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First published online September 7, 2007; 10.1104/pp.107.102806 Plant Physiology 145:905-918 (2007) © 2007 American Society of Plant Biologists
The Silver Lining of a Viral Agent: Increasing Seed Yield and Harvest Index in Arabidopsis by Ectopic Expression of the Potato Leaf Roll Virus Movement Protein1Institut für Pflanzengenetik und Kulturpflanzenforschung, D–06466 Gatersleben, Germany (K.K., T.R., M.-R.H.); Friedrich-Alexander-Universität Erlangen-Nürnberg, Lehrstuhl für Biochemie, 91058 Erlangen, Germany (F.V., U.S.); and Department of Molecular Biology, Copenhagen Biocenter, University of Copenhagen, 2200 Copenhagen N, Denmark (D.H.)
Ectopic expression of viral movement proteins (MPs) has previously been shown to alter plasmodesmata (PD) function and carbon partitioning in transgenic plants, giving rise to the view of PD being dynamic and highly regulated structures that allow resource allocation to be adapted to environmental and developmental needs. However, most work has been restricted to solanaceous species and the potential use of MP expression to improve biomass and yield parameters has not been addressed in detail. Here we demonstrate that MP-mediated modification of PD function can substantially alter assimilate allocation, biomass production, and reproductive growth in Arabidopsis (Arabidopsis thaliana). These effects were achieved by constitutive expression of the potato leaf roll virus 17-kD MP (MP17) fused to green fluorescent protein (GFP) in different Arabidopsis ecotypes. The resulting transgenic plants were analyzed for PD localization of the MP17:GFP fusion protein and different lines with low to high expression levels were selected for further analysis. Low-level accumulation of MP17 resulted in enhanced sucrose efflux from source leaves and a considerably increased vegetative biomass production. In contrast, high MP17 levels impaired sucrose export, resulting in source leaf-specific carbohydrate accumulation and a strongly reduced vegetative growth. Surprisingly, later during development the MP17-mediated inhibition of resource allocation was reversed, and final seed yield increased in average up to 30% in different transgenic lines as compared to wild-type plants. This resulted in a strongly improved harvest index. The release of the assimilate export block was paralleled by a reduced PD binding of MP17 in senescing leaves, indicating major structural changes of PD during leaf senescence.
Plant cells are interconnected by unique membrane-lined channels in the cell wall, termed plasmodesmata (PD), that provide cytoplasmic continuity for cell-to-cell transport and communication. PD form the symplastic network for photosynthate supply into the minor vein tissue of mature source leaves, and also contribute substantially to the unloading of assimilates into developing sink organs (Stitt, 1996  ; Imlau et al., 1999; Baluska et al., 2001; Haupt et al., 2001; Viola et al., 2001; Hoth et al., 2005). Although formerly seen as simple pores with a low molecular size exclusion limit (SEL) that allow only the passage of small metabolites, PD have now been shown to be dynamic structures that also facilitate the specific trafficking of large RNA and protein macromolecules involved in local and systemic signaling (Lucas and Lee, 2004; Oparka, 2004; Ruiz-Medrano et al., 2004; Kim and Zambryski, 2005). Beside the observation that apertures of individual PD fluctuate considerably in response to environmental cues, fundamental changes in plasmodesmal architecture and function have been detected during development and in different plant tissues (Crawford and Zambryski, 2001; Roberts and Oparka, 2003; Zambryski, 2004; Liarzi and Epel, 2005). Most remarkably, plasmodesmal permeability is considerably down-regulated during the sink-source transition in leaves, which is accompanied by a progressive conversion from simple to branched forms of PD (Oparka et al., 1999; Roberts et al., 2001). This close correlation between the importing-exporting transition for photoassimilates and changes in plasmodesmal conductivity suggested that PD may act as important control sites for symplastic assimilate transport and resource allocation between source and sink tissues (Lucas and Wolf, 1999; Lucas and Lee, 2004).
