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Journal of Experimental Botany 2007 58(11):3061-3069; doi:10.1093/jxb/erm163
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Differential temperature regulation of GA metabolism in light and darkness in pea

Jon Anders Stavang1, Olavi Junttila2, Roar Moe1 and Jorunn E. Olsen1,*

1Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, N-1432 Ås, Norway
2Department of Biology, University of Tromsø, N-9037 Tromsø, Norway

* To whom correspondence should be addressed. E-mail: jorunn.olsen{at}umb.no

Received 28 February 2007; Revised 18 June 2007 Accepted 25 June 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
In greenhouse production of a number of flowering plant species, a short diurnal temperature drop in the morning is commonly used to reduce stem elongation. Earlier studies of pea (Pisum sativum) exposed to different combinations of day and night temperature, indicate that light, temperature, and gibberellin (GA) interact in the control of stem elongation. However, the mechanisms behind the effects of short-term temperature drops and differential sensitivity depending on the timing of the drop treatment have not been reported. Here, the involvement of GA metabolism in this has been investigated by exposing pea to short-term temperature drops in light or darkness. A 2 h temperature drop from 21 °C to 13 °C in the middle of the light period rapidly reduced the rate of stem elongation temporarily by 55% and increased mRNA levels of the GA-deactivation gene PsGA2ox2 by 2-fold within 30 min and up to 4-fold within 1.5 h. GA1 levels were reduced by 36% after a 3–4 h time lag. A temperature drop in the night reduced stem elongation by 27%, but had no effect on transcript levels of PsGA2ox2. Instead, steady-state expression of the GA-biosynthesis genes NA, PsGA20ox1, and PsGA3ox1 was slightly stimulated, but there was no effect on GA1 level. In conclusion, the effect of a temperature drop on GA metabolism in pea is qualitatively different in light and dark. Light is required for deactivation of GA1 resulting from increased expression of PsGA2ox2. This suggests that GA-metabolism is a component in the short-term adaptation to changes in ambient temperature and putatively in low temperature-light stress responses.

Key words: Gibberellin, light, Pisum sativum, stem elongation, temperature drop, transcriptional regulation


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Temperature is an important environmental factor determining growth and developmental rate of plants as well as plant morphology. The ability of plants to discriminate between temperature during day and night in their response to growth, flowering, and fruiting is referred to as thermoperiodism (Went, 1944). In commercial greenhouse production a temperature drop concept has become an important tool to control stem elongation of a number of flowering short-day plants (SDP) such as Begoniaxhiemalis and poinsettia (Euphorbia pulcherrima), which is currently one of the economically most important flowering pot plants worldwide (Myster and Moe, 1995; Moe and Heins, 2000). This concept takes advantage of the fact that, in many species, plant stem elongation is sensitive to a short period of temperature drop. As a response to a diurnal 2 h temperature drop from 24 °C to 8 °C in the morning, stem elongation was reduced by more than 50% in poinsettia (Ueber and Hendriks, 1992). To increase the inhibitory effect of a temperature drop on stem elongation, one can either extend the period of the temperature drop or increase the degree of the drop treatment (Ueber and Hendriks, 1992; Moe and Heins, 2000).

In B.xhiemalis and poinsettia, the sensitivity to temperature drop seems to be strongest at the end of the night or during the first h of the photoperiod (Moe et al., 1992; Ueber and Hendriks, 1992; Grindal and Moe, 1994). A temperature drop in the middle of the night has very little effect on stem elongation in B.xhiemalis (Grindal and Moe, 1994). In some long-day plants (LDP) such as Fuchsiaxhybrida, Antirrhinum majus, and Petunia, as well as the day neutral Salvia splendens, a temperature drop at any time during the photoperiod may be equally effective in inhibiting stem elongation as a temperature decrease in the beginning of the photoperiod (Erwin and Heins, 1995). Furthermore, an increased temperature at the beginning or end of the night did not affect stem elongation in Salvia and Petunia (Erwin and Heins, 1995). However, a variety of experimental conditions were used in these experiments, and generalizations as to the sensitivity to short-term temperature drops during the light and dark period are thus difficult.

