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Keywords:

  • antisense;
  • Calvin cycle;
  • leaf growth;
  • phenotypic plasticity;
  • sedoheptulose-1;
  • 7-bisphosphatase

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The effects of reduced SBPase activity on growth and development were examined in a set of transgenic tobacco plants produced using an antisense construct driven by the ribulose bisphosphate carboxylase, small subunit promoter. Photosynthetic carbon assimilation rates and carbohydrate levels in source leaves were decreased in the antisense plants. Growth rate and total shoot biomass were reduced in the SBPase antisense plants, even in plants where SBPase activity was reduced by only 25%. Floral biomass also decreased in response to reductions in SBPase activity and the onset of flowering was delayed by 5–10 d. This is the first demonstration of a link between reproductive biomass and reductions in Calvin cycle enzyme activity using antisense plants. Furthermore, unexpected changes in the growth and development of the antisense plants were evident. Small reductions in SBPase activity (above 50% wild type) resulted in shorter plants with only a small decrease in stem biomass and specific leaf area. In contrast, plants with larger reductions in SBPase activity had an increase in specific leaf area and attained heights similar to that of the wild-type plants but with a much reduced stem biomass, largely due to a decrease in xylem tissue. This bi-modal response of growth to reductions in SBPase activity has similarities to changes in leaf and stem anatomy and morphology that accompany light acclimation.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant morphology changes during development as the young shoot progresses from the juvenile vegetative phase, through the mature vegetative, to the reproductive stage. Transition from the juvenile to mature vegetative phase is subtle but can involve changes in leaf shape and size and a number of genes involved in this process have now been identified (Poethig 1990; Poethig 2003). It has been suggested that the nutritional status of the plant plays a role in the regulation of the developmental programme. Carbohydrate, in the form of sucrose, is involved in the transition to flowering and increased translocation of sugars into the shoot apex has been implicated in floral induction (Bernier et al. 1993; McDaniel, Singer & Smith 1996; Corbesier, Lejeune & Bernier 1998). Shoot morphology is also affected by the environmental conditions that the plant experiences during development. For example in low light conditions leaves are thinner and the stems more elongate than those of plants grown in high light conditions (Björkman 1981). Changes in leaf geometry have also been noted in plants grown in elevated CO2 (Taylor et al. 2003). Both of these environmental parameters can affect photosynthetic performance and alter carbohydrate levels, but as yet the mechanism(s) responsible for mediating these changes in growth and development, have not been identified. Given that the photosynthesis in mature source leaves determines the availability of the carbohydrate essential for plant growth and development, it is possible that products of primary metabolism may play a role in regulating these responses. Antisense Rubisco plants with significant reductions in photosynthesis accumulated less carbohydrate, resulting in changes in root to shoot ratios and leaf geometry (Fichtner et al. 1993; Stitt & Schulze 1994). Interestingly, this decrease in source strength affected the normal timing of developmental events, resulting in a delay in the transition to the exponential phase of shoot growth. In addition, when compared to wild-type tobacco plants an increase in leaf longevity was observed, suggesting that senescence was delayed in the antisense Rubisco plants (Tsai et al. 1997; Miller et al. 2000). These results indicated that source strength may play an important role in determining the leaf developmental programme. However, the antisense plants used in this study of development had only 20% of wild-type Rubisco activity which implies that not only was the sink/source balance of these plants altered but, given that Rubisco protein constitutes approximately 25% of the leaf protein, the nitrogen status of the leaves would also be significantly different from wild-type plants (Fichtner et al. 1993; Masle, Hudson & Badger 1993).

