JXB Advance Access originally published online on November 28, 2003
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Journal of Experimental Botany, Vol. 55, No. 394, pp. 99-109, January 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
The histone-like protein H1-S and the response of tomato leaves to water deficit
Received 11 April 2003; Accepted 6 August 2003
1 Dipartimento di Scienze e Tecnologie per l’Ambiente ed il Territorio, Università degli Studi del Molise, via Mazzini 8, 86170 Isernia, Italia
2 Dipartimento di Biologia, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italia
3 Dipartimento di Scienze Chimiche, Fisiche e Matematiche ,Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi dell’Insubria, via Valleggio 11, 22100 Como, Italia
4 Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA
* To whom correspondence should be addressed. Fax: +39 0865 411283. E-mail: scippa{at}unimol.it
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Abstract |
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Linker histone protein variants are expressed in different tissues, at various developmental stages or induced by specific environmental conditions in many plant species. In most cases, the function of these proteins remains unknown. In the work presented here an antisense strategy has been used to study the function of the drought-induced linker histone, H1-S of tomato. Three independent H1-S antisense tomato mutants, selected for their inability to accumulate H1-S in response to water stress, were studied. These mutants have been characterized at the physiological and morphological levels. Histone H1-S antisense transgenic plants developed normally indicating that H1-S does not play an important role in the basal functions of tomato development. No differences were detected in chromatin organization, excluding a structural role for H1-S in chromatin organization. However, differences between the wild-type and antisense plants were observed in leaf anatomy and physiological activities. This analysis indicates that H1-S has more than one function, at different times, in controlling plant water status, highlighting the complexity of the water stress response.
Key words: Antisense, chromatin, H1 histone, relative water content, stomatal conductance, tomato, water-deficit stress.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The plant response to water deficit is articulated in a complex network of morphological, physiological and molecular changes. As plant relative water content decreases, plant growth is slowed and stomatal closure occurs which is paralleled by a decreased photosynthetic rate (Lawlor, 2002). These physiological changes occurring during the plant’s response to water stress have been correlated with the rapid translocation of ABA in the transpiration stream and the increase in ABA concentration in plant organs (Zeevaart and Creelman, 1988). Shoot growth is inhibited by water deficit stress, and evidence indicates that this is regulated metabolically rather than directly by the water status of the plant (Sharp, 2002). Although the molecular mechanism that controls growth in response to water stress is unknown, the plant hormones ABA and ethylene have both been implicated in this response. A wide number and variety of genes are induced by water stress (Bray, 2002; Ingram and Bartels, 1996; Seki et al., 2002). Many of these genes play important roles, not only in the protection of cellular structures from water loss, but also in signal transduction of the water-stress response (Shinozaki and Yamaguchi-Shinozaki, 1996, 2000). Some of these alterations may promote plant survival to water loss, whereas others represent an injury response to the stress (Bray, 1997). Many of the drought-inducible genes studied to date are induced by abscisic acid (ABA) (Bray, 1997; Shinozaki and Yamaguchi-Shinozaki, 1997, 2000). Although it is clear that ABA is one signalling molecule, there is evidence that other, yet to be identified, signalling molecules are also required to mount a response to cellular water-deficit stress.
In tomato, four genes were identified and characterized that require an increase in ABA content for expression during water-deficit stress (Bray et al., 1999; Cohen and Bray, 1990). One of these genes, le20, encodes a protein with the domains of H1 histone, thus it was renamed his1-s. This gene is expressed at the mRNA and protein levels in response to prolonged water-deficit stress (Kahn et al., 1993; Scippa et al., 2000). The mechanism of gene induction is at the post-transcriptional level (Cohen et al., 1999).
In plants, as in all eukaryotic organisms, genes and their promoters are complexed with histone proteins as chromatin. DNA is wrapped around an octamer of core histones to which, on average, one linker histone molecule is bound to form the basic unit of chromatin, the nucleosome. This, and higher order DNA packaging, organizes the chromosomes in the nucleus. Thus, all nuclear activities including transcription must take place in this context (Cartwright and Elgin, 1986; Reeves, 1984; Simpson, 1991). The core and linker histones must maintain their structural role as well as facilitate transcription. The linker histones are more variable than the core histones and may control specific processes during development and in response to the environment through alterations in expression of cell-type or stage-specific H1 variants (Jerzmanowski et al., 2000). Recent evidence indicates that H1 histone variants can have an important role in selective regulation of specific classes of genes in which genes may be repressed as well as induced by H1 and its variants (Bouvet et al., 1994; Pruss et al., 1995; Shen and Gorovsky, 1996; Tomaszewski and Jerzmanowski, 1997; Wolffe et al., 1997).
