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Oryzacystatin I expression in transformed tobacco produces a conditional growth phenotype and enhances chilling tolerance

Authors

  • Christell Van der Vyver,

    1. Forestry and Agricultural Biotechnology Institute, Botany Department, University of Pretoria, Pretoria 0002, South Africa
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  • Jörg Schneidereit,

    1. Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK
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    • Present address: University of Cologne, Botany, D-50931 Cologne, Germany.

  • Simon Driscoll,

    1. Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK
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  • Janice Turner,

    1. Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK
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  • Karl Kunert,

    1. Forestry and Agricultural Biotechnology Institute, Botany Department, University of Pretoria, Pretoria 0002, South Africa
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  • Christine H. Foyer

    Corresponding author
    1. Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK
      Correspondence (tel.: +44 01582 763133 extn 2636; fax: +44 01582 763010; e-mail: christine.foyer@bbsrc.ac.uk)
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Correspondence (tel.: +44 01582 763133 extn 2636; fax: +44 01582 763010; e-mail: christine.foyer@bbsrc.ac.uk)

Summary

A recent strategy for pest control in plants has involved transformation with genes encoding cysteine proteinase inhibitors (cystatins). Little is known, however, about the effects of constitutive cystatin expression on whole plant physiology. The present study using oryzacystatin I (OC-I) expression in transformed tobacco was designed to resolve this issue and also to test the effects on abiotic stress tolerance. All transformed plants expressing OC-I showed a conditional phenotype. A marked effect on stem elongation was observed in plants grown under low light intensities. After 7 weeks of growth at low light, the plants expressing OC-I were smaller with fewer expanded leaves and a slightly lower total biomass than empty vector controls or wild type plants. Maximal rates of photosynthesis (Amax) were also decreased, the inhibitory effect being greatest in the plants with highest OC-I expression. After 12 weeks of growth at low light, however, the plants expressing OC-I performed better in terms of shoot biomass production, which was nearly double that of the empty vector or wild type controls. All plants showed similar responses to drought, however photosynthesis was better protected against chilling injury in plants constitutively expressing OC-I. Photosynthetic CO2 assimilation was decreased in all plants following exposure to 5 °C, but the inhibition was significantly less in the OC-I expressing plants than in controls. The transformed tobacco plants expressing OC-I therefore show a phenotype–environment interaction with important implications for biotechnological applications.

Introduction

Cystatins bind tightly and reversibly to the papain-like group of cysteine proteinases. Several plant cystatins have been isolated and have been used to engineer better Coleopteran and nematode control in plants (Leple et al., 1995; Urwin et al., 2001). The best characterized of the plant cystatins, to date, is oryzacystatin-I (OC-I; Abe et al., 1987) and this rice cystatin has been also successfully expressed in tobacco (Gutiérrez-Campos et al., 2001; Masoud et al., 1993).

Cystatins are involved in the regulation of protein turnover in plants. Two observations suggest that they fulfil particularly important functions during seed development. First, plant cystatin mRNAs show a similar pattern of production to that observed for major seed storage proteins (Abe et al., 1987, 1992). Second, cystatins inhibit cysteine proteinase activity in developing seeds. More recently, cystatins have also been implicated in plant stress responses. Cystatins are specifically induced during cold and salt stress (Pernas et al., 2000) and also following wounding and/or treatment with methyl jasmonate (Botella et al., 1996). Prosystemin overexpression also stimulates cystatin production (Jacinto et al., 1998). These observations support the hypothesis that cystatins play a crucial role in plant defence mechanisms. To date, however, little is known of other additional benefits or disadvantages of cystatin production. In particular, strategies for improving abiotic stress tolerance by controlling cellular degradation processes by constitutive overexpression of cystatins, merit further examination. It is important to note, however, that a recent report has shown that expression of OC-I causes pleiotropic effects in transformed tobacco. In particular, OC-I expressing tobacco plants show increased growth rates, whole plant biomass and earlier flowering with increased numbers of flowers and seeds (Gutiérrez-Campos et al., 2001).

Many of the endogenous cysteine proteinases that are possible targets for cystatin inhibition have acidic pH optima in vitro, suggesting that they are localized to the vacuole in vivo (Callis, 1995). Also, cysteine proteinase expression has been intensively studied only in young and senescent leaves and flowers (Buchanan-Wollastan and Ainsworth, 1997; Guerrero et al., 1998; Xu and Chye, 1999). It is known, however, that cysteine proteinases accumulate in response to low temperatures and are induced by other stresses that increase oxidative stress (Schaffer and Fischer, 1988). The tobacco cysteine proteinase, CYP-8, for example, is involved in the wound response in tobacco (Linthorst et al., 1993). Recently, a role of cysteine proteinases in programmed cell death has also been proposed (Solomon et al., 1999; Xu and Chye, 1999). For example, cysteine proteinases are involved in developmentally regulated programmed cell death (Hadfield and Bennett, 1997; Penell and Lamb, 1997). The nature of the metabolic interactions between endogenous cysteine proteinases and endogenous cystatins is far from clear. Moreover, the effect of constitutive cystatin expression on the function and activity of cysteine proteinases remains to be elucidated.