Compelling evidence for this hypothesis has been provided by studies on virus-encoded movement proteins (MPs), which are well known to localize to secondary branched PD, to substantially increase the basal plasmodesmal SEL, and to facilitate cell-to-cell transfer of MP-bound viral genomes through PD (Wolf et al., 1989
In addition to the MPs of TMV, CMV, and TSWV, the 17-kD MP (MP17) of the phloem-limited potato leaf roll polerovirus (PLRV) has been used as another model system to study the consequences of altered PD function for symplastic assimilate transport and sink-source relations (Herbers et al., 1997 So far, studies on the mode(s) of action underlying the various MP-induced metabolic and phenotypic responses have mainly concentrated on solanaceous species, and the potential use of MP to manipulate resource allocation for the benefit of leaf biomass and seed production has not been addressed in detail. The aim of this study, therefore, was to establish how constitutive expression of PLRV-MP17 affects PD functioning in terms of carbon partitioning, resource allocation, and productivity in the Arabidopsis (Arabidopsis thaliana) plant model. We demonstrate that GFP-tagged MP17 localized to branched PD in various cell types, and caused alteration of carbohydrate status, biomass accumulation, and seed yield in an ecotype-independent but strict dosage-dependent manner. Low expression of MP17 resulted in increased vegetative biomass production, whereas high protein levels impaired assimilate export as evident by starch accumulation in source leaves and severe growth retardation. At later stages of plant development, however, the inhibition of assimilate export was released, resulting in improved seed yield and a considerably enhanced harvest index probably due to mobilization and allocation of excess carbohydrate pools. Importantly, the reversion of the symplastic transport block was paralleled by a reduced accumulation of MP17 protein at PD, suggesting changes of plasmodesmatal architecture and function during the progression of leaf senescence.
Association of MP17 with PD in Arabidopsis
To generate PLRV-MP17 expressing Arabidopsis plants, a binary construct harboring a MP17:GFP gene fusion under control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter (p35S-1; Hofius et al., 2001
MP17 Expression Alters Carbohydrate Levels, Biomass Accumulation, and Reproductive Growth in Different Arabidopsis Ecotypes
MP17-mediated alterations of carbohydrate status, biomass allocation, and phenotypic appearance were previously shown to be strictly dependent on MP17 protein amounts in transgenic tobacco plants (Hofius et al., 2001
Phenotype and growth analysis of transgenic lines at short-day (SD) conditions (6 weeks) revealed remarkable changes in biomass allocation in response to different MP17:GFP amounts (Fig. 2, A and B). Low-level accumulation of the fusion protein in lines Col-9 and C24-81 resulted in increased aboveground biomass production of 16% and 41% compared to the controls, respectively, but failed to reach statistical significance for C24-81. In contrast, high-level MP17 expression in Col-16 and C24-74 caused considerable growth retardation as evident by reduced biomass accumulation to only 13% (Col-16) and 30% (C24-74) of the respective wild-type plants (Fig. 2, G and H). To further analyze whether the MP17-induced growth penalty was due to impaired assimilate export as previously demonstrated for MP17 expressing tobacco plants (Hofius et al., 2001 ), steady-state levels of carbohydrates were determined in a plant set grown under SD conditions in parallel to the lines used for vegetative biomass analysis. As shown in Table I
, highest MP17 expression and lowest biomass accumulation in line Col-16 was accompanied by strong accumulation of soluble sugars and starch in source leaves, suggesting a MP17-mediated blockage of photosynthate export. Less severe growth inhibition in C24-74 was associated with only a minor increase in overall carbohydrate levels, whereas increased biomass production in low-level expressing lines Col-9 and C24-81 seemed to be paralleled by a slight reduction of both Suc and starch contents. These data suggested a dosage-dependent impact of MP17 expression on assimilate allocation from source leaves, which seem to have either beneficial or detrimental consequences for vegetative biomass accumulation at low and high expression levels, respectively.