Two well-defined clades of H1 histones have been identified by phylogenetic analyses (Jerzmanowski et al., 2000); one contains the major somatic forms of H1 and the other comprises H1 histone minor variants, which has been called the ‘drought-inducible’ clade. Three ‘drought-induced’ H1 histone minor variants of plants have been reported to date: H1-3 of Arabidopsis thaliana (Ascenzi and Gantt, 1997), H1-S of Lycopersicon esculentum (Bray et al., 1999; Scippa et al., 2000) and H1-D of L. pennellii (Wei and O’Connell, 1996). H1-C of tobacco, which is also grouped in the minor histone clade, is not induced by long-term water-stress treatment (Przewloka et al., 2002). Despite knowledge of their expression patterns, and cellular and subcellular localization, the role of the drought-inducible H1 histone variants remains obscure. Attempts have been reported in the literature to understand the function of the ‘drought-induced’ linker histones using under- and over-expression of the corresponding genes (Ascenzi and Gantt, 1999; Przewloka et al., 2002). Arabidopsis plants under-expressing his1-3 were tested for alterations in response to water deficit stress and no phenotypic effects were observed (Ascenzi and Gantt, 1999).
In previous work, two different possible roles for H1-S, the tomato H1 histone variant, have been proposed; a structural role in the protection of DNA from damage occurring during water deficit or a functional role in the regulation of gene expression (Scippa et al., 2000). By introducing an antisense transgene under the control of the CaMV 35S promoter to up-regulate the expression of his1-s antisense mRNA, tomato mutants that accumulate reduced amounts of H1-S during water stress were obtained. While the results of the analysis of chromatin organization seem to exclude the structural role of H1-S in the protection of chromatin organization, the analysis at the physiological and anatomical levels revealed multiple functions for H1-S during development and in response to soil water-deficit stress.
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Materials and methods |
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Plant material
For all experiments, seeds of Lycopersicon esculentum Mill. cv. UC82B and transgenic derivates were sown in a potting mixture of peat and vermiculite (3:1, v:v) and grown in the greenhouse at 20–24 °C with a relative humidity of 80%. Plants were watered every day with equal amounts of water and once a month with Miracle-Grow (Scotts, Port Washington, NY).
Construction of H1-S mutants
In order to decrease the expression of H1-S during water stress, an antisense strategy was used to produce transgenic tomato plants. The construct used for the transformation of tomato contained the entire coding sequence of his1-s (from which the untranslated sequences at the 3' end carrying potential regulatory signals were removed) in the antisense orientation and driven by the 35S cauliflower mosaic virus promoter for constitutive expression. To place the entire coding sequence for H1-S in the antisense orientation under the control of 35S cauliflower mosaic virus (35S CaMV) his1-s was removed from pBluescript plasmid (Stratagene) using HincII/SpeI digestion. The HincII/SpeI fragment containing his1-s was then ligated into pBI121 digested with SmaI/SstI to remove GUS and create compatible ends for HincII/SpeI ligation. The resulting 35S::antihis1-s construct was introduced into Agrobacterium tumefaciens, which was used for transformation of tomato cotyledons.
Tomato transformation
The tomato variety UC82B (Sunseeds Genetics, Hollister, CA) was used in the transformation experiments. H1-S antisense transgenic plants were regenerated from tomato cotyledons (Chao et al., 1999). Whole plants regenerated in media containing kanamycin, were screened using PCR amplification for the integration of the his1-s antisense construct into the plant genome. Genomic DNA was extracted from wild-type and transgenic leaves as previously described (Scippa et al., 2000). 1 µg of genomic DNA for each wild-type and transgenic leaf was used for the PCR screening with the 5'-CCCACAGATGGTTAGAGAGGC-3' primer that is complementary to the 35S promoter, and the 5'-ACGGCAATCGGAGA AGTTGAG-3' primer complementary to the his1-s sequence. The genotype of the T0 and T1 plants was determined.
In order to follow the accumulation of H1-S in transgenic (T0) and wild-type leaves in response to water-deficit stress, a chromatin preparation followed by immunoblotting with the H1-S antibody was carried out (Scippa et al., 2000).
