Journal of Experimental Botany, Vol. 51, No. 343, pp. 299-304,
February 2000
© 2000 Oxford University Press
Short Communication |
Dehydrin transcript fluctuations during a day/night cycle in drought-stressed sunflower
Biochimie et Physiologie Moléculaire des Plantes, INRA/CNRS (URA2133)/ENSA-M/UMII, place Viala, 34060 Montpellier cedex 1, France
Received 15 February 1999; Accepted 14 September 1999
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Abstract |
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To investigate environmental stimuli involved in the modulation of drought-induced gene expression, the influence of the day/night cycle on the expression of two dehydrin genes (HaDhn1 and HaDhn2) in leaves of sunflowers subjected to mild or severe drought stress has been studied. It was observed that the HaDhn1, but not HaDhn2, transcript oscillated in a diurnal fashion. In severely stressed plants, the peak of HaDhn1 mRNA accumulation occurred at midday.
Key words: Dehydrin transcript, drought stress, day/night cycle, sunflower
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Physiological and molecular responses to drought are under the control of whole plant signalling mechanisms (Bray, 1997
). The rapid translocation of abscisic acid (ABA) to the shoot via xylem flux and the increase of ABA concentration in plant organs correlate with many physiological and molecular changes occurring during the plant response to drought (Chandler and Robertson, 1994; Zeevaart and Creelman, 1988). However, there is evidence suggesting that additional signals are involved in these processes, and it is now well established that ABA is not the only factor that mediates the drought regulation of gene expression (for review, see Davies et al., 1994; Shinozaki and Yamaguchi-Shinozaki, 1997).
External stimuli, such as light, can modulate the expression of drought-induced genes. In Craterostigma plantagineum, light conditions were shown to influence the expression of dehydration-responsive genes encoding either cytosolic or chloroplastic proteins (Alamillo and Bartels, 1996; Bartels et al., 1992). The combined effects of light and ABA on drought- or salt-induced gene expression, suggest that light-dependent and light-independent pathways are involved in the ABA-mediated response (Alamillo and Bartels, 1996; Bartels et al., 1992; McElwain et al., 1992). Additionally, in Lemna gibba and Arabidopsis thaliana, dark treatments of light-grown plants were shown to increase ABA level (Weatherwax et al., 1996), suggesting that the effect of light might occur through modulation of the level of this plant hormone.
In this paper, the diurnal changes in the expression of HaDhn1 and HaDhn2, two drought-induced nuclear genes of sunflower encoding dehydrins (Ouvrard et al., 1996) were investigated. Among the drought-induced proteins so far identified, dehydrins, the D-11 subgroup of Late Embryogenesis Abundant [LEA] proteins (Dure III et al., 1989) are very frequently observed (Close, 1997). Dehydrins are highly abundant in desiccation-tolerant seed embryos, and accumulate during periods of water deficit in vegetative tissues. These proteins display particular structural features such as the highly conserved lysine-rich domain predicted to be involved in hydrophobic interaction leading to macromolecule stabilization (Close, 1996). However, very little is known about dehydrin functions in planta. In sunflower, increased HaDhn1 and HaDhn2 transcript accumulation is highly correlated with the decrease of midday leaf water potential during progressive drought (Cellier et al., 1998). Moreover, HaDhn1 and HaDhn2 transcripts were shown preferentially to accumulate in a drought-tolerant line (Cellier et al., 1998). Like other members of the dehydrin family, HaDhn1 and HaDhn2 genes are up-regulated in response to exogenous ABA (Ouvrard et al., 1996). To reveal whether the expression of these two genes was also affected by light, the accumulation of HaDhn1 and HaDhn2 transcripts was investigated during a day/night cycle in whole plants subjected to water deficit. Plant water status was characterized during the experiment by monitoring soil water content, leaf water potential and the concentration of ABA in the xylem sap.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material and treatments
The R1 genotype of sunflower (Helianthus annuus L) was supplied by Rhône-Poulenc Agrochimie (Lyon, France) and described previously (Ouvrard et al., 1996