To further elucidate whether MP17-induced changes of photosynthate export also influence the transition from vegetative to reproductive growth as well as the reproductive outcome, i.e. seed production, transgenic lines were shifted after 6 weeks from SD to long-day (LD) conditions. Determination of assimilate accumulation in leaves of the various lines grown under these flowering inducing conditions revealed similar results as demonstrated for the SD-grown plants (data not shown). Interestingly, flowering initiation in low and modestly expressing lines C24-81, C24-84, and Col-9 was significantly accelerated by approximately 6, 3, and 1 d compared to wild type, respectively (Fig. 2, J and K). In contrast, high-level accumulation of MP17 in lines Col-16 and C24-74 resulted in delayed flowering by 6 and 7 d relative to the controls, suggesting that the dosage-dependent impact of MP17 on carbohydrate partitioning and vegetative biomass accumulation was correlated with specific alterations of reproductive development. Consequently, seed yield was determined for the various lines after all plants were completely senescent and siliques on the main inflorescence started to open. Surprisingly, highest MP17 expression in the severely growth-retarded line Col-16 caused a consistent increase in seed yield by 12.6%, 37.8%, and 42.8% (in average 29.9%, P < 0.001) in three independent growth sets compared to the wild-type control, respectively, whereas seed production appeared to be largely unaltered in the lower expressing transgenic lines Col-9 and Col-14 (Fig. 2M). Reproductive outcome of C24 lines was either unaffected or even slightly reduced in line C24-81, which showed the highest leaf biomass accumulation under SD conditions (Fig. 2N). These results indicated an upper threshold level for the beneficial impact of transgenic MP17 protein accumulation on seed production and additionally suggested a close interrelationship between MP17-induced carbohydrate accumulation, growth penalty, and improved yield. To investigate whether enhanced seed production in Col-16 was solely the consequence of high MP17 expression level or rather due to ecotype-specific effects, we introduced the 35S-MP17:GFP construct into the Landsberg erecta (Ler) ecotype and screened for transgenic lines that showed a severe growth inhibition and comparably high MP17 amounts as Col-16. Two lines, termed Ler-1 and Ler-12, were identified that exhibited strong MP17:GFP-derived fluorescence at PD (data not shown) and exceeded the transgene protein level in Col-16 by 16% and 12%, respectively (Fig. 2F). Consequently, both lines showed dramatically reduced biomass production that reached only 6% and 8% of the wild-type level after 6 weeks under SD conditions, respectively (Fig. 2I). Consistent with the results in line Col-16, the severe growth phenotype in Ler-1 and Ler-12 was accompanied by assimilate export deficiency as revealed from source leaf-specific accumulation of soluble sugar and starch (Table I). Additionally, Ler-1 and Ler-12 showed a significantly altered transition from vegetative to reproductive growth as flowering initiation was observed approximately 4 and 6 d later than in the wild type under inducing LD conditions, respectively (Fig. 2L). Most remarkably, seed production in both lines was significantly enhanced by 49% (P < 0.05) and 26% (P < 0.05) relative to the wild-type control, respectively (Fig. 2O). These data suggested an ecotype-independent relationship between high protein expression, reduced leaf biomass accumulation, and higher seed production.
To directly link this dosage-dependent and inversely related impact of MP17 on biomass and seed production to changes in assimilate allocation, the capacity of source leaves for Suc export was assessed in the different transgenic lines during vegetative and reproductive growth phases. Consistent with improved vegetative biomass accumulation in low-level expressing lines Col-9 and C24-81, Suc efflux rates from detached petioles were slightly (20%), and in case of C24-81, significantly higher than in wild-type plants grown for 6 weeks under SD conditions (Fig. 3, A and C ). In contrast, growth-retarded lines Ler-1, Ler-12, Col-16, and C24-74 showed considerably reduced Suc concentrations in phloem exudates after the 6-h collection period that reached only 29% to 48% of the respective wild-type controls (Fig. 3, A, C, and E). However, after an additional growth period of 3 weeks under LD conditions, Suc efflux rates in lines Col-16, Ler-1, and Ler-12 tended to exceed the respective wild-type level by 15%, 25%, and 31%, respectively (Fig. 3, B and F), which was statistically significant for Ler-12 (P < 0.05). This strongly suggested the reversion of the assimilate export block during the reproductive growth phase.
Collectively, these results indicate that enhanced biomass as well as seed production in low- and high-level expressing lines is associated with growth stage-specific changes in Suc export capacity.