Stress treatment
For the water-stress experiments, wild-type plants and transgenic plants (T1) of each of three independent transgenic tomato lines were grown from seeds in 4.0 l pots, containing a potting mixture of peat:vermiculite (3:1, v:v). Homozygous transgenic T1 seedlings were identified by PCR analysis and then immunoblotted as described for the T0 plants. Seven transgenic seedlings were chosen from each of the three independent lines. Seven seedlings of the isogenic line cv. UC82B were used as the control.
Growth and physiological parameters were measured in well-watered conditions, in seedlings starting from germination before imposing water stress. Two-month-old plants were gradually subjected to water deficit by withholding water for a total period of 4 weeks. After the water-deficit period, plants were rewatered and the recovery response was analysed for the following 2 weeks. During and after the water-stress treatment the growth pattern was analysed and the physiological parameters relative water content (RWC), photosynthesis rate, stomatal conductance, and transpiration were measured once a week.
Growth and physiological parameters
Growth of the wild-type and transgenic lines was analysed by counting the number of leaves and measuring the height of the shoot. These two traits were measured weekly before and during the water-stress treatment.
Relative water content (RWC) was measured to follow plant water status. This value was calculated (Hewlett and Kramer, 1962)
RWC (%)=((FW–DW)/(TW–DW))x100
Four leaves were removed from each plant and immediately weighed to determine their FW (fresh weight). The same leaves were floated on ddH2O for 24 h, blotted dry and weighed to determine their TW (turgid weight). DW (dry weight) measurements were taken after the leaves were oven-dried (65 °C) in brown paper bags for 24 h.
The physiological parameters stomatal conductance, transpiration and net photosynthetic rate were measured using a computerized portable gas analyser (CIRAS-1, PP Systems, UK). Measurements were taken at the same time of day (11.00–13.00 h) when the water potential is more stable (Fischer and Sanchez, 1979). Six fully expanded leaves at the same height on the stem in each plant were used for the measurements of all physiological parameters.
Leaf anatomy and ultrastructural organization
The leaf anatomy and the ultrastructural organization were investigated in leaves of well-watered and stressed plants of the wild type and all three transgenic lines. For the analysis of leaf anatomy, four leaves were detached from each plant (wild type and transgenic) grown in well-watered and water-stress conditions for 2 weeks and fixed with 4% (v/v) formaldehyde in 0.1 M phosphate buffer (pH 6.8). After fixation, samples were dehydrated in an ethanol series and embedded in a methacrylate resin (Technovit 7100, Heraeus Kulzer, Germany). Sections 5 µm thick, obtained using a Leica SM2400 microtome, were stained with toluidine blue (0.03% w/v in water) and observed at 20x amplification on a Leica DMLB light microscope. Images were recorded and analysed using the software QuantiLite (Leica, Microsystems SPA). Ten different measurements of the leaf, palisade and mesophyll thickness were taken along the same section and four different sections of each sample were analysed.
For the ultrastructural analysis, leaves were fixed in cold 0.1 M cacodylate buffer, pH 6.89, containing 3% glutaraldehyde. After a few days, the samples were repeatedly rinsed in the 0.1 M cacodylate buffer, post-fixed in cacodylate buffer containing 1% osmium tetroxide, pH 6.89, for 3 h at 4 °C, dehydrated in ethanol, and embedded in Epon Araldite (Fluka). Ultra-thin sections, obtained using a Reichert Jung Ultracut E microtome (Reichert, Au), were stained with 3% uranyl-acetate and lead citrate, and observed with a transmission electron microscope (JEOL USA, Inc. Peabody, MA). Different magnification images were recorded on Electron Micrography film (Kodak).
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Results |
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Generation of H1-S antisense tomato plants
Whole tomato plants were regenerated from calli transformed with the construct 35S::antihis1-s. Three independent T0 kanR transformants were tested further by PCR analysis for the incorporation of the his1-s antisense gene. All three plants selected (A1, A2, A3), amplified a fragment with the size corresponding to his1-s plus the 35S promoter (data not shown). The efficiency of the antisense construct in reducing the accumulation of H1-S in transgenic plants under water-stress conditions was evaluated at the protein level. Chromatin preparations prepared from wilted leaves of all genotypes were subjected to SDS-PAGE electrophoresis and immunoblotted with the anti-H1-S polyclonal antibody (Scippa et al., 2000). H1-S accumulated in the wilted leaves of wild-type plants, but not in the wilted leaves of the three independent antisense plants that were tested (Fig. 1). The amount of protein accumulated in the antisense plants was similar to that found in wild-type leaves grown under well-watered conditions.