The enzyme sedoheptulose-1,7-bisphosphatase (SBPase: EC 3.1.3.37) functions in the regenerative phase of the Calvin cycle where it catalyses the dephosphorylation of sedoheptulose-1,7-bisphosphate. Transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun) with small reductions in SBPase activity were found to have decreased rates of photosynthetic carbon fixation and altered carbohydrate levels in mature source leaves (Harrison et al. 1998; Raines et al. 2000; Olcer, Lloyd & Raines 2001). Given the sensitivity of photosynthesis to reductions in SBPase activity and the fact that SBPase constitutes less than 1% of leaf protein, the SBPase antisense plants provide useful tools to investigate the role of decreasing source strength on the plant developmental programme without altering levels of leaf nitrogen content. The SBPase antisense plants used in previous studies were made using a construct the expression of which was driven by the CaMV 35S promoter and displayed a leaf veinal chlorosis when SBPase activity was reduced below 20–25% of wild-type levels (Harrison et al. 1998). A similar leaf chlorosis was observed in transgenic plants with altered activities of glutamate 1 semi-aldehyde aminotransferase and plastid transketolase (Hartel, Kruse & Grimm 1997; Henkes et al. 2001). In both of these cases the antisense construct was also driven by the CaMV promoter, suggesting that this phenotype was due to differential expression of the CaMV promoter in different cell types. To avoid this chlorotic phenotype we have produced a new set of antisense tobacco SBPase plants using the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS ssu) promoter. We have used this new set of plants with a full range of reductions in SBPase activity to investigate the impact of decreased photosynthetic capacity and source strength on vegetative and floral biomass and plant growth and development.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant transformation

Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum L. cv. Samsun leaf discs, with the antisense gene construct prepared in the binary vector pBin19, was carried out essentially as described by Horsch et al. (1985). Shoots were regenerated on MS medium containing kanamycin (100 µg mL−1) and carbenicillin (500 µg mL−1). Kanamycin resistant primary transformants (T0 generation) with established root systems were transferred to compost (Levington F2; Fisons, Ipswich, Suffolk, UK) and allowed to self fertilize. The nomenclature Tsa2 was used to denote transgenic plants containing this tobacco antisense construct regulated by the Rubisco promoter. All of the analysis was carried out using progeny (T3) of the primary transformants.

Growth of plants for physiological analysis

Wild-type and transgenic tobacco (Nicotiana tabacum L. cv. Samsun) seeds were germinated on sterile MS media containing 3% (w/v) sucrose (Murashige & Skoog 1962). For transgenic seedlings the media was supplemented with kanamycin (500 µg mL−1). Three-week-old-seedlings were transferred to soil (Levington F2; Fisons) or for measurement of root biomass plants were grown in a 3 : 1 mixture of sand and a 6 mm grit sand soil mixture and acclimatized in a propagator before transfer to 18 cm pots. Plants were grown a controlled environment greenhouse in light levels of between 400 and 1500 µmol m−2 s−1, a 16 h photoperiod at a minimum of 25 °C light/18 °C dark, maximum 35 °C light/25 °C dark. Plant positions were rotated daily. For plants used for photosynthetic analysis, leaves 11–15 were identified on emergence and their length and width measured every two days. This allowed the exact time at which full leaf expansion was reached to be determined and gas exchange measurements made at this point. Leaf discs (1.5 cm diameter) for protein, carbohydrate and chlorophyll analysis were harvested from the same leaves used for photosynthesis measurements and immediately frozen in liquid nitrogen and stored at −70 °C.

Carbohydrate analysis

Carbohydrates were extracted from frozen leaf discs (1.5 cm) in 1 mL of 80% buffered ethanol (50 mm HEPES-KOH, 5 mm MgCl2.6H2O pH 7.4) at 80 °C for 30 min, the supernatant was then removed and stored on ice. This process was repeated (typically between three and five extractions) until no green coloration remained in the tissue and the supernatants (3–5 mL in total) freeze dried before dissolving in distilled water (1 mL) and stored at −20 °C. The remaining ethanol insoluble material was stored at −20 °C for starch analysis. Concentrations of Glc, Fru and Suc were determined using an enzyme-linked assay (Stitt et al. 1989) using a microtitre plate reader (Dias; Dynatech Laboratory, St. Peter's Port, Sussex, UK). Enzymes were obtained from Boehringer Mannheim (Lewes, East Sussex) or Sigma (Poole, Dorset, UK). Starch was measured in the ethanol-insoluble fraction according to Stitt, Bulpin & ap Rees (1978), with the exception that samples were boiled rather than autoclaved.