The physiological mechanism mediating the effect of a short-term temperature drop on stem elongation has not been much investigated, but Nishijima et al. (1997) showed that the levels of active GA1 in stem tissue of Dendranthema grandiflorum were reduced by a diurnal 4 h temperature drop treatment at the beginning of the light period. Many of the genes encoding the enzymes involved in the GA biosynthetic pathway of, for instance, pea (Pisum sativum), have been cloned and characterized (Fig. 1). Developmental regulation of the expression of these genes plays an important role in controlling the many aspects of GA-regulated plant growth (reviewed by Hedden and Phillips, 2000). Furthermore, GA mediates certain environmental signals and induces physiological processes such as stem elongation and flowering. Recently it was reported that plants grown for an extended period (12 d) under a temperature regime with lower day temperature (DT) than night temperature (NT) had increased mRNA levels of PsGA2ox2 compared with a temperature regime with higher DT than NT or constant temperature (Stavang et al., 2005). It has been thoroughly demonstrated that, at the same average temperature, a whole range of plants including pea, grown at lower DT than NT develop shorter internodes and have lower GA-levels than plants grown at higher DT than NT or constant temperature (Erwin et al., 1989; Jensen et al., 1996; Grindal et al., 1998; Moe and Heins, 2000; Stavang et al., 2005). Thus, light, temperature, and GA appear to be interacting factors involved in controlling the stem elongation rate in pea.


Figure 1
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  		Fig. 1. Simplified 13-hydroxylation pathway of GA biosynthesis in vegetative tissue of pea. The enzymes that are cloned and characterized in pea, and for which mRNA levels were quantified here, are underlined. Corresponding mutants are given in parentheses.

 
The objective of the present study was to investigate the mechanisms behind the effects on stem elongation of short-term temperature drops. To separate the effects of temperature decrease in light and darkness more clearly than in the earlier studies in which plants were exposed to extended periods of different DT and NT, and in which temperature and light conditions were altered simultaneously (Grindal et al., 1998; Stavang et al., 2005), pea plants were exposed to a short-term temperature-drop in either light or darkness. The earlier observed lack of diurnal variation in levels of the active GA1 at any temperature treatment, in spite of reduced GA1 level after 12 d at lower DT than NT, compared with higher NT than DT or constant temperature, might suggest a more long-term adjustment of GA metabolism (Stavang et al., 2005). The primary aim here was to investigate the instantaneous effect of the short-term temperature drop in light and darkness on transcript levels of genes involved in GA biosynthesis and deactivation, and on levels of the main GAs in pea.

Here it is reported that a short, moderate temperature drop, regardless of light or darkness, temporarily reduced stem elongation rate. However, the reduction was greater in light than in darkness. A temperature drop in light increased mRNA levels of the GA deactivation gene PsGA2ox2 within 30 min, and after a lag time the level of GA1 decreased as well. A temperature drop in darkness had no effect on expression of GA-deactivation genes, but instead slightly stimulated the expression of three GA-biosynthesis genes, NA, PsGA20ox1, and PsGA3ox1. However, there was no consistent effect of a temperature drop in the dark on GA1 levels. The results indicate that GA-metabolism is involved in plant growth acclimation to changes in ambient temperature, and that acclimation to a lower growth temperature is qualitatively different in light than in darkness. Light is required for the deactivation of GA1 resulting from increased expression of PsGA2ox2. This might explain some of the observed differences in the effect of temperature drops on stem elongation in a number of greenhouse-grown plants, depending on the timing of the drop treatment.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Plant material and growth conditions
One seed per pot of pea (Pisum sativum L.) wild-type line 107 (cv. Torsdag) was sown in fertilized peat (Floralux, Nittedal Torvindustrier, Norway) and grown under controlled environmental conditions (Conviron Growth Chambers, Controlled Environments Ltd, Winnipeg, Canada). The humidity was adjusted to give 0.47±0.03 kPa water vapour deficit. The daily light period was 12 h with a photon flux density of 170±10 µmol m–2 s–1 at 400–700 nm (F96T12/CW/1500 fluorescent tubes, General Electric, Fairfield, CT, USA, enriched with light from incandescent lamps (OSRAM, Munich, Germany). The red/far red-ratio was 1.7±0.1. The seedlings were watered daily with a complete nutrient solution of EC=1.5 mS cm–1. The temperature was kept at 21 °C for 10 d prior to the start of the temperature drop experiments.