Using photosynthesis, respiration and growth characteristics as physiological markers we have examined the effects of OC-I expression on whole plant physiology in OC-I expressing tobacco. In the following experiments we have used two types of control. First, we used wild type untransformed plants. Second, we included a control that had been through the transformation process and expressed both the gus and nptII genes, but not OC-I, in the same construct in all experiments. These two types of plants showed identical responses in all the parameters measured in the experiments reported here, therefore we will refer to them together hereafter simply as controls. The hypothesis that cystatins, as the natural inhibitors of cysteine proteinases, modulate plant responses to abiotic stress, was explored in this study. We show that the key processes of photosynthesis and respiration are modified by OC-I expression. Of the large number of marked effects observed in the transformed tobacco plants, the most dramatic effects were found in general plant performance and chilling tolerance.

Results

OC-I expression

Three independent transformed OC-I expressing lines and kanamycin-resistant controls expressing β-glucuronidase (GUS), but not OC-I, were selected for physiological and metabolic analysis. The OC-I expressing lines were selected on the basis of the amount of free OC-I protein (arising from the transgene) present in the leaves (Figure 1). The three transformed lines contained different amounts of leaf OC-I protein as judged by detection with specific antibodies (Figure 1A and B). Polyclonal antibodies prepared against a GST-OCI fusion protein were used for these studies because OC-I alone is not a strong antigen. Antibodies raised against the GST portion of the fusion protein alone produced no reaction in leaf extracts. A band of the predicted size for the free OC-I protein (11.5 kDa) was present in all plants of the three transgenic lines (Figure 1B) but not in the controls (Figure 1A). Occasionally higher molecular weight bands could be detected. These bands are complexes formed between OC-I and endogenous cysteine proteinases in the crude extracts. These are often stable in the presence of SDS and reducing agents because many proteases are stable even in denaturing conditions and plants cystatins have no disulphide bonds.

Figure 1.

The relative OC-I protein contents of leaves from control lines and lines T4/3–1, T4/3–2 and T4/5. For the comparisons in (A) and (B), samples (25 µg) of soluble protein from leaves of each line were loaded. Polyclonal antibodies raised against OC-I purified by affinity-chromatography were used to determine the relative OC-I protein contents in leaf extracts. (A) A comparison of OC-I protein contents in the soluble protein fraction from wild type plants (Wt) and from the OC-I expressing line, T4/5. (B) A comparison of OC-I protein contents in the soluble protein fraction from lines T4/3–1 (A1, A2), T4/3–2 (B1, B2) and T4/5 (C1, C2). (C) Commassie Blue staining of concentrated heat-treated soluble proteins of wild type (Wt) and T4/5 leaves. In this case 40 µg of soluble protein was loaded in each case. Arrows indicate the position of OC-I and of a protein standard (Bio-Rad) in kDa.

The amount of free OC-I protein present in the leaves of the OC-I expressing plants was greatest in line T4/5 and least in line T4/3–2 (Figure 1B). Plants of the F2 generation of the three independent transformed lines also had significantly lower endogenous cysteine proteinase activity than controls, with the lowest cysteine proteinase activity being in line T4/5, which had the strongest OC-I expression (Table 1). Recently, it was suggested that OC-I is poorly expressed in transgenic plants (Womack et al., 2000). This conclusion perhaps arose because the detection of OC-I transgene expression (based solely on immunoblotting) can be problematic. For this reason, we have included two selectable marker genes (nptII and gus), in addition to OC-I, in the transgenic lines. This allowed us to avoid selection of false positives for kanamycin resistance and to increase selection efficiency by using GUS expression together with OC-I, because it is unlikely that the gus gene would segregate away from OC-I in the progeny. No significant cross-reaction of the antibody used in this study was found with endogenous tobacco cystatins in control plants (Figure 1). Moreover, the expressed OC-I protein could also detected on gels after heat treatment to remove heat sensitive proteins and give a 10-fold concentration of the extracts (Figure 1C). The immunoblot analysis showed that the amount of free OC-I protein present in the leaves of the OC-I-expressing plants was greatest in line T4/5 and least in line T4/3–2 (Figure 1B). In addition, OC-I expression (as shown in Figure 1) was related to the level of total cysteine proteinase activity present in the different lines (Table 1). Endogenous cysteine proteinase activity, measured by the fluorometric assay technique, was significantly lower in the OC-I expressing lines compared to controls (Table 1). It should be noted, however, that binding of the inhibitor to endogenous cysteine proteinases complicates accurate direct measurements of exogenous inhibitor levels in plant extracts.

