adaptation to water-deficit stress. As the genome of Arabidopsis is now completed, much more information can be exploited to characterize these responses.

Drought- and ABA-Induced Changes in Polypeptide and mRNA Accumulation in Tomato Leaves.

Drought stress triggers abscisic acid (ABA) biosynthesis resulting in ABA accumulation. The ABA-deficient tomato mutant, flacca (Lycopersicon esculentum Mill. cv Ailsa Craig), does not synthesize ABA in response to drought stress. This mutant has been used to distinguish polypeptides and in vitro translation products that are synthesized during drought stress in response to elevated ABA levels from those that are induced directly by altered water relations. A set of polypeptides and in vitro translation products was synthesized during drought stress in the wild type. These polypeptides and in vitro translation products were synthesized to a lesser extent in the drought-stressed ABA-deficient mutant. Treatment of flacca with ABA resulted in the synthesis of the drought-stress-induced polypeptides and in vitro translation products. These results support the hypothesis that many of the polypeptides that are synthesized during drought are regulated by alterations in ABA concentration. Similarly, the mRNA population was altered by ABA during drought stress.

Wild-type levels of abscisic Acid are not required for heat shock protein accumulation in tomato.

Levels of endogenous abscisic acid (ABA) in wild type were not required for the synthesis of heat shock proteins in detached leaves of tomato (Lycopersicon esculentum Mill., cv Ailsa Craig). Heat-induced alterations in gene expression were the same in the ABA-deficient mutant of tomato, flacca, and the wild type. Heat tolerance of the mutant was marginally less that the wild type, and in contrast, ABA applications significantly reduced the heat tolerance of wild-type leaves. It was concluded that elevated levels of endogenous ABA are not involved in the tomato heat shock response.

The Compartmentation of Abscisic Acid and beta-d-Glucopyranosyl Abscisate in Mesophyll Cells.

beta-d-Glucopyranosyl abscisate (ABA-GE) is synthesized in Xanthium strumarium L. leaves during water stress. Following recovery from stress, the amount of ABA-GE does not decline. These observations led to the hypothesis that ABA-GE is sequestered in the vacuole where it is metabolically inert. The localization of abscisic acid (ABA) and ABA-GE was investigated by a dimethyl sulfoxide (DMSO) compartmentation method and by direct isolation of vacuoles.With the DMSO compartmentation method it was shown that in Xanthium mesophyll cells ABA was in a compartment not accessible to DMSO, presumably the chloroplast, whereas ABA-GE was in a compartment accessible to DMSO, presumably the vacuole. Neutral red, which accumulates in the vacuoles, showed a similar DMSO concentration dependence for its release from the cells as ABA-GE.Vacuoles isolated from Vicia faba L. leaf protoplasts contained 22% of the total ABA and 91% of the ABA-GE. Some of the ABA in the vacuole preparations was probably due to cytoplasmic contamination. These findings indicate that ABA-GE is sequestered in the vacuoles of mesophyll cells where the conjugated form of ABA is removed from the active ABA pool.

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons.

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the beta-subunit of beta-conglycinin, one of the major storage proteins of soybean seeds. The amount of the alpha'-and alpha-subunits of beta-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the beta-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and beta-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against beta-conglycinin, there was an increased amount of pre-beta-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-beta-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of beta-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the beta-subunit of beta-conglycinin in developing cultured soybean cotyledons.

Characterization of three mRNAs that accumulate in wilted tomato leaves in response to elevated levels of endogenous abscisic acid.

The accumulation of abscisic acid (ABA) has been shown to regulate some of the changes in gene expression which occur during water deficit. In order to characterize these ABA-induced changes, we have identified and isolated three copy DNAs (cDNAs) that represent genes which are expressed in response to ABA during drought stress. The ABA-deficient mutant of tomato, flacca, synthesizes low levels of ABA during water deficit compared to the wild type (WT) (Lycopersicon esculentum Mill. cv. Ailsa Craig). The mutant flacca was used to distinguish cDNAs corresponding to mRNAs which accumulate during water deficit in response to elevated levels of ABA from those mRNAs which are not ABA responsive. A cDNA library representing the mRNA population of wilted WT tomato leaves was constructed and a series of differential screens was used to select cDNAs that represent putative ABA- and drought-induced mRNAs. Three cDNAs were isolated from the screens and were identified as pLE4, pLE16, and pLE25. The corresponding mRNAs were preferentially expressed in wilted WT leaves and were not exessed in wilted ABA-deficient mutant leaves. The inability of the mutant to accumulate these drought-induced transcripts was reversed with exogenously applied ABA. A correlation was observed between the accumulation of the drought-induced mRNAs and the endogenous ABA levels measured in WT leaves throughout increasing periods of water deficit. These results indicate that endogenous ABA regulates the accumulation of pLE4, pLE16, and pLE25 mRNAs in tomato leaves during water deficit.

Compartmentation and equilibration of abscisic Acid in isolated xanthium cells.