The effect of a 2 h temperature drop from 21 °C to 13 °C, either in the middle of the day or in the middle of the night, on GA-metabolism steady-state expression was compared with constant 21 °C. In each temperature drop experiment, the uppermost 5–6 cm of the stem and petiole tissue that included the apex from 10 randomly chosen seedlings were harvested in liquid nitrogen in the following time-course: –5, 15, 30, 60, 90, 115, (temperature increased to 21 °C at 120 min) 135, 150, 180, 210, and 240 min after the start of the temperature drop (time 0). Two independent experiments were performed in each case. Control plants of the same age were grown at a constant temperature of 21 °C and harvested in a similar time-course. All samples were stored at –80 °C until used in the analyses. Upon analyses, each sample was homogenized in liquid nitrogen.

The effects of temperature drop on the rate of stem elongation in light or darkness as described above, were measured every 10 s using an angular Displacement Transducer, series 604 (Trans-Tec. Ellington, Connecticut, USA) connected to a data logger, type CR10-AM416 (Campbell Scientific Inc., England) as previously described by Torre and Moe (1998). The values obtained were averaged over a 10 min period. The water vapour deficit could not be precisely controlled in these chambers and the water vapour deficit varied from 1.37–0.87 kPa.

Analyses of transcripts of GA biosynthesis genes
mRNA was extracted from 150–200 mg of homogenized tissue per sample using Dynal beads (Dynal Beads Kit 610.12, Dynal Biotech, Oslo, Norway). Any DNA was removed with DNA-freeTM (Ambion, Austin, Texas, USA). The concentration and integrity of the mRNA were analysed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California, USA). Ribosomal RNA contamination was subtracted before a total of 300 ng mRNA from each sample was reverse transcribed using TaqMan® Reverse Transcription Reagents (Applied Biosystems, Foster City, California, USA).

Primers and probes (Tamra-probes, Applied Biosystems) were designed using Primer Express 1.5 software (Applied Biosystems). Transcript levels were analysed using a real time PCR machine (ABI Prism 7700 Sequence Detection System, Applied Biosystems). All chemicals used in the PCR reactions followed the recommendations as specified in the ‘PCR Master Mix Protocol’ (Part Number 4304449 Rev. C, Applied Biosystems). However, instead of using a 50 µl reaction volume in each tube, a 25 µl reaction volume was used. Primer concentrations, sequences, and GenBank association numbers were as described in Stavang et al. (2005).

Relative mRNA levels were determined using separate tubes and the Comparative Critical threshold (Ct) method for LS, LH, NA, PsGA20ox1, PsGA3ox1, and PsGA2ox1 and the Relative Standard Curve method for PsGA2ox2 according to the User Bulletin 2, ABI PRISM Sequence Detection System, pages 7–15, 1997, PE Applied Biosystems). Actin was used as an endogenous reference gene. All mRNA values were normalized to the lowest mRNA value in the night.