Photosynthesis measurements

The response of CO2 uptake to intercellular CO2 concentration (Ci) was determined at a light level of 1000 µmol photons m−2 s−1 with a leaf temperature of 25 ± 1.5 °C, using an open gas exchange system (CIRAS-1; PP-Systems, Hitchin, Herts., UK), incorporating an infrared CO2 and H2O analyser calibrated against a known CO2 standard (Linde Gas Ltd, Stratford, London, UK). Photosynthetic carbon fixation rates were initially measured at the ambient CO2 concentration (Ca; 354 µmol mol−1) at which the plants had grown. To determine the initial slope of the ACi response, Ca was decreased in three steps to 50 µmol mol−1 and then returned to ambient levels to confirm the original rate could be regained. The Ca was subsequently increased stepwise to 1500 µmol mol−1 for completion of the curve. CO2 assimilation rate per unit leaf area and intercellular CO2 concentration (Ci) were determined using the equations of von Caemmerer & Farquhar (1981). Irradiance was measured with a quantum sensor (Sky Instruments Ltd, Llandridnod Wells, Wales).

Growth analysis

Plant heights were measured every two days from the base of the stem to the apical bud. After flowering was complete all of the above-ground material was harvested, dried to a constant weight at 80 °C and dry weight determined. Total leaf area was estimated using a flat bed scanner (ScanJet, Iicx; Hewlett-Packard, San Diego, CA, USA). Stem diameter was measured at the base and the mid point of the stem using vernier callipers. Samples for anatomical analysis were taken at the time of harvest and fixed in 4% gluteraldehyde, dehydrated using an alcohol series before embedding in LR white acrylic resin (Sigma). Ten-micrometre-thick cross sections were cut using a glass knife microtome (Reichert-Jung Ultracut; E.M. Systems Support, Kettleshume, High Peak, Cheshire, UK).

Western blot analysis

Soluble proteins were separated on 10% (w/v) sodium dodecyl sulphate (SDS)-polyacrylamide gels and transferred electrophoretically to Immobilon PVDF membrane (Millipore, Bedford, Beds., UK) (Laemmli 1970). Prior to incubation with antibody the membranes were washed in phosphate-buffered saline (PBS) containing 0.05 (v/v) Tween 20 (PBS-T) and then blocked in PBS-T containing 6% milk powder (w/v). The membranes were then incubated with primary antibody (SBPase, FBPase, PRKase and Rubisco holoenzyme) for 1–3 h at room temperature followed by ten 5 min PBS-T washes. Proteins were detected using horseradish peroxidase conjugated second antibody and an enhanced chemiluminiscence (ECL) kit (Amersham Health, Little Chalfont, Buckinghamshire, UK) (Harrison et al. 1998). Prior to re-probing of the membranes, antibodies were removed following ECL detection by immersing in buffer containing 100 mm 2-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCL pH 6.7 for 30 min at 50 °C.

Determination of SBPase activity

Frozen leaf discs were ground to a fine powder in liquid nitrogen using a mortar and pestle in 1.4 mL extraction buffer [50 mm Hepes, pH 8.2, 5 mm MgCl2, 1 mm ethylenediaminetetraacetic acid (EDTA), 1 mm ethylene glycol bis (2-amino ether)-N-N-N-N tetra acetic acid (EGTA), 10% glycerol, 0.1% Triton X-100, 2 mm benzamidine, 2 mm amino caprionic acid, 0.5 mm phenylmethysulfonylfluoride; 10 mm dithiothreitol; (DTT)], transferred to a prechilled 2 mL tube and spun for 1 min at 4 °C. The supernatant was desalted using a NAP-10 column (Pharmacia, Milton Keynes, Bucks., UK) and equilibrated with desalting buffer (extraction buffer omitting the Triton X-100). Proteins were eluted from the column with 1.5 mL desalting buffer and aliquots stored in liquid nitrogen. To start the reaction, 20 µL of thawed extract was added to 66 µL of assay buffer (50 mm Tris, pH 8.2, 15 mm MgCl2, 1.5 mm EDTA, 10 mm DTT, 2 mm SBP (Sigma, Poole, Dorset, UK) and incubated at 25 °C for 5 min. The reaction was stopped by the addition of 50 µL of 1 m perchloric acid and stored on ice. The samples were centrifuged for 5 min and the supernatant assayed for phosphate. Samples (50 µL) and phosphate standards (0–0.5 mm NaH2PO4) were incubated with 850 µL molybdate solution (0.3% ammonium molybdate in 0.55 m H2SO4) for 10 min at room temperature. Malachite green (0.035% malachite green, 0.35% polyvinyl alcohol) was added (150 µL) and the samples incubated for a further 45 min and the absorbance then measured at 620 nm (Leegood 1990; Harrison et al. 1998).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Production of antisense SBPase plants