Table 1.  The effect of OC-I expression on cysteine proteinase activity in OC-I/nptII/gus lines compared to nptII/gus controls. Plants were grown at 20 °C for 7 weeks at 300 µmol/m2/s irradiance
LineCysteine proteinase activity (%)
  1. Data are represented as reduction in total cellular cysteine proteinase activity relative to a control. One hundred per cent represents 28 fluorescence units/mg protein. Values represent the mean s.e. of 11 different plants.

Control100
T4/5 75 ± 3
T4/3–1 68 ± 2
T4/3–2 70 ± 5

Conditional phenotype and growth characteristics

A marked effect of growth irradiance on the phenotype of the plants was observed (Figures 2 and 3). When the OC-I expressing plants were grown for 7 weeks at a relatively low light intensity (300–350 µmol/m2/s) in the greenhouse at 20 °C, stem elongation was substantially decreased compared to controls (Figure 2A). This phenotype was observed in all the OC-I expressing (OC-I/nptII/gus) lines but not in the GUS (nptII/gus) control line, which showed a similar phenotype to the non-transformed wild type controls. This indicates that decreased stem elongation is linked to OC-I expression and was not due to somaclonal variation consecutive to the transformation or tissue culture processes. Analysis of a much greater number of transformed lines than those studied here would be required in order to establish the precise relationships between measured OC-I levels, cysteine proteinase activity and plant phenotype. Phenotypic differences were, however, evident in plants grown at low light intensity (300 µmol/m2/s) throughout the growth cycle (Figure 3). Such differences were much less apparent at any developmental stage in plants grown at higher growth light intensities (900 µmol/m2/s). High light favours shorter stems (for example, compare Figure 2A and B). This phenomenon was consistently found in all experiments.

Figure 2.

A comparison of the OC-I phenotype relative to that of the kanamycin-resistant β-glucuronidase-expressing (nptII/gus; GUS) and wild type (Wt) controls. In (A) OC-I expressing (OC-I/nptII/gus; GUS) lines T4/3–1, T4/3–2 and T4/5, a GUS expressing (nptII/gus) line and wild type (Wt) plants were grown for 7 weeks at low light intensity (300–350 µmol/m2/s). In (B) for comparison, the effect of 7 weeks of growth at a higher light intensity (900 µmol/m2/s) in OC-I expressing lines T4/3–1 and T4/5 are shown relative to the wild type plant (Wt) plants. In this study the GUS expressing (nptII/gus) line had similar growth characteristics to the wild type and is hence not shown.

Figure 3.

The effect of low light on the OC-I expression phenotype with plant development. OC-I expressing lines T4/3–1, T4/3–2 and T4/5, and control plants (Con) were grown for up to 12 weeks under low light intensity (300–350 µmol/m2/s).

Phenome analysis was undertaken in plants of the same age (at 7 and 12 weeks). Flowering was delayed by an average of 20 days in the OC-I/nptII/gus lines (data not shown). The OC-I expressing lines also had longer more pointed leaves and a larger leaf area resulting in a greater leaf dry weight (Figure 4). It is important to note, however, that the leaf number was similar in all lines at all time points. After 12 weeks each plant had about 23/24 leaves regardless of the line measured. The leaves of the controls expanded more rapidly under low light conditions than those expressing OC-I (P ≤ 0.05) after 7 weeks growth (Table 2). Total leaf area was thus slightly lower in the OC-I expressing lines than in the controls at 7 weeks. At 7 weeks the plants expressing OC-I also had lower total biomass (fresh and dry weight) than the controls (Table 2). After 12 weeks growth at a lower light intensity (300–350 µmol/m2/s), when control plants, but not OC-I expressing plants, had flowered, the stems of the OC-I expressing plants were still much shorter (55–70%) than the stems of the respective controls (Figure 4).

Figure 4.

The effects of growth under low light intensity on stem length (A) and shoot biomass (B) at the end of the growth cycle (12 weeks). The average stem height and stem and leaf biomass was compared in OC-I expressing lines T4/3–1, T4/3–2 and T4/5, and the controls (Con). Ten plants per line were grown for 12 weeks under low light intensity (300–350 µmol/m2/s).

Table 2.  The effect of OC-I expression on plant height, leaf area and biomass (fresh and dry weights). Plants were grown at 20 °C for 7 weeks in the greenhouse at 300–350 µmol/m2/s irradiance
LinePlant height (cm)Leaf area (cm2)Fresh weight (g)Dry weight (g)
  1. In each case values represent the mean ± s.e. of three different plants.