Analyses of GAs
Each sample contained the uppermost 5–6 cm of the stem and petiole tissue that included the apex from 10 randomly chosen seedlings. The plant material was harvested in liquid nitrogen. In total, 32 samples from two independent experiments were analysed. The samples were extracted at 4 °C in 75 ml of cold methanol. [17, 17–2H]GA44, [17, 17–2H]GA53, [17, 17–2H]GA19, [17, 17–2H]GA20, [17, 17–2H]GA29, [17, 17–2H]GA1, and [17, 17–2H]GA8 (LN Mander, Australian National University, Canberra, Australia), were used as internal standards. The ratio of internal standards to endogenous GA was kept near 1:1. Purification of samples and gas chromatography–mass spectrometry-selected ion monitoring analysis were performed according to Olsen et al. (1994, 1995) and Olsen and Junttila (2002). This included partition against ethyl acetate, use of QAE-Sephadex A25 (Pharmacia, Uppsala, Sweden) anion-exchange columns combined with 0.5 g Sep-Pak Vac C18 cartridges (Varian, Harbor City, CA, USA), methylation and purification on 0.1 g bond elute aminopropyl cartridges (Varian), followed by reverse-phase HPLC. Combined HPLC fractions were trimethyl silylated and subjected to gas chromatography-selected ion monitoring analysis. For each GA, two characteristic ions and their deuterated analogs were recorded.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
A temperature drop from 21 °C to 13 °C reduces stem elongation rate within 10 min
Several experiments were conducted with angular transducer equipment to study the effect of short-term temperature drop treatments in the light or dark period on stem elongation rate of pea seedlings. A short-term temperature drop (from 21 °C to 13 °C) of 2 h in the light period rapidly reduced stem elongation rate temporarily by 55% (Fig. 2A). A short-term temperature drop in the night reduced stem elongation by about 27% within 30 min (Fig. 2B). Thus, the effect of a temperature drop on stem elongation in pea is more severe in the light period than in the dark. When the temperature increased to 21 °C again after 2 h, in both cases there was an after-effect of the temperature drop and it took several hours until the stem elongation rate fully recovered.


Figure 2
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  		Fig. 2. Stem elongation rate of 10-d-old seedlings of pea as affected by a short-term 2 h temperature drop from 21 °C to 13 °C in either light (A) or darkness (B). The control plants were grown at a constant temperature of 21 °C. Seedlings were grown for 10 d at 21 °C prior to start of the temperature drop treatments. {downarrow} and {uparrow} indicate start and end of the drop treatment, respectively. Results are the average of four plants.

 
Effects of a 2 h temperature drop on GA metabolism gene expression
To test whether a temperature drop affected steady-state expression of GA metabolism genes in darkness or in light, detailed time-course experiments were conducted. mRNA levels were analysed by real time RT-PCR and all responses discussed here were consistent in both replicate experiments.

A profound effect of a short-term temperature drop in light was found on steady-state expression of the GA-deactivating gene PsGA2ox2 (Fig. 3). As a response to a moderate temperature drop of 8 °C, there was a significant increase in PsGA2ox2 mRNA as measured only 30 min after the start of the temperature drop. After 1.5 h the steady-state expression reached a plateau, on average at a 4-fold higher value than the control. When the temperature increased to 21 °C again after 2 h temperature drop, the mRNA levels dropped back to the levels of the control within 1 h. In the middle of the dark period the levels of PsGA2ox2 mRNAs were 7–8 times lower than in the daytime. This is in agreement with the rhythmic steady-state expression pattern of this gene reported in previous work (Stavang et al., 2005). By contrast with the effect of temperature drop in the light, a temperature drop in the dark period did not affect steady-state expression levels of PsGA2ox2 (Fig. 3). There was no consistent effect of a temperature drop in the middle of the light or dark period on steady-state expression levels of the other GA-deactivating gene PSGA2ox1.


Figure 3
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  		Fig. 3. Effects of a short-term 2 h temperature drop from 21 °C to 13 °C on steady-state expression of late stage GA-metabolism genes in 10-d-old seedlings of pea. The control plants were grown at a constant temperature of 21 °C. The mRNA levels were normalized to mRNA of actin. Results are the average of two independent experiments ±SE. In each experiment the sample at each time point consisted of 10 plants. {downarrow} and {uparrow} indicate start and end of the drop treatment, respectively.

 
The effect of a temperature drop on steady-state expression levels of five GA-biosynthesis genes was investigated. There was no clear or only very small effects of a temperature drop in the light period on steady-state expression of LS, LH, and PsGA3ox1 (Figs 3, 4). As reported previously (Stavang et al., 2005), NA steady-state expression varies rhythmically at constant temperature with the highest levels at the end of the night and the lowest levels by the end of the day. This explains the tendency of higher steady-state expression at the beginning of the time-course in the light and lower values by the end of the time-course (Fig. 4). A temperature drop in the light period systematically affected steady-state expression of NA, and 30 min after the drop there was a slight increase in steady-state expression of this gene. After 1–2 h, steady-state expression was, on average, a 1.8-fold higher than the control. When the temperature increased again, steady-state expression was comparable with the control again after 1 h. Steady-state expression of PsGA20ox1 was reported earlier to be higher in light than in darkness (Garcia-Martinez and Gil, 2002; Stavang et al., 2005). This is obvious in these data here as well, with a 5-fold higher level in light than in the dark. A temperature drop in the light did not immediately affect steady-state expression of PsGA20ox1, but there was an after-effect of the temperature drop and steady-state expression of this gene was reduced by 50% when the temperature had increased again after the drop (Fig. 3).


