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Keywords:

  • Arabidopsis;
  • invertase;
  • flowering;
  • seed production;
  • branching;
  • apical meristem

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Resource allocation is a major determinant of plant fitness and is influenced by external as well as internal stimuli. We have investigated the effect of cell wall invertase activity on the transition from vegetative to reproductive growth, inflorescence architecture, and reproductive output, i.e. seed production, in the model plant Arabidopsis thaliana by expressing a cell wall invertase under a meristem-specific promoter. Increased cell wall invertase activity causes accelerated flowering and an increase in seed yield by nearly 30%. This increase is caused by an elevation of the number of siliques, which results from enhanced branching of the inflorescence. On the contrary, as cytosolic enzyme, the invertase causes delayed flowering, reduced seed yield, and branching. This demonstrates that invertases not only are important in determining sink strength of storage organs but also play a role in regulating developmental processes.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Cell wall invertases are hydrolytic enzymes that cleave sucrose into the monosaccharides glucose and fructose. Their role in re-directing photoassimilates to storage organs of plants has been demonstrated in various species (Weschke et al., 2003). As sugars not only serve as source of carbon and energy but also have important signaling function in plants, it is expected that, in concert with monosaccharide transporters, cell wall invertases play a role in regulating growth and development of plants (Sherson et al., 2003). In the model plant Arabidopsis thaliana, eight invertase genes have been identified, six of which encode cell wall invertases while the other two encode soluble vacuolar enzymes (Haouazine-Takvorian et al., 1997; Sherson et al., 2003). Five of the cell wall invertase genes are expressed in an organ-specific and developmentally regulated manner, with cell wall invertase gene 1 (AtcwINV1) and AtcwINV6 being preferentially expressed in cotyledons and mature leaves, while AtcwINV2, AtcwINV5, and to some extent AtcwINV4 are specific for flowers and seeds (Sherson et al., 2003; Tymowska-Lalanne and Kreis, 1998). Although four of the cell wall invertases show expression in seedlings, it is so far unclear if one of these invertases is involved in assimilate unloading at the apical meristem and would thus contribute to the regulation of assimilate partitioning within the plant.

One of the most important developmental switches in a plant's life cycle is the transition from vegetative to reproductive growth, i.e. the initiation of flowering, which has a major impact on reproductive output in many crop species (Bernier et al., 1993; Yin et al., 1997). This transition is controlled by not only endogenous programs but also a variety of environmental signals, e.g. not only by photoperiod and temperature but also by nutrient availability (reviewed by Koornneef et al., 1998; Reeves and Coupland, 2000; Sheldon et al., 2000; Simpson et al., 1999). Based on grafting experiments in many species of various families (reviewed by Zeevaart, 1976), it has been worked out that, upon flowering induction by photoperiod, a transmissible signal is produced in the leaves and transported to the apical meristem probably in the phloem together with the assimilates, which then in turn induces the transition from vegetative to generative growth (Bernier, 1988). The nature of this signal is not understood, but because it is transported in the phloem, streaming of assimilates has a strong impact on transmitting the signal.

Three mutually not exclusive models have been put forward in the last decades to explain the chemical control of flowering: (i) the florigen/antiflorigen concept assuming simple and specific hormone-like substances to promote or inhibit flowering (Colasanti and Sundaresan, 2000); (ii) the multifactorial control hypothesis assuming a complex mixture of several compounds, e.g. assimilates and known phytohormones, to induce flowering (Bernier, 1988); and (iii) the nutrient diversion hypothesis (Sachs and Hackett, 1983). In the latter hypothesis, no assumption is made regarding the nature of the chemical signals. Rather a change in the sink–source interactions is postulated that results in the shoot apex to become a stronger sink under flowering-inducing conditions, thus receiving a better supply of assimilates.

Molecular parameters governing sink strength are still poorly understood. Efficiency of unloading of photoassimilates from long-distance transport systems such as the phloem, however, is seen as a major parameter. Several hypotheses suggest that unloading is driven by a chemical and/or osmotic gradient created at the unloading site with apoplastic invertase (AI) being identified by several authors as the prime candidate to govern this gradient (Sonnewald et al., 1995; Weber et al., 1998).