An SBPase antisense gene construct was prepared by inserting the promoter from a tobacco small subunit gene (Mazur & Chui 1985) upstream of a tobacco SBPase cDNA fragment in the vector pBin19 (Bevan 1984) (Fig. 1a). The same cDNA was used previously to prepare an antisense construct containing the CaMV 35S promoter (Harrison et al. 1998). A total of 64 kanamycin resistant primary transformants were produced and Southern blot analysis used to confirm the presence of the transgene (data not shown). SBPase protein levels in the primary transformants were determined using western blot analysis and revealed that transgenic plants with reduced levels of SBPase had been produced and that no decrease in the levels of the Calvin cycle enzymes FBPase, PRKase or Rubisco small subunit protein was evident (Fig. 1b). SBPase activity was measured in the two newest fully expanded leaves (12 and 14), of wild-type tobacco and T1 progeny resulting from the selfing of selected T0 SBPase antisense lines (22, 20, 29 and 10). These data showed clearly that plants with a range of SBPase activity had been produced (Fig. 1c). The veinal chlorosis observed in some of the CaMV-SBPase antisense lines (Harrison et al. 2001) was not evident in any of the rbcS-SBPase plants produced in this experiment.

image

Figure 1. (a) SBPase antisense gene construct. A tobacco SBPase cDNA fragment was cloned in the reverse orientation between a tobacco rbcS promoter and the termination sequences of the nopaline synthase gene (nos), LB is the left border sequence of the T-DNA. (b) Calvin cycle protein levels in leaves of wild-type (WT) and SBPase antisense primary transformants (T0). Samples were taken from the newest fully expanded leaves (leaf 12 and 14) of plants in the mature phase of vegetative growth, proteins were then extracted, loaded on an equal leaf area basis and separated by SDS-PAGE and blotted onto nylon membrane. The resulting Western blots were probed with polyclonal antibodies raised against SBPase, FBPase, PRKase and Rubisco LSU and proteins detected using enhanced chemiluminescence (Amersham). (c) SBPase activities determined in T1 progeny, data points for the wild-type plants are the mean ± SE (n = 4, leaf 12); and for the transgenic SBPase antisense plants are the mean ± SE of triplicate measurements of single extracts from two individual leaves (12 and 14) on each plant.

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Photosynthesis and carbon accumulation in the SBPase antisense plants

A cohort of wild-type tobacco and progeny from four independent SPBase antisense lines were grown under controlled environmental conditions for photosynthetic analysis with light levels on the plant of between 400 and 1000 µmol m−2 s−1 and SBPase activity determined as described above. The response of photosynthetic CO2 assimilation rate (A) to increasing intercellular CO2 concentration was measured under saturating light, in wild-type and antisense SBPase plants. The effect of reduced SBPase activity on A measured at both ambient (400 µmol mol−1) and saturating (1000 µmol mol−1) CO2 concentrations, under high light (1000 µmol m−2 s−1) were determined from the ACi response curves. Reductions in SBPase activity resulted in a decline in the rate of carbon assimilation in the antisense plants, and this reduction was greater under saturating CO2 (Fig. 2a). The chlorophyll content of the leaves used for photosynthetic analysis was similar in wild-type and antisense SBPase plants, with the exception of the plants with less than 20% of wild-type SBPase activity when chlorophyll levels declined (data not shown).