Figure 4
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  		Fig. 4. Effects of a short-term 2 h temperature drop from 21 °C to 13 °C on steady-state expression of LS, LH, and NA in 10-d-old seedlings of pea. The control plants were grown at constant temperature of 21 °C. The mRNA levels were normalized to mRNA of actin. Results are the average of two independent experiments ±SE. In each experiment the sample at each time point consisted of 10 plants. {downarrow} and {uparrow} indicate start and end of the drop treatment, respectively.

 
A temperature drop did not affect steady-state expression of LS and LH in the dark. By contrast with the GA-deactivation genes, which were not affected by a temperature drop in the dark, the GA-biosynthesis genes NA, PsGA20ox1, and PsGA3ox1 were all stimulated 2–3-fold by a temperature drop in the night (Figs 3, 4). When the temperature increased to 21 °C, steady-state expression returned to levels comparable to the control.

Differential regulation of GA1 levels by a temperature drop in light and darkness
It was also investigated whether the short-term temperature drop-mediated changes in mRNA levels of genes involved in late-stage GA metabolism were accompanied by corresponding changes in GA levels (Figs 5, 6). Analyses of the levels of GA53, GA44, GA19, and GA20 gave no indications of any effect of a temperature drop on the levels of these metabolites (Fig. 5). However, GA1 levels were reduced by 36% by a temperature drop in the light (Fig. 5), whereas in darkness there was no consistent effect of the drop on GA levels (Fig. 6). Thus, upon a short-term temperature drop in the light, the decrease in level of GA1 after a lag time correlates with the steady-state expression of the GA deactivation gene PsGA2ox2. This gene is likely to be a key gene in mediating the temperature drop-mediated reduction of GA1 levels in the light. The levels of the GA catabolites GA29 and GA8 were not much affected by a temperature drop. However, the ratio of GA8/GA1 increased after a temperature drop in the light, indicating that the deactivation of GA1 to GA8 is higher in the plants exposed to a temperature drop in light than at a constant temperature or after a temperature drop in the dark. An increase in GA8 in response to the up-regulation of PsGA2ox2 upon a temperature drop in the light might have been expected, but this catabolite might have been converted further to GA8-catabolite.


Figure 5
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  		Fig. 5. Effects of a 2 h short-term temperature drop from 21 °C to 13 °C in the light on GA levels in apical stem tissue in 10-d-old seedlings of pea. The control plants were grown at 21 °C. Results are the average of two independent experiments ±SE. In each independent experiment the sample at each time point consisted of 10 plants. {downarrow} and {uparrow} indicate start and end of the drop treatment, respectively.

 