Control51.2 ± 4.82400 ± 166110 ± 316.8 ± 1.2
T4/512.0 ± 0.82100 ± 83 96 ± 713.0 ± 1.1
T4/3–111.6 ± 1.22166 ± 167 95 ± 512.1 ± 1.1
T4/3–215.6 ± 0.42116 ± 103103 ± 412.4 ± 1.2

Photosynthesis and respiration

Photosynthesis was lower (P ≥ 0.05) in OC-I expressing tobacco plants grown at 20 °C with an irradiance of 300–350 µmol/m2/s for 7 weeks, than in the nptII/gus line or the wild type controls (Table 3). Maximal rates of photosynthesis (Amax; 18.1 ± 0.2 µmol CO2/m2/s) were decreased as a result of OC-I expression (Table 3). All OC-I expressing plants had significantly lower (P ≤ 0.05) rates of CO2 assimilation than control plants. The inhibitory effect was greatest (11.0 ± 0.6 µmol CO2/m2/s) in OC-I expressing plants of line T4/5, which also had the highest level of OC-I protein. However, the apparent quantum efficiencies of photosynthesis (AQE) were in similar all lines. This observation indicates that while the absolute amounts of chlorophyll are increased (Table 4) and photosynthetic capacity is decreased (Table 3) in the OC-I expressing plants there is no photoinhibition (Table 3).

Table 3.  The effect of OC-I expression the apparent quantum efficiency (AQE) of photosynthesis, maximal CO2 assimilation rates (Amax) and dark respiration rates (R). Plants were grown for 7 weeks at 20 °C in the greenhouse at 300–350 µmol/m2/s irradiance
LineAQE (mol [CO2]/mol [light]) × 10−2Amax (µmol [CO2]/m2/s)R (µmol [CO2]/m2/s)
  1. In each case values represent the mean ± s.e. of samples of six different plants.

Control3.56 ± 0.3618.1 ± 0.20.82 ± 0.10
T4/52.92 ± 0.2411.0 ± 0.60.44 ± 0.21
T4/3–12.88 ± 0.1615.6 ± 1.40.89 ± 0.19
T4/3–23.12 ± 0.2415.8 ± 1.81.34 ± 0.09
Table 4.  The effect of OC-I expression on total leaf soluble protein, chlorophyll and the ratio of soluble protein to chlorophyll. Plants were grown for 7 weeks at 20 °C in a greenhouse at 300–350 µmol/m2/s irradiance
LineProtein (µg cm−2)Chlorophyll (µg cm−2)Ratio
  1. In each case values for protein and chlorophyll represent the mean ± s.e. of samples from six different plants.

Control348 ± 2446 ± 47.6
T4/5480 ± 5657 ± 58.4
T4/3–1492 ± 6061 ± 38.1
T4/3–2516 ± 2054 ± 29.6

Dark respiration rates varied between the plants of the different lines (Table 3). Leaves from line T4/3–1 had similar rates of dark respiration to those of control plants. Respiration rates in T4/5 plants with the highest OC-I expression were half (P ≤ 0.05) those of the controls. Moreover, respiration was significantly higher in transformed plants of line T4/3–2 (P ≤ 0.05; Table 3).

Responses to low temperature and drought stress

Photosynthetic CO2 assimilation was decreased following exposure to 5 °C for 2 days, with all plants showing lower and AQE (20–75% lower; Figure 5) values and Amax (65–80% lower; Figure 6). The decline in AQE in two OC-I expressing lines (T4/3–1 and T4/3–2), however, was significantly less (20% and 31%, respectively; P≤ 0.05) than that measured in control plants, in which AQE declined by 75% (Figure 5A). While the low-temperature induced decline in AQE (65%) was also less in T4/5 plants (with the highest OC-I expression), this was not significantly different (P ≥ 0.05) to that observed in the controls. In all cases, low-temperature induced changes in measured values for photochemical and non-photochemical quenching of chlorophyll α fluorescence were in agreement with the changes in AQE (data not shown). Following the 2 days of exposure to low growth temperatures, plants were allowed to recover at 20 °C for 2 days (Figure 5B). After 2 days recovery, AQE had returned to values measured in non-chilled plants in all lines (Figure 5B). There were no significant differences in AQE between plants of the different lines (P ≥ 0.05) in the recovery phase (Figure 5B).

Figure 5.

The effect of chilling on the apparent quantum efficiency of photosynthesis (AQE) in OC-I expressing lines T4/5, T4/3–1 and T4/3–2, and in control plants (Con). All plants were grown for 7 weeks at a light intensity of 300–350 µmol/m2/s. Measurements were made after 2 days at 5 °C (A) and after 2 days subsequent recovery at 20 °C (B). Values are expressed as percentages of those measured before cold treatment. These were 3.56 ± 0.36 mol [CO2]/mol [light] × 10−2 for wild type plants, 2.92 ± 0.24 mol [CO2]/mol [light] × 10−2 for T4/5, 2.88 ± 0.16 mol [CO2]/mol [light] × 10−2 for T4/3–1, and 3.12 ± 0.24 mol [CO2]/mol [light] × 10−2 for T4/3–2. In each case values represent the mean ± s.e. of leaves of six different plants.

Figure 6.

The effects of chilling and recovery on photosynthesis in 7-week-old control (Con) and OC-I expressing lines T4/5, T4/3–1, and T4/3–2. Measurements were made after 2 days at 5 °C (A) and after 2 days subsequent recovery at 20 °C (B). Values are expressed as percentages of those measured before cold treatment. In each case values represent the mean ± s.e. of leaves of six different plants.