The astonishing finding of an inverse relationship between MP17-mediated alterations in vegetative biomass and yield production implies the potential of substantial changes in harvest index at high expression level. Harvest index is defined as ratio of seed mass and total plant mass and as such serves as indicator of resource allocation toward harvestable organs and important agricultural parameter of plant productivity. To assess changes in harvest index in high-level expressing lines Col-16, Ler-1, and Ler-12, plants were grown for 6 weeks at SD before they were shifted to LD conditions to induce reproductive development. Total dry matter production was determined after plants were fully senescent, demonstrating that Col-16, Ler-1, and Ler-12 accumulated biomass to approximately 44%, 69%, and 70% of the wild type, respectively (Fig. 4A ). This suggested that the induction of reproductive growth partially compensated the dramatic repression of vegetative biomass accumulation in the transgenics under SD conditions and agrees with the observed shift toward enhanced Suc export capacity under prolonged LD conditions. Consistent with the previous results, improved reproductive growth was also manifested by enhanced seed production in all three lines (Fig. 4A), giving rise to a considerably increased harvest index by 2.8-fold in Col-16 (P < 0.01) and by 1.8-fold in both Ler-1 (P < 0.01) and Ler-12 (P < 0.01) compared to the ecotype-specific control plants (Fig. 4A).
To explain the higher seed yield upon high-level MP17:GFP expression, we determined the 1,000-seed weight of the three plant lines and found no major changes for all lines compared to the wild-type controls (Fig. 4B), indicating an increase in seed number in the transgenics. Counting the number of siliques per plant revealed a consistent decrease in the transgenic plants relative to the wild types (498 in Col-16 versus 516 in Col-wild type; 302 in Ler-1 and 298 in Ler-12 versus 320 in Ler-wild type, respectively). Thus, the average number of seeds per silique, calculated from 1,000-seed weight, the total seed yield, and the number of siliques is considerably enhanced from approximately 12 in Col-wild type to 15 in Col-16, and from 20 in Ler-wild type to 25 in both Ler-1 and Ler-12 (Fig. 4B).
The increased reproductive output in high-level MP17:GFP expressing Col-0 and Ler lines in association with enhanced Suc export capacity of source leaves during the reproductive growth phases suggests an improved availability and supply of assimilates for generative development. To support this hypothesis, we speculated that the release of MP17-induced export deficiency might also occur as a consequence of progressing leaf age. To test this possibility, fully expanded source leaves (Max) and different leaf stages with progressing senescence from SD-grown Col-16 plants (leaves -6 until -1 with the latter being defined as oldest leaf within the rosette [Fig. 5A ]) were assessed for starch accumulation by iodine staining at the end of dark period. As expected, source leaves (Max) of Col-16 exhibited a severe starch excess phenotype compared to the wild-type control. However, starch accumulation seemed to progressively decrease in senescing leaf stages, almost approaching wild-type level in the oldest rosette leaf (-1; Fig. 5B). To further verify these observations, soluble sugars and starch were quantitatively assayed at the indicated leaf stages from 66-d-old Col-16 and wild-type plants at the end of an 8 h light period. A strong decline in starch levels could be confirmed for the different senescing leaf stages compared to maximal amounts reached in the export-deficient source leaf (Max) of Col-16 plants (Fig. 5C). While rosette leaf -6 still exhibited a considerably increased starch level, older leaf stages showed starch accumulation in a comparable range as detected in the presumably export-competent wild-type Max leaf, suggesting a successive reversion of the MP17-mediated export block. Interestingly, in contrast to starch levels, soluble sugars seemed to be increased in older leaf stages (-6 until -4) in relation to Max leaves of line Col-16, and were consistently higher throughout all leaf stages compared to the wild-type level in Max leaves. Importantly, similar changes of starch and soluble sugar levels were also detected upon progressing leaf senescence in Col-16 plants under reproductive, i.e. LD conditions (data not shown). This might indicate an increased turnover of the previously accumulated starch in senescing leaves of Col-16, leading to an enhanced availability of soluble sugars for resource allocation.
Recent evidence suggests that maltose serves as a major metabolic intermediate in conversion of starch to Suc during the night (Chia et al., 2004 ; Niittyla et al., 2004; Lu and Sharkey, 2006). Hence, elevated levels of maltose are regarded as valuable indicators of impaired starch mobilization and disturbed assimilate export capacity in source leaves. Consequently, maltose levels were determined in the different leaf stages of Col-16 and wild type at the end of the 16-h dark period. In agreement with the observed starch excess phenotype and proposed assimilate export block, Max source leaves of Col-16 showed 2.4-fold higher maltose concentrations than the comparable leaf stage of the wild-type control (Fig. 5C). In contrast, rosette leaf stages -6 until -1 showed decreased levels of maltose that were consistently lower than the level in the wild-type Max leaf. This decrease could not be explained by lowered starch levels, since leaf stage -6 accumulated considerably more starch than the wild-type max leaf. Thus, it rather suggests the release of the MP17-induced export block resulting in successive mobilization of starch reserves upon leaf senescence.