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Morphological appearance and growth pattern of H1-S antisense plants
Transgenic tomato plants unable to accumulate H1-S in response to water deficit were first analysed for their phenotype and development. The T0 transgenic plants did not have visible alterations in overall phenotype and developed normal fruits and seeds. Seeds from each transgenic line were collected, germinated and the seedlings obtained were screened by PCR for T1 progeny containing the transgene. T1 plants from each of three transgenic lines were used for the analysis of the growth pattern in a normal watering regime and in response to water-deficit stress. In this regard, the growth of transgenic and wild-type plants was investigated with a particular focus upon two parameters: stem height and number of leaves (Fig. 2). Both parameters were measured at weekly intervals for 90 d from the onset of germination. In the first 45 d of growth, no differences were observed in the height of wild-type and transgenic plants under well-watered conditions. However, after day 45, independently of the water regime, the stem height of the three transgenic lines was reduced by 30% compared with the wild type (Fig. 2A). This reduction appeared to have resulted from a shortening of the stem internodes and it remained visible when water was withheld from the plants (Fig. 2A). After day 62 when the water was withheld, the range in leaf number between the wild type and the three transgenic lines increased. Contrary to the stem heights, transgenic plants appeared to have a higher number of leaves than the wild type at the same age, however, the difference was not supported by a statistical analysis (Fig. 2B).
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Water status and physiological analyses
To determine if H1-S plays an important role in the response to water deficit, water relations and physiological activities of wild-type and antisense plants were measured. The water status, in well-watered conditions and during the water-stress treatment, was evaluated by measuring leaf relative water content (RWC). Wild-type and transgenic plants had similar RWC values when well-watered. During the first two weeks of water-stress treatment (day 62–76), there was a similar decrease in RWC in all genotypes (Fig. 3). Differences in the kinetics of RWC decrease between the wild type and the antisense lines were present only after more than 2 weeks of water-stress treatment. Antisense plants had a faster decrease in leaf RWC reaching the lowest value 1 week earlier than the wild type (Fig. 3). When the plants were 82-d-old and water had been withheld for 3 weeks, the RWC of the transgenic lines was 61.9% and the wild type was 71.3%. During the rewatering period, the trend of RWC of all genotypes was similar, with the exception of the transgenic line A2 which was slower to recover RWC (Fig. 3).
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To characterize the physiological activities of plants containing an antisense his1-s construct, stomatal conductance, and transpiration and net photosynthetic rate were measured, since they are among the first physiological processes affected by water loss (Hsiao, 1973). In well-watered conditions, all three physiological parameters had higher values in all three transgenic lines than the wild type, with the exception of the transpiration rate of line A1 on day 62 (Fig. 4A, B, C). When a water deprivation regime was applied the kinetics of decrease of the three parameters was different between the wild type and the antisense lines. During the first week of water-stress treatment, all three physiological parameters were decreased in wild-type plants; stomatal conductance dropped to 49% of the initial value (Fig. 4A), transpiration rate to 43% (Fig. 4B) and net photosynthesis rate to 27% (Fig. 4C). By contrast, 1 week of the water-stress treatment did not alter the physiological activities of the three independent antisense lines. In the third week of the water-stress treatment, all genotypes reached similar values that did not change for the remaining week of the water-stress treatment. During the rewatering period, wild-type and transgenic plants showed a similar trend for recovery of physiological activities (Fig. 4A, B, C).
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Anatomical and ultrastructural analyses
The morphology of H1-S antisense plants was characterized by analysing the leaf anatomy and the cell ultrastructure. No differences in leaf shape, colour and overall morphology, were observed between wild-type and H1-S antisense plants either in well-watered or water-stress conditions. However, when transverse sections of the leaf lamella were analysed, differences were noted between the wild-type and transgenic plants subjected to both environmental conditions. In response to well-watered conditions, the thickness of the leaf blade of the antisense plants was reduced by 20% compared to the wild type. After 2 weeks of water stress treatment, the thickness of the leaf lamella of the wild-type plants was reduced by 48.4% compared to the non-stressed controls. The decrease in thickness was attributed to both a decrease in palisade cell length and a strong compression of the spongy mesophyll cells (Table 1). The decrease in thickness of the spongy mesophyll was visible in the sections and was caused by a loss of intercellular spaces and a reduction in cellular diameters (Fig. 5). These anatomical alterations were less evident in the leaf lamella of H1-S antisense plants under water-stress conditions, where the reduction in thickness of palisade and spongy mesophyll layers in response to water stress was only 23.7% and 30.3 %, respectively (Table 1; Fig. 5). The anatomical differences observed with the light microscope between the genotypes were confirmed by electron microscopy analyses. In response to water-deficit stress, the intercellular spaces between mesophyll cells were greater in the leaves of the H1-S antisense plants than the wild type (Fig. 6).