). Seeds were surface-sterilized with sodium hypochlorite 1% (w/v) and germinated on water-moistened filter paper for 48 h in the dark at 25 °C. Seedlings were transferred to composit soil (peat compost/vermiculite, 1 : 1, by vol.) and grown in a greenhouse under natural light, 24/25 °C (night/day) and 60–70% humidity. Natural illumination corresponded to 16 h of light at the period of year the experiment was carried out. Plants were grown initially for 15 d in a 0.5 l pot, and then transferred to a 3.0 l pot. Plants were watered daily, and fertilized weekly with a complete nutrient solution. One-month-old plants were submitted to progressive drought by withholding water. Control plants were watered daily. After the last watering, gravimetric soil water content was measured daily and expressed as g water g-1 oven-dried soil. Depending on their location in the greenhouse, the irradiation received by each plant was variable and, consequently, the decrease in soil water content displayed differences within the plant population. This allowed the selection of two sets of plants, homogeneous in size and development, experiencing different stress intensities (mild or severe) with regard to soil water content, as described in the results section.
Physiological measurements during the day/night cycle
The leaf water potential was measured on young fully expanded leaves using a pressure chamber, and 100 µl of xylem sap was then extracted at a pressure of about 0.5 MPa above the balancing pressure. Sap samples were stored at -80 °C for subsequent ABA analysis by radioimmunoassay (Quarrie et al., 1988). The day/night experiment started at 20.00 h of day 0 and ended at 21.00 h of day 1. Days 0 and 1 started at 00.00 h and ended at 24.00 h. All the hours indicated refer to solar time. Solar time is defined as follows: when the sun was at the zenith on the date of the experiment, this time was labelled 12.00 h. During the experiment, the dark period started at 19.00 h and ended at 03.00 h. During the experiment, the light intensity measured at midday (12.00 h) of day 0 and day 1 was between 700–800 µE m-2 s-1. A green-light lamp was used for sampling in the dark.
Northern analysis
Total RNA was extracted as described (Ausubel et al., 1991). Northern blot analysis was performed as reported (Cellier et al., 1998).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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One-month-old plants were submitted to progressive drought by withholding water. Each pot was weighed daily at 09.00 h (solar time) in order to monitor gravimetric soil water content. Eleven days after the last watering (day 0 of the experiment), plants were separated into two groups referred to as ‘mild’ and ‘severe’ drought stress, depending on their gravimetric soil water content values (Table 1
). This allowed the study of plants experiencing contrasting soil water status but sampled on the same day. Periodically, from 20.00 h of day 0 to 21.00 h of day 1, two opposite, young, fully expanded leaves were collected from 3–4 individual plants of each group. For each plant, one leaf was used to measure leaf water potential and the concentration of ABA in xylem sap ([ABA]xyl) while the opposite one was frozen separately for RNA extraction.
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Leaf water potential of plants subjected to a ‘mild’ or ‘severe’ drought treatment, fluctuated during the daytime (Fig. 1
). The maximal value was observed before dawn as the result of the equilibrium between soil and plant water potentials. The minimal value was observed in the late afternoon due to the increase of evaporative demand. There were large differences between leaf water potential values of plants subjected to ‘severe’ and ‘mild’ drought stress. Therefore, the differences in gravimetric soil water content led to differences of leaf water status. In addition to diurnal fluctuation, as a result of the increase of water deficit intensity, the leaf water potential of plants experiencing ‘severe’ water deficit was lower at 20.00 h of day 1 than those measured at 20.00 h of day 0. The leaf water potential of control plants, measured at midday of day 1 and day 0 was -3.85 (±0.33) MPa. Similar variation of leaf water potential during the day was reported in field-grown sunflower subjected to ‘mild’ or ‘severe’ water deficit (Tardieu and Simonneau, 1998).