We have investigated whether cell wall invertases could be involved in modification of sink strength at the apical meristem by altering invertase activity at the apex. We used the meristem-specific promoter of the KNOTTED-1 gene homolog of A. thaliana (Lincoln et al., 1994) to drive expression of a yeast invertase that was targeted to either the apoplastic space using the potato proteinase inhibitor II (PIN II) signal sequence (Sonnewald et al., 1991) or the cytosol. The results demonstrate that cell wall invertases have the potential to modify sink strength in vegetative tissues, thereby influencing resource allocation and developmental decisions in the plant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Meristem-specific expression of invertase

The KNOTTED-1 gene has been reported to confer meristem-specific expression in case of corn (Smith et al., 1995), and the transcript of the A. thaliana homolog (KnAT1) could be detected in meristematic tissues (Granger et al., 1996). Therefore, the KnAT1 gene promoter seemed a suitable candidate for meristem-specific gene expression. We used a 1.5-kbp fragment of the promoter to drive the uidA gene of Escherichia coli as a reporter. In 17 independent transformants, β-glucuronidase (GUS) activity could be found. Only one line showed GUS activity in the hypocotyl and vasculature. In the other 16 lines, expression of the GUS gene was highly meristem specific with no expression in any other parts of the plant except adventitious root initials (Figure 1a–c). Only after induction of flowering, the KnAT1 promoter shows additional activity in the inflorescence (Figure 1f).

image

Figure 1. Histochemical analysis of transgenic A. thaliana plants expressing the GUS (uidA) gene under the control of the KnAT1 promoter.

(a) Eight-day-old seedling.

(b) Four-leaf stage.

(c) Six-leaf stage.

(d) Section of the apical meristem.

(e) Close-up of GUS activity in lateral root buds.

(f) Ten days after flower induction.

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We next fused a chimeric gene encoding the invertase from yeast with a signal sequence for apoplastic targeting from the potato PIN II to this promoter (AI construct). In order to test whether or not the apoplastic localization of the invertase is of importance, we targeted the same invertase also to the cytosol of meristematic cells. To this end, the same construct was transferred into A. thaliana plants except the fact that the signal peptide leading to the apoplastic localization was deleted (cytosolic invertase (CI) construct).

Transgenic plants were screened for expression of the invertase gene on RNA level (data not shown), and activity of the ectopic invertase in the meristematic region was monitored by in situ localization of glucose release from sucrose by invertase in lines selected for further experiments (see below). A representative example of elevated AI activity in the meristem zone is shown in Figure 2 for line KnAT-AI2. Invertase activity in CI lines was considerably lower. This may be related to detrimental effects of CI as demonstrated by others (Sonnewald et al., 1991), but can also be an artifact of the staining method.

image

Figure 2. In situ localization of sucrolytic activity in the meristematic region of A. thaliana wild-type (a) and KnAT-AI line 2 (b).

Soil-grown plants were defoliated and immersed in a sucrose solution. The glucose released from sucrose by the activity of AI was visualized by subsequent incubation with glucose oxidase and phenazine methosulfate.

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Early flowering and increased seed yield upon AI expression

Six independent transformants of the AI construct and five of the CI construct were chosen for analysis of their flowering behavior under both photoperiodically inductive (i.e. long day) and non-inductive (i.e. short day) conditions (Figure 3). While there was no significant difference in time to flowering under short-day conditions, under long-day treatment, the AI transformants flowered on average 3–4 days earlier than the wild type. In contrast, flowering was delayed by 5 and 10 days in CI transformants under long days and short days, respectively.

image

Figure 3. Flowering phenotype of wild type (WT) and transgenic plants grown under inductive conditions.

Upper panel: flowering of A. thaliana lines expressing AI (lines no. 2, 9, 14, 20, 53, and 54 from left to right) at day 20 after transfer to a long-day growth cabinet in comparison with a representative WT plant grown under identical conditions. Lower panel: transgenic A. thaliana lines expressing CI (plant no. 4, 10, 52, 56, and 67 from left to right) at day 28 after transfer to the long-day growth cabinet in comparison with a representative WT plant grown under identical conditions. Because of the earlier flowering of the AI transgenics, the photos of the upper and the lower panels were taken at different time points.

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The general phenotypic appearance of the transgenic AI plants did not differ from the wild types until start of the flowering phase, while the CI caused moderate growth retardation in CI transformants.