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280) from measurements taken on antisense line A1.
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The electron microscopy analyses showed that chloroplast shape, size, and thylakoid organization were not changed by water deficit in both genotypes (Fig. 6B, D). Differences were observed in the number and dimensions of starch granules which were more significantly reduced in the chloroplasts of wild-type leaves than in the chloroplasts of H1-S antisense leaves after 2 weeks of water-deficit conditions (Fig. 6). No significant differences in nuclei and chromatin organization were observed between wild-type and transgenic plants in either well-watered or water-stress conditions. Both genotypes showed an increase of heterochromatic regions around the inside of the nuclear membrane when water stress was imposed (Fig. 7, arrows).
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Discussion |
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The gene his1-s (originally called le20), which encodes a variant of H1 histone in tomato, H1-S, is induced by prolonged water-deficit stress (Bray et al., 1999; Kahn et al., 1993; Scippa et al., 2000) and the accumulation of the RNA is also modulated by the day/night cycle with greatest expression at the end of the light period (Corlett et al., 1998; Thompson and Corlett, 1995). This multiple regulatory mechanism has been interpreted to indicate that his1-s has roles at multiple times in plant growth and development and/or the gene product has multiple roles (Corlett et al., 1998). H1 histones, originally thought to play a structural role in the packaging of DNA necessary for organism viability, have more recently been shown using knockout technology to have limited effects or no discernible effects on the growth and development of several different organisms. For example, a knockout of the single H1 histone gene in Aspergillus resulted in no change in observable phenotype (Ramon et al., 2000), and the elimination of all H1 histones alters the transcription of specific genes in Tetrahymena, but did not alter viability (Shen and Gorovsky, 1996; Shen et al., 1995). An antisense strategy combined with a physiological and anatomical analysis was used to investigate the function of the water-deficit-induced gene, his1-s (Bray et al., 1999; Scippa et al., 2000). Antisense resulted in physiological and anatomical changes that can be identified prior to and during water-deficit stress imposition, indicating that H1-S has multiple roles in the tomato plant.
The phenotype of the his1-s antisense plants indicates that H1-S functions in plants exposed to well-watered conditions. The analyses at the physiological level indicated that the antisense plants had a stomatal conductance, transpiration and photosynthetic rate approximately 37% greater than the wild type (Fig. 4A, B, C), implicating H1-S in the stomatal regulatory mechanisms. Thus in wild-type plants, in which H1-S has not been altered by antisense mechanisms, stomata may be more sensitive to environmental conditions. A potential role of H1-S under non-stress conditions may be in regulating the phenomenon of midday stomatal closure. Despite the greater stomatal conductance of the transgenic plants, the leaf RWC was similar in the wild-type and transgenic plants (Fig. 3). Two-week-old Arabidopsis plants, expressing an antisense construct of the Arabidopsis ‘drought-inducible’ histone H1-3, subjected to progressive soil water deficit (Ascenzi and Gantt, 1999) had the same relative water content (81–84%) as the wild type. It was concluded that H1-3 does not play a role in the stress response. However, measurements of stomatal conductance prompted the proposal that the antisense plants have a mechanism to compensate for the loss of water from the increased stomatal conductance. Although this mechanism was not directly investigated, decreased transpirational area, increased water retention in the leaf tissues or increased root hydraulic conductivity are possible mechanisms that may be involved in the control of the water status of the transgenic plants (Kramer and Boyer, 1995). At day 54, the first date when RWC and stomatal conductance was measured, the leaf number was the same in all of the genotypes studied, but the height of the shoot was reduced in the transgenic lines compared with the wild type. Since whole plant transpiration rate was not measured, it is unknown if this difference is significant with respect to plant water status.