The strict dosage-dependent impact of PD-localized MP17 on carbon levels and export capacity in source leaves raises the possibility of reduced MP17 accumulation in senescent leaves. Therefore, MP17:GFP protein levels were determined by means of western-blot analysis in the different leaf stages of Col-16. As demonstrated in Figure 5D, MP17:GFP protein levels continuously decreased upon leaf aging, suggesting either reduced transcriptional activity of the CaMV 35S promoter or proteolytic degradation of the MP17:GFP fusion protein with progressing leaf senescence. Analysis of MP17:GFP expression by northern-blot analysis indicated no major changes in transcript accumulation throughout all leaf stages (Fig. 5E), thereby excluding the possibility of reduced transgene expression. Consequently, we speculated that MP17:GFP showed reduced affinity to PD of older leaf stages and that nontargeted protein might be subjected to proteolytic degradation in these leaves. To test this hypothesis, plasmodesmal localization of MP17:GFP in epidermal cells was monitored throughout the different leaf stages of Col-16 by confocal laser scanner microscopy. As demonstrated in Figure 6 , accumulation of MP17:GFP at PD continuously decreased with leaf age, suggesting the occurrence of significant structural and, potentially, also functional changes of PD in senescing leaves.
To assess alterations of PD function and symplastic transport capacity in different leaf stages upon MP17:GFP expression, we monitored the movement and phloem unloading of the membrane-impermanent fluorescent solute carboxyfluorescein (CF) in sink, source, and senescing leaves of Col-16 plants in comparison to the wild-type control. The tracer was loaded through cut petioles and allowed to distribute throughout the leaves, respectively. As shown in Figure 7, A to C , CF unloading from the phloem was clearly detectable in all three wild-type leaf stages. In contrast, symplastic trafficking out of the minor vein network in source leaves appeared to be clearly restricted in MP17:GFP expressing Col-16 plants, strongly indicating a MP17-mediated inhibition of symplastic continuity between cells of the vascular bundles and the surrounding mesophyll tissue (Fig. 7E). However, most likely due to the absence of MP17 from simple PD (see Fig. 1), massive movement of the fluorescent dye was observed in sink leaves of Col-16 (Fig. 7D). Importantly, a wild-type-like unloading pattern was also detectable in senescent leaves of the transgenic line (Fig. 7F), indicating that the reduced accumulation of MP17 protein at PD is sufficient to release the MP17-induced symplastic transport block detectable in source leaves.
Stable expression of MPs from different viruses in transgenic plants has previously been used to elucidate PD structure and functioning as well as the contribution of the symplastic pathway to assimilate transport and biomass partitioning. From these studies, the overall picture had emerged that association of MPs with branched PD significantly increases plasmodesmal conductivity in source leaves, however, with varying consequences for carbon metabolism, photoassimilate export, and growth performance. MP-induced effects appeared to be largely dependent on the selected MP, plant species, and cell types used for transgene expression and were also shown to be developmentally regulated (Olesinski et al., 1996 ; Almon et al., 1997; Hofius et al., 2001; Shalitin et al., 2002; Mansilla et al., 2006). In this study, we constitutively expressed PLRV-MP17 fused to GFP in different Arabidopsis ecotypes to alter PD-mediated assimilate transport processes and observed significant changes of carbohydrate levels, vegetative biomass accumulation, and reproductive growth in a dosage-dependent manner. Low-level expression of MP17 increased leaf biomass production whereas high MP17 protein accumulation induced a severe growth penalty due to assimilate export deficiency of source leaves. However, reduced leaf biomass accumulation at high MP17 level was accompanied by enhanced seed production, giving rise to a strongly increased harvest index. This surprising effect seemed to be mediated by the release of the symplastic transport block in senescing leaves most likely due to decreasing MP17 accumulation at PD, which allowed the accumulated starch to be metabolized and the excess carbon pools to be allocated for the benefit of seed set and development.