image

Figure 2. (a) Effect of reductions in SBPase activity on photosynthetic carbon assimilation under conditions of high light and ambient CO2 (circles) or saturating CO2 (squares). For wild-type plants, each point is the mean ± SE (n = 4, leaf 12) (open symbols) and for the SBPase antisense plants is the average of measurements on two separate, newly fully expanded, leaves (12 and 14) on individual plants (closed symbols). (b) Diurnal carbohydrate accumulation in SBPase antisense plants. Sucrose and starch were measured in samples harvested at the end of the light period and at the end of the dark period, from the same leaves used for SBPase activity and photosynthesis measurements. Data points for the wild-type plants (open circles) are the mean ± SE (n = 4, leaf 12); and for the transgenic SBPase antisense plants (closed circles) are the mean ± SE of triplicate measurements of single extracts from two individual leaves (12 and 14) on each plant. For each transgenic plant SBPase activities were determined in leaves 12 and 14 and the mean ± SE is presented.

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The diurnal turnover of sucrose and starch was determined in the leaves used for photosynthetic analysis. The majority of the carbohydrate in the mature leaves of both wild-type and SBPase antisense tobacco plants accumulated as starch (Fig. 2b). In wild-type plants the levels of both starch and sucrose were highest at the end of the photoperiod, and declined to low levels at the end of the dark period. Small reductions in SBPase activity led to a large decrease in the amount of starch accumulated at the end of the day. In the plant with 20% wild-type SBPase activity little or no accumulation of starch occurred during the photoperiod. Reductions in sucrose levels were also seen in all of the antisense plants, although this was less pronounced than for starch (Fig. 2b). These analyses clearly showed that the source capacity of newly fully expanded leaves was reduced in response to decreased SBPase activity.

The effects of reduced SBPase activity on total plant biomass

To obtain data on root biomass, SBPase antisense plants was grown in greenhouse conditions in a sand and grit medium. At the onset of flowering, wild type and SBPase antisense plants were harvested and the dry weight of roots and shoots determined. Total vegetative biomass decreased in response to reductions in SBPase activity (Fig. 3a). This was due to a decrease in both the shoot and root biomass (Fig. 3b & c). No change in the shoot-to-root ratio occurred in response to reductions in SBPase activity (Fig. 3d).

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Figure 3. Biomass of wild-type and SBPase antisense plants. (a) Total plant biomass; (b) shoot biomass; (c) root biomass; and (d) shoot/root ratio. Plants were grown in a 3 : 1 mixture of sand and grit (6 mm). Plants were harvested at flower initiation. The values are means ± SE for seven wild-type plants (open squares) and measurements for individual SBPase antisense plants (closed circles).

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A further cohort of SBPase antisense plants was grown in a controlled environment greenhouse in compost and used for growth analysis. Measurements of plant height revealed that reductions in SBPase activity resulted in a decrease in the rate of growth of the transgenic plants compared with wild-type plants (Fig. 4a). The wild-type plants entered the rapid exponential phase of vegetative growth 40 d after planting (DAP) but, in the SBPase antisense plants, this phase change was delayed and occurred 5–10 d later. As a result of this all of the antisense plants were shorter than the wild-type plants at 60 DAP (Fig. 4a & b). Unexpectedly, the decreased growth rate of the SBPase antisense plants did not always result in a reduction in the maximum height attained and this was dependent on the level of SBPase activity (Figs 4a & 5a). Antisense plants with small reductions in SBPase activity were shorter than the wild-type plants, while the antisense plants with less than 30% wild-type SBPase activity, grew to heights similar to wild-type tobacco plants (Fig. 5a). In general the antisense plants had a reduced stem biomass in comparison with wild-type tobacco (Fig. 5b). The relationship between stem length and biomass indicated that stem thickness may be reduced in plants with the lowest SBPase (Fig. 5c). This was confirmed by measurements of the stem diameter that showed that the antisense plants with the lowest SBPase activity had substantially thinner stems than wild-type plants (Figs 6a & b). Transverse sections taken from the base of these plants revealed that reductions in SBPase activity resulted in the production of significantly less xylem tissue in the antisense plants (Fig. 6c & d) compared with wild-type plants (Fig. 6e). In plants with moderate reductions in SBPase activity no difference in the amount of phloem and cortical tissues was evident. However, in plants with less than 40% wild-type SBPase activity the cortex was also reduced and appeared to have smaller cells (Fig. 6c).