Amax was reduced by 84% in wild type plants after 2 days at 5 °C (Figure 6A). The chilling-induced decrease in Amax was, however, less in the OC-I expressing lines. Compared to values measured at 20 °C, Amax was decreased by 72% in plants of lines T4/3–1 and T4/5 and by 65% in line T4/3–2 (Figure 6A). Two days after return to 20 °C, Amax had recovered to 70% of the original values in line T4/3–1 and in the controls. A trend to higher recoveries was observed in plants of lines T4/3–2 (78%) and T4/5 (85%), but Amax was significantly higher in line T4/5 with the highest OC-I expression (P ≤ 0.05; Figure 6B).

Plants were subjected to water deficits for a period of 7 days. Soil water content measurements were performed to assess the degree of drought experienced by the plants. Amax and transpiration rates decreased to almost zero in all lines after 7 days of drought, but no differences between the OC-I expressing plants and control plants were observed in the relationships between the decline in Amax and water loss (Figure 7). Since all three lines (T4/5, T4/3–1 and T4/3–2) showed similar drought responses to each other and to controls, data obtained from controls (•) and line T4/5 (▴) plants only are shown in Figure 7.

Figure 7.

The effect of drought on photosynthesis in an OC-I expressing line (OC-I/nptII/gus; T4/5; •) and a GUS expressing control (nptII/gus) line (▴). The relationships between photosynthesis and soil water loss were compared in 7-week-old plants. Plants were deprived of water on day 0 and were not watered for 7 successive days. Soil water contents and photosynthetic CO2 assimilation (Amax) measurements were made at the same point in the photoperiod on each successive day of drought. Values represent the mean ± s.e. of leaves of six different plants. Data obtained from control plants and line T4/5 only are shown.

Protein and chlorophyll content

When the OC-I expressing plants were grown at a lower light intensity (300–350 µmol/m2/s) for 7 weeks their leaves were darker green with a more narrow shape at the base than the leaves of the nptII/gus line and the wild type controls. The OC-I expressing lines contained significantly higher leaf chlorophyll contents than those of the controls (P ≤ 0.05). Leaves from lines T4/5, T4/3–1 and T4/3–2 had between 1.17 and 1.32 times as much chlorophyll as the controls (Table 4). The OC-I expressing leaves also had significantly higher (1.37 and 1.48 times) amounts of soluble protein (P ≤ 0.05) than leaves of controls under the same growth conditions. There was no significant difference between the three OC-I expressing lines in soluble leaf protein (P ≤ 0.05; Table 4). The leaf soluble protein to chlorophyll ratio was higher in the OC-I expressing lines than in the nptII/gus controls (Table 4).

Discussion

This is the first detailed report concerning the effects of abiotic stress on transformed plants expressing OC-I. The data presented here provides evidence of environment-dependent effects of OC-I expression on plant phenotype. Constitutive OC-I transgene expression has both disadvantages and benefits. For example, one obvious disadvantage is that flowering is delayed in the OC-I/nptII/gus lines. OC-I expression, however, offers benefits in terms of better stress tolerance (Figure 4). While all lines showed similar sensitivity to drought, photosynthesis was better protected against chilling-induced photoinhibition in OCI-expressing plants compared to control plants. Constitutive OC-I expression also facilitated better postchilling recovery of CO2 assimilation. Indeed, measured rates of photosynthesis were higher in the recovery phase than before cold treatment. Cysteine proteinases have been found to be expressed during chilling (Schaffer and Fischer, 1988). The presence of OC-I in the cytosol of transformed plants clearly provides improved acclimation characteristics. Moreover, after 12 weeks of growth at low light intensities all the plants of the three transformed lines had out-performed the controls in terms of biomass accumulation and leaf area (Figure 4). This result is similar to that recently reported by Gutiérrez-Campos et al. (2001).

There is no evidence to suggest that transgene expression caused an undue demand on carbohydrate reserves or associated energy status. The nptII/gus controls have a similar phenotype to the wild type (Figure 2). Moreover, the OC-I/nptII/gus plants had more total leaf protein and chlorophyll than controls (Table 4). The content and composition of many major proteins such as ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) was comparable in all lines although the composition of lower abundance proteins was modified in the OC-I/nptII/gus lines compared to nptII/gus controls (data not shown).

The highest recoveries following chilling were observed in transformed plants with high OC-I expression (line T4/5). These plants had rather lower CO2 assimilation rates than controls after 7 weeks of growth, however they also had the lowest dark respiration rates, which may suggest better utilization of resources (Lewis et al., 2000). Leaf chlorophyll contents were increased on a surface area basis at 7 weeks (Table 4). This may result from the shorter stems and hence closer proximity of leaves in the plants from all three transformed lines compared to controls (Figure 3). The shorter stem length that was characteristic of the transformed plants would lead to greater mutual shading of leaves in individual plants. More chlorophyll will be required for light harvesting in these circumstances. This is similar to growth in shaded environments or within crowded plant communities (Ballaréet al., 1994). The rate of photosynthesis expressed on a chlorophyll basis is therefore also much lower in the transformed plants than in the controls. These potentially negative effects do not, however, have long-term consequences for yield, which is higher in the transformed lines compared to controls (Figure 4).