Figure 6
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  		Fig. 6. Effects of a 2 h temperature drop from 21 °C to 13 °C in darkness on GA levels in apical stem tissue in 10-d-old seedlings of pea. The control plants were grown at 21 °C. Results are the average of two independent experiments ±SE. In each independent experiment the sample at each time point consisted of 10 plants. {downarrow} and {uparrow} indicate start and end of the drop treatment, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
It has been shown here that a moderate short-term temperature drop of 8 °C in the light significantly increases steady-state expression of the GA-deactivation gene PsGA2ox2 by 2-fold within 30 min and by 4-fold within 1.5 h (Fig. 3). By contrast, a temperature drop in darkness did not affect steady-state expression of this gene, but instead slightly stimulated the GA-biosynthesis genes PsGA20ox1, PsGA3ox1, and NA (Figs 3, 4). The temperature drop-mediated change in steady-state expression of the GA inactivation gene PsGA2ox2 in the light correlated, after a lag time, with reduced levels of GA1 by 36% (Figs 5, 6). Apparently light is an important component in temperature drop-mediated regulation of GA metabolism in pea. In earlier work, in which plants were exposed to lower DT and higher NT for 12 d, GA1 levels were lower than under higher DT and lower NT or constant temperature, but the diurnal GA1 levels were stable and could not explain differences in temperature-mediated changes in diurnal stem elongation rate rhythms (Stavang et al., 2005). This might have been due to a more long-term adjustment of GA metabolism and signalling. In the present study GA1 levels decreased and the ratio of GA8/GA1 increased after a short lag time after initiation of a temperature drop, i.e. the changes were observed 1–2 h after end of the 2 h temperature drop. This might reflect a lag between an increase in transcript level of PsGA2ox2 and an increase in its protein activity. The rapidly reduced rate of shoot elongation prior to the decrease in level of GA1 upon a temperature drop in the light indicates that the growth reduction is not GA-dependent.

At this stage, it is not clear whether temperature regulated steady-state expression of PsGA2ox2 is dependent on light only, or if it depends on input from the circadian clock. In poinsettia and Begonia, which are both SDP, a temperature drop reduced plant height somewhat more when given at the beginning or at the end of the dark period than in the middle of the dark period (Moe et al., 1992; Grindal and Moe, 1994). In poinsettia, a temperature drop early in the morning also resulted in shorter stems compared with a drop later in the day (Ueber and Hendriks, 1992). These results might indicate a circadian component in mediating the effects of temperature drops on GA metabolism and stem elongation. However, pea is a LDP and in the LDP Fuchsiaxhybrida, Antirrhinum majus, and Petunia as well as the day neutral plant Salvia splendens stem elongation has been reported to be similarly reduced by a temperature drop at any time during the photoperiod (Erwin and Heins, 1995). Also, increased temperature at the beginning or end of the night did not affect Salvia and Petunia (Erwin and Heins, 1995). However, in the different experiments with different species, a variety of photoperiods and light conditions were used, and thus generalizations are difficult. These results here show that the effect of a temperature drop differs in the light and dark in a LDP like pea also, at least when given in the middle of a relatively short photoperiod as compared to in the middle of the night.

Except for a possible small effect on transcription levels of the NA gene, a temperature drop in the light period essentially did not affect steady-state expression of the GA biosynthesis genes examined in this study. The tendency for a stimulation of NA steady-state expression by a temperature reduction has also been observed in a previous study (Stavang et al., 2005), but the functional significance of this is, at present, unclear. When comparing the increased expression of NA with the stimulation of the GA deactivation gene PsGA2ox2 by a temperature drop in light, increased GA1 deactivation could be expected as a result of the temperature drop. And indeed, after a lag time, this occurs (Fig. 5). By contrast, a temperature drop in the dark did not affect steady-state expression of the GA-deactivating genes, but slightly stimulated expression of the GA biosynthesis genes NA, PsGA20ox1, and, to a certain extent, PsGA3ox1 (Figs 3, 4). It is noteworthy that even though a temperature drop in the night slightly stimulated PsGA20ox1 steady-state expression, the mRNA levels were still only half of the levels in the light period (Fig. 3). Due to higher levels of NA mRNA in the middle of the night than in the middle of the day, the stimulation of NA gene expression by a temperature drop has probably a greater effect on absolute levels in the night period, even though the relative effect is somewhat larger in the light (2–3-fold increase compared with 1.8-fold increase). This might imply that, in addition to light and dark, timing of the temperature drop within the dark/light periods might affect the degree of stimulation of NA gene expression. However, the increase in NA mRNA levels after a short-term temperature drop in the dark was not reflected in the levels of GA1, which were relatively stable. The lack of effect on levels of GA1 and expression of PsGA2ox2 after a 2 h temperature drop in the dark, in spite of a certain degree of temporary decrease in rate of stem elongation, might also suggest some non-GA-related effect of cooling the tissue. Also, the reduction in growth rate prior to a decrease in the level of GA1 upon a temperature drop in the light, indicates that the growth reduction is not dependent on GA. Although the growth reduction might be due a non-hormonal-related effect of low temperature, temperature might also affect other hormones than GA or possibly hormone interactions. Increasing the temperature from 20–29 °C after germination was shown to promote auxin-mediated hypocotyl elongation in Arabidopsis (Gray et al., 1998). However, in this study, only the situation after several days of temperature treatment was studied. The possibility also exists that the sensitivity of the tissue to GA or other hormones might be affected by a temperature drop treatment.