Fluorescence and immunoelectron microscopy of transgenic Arabidopsis lines revealed localization of MP17:GFP to branched PD of various vascular and nonvascular cell types in source leaf and root tissue (Fig. 1, C–H), whereas simple PD of sink leaves were not targeted by MP17:GFP (Fig. 1, A and B). This localization pattern is very similar to the situation observed in MP17:GFP expressing tobacco plants (Hofius et al., 2001
In contrast to the apparent detrimental effect of high MP17 amounts on the Suc efflux pathway in source leaves leading to sugar accumulation and severe growth retardation, the increased leaf biomass production in low-level MP17 expressing Arabidopsis lines (Col-9 and C24-81; Fig. 2, G and H) seemed to be caused by improved capability for photoassimilate export and allocation. This assumption is supported by increased Suc efflux (Fig. 3, A and C) and concomitantly decreased levels of Suc and starch in source leaves (Table I), the latter has previously been observed in low-level expressing MP17N and MP17:GFP transgenic tobacco lines (Hofius et al., 2001
Additional support for the assumption of increased nutrient flow to consuming sinks like the vegetative meristem in low-level MP17 expressing Col-0 and C24 seems to be provided by significantly accelerated floral transition under flower promoting, i.e. LD conditions (Fig. 2, J and K). It is well established that Suc has a stimulatory role on flowering induction at the shoot apical meristem in Arabidopsis and other plant species (Bernier et al., 1993
Although high-level MP17 expressing transgenic lines were able to attenuate their dramatic vegetative growth retardation after floral induction, total biomass accumulation at harvest point reached only 60% to 70% of the respective wild type. Together with the concomitant increase in seed yield, a strongly improved harvest index could be achieved (Fig. 4A), which additionally demonstrated the MP17-mediated success in partitioning photoassimilate resources to the harvestable component. Changes in harvest index of cereal crops were formerly shown to be responsible for the large increases in global wheat (Triticum aestivum) and rice (Oryza sativa) yield during the green revolution (Hay, 1995
The beneficial effect of MP17 on either vegetative or generative productivity at low and high expression levels, respectively, seems to be likewise mediated by alterations of the symplastic transport pathway via interaction with PD. However, the modes of action appear to be rather different. As discussed above, improved assimilate allocation at low-level MP17 amounts are directly caused by increased symplastic permeability and Suc efflux in mesophyll and/or vascular cells of source leaves. In contrast, accumulation of large amounts of MP17 at PD of vascular-associated cells initially interrupted symplastic permeability and the Suc transport pathway into the minor vein phloem, resulting in excess carbohydrate accumulation in source leaves. However, this deleterious impact of MP17 appeared reversible upon progression of plant age as indicated by the switch to increased Suc efflux after a prolonged growth period under LD conditions. Additional support for a senescence-associated alleviation of the MP17-induced disruption of symplastic transport and assimilate export was provided by reduced staining for starch accumulation (Fig. 5B), decreased amounts of PD-targeted MP17:GFP (Fig. 6), and unrestricted diffusion of CF from the minor vein network (Fig. 7) in older leaf stages compared to mature source leaves. Quantification of soluble sugars, starch, and maltose levels in these leaf stages further indicated that the accumulated starch was successively metabolized (Fig. 5C) and thus additional carbon resources made available for translocation into reproductive sinks after floral induction.
The observed decline in plasmodesmal localization and overall protein accumulation of MP17:GFP (Figs. 5D and 6) suggested the absence or reduced affinity of putative PD receptor proteins and subsequent proteolytic degradation of nontargeted MP17 in senescent leaves, strongly resembling the situation in sink leaves (Fig. 1; Vogel et al., 2007
Our work demonstrates the ability of PLRV-MP17 to alter PD functioning in terms of symplastic assimilate transport and resource allocation in Arabidopsis, and thus to improve leaf biomass and seed production in an inversely related and dosage-dependent manner. Furthermore, due to the penetrance of the MP17-mediated impact in different Arabidopsis ecotypes, the transgenic lines provide a novel and powerful tool for forward genetic approaches to identify cellular factors required for plasmodesmal targeting and trafficking as well as regulatory components involved in sink-source relations. Finally, our data indicate structural and potentially functional changes of PD during leaf senescence that might be related to the requirement for efficient and unrestricted relocation of recycled and mobilized resources for reproductive success.