For each construct, three transgenic lines carrying a single copy of the transgene were selected for a detailed statistical analysis of the time to flower and reproductive output. As a measure of time to flowering, we counted the number of rosette leaves, which is highly correlated to the duration of the vegetative phase but can be determined more accurately (Koornneef et al., 1998). Only true rosette but not cauline and axillary leaves were counted in flowering experiments. A strong and highly significant difference between both sets of plants and wild-type control was observed under inductive conditions. The AI plants displayed a much shorter vegetative phase, forming about four leaves less than the wild type, while the CI transformants produced about five leaves more (Figure 4). Given the differences in the time to flower, this means that the rate of leaf production was not altered in the transgenic lines and that one rosette leaf was initiated per day at the end of the vegetative phase. Similar leaf initiation rates were observed by others (Telfer and Poethig, 1998).

image

Figure 4. Number of rosette leaves at flower induction (visibility of inflorescence) for transgenic and wild-type (WT) lines grown for 14 days under short-day conditions and then for additional 86 days under long-day conditions before harvest (see Experimental procedures).

The effect of line number on the number of rosette leaves was highly significant (F6,256 = 86.95; P ≤ 0.0001). Lines were grouped into significance groups according to Duncan's Multiple Range Test. Lines with the same letter are not significantly different.

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To determine the effect of flowering time on reproductive output, seeds of five plants of each line were harvested after a growth period of 100 days (14 days short-day treatment followed by long-day conditions, see Experimental procedures). By that time, plants were completely senescent, and siliques on the main inflorescence started to open. Total seed yield per plant is given in Figure 5. The transgenic plants displayed a striking difference in seed yield. Under the conditions applied, the control plants yielded on average 373 mg seed per plant, while the AI lines yielded between 518 and 533 mg seed per plant, which is an increase by 28%. On the contrary, two of the three CI transformants were reduced in seed yield, producing 120 and 211 mg seed per plant, respectively. While all AI lines had a significantly higher seed yield than the wild type, the yield reduction in CI lines was less consistent. These data convincingly demonstrate that the apoplastic localization of the invertase is of crucial importance for the acceleration of flowering and the increase of seed yield.

image

Figure 5. Seed yield of transgenic and wild-type (WT) lines in gram per plant.

The effect of line number was highly significant (F6,24 = 12.76; P < 0.0001).

According to Duncan's Multiple Range Test, lines were grouped into significance groups. Lines with the same letter are not significantly different. Growth conditions were as described in Figure 4.

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Increased seed yield results from enhanced axillary inflorescence formation

In order to explain changes in seed yield, we determined the 100-seed-weight of the transgenic AI and CI lines and found no change for AI plants as compared to the wild type (1.7 mg versus 1.63 mg per 100 seeds), while the 100-seed-weight was even increased in CI transformants (2.4 mg per 100 seeds), indicating a reduction in seed number of CI plants and an increase in AI transgenics. To answer the question whether changes in seed number resulted from changes in the number of seeds per silique or the number of siliques per plant, we conducted an experiment in which we counted siliques on the main inflorescence as well as on secondary flowering shoots (axillary inflorescences) and their branches (Figure 6). The number of siliques per flowering shoot showed only little variation between the lines. Interestingly, the maximal number of axillary inflorescences was the same for all three genotypes, and the maximum number of branches on the main inflorescence was even the highest in CI plants. However, the AI plants produced on average more axillary inflorescences, while the CI plants produced less, and the AI plants produced on average more branches per axillary inflorescences than did the wild type and the CI transformants (Table 1). This is reflected in the higher ‘weighted mean’ number of siliques per inflorescence, which is the average number of siliques per inflorescence multiplied by the relative abundance of the respective branch (Figure 6, numbers in parentheses). It should be noted that the maximal number of siliques per stem rarely exceeded 35, which means that inflorescences having higher numbers of siliques showed higher order branching. The average number of branches of the main as well as of the axillary inflorescences is given in Table 1 and shows significant differences between the genotypes. The average number of seeds per silique, calculated from 100-seed-weight, the total seed yield, and the number of siliques were nearly identical in AI plants and the wild type, and were reduced in CI plants (Table 1).

image

Figure 6. Average number of siliques on the main inflorescence, its branches, and secondary inflorescences.

For clarity, only the sum of siliques per secondary inflorescence is shown. The number of siliques multiplied by the relative abundance of the respective branch is given in parentheses. Where these numbers are identical, the branch was present in all the plants analyzed. If a branch did not occur on any of the plants, it is shown as a dotted line. Growth conditions were as described in Figure 4.