As an environmental stress, chilling in the light has been recognized for at least 70 years (Faris, 1926; Wise, 1995). However, pea is regarded as a classic chilling-resistant species, and is able successfully to detoxify superoxide at significant rates even in the cold (Wise and Naylor, 1987). A moderate temperature drop in the light, like the one used in this experiment, was accordingly not expected to cause damage to the plants, although a reduced growth potential was expected that should be reflected in a change in GA-metabolism. As shown here in pea, a temperature drop in the light increases expression of a GA-2 oxidase gene that deactivates GA1 (Fig. 3). It has recently been shown that high salinity stress or drought stress in Arabidopsis induces the DDF1 gene, a transcription factor putatively involved in stress responses and regulation of GA metabolism, resulting in reduced levels of GA. The reason for the reduced GA levels was an up-regulation of a GA 2-oxidase gene (Magome and Oda, 2004). The question then arises as to whether reduced levels of GA reflect reduced growth potential in stress situations, or if reduced GA levels actually help to reduce environmental stress imposed on a plant. The findings that reduced levels or reduced sensitivity to GA are found to increase stress tolerance in barley (Sarkar et al., 2004), might imply that strict control of GA-metabolism is important in reducing the negative effects of stress imposed on plants by environmental factors, and thus crucial to increase survival.

In conclusion, the effect of a temperature drop on GA metabolism in pea is qualitatively different in light and dark. Light is required for deactivation of GA1 resulting from increased expression of PsGA2ox2. The results presented here and in previous work on temperature and GA-metabolism (Stavang et al., 2005) and the recent findings that environmental stress like drought and salt-stress all involve the regulation of transcript levels of GA deactivation genes, might imply a general role of GA deactivation in coping with stress. Furthermore, common methods in commercial greenhouse production, like temperature drop treatments and drought stress, can be considered as a way of utilizing general physiological plant stress responses in order to control plant form. Thus, further understanding of environmental control of GA-metabolism provides an important tool for environmentally sustainable manipulation of plant growth without using environmentally detrimental chemical growth retardants.


   Acknowledgements
 
We thank Marit Siira, Linda Ripel, Rigmor Reiersen, and Bente Lindgård for skilful technical assistance. This study has been carried out with financial support from the Norwegian Research Council (project no. 140322/110 and NFR 167890/110) and The Norwegian University of Life Sciences.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Erwin JE, Heins RD. Thermomorphogenic responses in stem and leaf development. HortScience (1995) 30:940–949.[Free Full Text]

Erwin JE, Heins RD, Karlsson MG. Thermomorphogenesis in Lilium longiflorum. American Journal of Botany (1989) 76:47–52.[CrossRef][Web of Science]

Faris JA. Cold chlorosis of sugar cane. Phytopathology (1926) 16:885–891.[Web of Science]

Garcia-Martinez JL, Gil J. Light regulation of gibberellin biosynthesis and mode of action. Journal of Plant Growth Regulation (2002) 20:354–368.

Gray WM, Östin A, Sandberg G, Romano CP, Estelle M. High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis. Proceedings of the National Academy of Sciences, USA (1998) 95:7197–7202.[Abstract/Free Full Text]

Grindal G, Ernstsen A, Reid JB, Junttila O, Lindgård B, Moe R. Endogenous gibberellin A1 levels control thermoperiodic stem elongation in Pisum sativum? Physiologia Plantarum (1998) 102:523–531.[CrossRef]

Grindal G, Moe R. Effects of temperature-drop and a short dark interruption on stem elongation and flowering in BegoniaxHiemalis Fotsch. Scientia Horticulturae (1994) 57:123–132.[CrossRef]

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