The above results suggest that whole plant physiology and metabolism are modified by the presence of OC-I. OC-I could modify protein turnover rates in the cytosol by interaction with endogenous cysteine proteinases. While cysteine proteinases are generally considered to reside in the vacuole the possibility of cytosolic isoforms has not been thoroughly explored. Recent evidence indicates that the endoplasmic reticulum houses a novel proteinase-storing system that assists in cell death under stress conditions (Hayashi et al., 2001).

Plant growth

When plants were grown under low light in the greenhouse stem elongation was suppressed in OC-I expressing lines compared to controls. This indicates a direct link between OC-I expression and growth (Figures 2A and 3). No effects of OC-I expression on growth were observed in the nptII/gus controls (Figure 2), which had a similar phenotype to the wild type. OC-I expression appeared to modify stem elongation more than any other parameter measured. The short stem phenotype was dependent on the light environment in which the OC-I/nptII/gus plants were grown. Differences in stem elongation were much less obvious when OC-I/nptII/gus plants were grown at high light intensities. High light favours slower stem elongation rates. While earlier studies on OC-I expression in tobacco (for example that of Masoud et al., 1993) did not report phenotypic effects, a more recent evaluation by Gutiérrez-Campos et al. (2001) found increased growth rates, greater biomass and earlier flowering as well as increased numbers of flowers and seeds in OCI-expressing lines. Such discrepancies may be explained, at least in part by the different tobacco cultivar used in each study as well as growth conditions. Masoud et al. (1993) and Gutiérrez-Campos et al. (2001) used Xanthi while the cultivar Samsun was used in the present study. The two cultivars have been reported to respond differently in terms of transgene expression (Caligari et al., 1993)

Agronomic value

Through constitutive OC-I expression we have produced plants that offer improved biomass and acclimation to chilling stress. In addition, it may also be advantageous to have smaller plants with higher leaf area and protein contents under shade conditions. For example, this may be useful for grazing animals (if the cystatin does not impair digestion). It should also be noted that much of the increase in wheat yields over the last 100 years was specifically achieved by decreased stem elongation. Taken together, the results presented here demonstrate that the OC-I expressing plants have an interesting phenotype with potential agronomic value. The conditional plant phenotype of reduced stem elongation under shade conditions with the additional benefit of increased chilling tolerance might therefore form a very useful basis for further biotechnological applications. They may also be useful for the production of exogenous proteins of commercial value in Planta, particularly with regard to proteins that are degradation-sensitive (Michaud, 1997).

Finally, our data also confirm that the selection of transformed plants for study and application should be carried out with extreme care. A thorough evaluation of whole plant physiology and performance, particularly under suboptimal growth conditions should always be included to exclude possible negative interactions with endogenous plant components or the occurrence of unusual phenotypes created either by transgene expression or transformation.

Experimental procedures

Plant material

Tobacco seeds of the cultivar Samsun were obtained from the Tobacco and Cotton Research Institute at Rustenburg/South Africa. Plants of the same age were compared in all of the experiments outlined below.

Plasmid vectors and constructs

The binary plasmid pKYOC-I was used for tobacco transformation. The pKYOC-I plasmid, which was generously provided by our colleague L. Jouanin (INRA Versailles, France), encodes OC-I coding sequence under the control of a double 35S promoter (P70) from cauliflower mosaic virus between the left border (LB) and right border (RB). Also present on the T-DNA is an Ω leader sequence for gene expression enhancement. In addition, the construct contains the nptII gene under the control of a 35S promoter (P35SNPTII), which was used as a selectable marker, and an intron-containing gus gene (GUS) encoding β-glucuronidase (P35S GUSint) under the control of a 35S promoter.

Plant transformation

The standard procedure as outlined by Horsch et al. (1985) was applied for transformation tobacco with Agrobacterium tumefaciens (C58pMP90) carrying the binary vector pKYOC-I. Regeneration of transformed shoots carrying the OC-I coding sequence was carried out on a Murashige and Skoog (MS) medium (Murashige and Skoog, 1962). This consisted of 0.8% agar supplemented with 0.1 mg/L BAP, 100 mg/L kanamycin for selection of transformed shoots, and 500 mg/L cefotaxime to prevent further growth of Agrobacterium. After 4 weeks cultivation in a growth room at 25 °C with a 16/8 h light/dark cycle, leaf discs were cut and transferred to new MS medium containing BAP, kanamycin and cefotaxime. Putative transformants were placed on half-strength MS medium, containing 100 mg/L kanamycin and 300 mg/L cefotaxime to stimulate root formation. Shoots that grew on the kanamycin-containing medium and expressed GUS (Jefferson et al., 1987) were transferred to soil. They were grown in the greenhouse for 2 weeks and then tested for OC-I expression.