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Table 1. ANOVA for seed yield, number of siliques, and branching of transgenic versus wild-type plants grown for 14 days under short-day conditions and then for additional 86 days under long-day conditions before harvest (see Experimental procedures)
 AIWild typeCI
  1. The three transgenic lines of each construct (AI, apoplastic invertase; and CI, cytosolic invertase) were treated as one genotype. Of each of the three transgenic lines under investigation, eight plants were analyzed, i.e. branches and axillary inflorescences were counted on 24 plants per construct and means are given in the table. For a more detailed presentation of the branching pattern, see Figure 6. According to Duncan's Multiple Range Test, lines were grouped into significance groups given in parentheses. Lines with the same letter are not significantly different. For the number of seeds per silique, no statistical calculations were made, as 100-seed-weight was determined for only 3 out of 24 individual plants.

Siliques1234.4 (A)1009.0 (B)819.3 (B)
Branches on main inflorescence7.1 (B)6.8 (B)8.7 (A)
Axillary inflorescences14.8 (A)13.0 (A)10.0 (B)
Branches on axillary inflorescences40.5 (A)31.1 (B)23.9 (C)
Total number of branches63.4 (A)52.0 (B)43.8 (B)
Siliques/branch19.5 (A)19.4 (A)20.0 (A)
Seeds/silique40.242.033.3

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Using ectopic invertase expression as a tool to study resource allocation, we found that cell wall invertases, which are known to be fundamental in establishing storage sinks like seeds (Weber et al., 1998) or tubers (Sonnewald et al., 1995), can also exert strong effects on consuming sinks like the vegetative meristem. While increased cell wall invertase activity caused accelerated floral transition, an artificial CI led to the opposite effect. Considering the stimulatory role of cell wall invertases on assimilate unloading from the phloem, the results obtained are in support of the ‘Nutrient Diversion’ hypothesis of floral induction, which assumes that changes of relative sink strength favoring assimilate supply cause the shoot apical meristem (SAM) to undergo floral transition (Sachs and Hackett, 1983). The complementary effect of reduced assimilate supply and delayed flowering is achieved through CI expression, which has been demonstrated to severely reduce sink strength (Sonnewald et al., 1995). As the KnAT1 promoter showed some activity also in lateral root initials, we cannot exclude that ectopic invertase expression in the roots contributed to the observed phenotypes. However, promoter activity in this tissue was much lower than at the SAM (see Figure 1a–c).

A flower-promoting effect of sucrose at the SAM has been reported for A. thaliana and other species (Bernier et al., 1993 for review), and it was demonstrated for various ecotypes of A. thaliana as well as mutants affected in floral regulation that sucrose applied at the aerial parts of the plant together with daylength is involved in floral induction (Roldan et al., 1999). While we found flowering to be delayed in CI lines under both long- and short-day conditions, the early flowering of AI lines was observed only under flower-promoting, i.e. long-day conditions. From the work of Roldan et al. (1999), it appeared that sucrose is especially effective in promoting flowering via the so-called autonomous promotion pathway, while other regulators superimpose its effect during photoperiod-induced flowering, involving the FT and FWA gene function. The absence of an effect of AI under non-inductive photoperiodic conditions is in agreement with this observation: floral repression involving the PHYB gene would suppress the stimulating effect of increased assimilate supply under short day. Alternatively, the invertase effect could be related to the photosynthetic activity of the plants. As invertase is a sucrose-cleaving enzyme, its substrate supply is directly influenced by daylength. To discriminate between these possibilities, dark-grown plants with exogenously supplied sucrose, as described by Roldan et al., could be investigated. However, exogenous sucrose applied to aerial plant parts would interfere with the proposed assimilate-unloading effect of AI.

High exogenous sugar concentrations of synthetic media have also been reported to suppress floral induction by extending the late vegetative growth phase (Ohto et al., 2001). Additionally, in plants grown on solidified synthetic media containing 1% sucrose, repression of genes FT and AGL20 involved in the photoperiod promotion pathway, and the integration of the other pathways of floral induction is demonstrated by Ohto et al. (2001). As the sugar is applied to the roots of the plants, it could stimulate cell divisions in this competing sink tissue, thus lowering assimilate supply to the apical meristem, while the AI at the SAM would have the opposite effect. However, it is difficult to compare exogenous sucrose application experiments with the invertase effects described here because of their conflicting effects in different ecotypes, the dependence of the time and duration of application, and the sugar concentrations (Ohto et al., 2001; Roldan et al., 1999).