Prior to the stress treatment, transgenic tomato plants antisense for H1-S were reduced in height and leaves were reduced in thickness by approximately 20% compared with the wild type (Fig. 2; Table 1). This was similar to results obtained for tobacco plants overexpressing the Arabidopsis major H1 histone H1-2 (Prymakowska-Bosak et al., 1996; Slusarczyk et al., 1999) and for plants antisense for the tobacco variant histone H1C (Przewloka et al., 2002). Both of these conditions decreased the content of the minor variants of histone H1 in the ‘drought-inducible’ clade, strengthening the evidence that H1-S functions in plant growth. In addition, leaf thickness was altered in the transgenic plants overexpressing H1-2 from Arabidopsis, which was attributed to decreased growth (Slusarczyk et al., 1999). The leaf lamella was reduced to the same extent in the tobacco plants overexpressing H1-2 from Arabidopsis as in the antisense tomato plants. Plants with an increased amount of H1C and H1D, which occurred when the expression of the tobacco H1B was reduced by antisense RNA, do not have altered stature or leaf blade thickness (Prymakowska-Bosak et al., 1999).
When transpiration exceeds water absorption, there is a decrease in leaf RWC accompanied by physiological and metabolic changes. The wild-type and transgenic plants responded differently at the initial stages of soil water limitation. During the first week of water-deficit stress, the stomatal conductance, transpiration and photosynthetic rate of the wild-type plants were significantly reduced (Fig. 4). By contrast, the antisense plants were not affected by the first week of water-stress treatment (Fig. 4). During the first week of stress, a large number of starch granules were maintained in the chloroplasts of the antisense plants compared with the wild type (Fig. 6). Thus, a potential role for H1-S is to promote sensitivity to water stress. Since the effect was also apparent prior to the stress treatment, the H1-S may have a similar role under well-watered and water-deficit conditions.
It has previously been shown that H1-S can be detected in tomato leaves after a prolonged water-stress treatment of whole plants and detached leaves (Scippa et al., 2000). Using a specific antibody to tomato H1-S, the H1 histone could first be detected in the leaves of whole plants 9 d after water was withheld, when the RWC reached approximately 70%, and could not be detected in plants grown under well-watered conditions. Thus changes in physiological acitivites reported here occur in plants under conditions in which the protein can not be detected. However, the mRNA can be detected under non-stress conditions and Corlett et al. (1998) have shown that mRNA abundance increases at the end of the light period and decreases during the dark period. Unfortunately, it is unknown if the amount of H1-S protein also cycles with the light condition. There are a number of reasons that may explain the inability to detect H1-S protein under non-stress conditions including that the protein may only be present in specific cell types such as the guard cells, the protein may be active at a low level, or that H1-S is post-translationally modified preventing its recognition by the antibody produced against a protein expressed in E. coli (Scippa et al., 2000). During the time period of the stress treatment when the H1-S protein is most abundant, the RWC of the antisense plants was lower than the wild type. This may implicate H1-S in the maintenance of water status during a specific window of the water-stress treatment.
When the amount of specific plant H1 histones are altered, specific changes in plant development have been documented (Prymakowska-Bosak et al., 1996, 1999; Przewloka et al., 2002). When the tobacco histones H1A and B were reduced to 25% of the control amounts, there was an increase in the four minor variants H1 C, D, E, and F and the chromosomes were less tightly packed (Prymakowska-Bosak et al., 1999). These plants had normal vegetative development, but were male sterile, which was linked to abnormal flower development. When the Arabidopsis histone H1-2 was overexpressed in tobacco, there was an increase in heterochromatinization in the nuclei (Prymakowska-Bosak et al., 1996; Slusarczyk et al., 1999). However, no alterations in the ultrastructure of the nuclei were observed when H1-S protein was reduced.
In conclusion, the characterization of the antisense transgenic plants highlights the complexity of water-stress responses and the function of factors involved. Although more work is needed to understand H1-S function fully, some conclusions can be made; the role of H1-S in the structural protection of the chromatin can be excluded, as shown by the absence of differences in chromatin organization in the transgenic compared to the wild type. It is proposed that H1-S has a role in regulating and modulating important mechanisms involved in the regulation of stomatal function.
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Acknowledgement |
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The authors are grateful to the Italian MIUR (Ministero dell’Istruzione, Università e Ricerca) that partially supported this study (Progetti di rilevanza Nazionale, ex 40%).
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References |
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