Plants were selected on the basis of cysteine proteinase activity measured as described by Barrett and Kirschke (1981) and by OC-I protein content. D. Michaud (Laval University, Quebec, Canada) kindly provided the specific OC-I antibodies, used for immuno-blotting. On this basis three transformed plants were selected for self-fertilization. These transformed plants had the lowest endogenous cysteine proteinase activity and showed a band of the predicted size of the OC-I protein after immuno-blotting. F1 generation seeds (20 for each independent line) were tested for antibiotic resistance on kanamycin. Seeds that germinated and produced rooted dark-green plantlets on the antibiotic-containing medium were again tested for expression of GUS, endogenous cysteine proteinase activity and the presence of the OC-I protein by immuno-blotting. One plant, expressing GUS and with the highest OC-I expression and the lowest endogenous cysteine proteinase activity, was selected from each of the independent lines, T4/3–1, T4/3–2 and T4/5. These three plants were then self-fertilized to produce the F2 generation. Finally, 40 seeds from each of the independent lines were again tested for kanamycin resistance. Over 80% of these seeds germinated and produced seedlings on kanamycin-containing media. Only plants expressing GUS and the OC-I transgene were used in the following experiments.

Plant growth

For all experiments, plants of the same age were compared. These were grown under identical controlled environment conditions where light, temperature and water were modified as indicated in the legends to the Figures. Seeds of the wild type controls, controls expressing nptII/gus and the F2 generation of the three independent transformed expressing OC-I/nptII/gus lines were germinated on wet filter paper without selection. Germinated transformed seedlings that expressed GUS were transferred to pots along with the controls and grown in compost. They were grown under controlled environment conditions with a 16-h photoperiod at either a light intensity of 300–350 µmol/m2/s or 900 µmol/m2/s supplied by incandescent lamps. A light intensity of 350 µmol/m2/s is about five fold lower than full sunlight, while 900 µmol/m2/s is about half of full sunlight. The growth temperature was 20 °C during the day and 15 °C during the night period. The relative humidity was 70 ± 5%. After the seedlings were transferred to compost, they were watered with tap water every day.

Large scale growth and biomass experiments

Ten plants of the control and three independent transformed tobacco lines were grown in pots containing compost, in a greenhouse with a 16-h photoperiod at a light intensity of 300–350 µmol/m2/s. The growth temperature was 20 °C during the day and 15 °C during the night. Plants were harvested at 12 weeks when the controls had started to flower.

For each experiment morphological parameters such as leaf number, leaf area, stem height, fresh and dry weights of upper plant organs, were compared in 7 and 12-week-old tobacco plants from each line. Leaf number was measured by counting the number of leaves from base to tip. Leaf area was measured by using the leaf area meter (Paton Industries Australia) technique. Stem height was determined by measuring the distance from ground surface to the apical meristem and fresh weight was taken by weighing the upper plant material without the root material directly after collection. For determination of the dry weight the plant material was dried for 24 h in an oven at 90 °C and then weighed.

Cold stress and drought treatments

For cold stress studies plants, which were grown for 7 weeks at a light intensity of 300–350 µmol/m2/s, were subjected to 5 °C for 2 days and then returned to 20 °C for a further 2 days to allow recovery. Photosynthesis and respiration measurements were made each day on intact leaves throughout the period of the experiment.

For drought experiments plants, which had been grown for 7 weeks at a light intensity of 300–350 µmol/m2/s, were deprived of water for 5 days. Photosynthesis and respiration measurements were made on intact leaves each day throughout the period of the experiment. Soil water content measurements were performed on soil cores taken from the pots throughout the experiments. Each core taken from pots of well-watered plants contained 2 g fresh weight of soil. The cores were dried for 24 h at 80 °C and the dry weight was measured to determine the soil water content.

Gas exchange measurements

Gas exchange measurements were performed on whole, attached leaves using an automated multichamber open-circuit gas exchange system. The system comprises an infrared gas analyser (IRGA; WA-225-MK3, ADC, Hoddesdon, UK), a gas handling (WA-161 2K, ADC, Hoddesdon, UK) and a mode-switching unit (WA-357-MK3, ADC, Hoddesdon, UK). The O2 concentration in the gas phase was measured with a gas analyser (Series 80, Ox-An Systems, Huddersfield, UK). A gas blender (Signal Instruments Co., Croydon, UK) regulated the CO2 and O2 composition of the air entering the leaf chambers. The flow rate was kept at 9 cm3/s by mass flow meter and controllers (Bronkhorst HI-Tech B.V., Holland). Temperature and relative humidity of the air in the chamber was regulated by bubbling the gas stream through water either at 20 °C (optimal experimental conditions) or 5 °C (chilling conditions), and then through a condenser set to the required dew point. Chamber temperature was measured with thermocouples. A capacitance humidity sensor (Vaisala, Helsinki, Finland) was used to measure water content of the air before and after passing through the leaf chamber. White light was supplied by metal-halide lamps (Wotan, Philips, Holland) and was measured with selenium sensors (Megatron, London, UK).