As the transition to flowering is an important life history decision with a strong impact on reproductive success, flower initiation is under tight genetic control in A. thaliana (cf., e.g., Koornneef et al., 1998; Pineiro and Coupland, 1998; for reviews; Reeves and Coupland, 2000; Simpson et al., 1999), and a number of genes have been identified, the products of which participate in induction or repression of flowering directly at the apical meristem. Mutation of these genes interferes with floral initiation and development, as in the case of CONSTANS (Putterill et al., 1995) or LEAFY (Weigel et al., 1992). Transgenic plants overexpressing either CONSTANS or LEAFY display strongly accelerated flowering and a rapid completion of their life cycle (Simon et al., 1996; Weigel and Nilsson, 1995), indicating that these regulators can override the default developmental program of A. thaliana causing early senescence. This contraction of the life cycle is associated with reduced reproductive output, thus imposing a clear selective disadvantage (He et al., 2000).

Under natural conditions, the life cycle of A. thaliana is largely constant and limited by exogenous factors, e.g. climate or competition, resulting in a trade-off between vegetative and reproductive allocation (Pigliucci and Schlichting, 1995). As a consequence, a negative correlation exists for vegetative traits (i.e. leaf initiation) and reproductive success in natural populations of A. thaliana, i.e. early flowering accessions produce more seeds and less leaf biomass than late flowering ones (Pollard et al., 2001). Using our transgenic plants, we can directly affirm this interaction in genetically otherwise identical plants. This behavior of A. thaliana seems to be a consequence of a long-term selection in this species for a roughly constant life cycle, which is itself caused by the poor competitiveness of this small weed (Pigliucci and Hayden, 2001). In the invertase-expressing lines, the duration of the life cycle, i.e. the time from sowing until senescence, was not different from that of the wild type, and thus variation in reproductive output was not caused by differences in the developmental status of the plants. The higher yield of AI lines was not caused by a competitive advantage, as lines were not mixed but grown individually in separate pots. Neither was nutrient depletion of the soil during the reproductive growth phase responsible for the difference in productivity, because plants were fertilized at the eight-leaf stage and immediately after bolting (see Experimental procedures). In the case of the CI lines, it cannot be excluded that the reduction in seed yield was at least partly caused by detrimental effects of the CI. For tobacco, it has been reported that CI causes stunted growth and reduced photosynthesis when expressed in leaves (Sonnewald et al., 1991). Although we never observed KnAT promoter activity in leaves and could not find increased invertase activity in leaves of CI plants (data not shown), we noticed a slight growth reduction in CI lines. However, the modification of plant architecture in AI and CI lines (see below) indicates a more complex effect of the meristem-specific CI.

A characteristic feature of A. thaliana is the strong correlation of seed yield with the number of secondary flowering shoots (paraclades) either initiated at the main inflorescence or arising from axillary buds of the rosette (Pigliucci and Marlow, 2001). Investigations in a large set of ecotypes of the average number of seeds per silique (Mauricio and Rausher, 1997; Westermann and Lawrence, 1970) as well as siliques per inflorescence (Pigliucci and Marlow, 2001) showed that there is only minor plasticity for these traits. Hence, the only way of increasing seed production under conditions of a prolonged reproductive phase resulting from early flowering is the initiation of additional inflorescences or a higher branching of the inflorescences. Both phenomena are observed in the AI lines. They produced some more axillary inflorescences than the wild type, and these inflorescences had a significantly higher number of branches, resulting in a significantly higher number of total branches as compared to the wild type.

The increased cell wall invertase activity in meristematic tissues of the AI plants could also have directly stimulated lateral inflorescence formation. The fact that the invertase expression exerted its effect on branching only under conditions, when it also caused expansion of the floral phase, i.e. in the long day, may argue against such a stimulatory effect. However, an interaction of the invertase effect with the duration of the light phase that also influences the total photosynthetic sucrose production cannot be excluded.

In contrast to the AI lines, CI lines had on average a lower number of axillary inflorescences, which also showed reduced branching. In contrast, branching of the main inflorescence was increased. Such a complex change of plant architecture argues against a basic reduction of vigor of the CI plants. Instead, it suggests an effect of altered invertase activity on pattern formation at the apical or axillary meristems of the inflorescence, or on sink activity of the inflorescence. For potato, it was shown that tuber-specific expression of AI and CI had contrasting effects on tuber size concomitant with increased and reduced tuber yield, respectively (Sonnewald et al., 1995). It was hypothesized that extracellular glucose could stimulate cell divisions in the AI plants, while increased cell turgor in CI plants would reduce assimilate supply. As similar effects are observed in the AI and CI lines, such mechanisms may also apply to the inflorescence.