image

Figure 4. Growth characteristics of the SBPase antisense plants. (a) Time course of plant height, measured on alternate days until flowering. The values are means ± SE for four wild-type plants (filled circles) and for the antisense plants with reductions in SBPase activity of (open symbols) 35% (circles, n = 6), 55% (squares, n = 5) and 75% (triangles, n = 5). (b) Height of antisense and a wild-type plant (far left) at 60 d after planting (DAP).

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image

Figure 5. Plant height and stem biomass as a function of SBPase activity. (a) Plant height, from the base of the stem to the apex, was determined after flowering; (b) stem biomass; (c) ratio of stem height to stem biomass. The values are for four wild-type plants (mean ± SE) (open squares) and single measurements for individual SBPase antisense plants (closed circles). SBPase activities were determined in triplicate, in samples from two individual leaves (12 and 14) (mean ± SE).

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image

Figure 6. Stem diameter as a function of SBPase activity. Stem diameters were measured at the base (a) and mid-stem (b). The values are means ± SE for six wild-type plants (open squares) and single measurements for individual SBPase antisense plants (closed circles). Transverse stem sections taken from the base of two antisense plants with reductions in SBPase activity of (c) 60% and (d) 37% and a wild-type plant (e) showing X, xylem deposition and C, cortex. The scale bar represents 1 mm.

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Leaf biomass and leaf area

When the effect of reductions in SBPase activity on total leaf area was measured, a bi-modal response similar to that obtained for stem height was also observed (Fig. 7a & b). Moderate reductions in SBPase activity led to a decrease in leaf area. However, when SBPase activity levels were reduced to below 30% wild type, the leaf area did not continue to decrease but instead was similar to wild-type plants (Fig. 7a). In contrast to leaf area, total leaf biomass declined with reduced SBPase activity (Fig. 7b). These data showed that small reductions in SBPase activity resulted in a decrease in specific leaf area compared with wild-type plants (Fig. 7c). However, with further reductions in SBPase activity a linear increase in the specific leaf area was evident and, in some of the antisense plants, the specific leaf area was greater than in wild-type tobacco.

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Figure 7. Effect of reduced SBPase activity on (a) total leaf area; (b) total leaf biomass; and (c) specific leaf area. The values are means ± SE for four wild-type plants (open squares) and single measurements for individual SBPase antisense plants (closed circles). SBPase activities were determined in triplicate, in samples from two individual leaves (12 and 14) (mean ± SE).

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To investigate the impact of reductions on SBPase activity on individual leaves, areas for all the leaves on the plants at floral determination were measured. At this stage all the leaves on both the antisense and wild-type plants had reached full expansion. The leaf area profiles for the antisense plants were similar to the wild-type plants with the largest leaves being produced between nodes 6 and14 (Fig. 8). However, some differences were evident. The area of the largest leaves (leaves 8–14) on some of the SBPase antisense plants was greater than the equivalent leaves on the wild-type plants. In contrast, leaves produced later in the vegetative phase of development (nodes 16–28) on the antisense plants were significantly smaller than wild-type plants. During this phase of growth a correlation between average leaf area and SBPase activity was evident and plants with the most severe reductions had the smallest average leaf area (Fig. 8).

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Figure 8. Average area of leaves at each node at the stage of flower induction for wild-type and SBPase antisense plants. Values are the mean ± SE for wild-type plants (open symbols, n = 7) and for the antisense plants with reductions in SBPase activity of (filled symbols) 20% (triangles, n = 6), 40% (circles, n = 5) and 60% (squares, n = 5).