Photosynthesis measurements

The CO2 assimilation rate was measured in the youngest leaves (6 cm width) of 7-week-old plants. Leaves were allowed to attain steady state photosynthesis for 20–30 min at constant conditions prior to measurement. Measurements were taken over a 1-h period. Leaf chamber CO2 concentrations (Ca) were maintained at 350 ± 10 µmol/mol and O2 at 210 mmol/mol. Relative humidity in the leaf chamber was 50–60% and the light intensity was set at 800 ± 35 µmol/m2/s. The temperature of the leaf chambers was set at 20 ± 0.5 °C and calculations of CO2 assimilation rate (Pn were performed as described by von Caemmerer and Farquhar, 1981). Steady state CO2 assimilation measurements of single leaves were taken to assess the average steady state assimilation for the respective plant lines.

The apparent quantum efficiency (AQE) was determined from the light response curves for photosynthesis measured from 0 to 1500 ± 100 µmol/m2/s. Irradiance was varied using neutral density filters (Lee Filters, A.C. Lighting, Bucks, UK). Leaf chamber CO2 concentrations were maintained at 350 ± 10 µmol/mol. Linear regression of the initial slope of the hyperbolic light response curves allows AQE calculation at points approximately 50 µmol/m2/s.

SDS-PAGE and immunoblotting

Polyclonal OC-1 antibodies were prepared according to the method of Nguyen-Quoc et al. (1995). The OC-I coding sequence was fused in frame with the GST coding sequence. The fusion protein was expressed in E. coli and purified via affinity chromatography using glutathione sepharose. The antibody was then prepared against the purified fusion protein.

Leaf discs (60 mg fresh weight) were excised using a cork borer and immediately frozen in liquid nitrogen. Two frozen leaf discs were homogenized in 0.3 mL 50 mm Tris HCl (Tris [hydrodymethyl]amonomethane) buffer (pH 7.0) containing 1 mm PMSF (phenylmethylsulphonyl fluoride) to avoid protein degradation. The soluble protein fraction produced by centrifugation at full speed in an Eppendorf bench centrifuge, was used for protein analysis. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS) was carried out as outlined by Laemmli (1970) with 25 µg of total protein. Detection of OC-I protein on SDS-PAGE was carried out as described by Masoud et al. (1993). Immuno-blots were performed as described by Sambrook et al. (1990). The OC-I antiserum prepared as above was used as the primary antibody to detect OC-I on Hybond C extra membranes (Amersham, UK). Anti-rabbit IgG horseradish peroxidase conjugate (Amersham, UK) was used as the secondary antibody. The protein was detected with the help of the ECL KIT (Amersham, UK) through the initiation of a photoreaction and fluorescence detection on a Biomax MR film.

Cysteine proteinase assay

Cystatin activity was measured according to the method of Barrett and Kirschke (1981). Plant extract (10 µL) was diluted in 500 µL of a solution containing 0.1% Brij 35 and 250 µL of a proteinase reaction buffer. For temperature equilibration and activation of the enzyme, the solution was placed at 30 °C for 1 min and after equilibration, 250 µL of 20 µm of the cysteine proteinase substrate Z-Phe-Arg-Nmec was added to release after proteinase action the fluorescent compound 7-amino-4-methylcoumarin. After incubation for 10 min at 30 °C, 1 mL of monochloroacetate stopping reagent was added and the fluorescence of the free aminomethylcoumarin was determined in a fluorometer using 370 nm for excitation and 460 nm for emission.

Total soluble protein and chlorophyll analysis

The protein content of leaf discs (60 mg) was determined using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). The absorbance at 595 nm was measured with a spectrophotometer (ULTROSPEC II, LKB-Biochrom). The protein concentration of the extract was calculated from a standard curve with dilutions from 0 to 25 µg (BSA)/mL.

The chlorophyll content of freeze-clamped leaf samples (2 × 5.31 cm2) was determined according to the method described by Arnon (1949) after extraction of chlorophyll from leaf samples with 80% acetone.

Statistical analysis

All measurements were performed on a minimum of three to five plants depending on the experiment (see figure legends). The statistical significance of the difference between the mean values was determined by Student's two-tailed t-test. P-values ≤ 0.05 were considered to be significant.

Acknowledgements

The authors are grateful to Dr L. Jouanin for providing the binary plasmid pKYOC-I and Dr D. Michaud for providing an antiserum against OC-I. We thank Rieckert van Heerden from Potchefstroom University, South Africa, for growing tobacco plants under artificial light of high intensity. J. Schneidereit was supported by an EU Leonardo Da Vinci fellowship. This work was supported by the BBSRC (UK) and National Research Foundation of South Africa and by a grant to C.V.d.V. and K.J.K. from the Mellon Foundation. We are indebted to Guy Kiddle and Graham Noctor for their expert advice and assistance in optimizing methods.

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