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) or ‘severe’ (•) water-stress during a day/night cycle. Data are means of 3–4 replicates. Standard error is indicated. Bar above the graph indicates the light/dark conditions during the experiment (open box, light conditions; black box, dark conditions). Hours indicated above the bar refer to solar time. ‘Mild’ and ‘severe’ drought stress refer to groups of plants described in Table 1.HaDhn1 and HaDhn2 transcripts displayed higher levels of accumulation in plants subjected to ‘severe’ water deficit compared to ‘mild’ water deficit (Fig. 2
). At any time during the day/night experiment, these transcripts were never detected in irrigated plants. The expression of both genes, observed at midday, was previously reported to be highly correlated with the decrease of soil water content and leaf water potential (Cellier et al., 1998). During the day/night cycle, dehydrin transcripts HaDhn1 and HaDhn2 displayed different patterns of accumulation (Fig. 2). The amount of HaDhn2 transcripts did not fluctuate with the time of the day and, beside slight variations, increased as a function of the intensity of water deficit, independently of day/night periods. The steady-state level of HaDhn1 transcripts displayed marked diurnal changes, regardless of the stress intensity. In plants subjected to ‘severe’ water deficit, the amount of HaDhn1 transcripts was strongly enhanced at the onset of the light period, reached a maximum around midday, and decreased at the beginning of the dark period. However, additional experiments are needed to determine whether the decrease of HaDhn1 transcript accumulation precedes the beginning of the dark period or not. A similar pattern of accumulation was observed in plants subjected to ‘mild’ drought stress, however, the maximum of accumulation of transcripts occurred earlier, and the decline preceded the beginning of the dark period. For both treatments, the amplitude of oscillation was approximately 10-fold. In severely stressed plants, as a result of the increase of water deficit intensity, the amount of HaDhn1 transcripts was higher at 20.00 h of day 1 than at 20.00 h of day 0. For both treatments, the increase of HaDhn1 transcripts during the first part of the light period was strongly correlated with the decrease of the leaf water potential. However, the decline of HaDhn1 transcript accumulation during the second part of the light period was not correlated to variation of leaf water potential.
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The concentration of ABA in xylem sap ([ABA]xyl) fluctuated with the time of the day (Fig. 3
). In control plants, [ABA]xyl measured at midday of day 0 and day 1 was 21.7±4.5 µmol m-3. In severely stressed plants, a decrease was observed in [ABA]xyl during the second half of the dark period and the beginning of the light period, followed by an increase in [ABA]xyl at the beginning of the night. However, these results do not allow the determination as to whether the rise in [ABA]xyl preceded the beginning of the dark period or not. In plants subjected to ‘mild’ drought stress, [ABA]xyl increased transiently during the dark period and remained nearly constant during the light period. As expected, [ABA]xyl was higher in plants subjected to ‘severe’ water deficit than in plants subjected to ‘mild’ water deficit. Observations concerning variation of [ABA]xyl during the day in drought-stressed sunflowers were previously reported. In growth chamber-cultivated plants, [ABA]xyl was shown to fluctuate during the light period with the lowest value at midday (Neales et al., 1989; Schurr et al., 1992). In field-grown sunflower subjected to ‘mild’ or ‘severe’ water deficit, [ABA]xyl displayed a similar behaviour, but with a lower amplitude of variation (Tardieu and Simonneau, 1998). The HaDhn1 gene was shown to be up-regulated in response to exogenous ABA (Ouvrard et al., 1996), suggesting that ABA could mediate drought-induced expression of HaDhn1. During the day/night cycle, HaDhn1 transcript fluctuations were not directly correlated with those in [ABA]xyl. Additionally, it was also observed that in ABA-treated plants, HaDhn1 transcript levels displayed diurnal changes (Cellier et al., 1998). Therefore the daily oscillating pattern of HaDhn1 transcript accumulation in plants subjected to water deficit is unlikely to be ABA-mediated. However, the possibility cannot be ruled out that changes in the total leaf ABA content could explain the HaDhn1 transcript fluctuation during the day/night cycle.