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Floral biomass and development

Floral biomass decreased with reductions in SBPase activity and in some plants it was less than one-third of that of wild-type plants (Fig. 9a). The antisense plants were delayed in reaching reproductive maturity by between 5 and 10 d (Fig. 9b). The antisense plants also took longer to reach anthesis than the wild-type plants and the length of delay correlated with the extent of the reduction in SBPase activity (Fig. 9b). The number of nodes produced prior to floral determination and anthesis in Nicotiana tabacum (Samsun) has been shown to be between 25 and 32 and the wild-type plants used in this study produced an average of 29 nodes (McDaniel & Hsu 1976; McDaniel et al. 1996). In the SBPase antisense plants the number of nodes produced prior to floral determination varied, but was dependent on the level of SBPase activity. Plants with more than 32% wild-type SBPase activity, produced a greater number of nodes before floral determination, compared with wild type. However, plants with less than 26% wild-type SBPase activity had wild-type number of nodes at floral determination (Table 1). Interestingly, all of the SBPase antisense plants had a significant increase in the number of nodes at anthesis (Table 1).

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Figure 9. Impact of reductions in SBPase activity on (a) total floral biomass and (b) flowering time; number of days after planting until floral initiation (squares) and anthesis (circles). The values are means ± SE for four wild-type plants (open symbols) and single measurements for individual SBPase antisense plants (closed symbols). SBPase activities were determined in triplicate, in samples from two individual leaves (12 and 14) (mean ± SE).

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Table 1.  Effects of reduced SBPase activity on the node number at floral determination and at anthesis
SBPase activity Per cent of wild typeNode number at floral determinationNode number at anthesis
  1. SBPase activity is expressed as a percentage of wild-type activity. Node numbers are the means ± SE for afour individual wild-type plants, b12 and csix individual SBPase antisense plants. SBPase activities were determined in triplicate, in samples from two individual leaves (12 and 14) (mean ± SE).

100a29 ± 0.530 ± 1.0
32–77b34 ± 1.138 ± 0.6
9–26c29 ± 1.241 ± 1.1

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The aim of the work presented here was to use transgenic plants with reduced levels of SBPase to investigate the impact of reductions in photosynthetic carbon flux on plant growth, development and biomass production. In order to do this we have produced a new set of antisense plants with a range of SBPase activities using an antisense construct driven by a tobacco rbcS gene promoter (Mazur & Chui 1985). In contrast to the SBPase antisense plants produced using the CaMV 35S promoter, the rbcS-SBPase antisense plants had no leaf veinal chlorosis, even in plants with less than 5% wild-type SBPase activity (data not shown). The data in this paper has shown clearly that, in these antisense plants, photosynthetic carbon fixation rates and carbohydrate accumulation were sensitive to small reductions in SBPase activity. This confirms our earlier analysis of the CaMV-SBPase antisense plants where flux control coefficient values for SBPase over carbon fixation of 0.5 were obtained (Harrison et al. 1998; Raines et al. 2000; Olcer et al. 2001). Diurnal carbohydrate measurements revealed that starch accumulation was decreased significantly in all the SBPase plants examined. Despite the fact that these plants were grown under high light in long days, the antisense plants with less than 30% wild-type SBPase activity accumulated almost no starch at the end of the light period. The reductions in the magnitude of the diurnal turnover of starch and sucrose, reported here, showed clearly that decreased SBPase activity leads to a severe source limitation.

In the SBPase antisense plants shoot, root and leaf biomass were reduced, even when SBPase activity was reduced by only 25%, compared with wild-type plants. A decrease in biomass has also been noted in antisense plants where other Calvin cycle enzymes have been targeted. Analysis of antisense Rubisco, aldolase and transketolase tobacco plants grown in a range of environmental conditions has shown that a reduction of more than 40% in the activities of these enzymes was needed to bring about a change in biomass similar to that observed in the SBPase antisense plants (Quick et al. 1991; Fichtner et al. 1993; Masle et al. 1993; Haake et al. 1998; Henkes et al. 2001). In antisense plants with reduced levels of PRKase growth under a range of environmental conditions was essentially unaffected until enzyme levels were less than 15% wild type (Banks et al. 1999).