As was demonstrated for potato, the data clearly demonstrate that turnover of the primary metabolite sucrose at the apical meristem influences morphological traits, probably by modulating sink/source interactions. This finding is of prime importance to plant breeding, where most significant increases in crop yield have been obtained through alterations of the harvest index, i.e. the proportion of resources delivered to harvestable organs.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant transformation and growth

A 1456-bp fragment of the KnAT promoter (EMBL Accession AJ131822, nucleotides (nt) 3–1459) carrying a mutation at nt 1456–1459 to remove the ATG start codon was used to drive expression of either the uidA (GUS) gene or the yeast invertase gene (suc2; EMBL Accession M13627, nt 785–2383), fused to the Solanum tuberosum PIN II signal sequence in the case of the AI construct (von Schaewen et al., 1990). The constructs are analogous to the invertase expression constructs described by Sonnewald et al. (1991), except that the KnAT promoter was used instead of the constitutive CaMV 35S promoter. A. thaliana plants (cv. C24) were transformed using the binary vector pGPTV-HPT (Becker et al., 1992) for Agrobacterium tumefaciens-mediated gene transfer.

Seeds were sown on soil/vermiculite (1 : 1) and kept under short-day conditions (8 h light) for 14 days until five seedlings of each line were transferred to new pots. Afterwards, plants were grown in a fully climate controlled greenhouse at a day/night rhythm of 16/8 h for long-day or 8/16 h for short-day treatments. During the day, a light intensity of at least 250 µmol m−2 sec−1 was provided by fluorescent lamps; the temperature was 20°C, and relative humidity was 70%. During the night, the temperature was lowered to 17°C. In flowering experiments, homozygous plants of the F4 generation were grown on soil/vermiculite (1 : 1) with five plants per pot (diameter 10 cm, substrate content 235 g). Pots were fertilized twice, at the eight-leaf stage and immediately after bolting, with 30 mg of fertilizer (16% N2, 8% P2O5, 22% K2O, and 3% MgO) per pot. Experimental design was block-wise with 10 blocks consisting of one pot with five plants per line. Plants were grown for a period of 100 days, after which plants were completely senescent and fruits on the main inflorescence started to open. After the growth period, plants were put in bags, watered for further 14 days, and then kept dry. After additional 14 days, bags were removed from the plants and seeds dried at 12°C per 30% humidity for 2 weeks. Thereafter, total seed yield per pot was measured and the average seed weight per plant was calculated by dividing total yield by the number of plants per pot.

Statistical evaluation

Complete analysis of variance (anova) with F-test for the effect of line and block number on the number of rosette leaves and on seed yield was performed using the sas software package. Duncan's Multiple Range Test was performed for lines at the α = 0.05 level.

Staining procedures

UidA activity was monitored by incubating seedlings and plants of different age in 1 mg ml−1 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-Gluc) in 50 mm sodium phosphate buffer, pH 7.2, containing 10 mm EDTA, 0.33 mg ml−1 potassium hexacyanoferrate (III), 0.1% Triton X-100, and 0.1% Silwet L77 for 12 h at 37°C. Afterwards, chlorophyll was extracted in 80% ethanol. For sections, seedlings at the two-leaf stage were fixed in 0.2% glutaraldehyde, 4% formaldehyde, 0.5% Triton X-100, and 100 mm sodium phosphate (pH 7.0), dehydrated with increasing concentrations of ethanol, and embedded in Technovit 7100. Invertase activity was visualized following a modified method provided by Dahlquist and Brun (1961). Briefly, plants grown on soil for 3 weeks under short-day conditions were washed in 50 mm sodium phosphate (pH 7.2), and glucose released from sucrose was stained in a solution containing 0.1 mg ml−1 glucose oxidase, 0.15 mg ml−1 phenazine methosulfate, and 0.1% sucrose in 50 mm sodium phosphate (pH 6.0). The reaction was stopped with 5% formaldehyde at the time when background staining became visible in the wild type.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We would like to thank Dr Karin Köhl for help with statistical analysis and valuable discussions of the manuscript. We gratefully acknowledge the assistance of Michaela Hundertmark.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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