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The results presented demonstrate that the day/night cycle may modulate drought-induced gene expression and, therefore, have to be considered when examining the expression of stress-responsive genes. Specifically, these results indicate the importance of taking into account the time of day of tissue sampling when interpreting studies of drought- responsive genes.
To the best of the authors' knowledge, this is the first evidence showing diurnal fluctuation of a gene induced in response to water deficit, but never expressed in irrigated plants. In tomato, diurnal expression of the drought- responsive gene le20 was reported (Thompson and Corlett, 1995). The maximum accumulation of transcripts was observed at the end of the light period. However, diurnal fluctuations of le20 were also shown to occur in irrigated plants, and therefore are independent of drought stress (Corlett et al., 1998). In A. thaliana, CCR2, a gene overexpressed in response to cold, drought or ABA treatment, was shown to be diurnally expressed (Carpenter et al., 1994). This gene displays a circadian rhythm in control and cold-treated plants, although with a reduced amplitude in plants subjected to cold treatment (Kreps and Simon, 1997). However, the behaviour of CCR2 during drought was not reported.
The diurnal expression of HaDhn1 was not correlated with daily changes in leaf water potential or [ABA]xyl. Day/night expression of HaDhn1 might be related to other physiological processes known to be diurnaly regulated and affected by drought or ABA treatment, such as stomatal control or photosynthetic light harvesting (McClung and Kay, 1994). Dehydrins are localized in the cytosol and nucleus and were never reported to be associated with photosynthesis activity (Close, 1996). In sunflower, stomatal control was shown to depend on [ABA]xyl (Tardieu et al., 1996), and to display diurnal fluctuation (Tardieu and Simonneau, 1998). It was observed that stomata of drought-stressed sunflower were closed when [ABA]xyl reached 150 µmol m-3 (F Cellier, unpublished data). Therefore, in severely stressed plants stomata are likely to be closed (Fig. 3). The diurnal expression of HaDhn1 is therefore unlikely to be related to stomatal control. The diurnal expression of HaDhn1 might be related to climate variables such as irradiance. On the other hand, the periodic increase and decrease in the level of HaDhn1 transcripts might also result from a circadian control. Further investigations are needed to determine which processes may have driven the rhythm of HaDhn1 expression.
The functional significance of the oscillation in HaDhn1 expression is not known. In severely stressed plants, the peak of transcript accumulation occurred around midday, when the water-stress intensity sensed by leaves is high because the evaporative demand is maximal. This pattern of expression is consistent with the predicted role of dehydrin in protecting cellular function from the effects of dehydration (Close, 1997). However, the accumulation of dehydrin transcripts does not necessarily correlate with the content of the corresponding proteins. Although HaDhn1 and HaDhn2 belong to the same protein family, the corresponding transcripts accumulate differently during day/night cycles. They also respond differently to applied ABA (Cellier et al., 1998). This suggests that the various members of this family may have different functions in the plant in response to drought, or that they are involved in various regulatory processes concerning the same function.
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Acknowledgments |
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We are very grateful to Drs Marc Lepetit and Alain Gojon for helpful discussions and Dr Tim Tranbarger for critical reading of the manuscript. We wish to thank Philippe Barrieu (Ecophysiologie des Plantes sous Stress Environnementaux, INRA Montpellier, France) for endogenous ABA measurements, and Hugues Baudot for help in raising the plants. This work was financially supported by AIP no. 924840 from ‘Institut National de la Recherche Agronomique’.
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Notes |
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1 To whom correspondence should be addressed. Fax: +33 4 67 52 57 37. E-mail:cellier{at}ensam.inra.fr
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Abbreviations |
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[ABA]xyl, concentration of ABA in xylem sap.
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