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 Escherichiacoli. 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::β-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. Received: 1 October 1999 / Accepted: 3 December 1999     

Expression of a GALACTINOL SYNTHASE gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.     

Expression and molecular cloning of drought-induced genes in the wild tomato Lycopersicon chilense.

Protein synthesis and translatable mRNA population changes induced during water stress were studied in leaves of a drought-resistant wild relative of tomato, Lycopersicon chilense, using one- and two-dimensional polyacrylamide gel electrophoresis. Under our experimental conditions, water deficit did not significantly affect total protein synthesis capacity. However, it induced biphasic synthesis of a new set of proteins. These newly synthesized proteins resumed to control levels upon rehydration of the plants. Certain drought-induced proteins also accumulated in leaves subjected to heat shock (39 degrees C) or exogenous abscisic acid (ABA, 1 mM) treatments. A cDNA library was constructed using poly(A)+ RNA from leaves of plants exposed to drought stress for 4 days. Differential screening of the library identified three groups of clones corresponding to drought- and ABA-induced mRNAs. Northern blot analysis showed that the genes of selected clones respond differently to the different environmental stresses. Our data clearly demonstrate that water stress alters gene expression in L. chilense plants resulting in the synthesis of new proteins, of which several respond to high temperature stress and others to an osmotic effect. These responses are in part modulated by ABA.     

A drought-stress-inducible histone gene in Arabidopsis thaliana is a member of a distinct class of plant linker histone variants

We have isolated and characterized a gene, His1-3, encoding a structurally divergent linker histone in Arabidopsis thaliana. Southern and northern hybridization data indicate that A. thaliana expresses three single-copy linker histone genes, each encoding a structurally distinct variant. H1-3 is a considerably smaller protein (167 amino acids with a mass of 19.0 kDa) than any other described linker histone from higher eukaryotes. We examined the expression of His1-3 at the RNA and protein levels and found that it is induced specifically by water stress. In contrast, expression of His1-1, His1-2 and His4 appear unaffected by water stress. Furthermore, the primary structure of the protein possesses distinct characteristics that are shared with another drought-inducible linker histone, H1-D, isolated from Lycopersicon pennellii. Based on structural characteristics of the deduced protein and its inducible expression, we hypothesize that H1-3 and H1-D are linker histone variants that have specialized roles in the structure and function of plant chromatin and therefore they can be considered to be members of a unique subclass of plant histones. Immunoblotting with an antibody produced against a short polypeptide in the conserved domain of this subtype indicates that similar proteins may exist in other plants.     

Water Stress in Plants

The abscisic acid (ABA) content was determined quantitatively in the leaves from wilted and unwilted tomato plants (Lycopersicon esculentum Mill. CV. Revermun) by the use of the wheat coleoptile test and gas-liquid chromatography (GLC). Plants which have received an insufficient daily water supply for 18 days showed adaptation to wilting conditions. The plants adjust to the added amount of water by regulating their water loss through transpiration. The concentration of ABA was not higher in the leaves of plants adapted to water stress than in plants that were watered abundantly. Wilted detached leaves and leaves from rapidly wilted intact plants showed the well-known reaction by increasing the ABA level. A possible role of ABA in the early stages of the adaptation process is discussed.     

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.     

Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles,water stress and abscisic acid

Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.     

Molecular characterization and expression of a tobacco histone H1 cDNA

We have isolated a 1104 bp tobacco cDNA clone (H1c12) which includes an 846 bp open reading frame. This encodes a polypeptide of 282 amino acid residues and represents the largest plant H1 histone identified so far. The structure of the deduced protein shows the classical tripartite organization of the H1-type linker histones. The expression of the tobacco H1 histone gene(s) corresponding to the H1c12 cDNA clone was examined during different developmental stages. We found that, at the level of steadystate mRNA, expression of gene(s) encoding this H1 histone was rapidly induced in germinating seeds. The H1 gene was expressed in all tissues examined. However, its expression was higher in tissues known to contain meristematic cells. Furthermore, in the leaves of mature plants accumulation of the H1 mRNA exhibits a very characteristic oscillation. This latter finding indicates that, at least in fully developed plants, the expression of this type of H1 histone gene(s) is modulated by a diurnal cycle.     

A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks.     

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.     

Methylation of histone H3 at lysine 4 and expression of the maltase-glucoamylase gene are reduced by dietary resistant starch

Methylated histone H3 at lysine 4 (K4) is associated with euchromatin and is involved in the transactivation of genes. However, it is unknown whether histone methylation is involved with changes in gene expression induced by nutrients. In this study, we examined whether methylations of histone H3 at K4 on maltase-glucoamylase (Mgam), which is responsible for the digestion of starch in the small intestine, as well as Mgam expression were altered by feeding rats an indigestible starch (resistant starch, RS). The mRNA and protein levels and the activities of MGAM were reduced in rats fed an RS diet compared with those fed a regular starch diet. Furthermore, we found that decreases in di- and tri-methylation of histone H3 at K4, as well as reduced acetylation of histones H3 and H4 on the Mgam gene were associated with a reduction of Mgam gene expression. These results suggest that the reductions of jejunal MGAM levels and activities caused by the RS diet are regulated at the mRNA level through a decrease in methylation of histone H3 at K4 and reduced acetylation of histones H3 and H4 on the Mgam gene.     

Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA

We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.