The compartmentation of endogenous abscisic acid (ABA), applied (+/-)-[(3)H]ABA, and (+/-)-trans-ABA was measured in isolated mesophyll cells of the Chicago strain of Xanthium strumarium L. The release of ABA to the medium in the presence or absence of DMSO was used to determine the equilibration of ABA in the cells. It was found that a greater percentage of the (+/-)-[(3)H]ABA and the (+/-)-trans-ABA was released into the medium than of the endogenous ABA, indicating that applied ABA did not equilibrate with the endogenous material.Therefore, in further investigations only the compartmentation of endogenous ABA was studied. Endogenous ABA was released from Xanthium cells according to the pH gradients among the various cellular compartments. Thus, darkness, high external pH, KNO(2), and droughtstress all increased the efflux of ABA from the cells. Efflux of ABA from the cells in the presence of 0.6 m mannitol occurred within 30 seconds, but only 8% of the endogenous material was released during the 20 minute treatment.

NPR genes, which are negatively regulated by phytochrome action in Lemna gibba L. G-3, can also be positively regulated by abscisic acid.

We have found that NPR1 and NPR2, two genes from Lemna gibba L. G-3 that can be negatively regulated by phytochrome action, can also be positively regulated by the plant hormone abscisic acid (ABA). Both genes were responsive to low concentrations of exogenous ABA; an increase in NPR1 RNA could be detected in response to concentrations as low as 10 nM. We have also tested phytochrome responsiveness of 5' promoter-deletion constructs of one of these genes, NPR1, in transient assays utilizing particle bombardment. This analysis demonstrated that DNA sequences important for phytochrome regulation are present downstream of -198 from the transcription start site. A response to ABA treatment could also be observed in the transient assay system. When intact plants were placed in darkness, there was an increase in ABA levels as well as increased levels of NPR1 and NPR2 RNA.

Nucleotide sequence and spatial expression pattern of a drought- and abscisic Acid-induced gene of tomato.

The nucleotide sequence of le16, a tomato (Lycopersicon esculentum Mill.) gene induced by drought stress and regulated by abscisic acid specifically in aerial vegetative tissue, is presented. The single open reading frame contained within the gene has the capacity to encode a polypeptide of 12.7 kilodaltons and is interrupted by a small intron. The predicted polypeptide is rich in leucine, glycine, and alanine and has an isoelectric point of 8.7. The amino terminus is hydrophobic and characteristic of signal sequences that target polypeptides for export from the cytoplasm. There is homology (47.2% identity) between the amino terminus of the LE 16 polypeptide and the corresponding amino terminal domain of the maize phospholipid transfer protein. le16 was expressed in drought-stressed leaf, petiole, and stem tissue and to a much lower extent in the pericarp of mature green tomato fruit and developing seeds. No expression was detected in the pericarp of red fruit or in drought-stressed roots. Expression of le16 was also induced in leaf tissue by a variety of other abiotic stresses including polyethylene glycol-mediated water deficit, salinity, cold stress, and heat stress. None of these stresses or direct applications of abscisic acid induced the expression of le16 in the roots of the same plants. The unique expression characteristics of this gene indicates that novel regulatory mechanisms, in addition to endogenous abscisic acid, are involved in controlling gene expression.

Organ-Specific and Environmentally Regulated Expression of Two Abscisic Acid-Induced Genes of Tomato : Nucleotide Sequence and Analysis of the Corresponding cDNAs.

The cDNAs, pLE4 and pLE25, represent mRNAs that accumulate in response to water deficit and elevated levels of endogenous abscisic acid in detached leaves of drought-stressed tomato (Lycopersicon esculentum Mill., cv Ailsa Craig) (A Cohen, EA Bray [1990] Planta 182: 27-33). DNA sequence analysis of pLE4 and pLE25 showed that the deduced polypeptides were 13.9 and 9.3 kilodaltons, respectively. Each polypeptide was hydrophilic, cysteine- and tryptophan-free, and found to be similar to previously identified proteins that accumulate during the late stages of embryogenesis. pLE4 and pLE25 mRNA accumulated in a similar organ-specific pattern in response to specific abiotic stresses. Yet, expression patterns of the corresponding genes in response to developmental cues were not similar. pLE25 mRNA accumulated to much higher levels in developing seeds than in drought-stressed vegetative organs. pLE4 mRNA accumulated predominantly in drought-stressed leaves. The similarities and differences in the accumulation characteristics of these two mRNAs indicates that more than one mechanism exists for the regulation of their corresponding genes.

The interaction of light and abscisic acid in the regulation of plant gene expression.