Reductions in SBPase activity were also found to have an impact on floral biomass. In addition, a shift in the timing of reproductive development was evident, with flowering occurring 5 to 10 d later than in wild-type plants, due to the delay in the early phase of growth (Fig. 9). Reductions in SBPase activity perturbed the transition to flowering and, in contrast to wild-type tobacco, which stopped producing new leaves after floral determination, all of the antisense SBPase plants in this study continued to produce additional leaves. This resulted in an increase in the number of nodes produced by the SBPase antisense plants by anthesis. The possibility that the reduction in carbohydrate supply to the shoot apex was disrupting the normal switch from vegetative to reproductive phase in these plants is supported by work which has shown that sucrose availability at the shoot apex influences the onset of flowering (Bernier et al. 1993; Corbesier et al. 1998). This transgenic analysis has shown a clear relationship between carbon flux through the Calvin cycle and the transition to flowering and reproductive capacity.

The rates of growth of all the SBPase antisense plants were slower when compared with wild-type plants and this appeared to be largely due to a 7 to 10 d delay in the transition from the juvenile to adult stages of vegetative growth. A delay in the timing of the transition from the early to late phase of vegetative growth was also observed in Rubisco antisense plants. However, in contrast to the SBPase antisense plants, the leaves produced early in development on the Rubisco antisense plants displayed a different morphology from equivalent leaves at the same node on wild-type plants (Tsai et al. 1997). The Rubisco antisense plants displayed a delay in senescence, attaining maximum photosynthetic rates later in maturity which was then sustained for longer (Jiang & Rodermel 1995; Miller et al. 2000). In contrast, the SBPase antisense plants, reached maximum photosynthetic rates more rapidly than wild-type plants but this was not sustained and the decline of photosynthesis in mature leaves was accelerated, suggesting that no delay in senescence occurred in the SBPase antisense plants (Olcer et al. 2001). If source strength was the only factor involved in determining the developmental changes observed in the Rubisco antisense plants, then it would be expected that the SBPase antisense plants would display a similar phenotypic response. It has been shown that reductions in Rubisco result in changes in the N status of the leaves therefore it is possible that the changes in leaf development, observed in the Rubisco antisense plants, were due to a change in the C/N balance (Fichtener et al. 1993; Masle et al. 1993; Matt et al. 2002). Further support for this suggestion comes from the finding that nitrogen re-allocation occurs in the Rubisco antisense plants grown under low light conditions (Lauerer et al. 1993).

One interesting observation from this growth study was the bimodal response of leaf and stem morphology to reductions in SBPase activity. Plants with large reductions in SBPase activity had an increased specific leaf area and were the same height, or taller, and had thinner stems when compared with wild-type plants. The reduction in stem diameter was due to a decrease in the amount of xylem tissue produced, indicating a change in allocation and partitioning of carbon. In antisense plants with small reductions in SBPase activity the plants were shorter and had reduced specific leaf area when compared with wild-type plants. Although a bimodal response has not been reported for other Calvin cycle antisense plants, large reductions in Rubisco activity led to increased specific leaf area (Stitt & Schulze 1994; Raines 2003). In addition antisense Rubisco plants with only 20% wild-type activity were the same height at flowering as wild-type plants (Tsai et al. 1997). These data suggest that reductions Calvin cycle activity that result in a limitation of source capacity can impact on plant development. Similar changes in leaf and stem morphology occur in some species in response to light levels in the growth environment (Björkman 1981; Evans 1996). For example, in growth conditions in which light limits photosynthesis plants optimize carbon utilization by producing thinner, larger leaves and longer, thinner stems, to maximize light capture. It is possible that the reductions in source capacity in the SBPase antisense plants alter the metabolic signals that mimic the acclimation responses to different light environments.

In conclusion this paper shows for the first time that plant growth and development are sensitive to reductions in SBPase activity. This work has also shown that in tobacco there is a clear link between photosynthetic capacity, source strength and plant yield.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

This work was supported by funding from the Biotechnology and Biological Sciences Research Council, United Kingdom (grant no. P01723 to C.A.R and J.C.L and studentship P1244RS to B.B). We would like to thank James Morison for critical reading of the manuscript.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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