Extended dark treatments of light-grown plants of both Lemna gibba and Arabidopsis thaliana resulted in substantial increases in abscisic acid (ABA) concentrations. The concentration of ABA could be negatively regulated by phytochrome action in Lemna. As has been noted in other species, ABA treatment reduced Lemna rbcS and Lhcb RNA levels, which are positively regulated by phytochrome in many species. In view of these observations, the possibility that phytochrome effects on gene expression may be mediated primarily by changes in ABA was tested using a transient assay in intact plants. The phytochrome responsiveness of the Lemna Lhcb2*1 promoter was still apparent in the presence of exogenous ABA. Additionally, when 2-bp mutations were introduced into this promoter so that phytochrome responsiveness was lost, a response to exogenous ABA was still present. We conclude that phytochrome- and ABA-response elements are separable in the Lhcb2*1 promoter. We tested whether the effects of ABA on RNA abundance could be inhibited by treatment with gibberellin and found no evidence for such an inhibition. We have also found that the ABA-responsive Em promoter of wheat can be negatively regulated by phytochrome action. It is likely that this regulation is mediated at least in part by phytochrome-induced changes in ABA levels. Our results demonstrate that it is essential to take into account that dark treatments and the phytochrome system can affect ABA levels when interpreting studies of light-regulated genes.

The H1 histone variant of tomato, H1-S, is targeted to the nucleus and accumulates in chromatin in response to water-deficit stress.

Water deficit has a significant impact on patterns of gene expression. Based on the deduced amino acid sequence, it has been proposed that the drought and abscisic acid-induced gene (his1-s) of tomato (Lycopersicon esculentum Mill.) encodes an H1 histone variant. To study the role of H1-S it is important to understand the expression characteristics of the protein. To identify the his1-s product in vivo the his1-s cDNA was fused to a (His)6 tag and overexpressed in Escherichia coli. The H1-S fusion protein was used to generate an antibody that recognized a protein with an apparent molecular weight of 31 kDa that accumulates in response to water deficit in the whole plant and detached leaves. A time course of his1-s expression showed that protein accumulation is delayed compared to the mRNA accumulation in both the whole plant and detached leaves. Cellular fractionation, immunofluorescence and H1-S::beta-glucuronidase fusion analyses in transgenic tissues were used to determine the cellular localization of H1-S. The results showed that H1-S accumulates in nuclei and is associated with chromatin of wilted tomato leaves. The drought- and abscisic acid-induced gene his1-s encodes a linker-histone subtype specifically accumulated in the nuclei and chromatin of tomato leaves subjected to water-deficit conditions. Although the molecular mechanism of H1-S function is still unclear, the expression characteristics of H1-S are consistent with a potential role of this protein in the regulation of gene expression in response to water deficit.

Expression of the ß-subunit of ß-conglycinin in seeds of transgenic plants.

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein ß-conglycinin, encoded by a multigene family. ß-Conglycinin consists of three subunits; α', α, and ß. A genomic clone for a ß-subunit of ß-conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this ß-subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The ß-subunit expressed in seeds of petunia and tobacco was recognized by anti-ß-conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the ß-subunit were produced. There was approximately a twofold variation in the accumulation of the ß-subunit protein in the mature seeds of transgenic petunia plants, each containing a single ß-subunit gene. However, the level of protein accumulation in mature seeds and the amount of ß-subunit mRNA in developing seeds was not correlated. Accumulation of the ß-subunit protein in transgenic seeds was less than the α'-subunit protein that accumulated in transgenic petunia seeds containing a single α'-subunit gene and less than the amount of the ß-subunit in mature soybean seeds which contain 8-13 ß-subunit genes. In transgenic tobacco plants, the accumulation of the ß-subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.

Isolation and characterization of a genomic clone encoding the ß-subunit of ß-conglycinin.

ß-Conglycinin, and abundant storage protein in soybean (Glycine max (L.) Merr.) seeds, is a trimeric protein consisting of various isomers containing the three subunits, α', α and ß. Accumulation of the ß-subunit is unique because it appears to be regulated by a variety of developmental and environmental signals. In this paper we describe the isolation and characterization of a genomic clone encoding the ß-subunit of ß-conglycinin. The genomic clone was characterized by restriction-enzyme mapping and partial DNA sequence analysis, by immunoprecipitation of a hybrid-selected invitro translation product, and by RNA blot hybridization reactions. An mRNA of approx. 1700 nucleotides hybridized to an internal 2-kilobase (kb) region of this 4.4-kb cloned DNA restriction fragment and was translated to yield a polypeptide with an approximate molecular weight of 48 kilodalton. This polypeptide is immunoprecipitable by antibody against ß-conglycinin and is of appropriate size to represent the precursor polypeptide of the ß-subunit. When this sequence was used as a probe in RNA blot hybridization experiments, the ß-gene transcript was first detected by stage K and accumulated through stage O during soybean seed development, coincident with appearance of the ß-subunit. Partial DNA sequence analysis of the 5' end of the gene confirmed that the isolated gene encoded a ß-subunit, based upon the previously reported amino terminal sequence for this protein. Genomic DNA blot hybridization analyses indicate that multiple DNA restriction fragments are highly homologous to this cloned ß-gene sequence.