Plant Material and Growth Conditions Arabidopsis (Arabidopsis thaliana) ecotypes Col-0, C24, and Ler were maintained in Klasman substrate number 1 compost (Klasmann-Deilmann GmbH) under controlled SD condition in a growth chamber (8 h day/16 h night regime with 20°C/18°C, 60% humidity, 120 µM m–2 s–1 light). For induction of reproductive growth, plants were transferred to LD conditions in a temperature-controlled greenhouse (20°C day/18°C night) with 16 h supplementary light (100 µM m–2 s–1) and 8 h darkness. For selection of transgenic plants, seeds were sterilized and sown onto Murashige Skoog medium (Sigma) supplemented with Gamborg's vitamin solution (1:1,000) and 50 µg/mL kanamycin. Plants were given cold treatment for 2 d to synchronize germination before incubation under a 16-h-light/8-h-dark photoperiod (150 µM m–2 s–1 light, 21°C) at 50% relative humidity.
Transformation of different Arabidopsis ecotypes with the previously described binary construct p35S-1, harboring an in-frame gene fusion of PLRV-MP17 and GFP under control of the CaMV 35S promoter (Hofius et al., 2001
Samples of Arabidopsis leaves corresponding to 0.81 cm2 were homogenized in 2x SDS sample buffer containing 50 mM Tris-HCl, 5% (v/v)
Extraction of total RNA from leaf material and northern-blot analysis was performed as described by Chen et al. (2003)
Soluble sugars and starch levels were determined in leaf samples extracted with either 80% (v/v) ethanol/20 mM HEPES, pH 7.5 (Table I), or 80% (v/v) ethanol/10 mM EDTA, pH 7.5 (Fig. 4D) as described (Sonnewald, 1992
To assay vegetative and reproductive growth, seeds were sown in soil and kept under controlled SD conditions (see above) for 10 d until seedlings were transferred individually to new pots. Vegetative biomass accumulation corresponding to the fresh weight of the entire leaf rosette without the root system was determined after an additional period of 32 d under SD (see above). To measure reproductive growth, an additional set of plants grown under the same conditions for 6 weeks was transferred to LD conditions (see above) and flowering induction of the transgenic lines was recorded by counting the days from the date of transfer until the appearance of the first open flower. Plant lines were subsequently maintained for a period of 40 to 50 d under temperature-controlled greenhouse conditions (see above) until all plants were completely senescent and fruits on the main inflorescence started to open. After plants were kept dry for additional 2 weeks, total dry weight, number of siliques, total seed weight, and 1,000-seed weight was determined for each individual plant and the harvest index (ratio between seed weight and total biomass) and the average number of seeds per silique was calculated.
Collection of phloem exudates for Suc efflux analysis was performed at two different time points, with plants grown for 6 weeks under SD condition (vegetative stage) and additional 3 weeks under LD condition (reproductive stage). At the latter growth stage, all plants had developed stems and flowers and initial signs of leaf senescence could be detected. Two mature source leaves per plant were detached by cutting petioles close to the stem. Excised petioles were immediately placed in a 1.5 mL test tube containing 200 µL of 5 mM EDTA, and exudates were collected for 6 h under conditions of 150 µM m–2 s–1 supplementary light and water-saturated atmosphere (Corbesier et al., 1998
Detection of MP17:GFP fusion protein-derived fluorescence signals in manual sections of leaf and root tissue from different transgenic lines was performed by confocal laser scanning microscopy according to Hofius et al. (2001)
Fixation, embedding, and immunogold labeling of leaf and root sections with anti-MP17 as primary antibody followed exactly the protocol described in Hofius et al. (2001)
To monitor symplastic unloading of CF from vascular bundles, different leaf stages (sink, source, and senescent leaves) of line Col-16 and wild type were cut and inserted with their petioles in capillaries containing 2 mM carboxyfluoresceindiacetate (prepared by diluting a stock solution of 13 mM in acetone with water/5 mM KOH; Molecular Probes Inc.). CF-specific fluorescence was detected 10 min after loading using a Leica MZ16F stereomacroscope, a Leica DFC480 fluorescence camera, and Leica IM500 Image Manager software.
Differences between transgenic lines and the corresponding wild type were compared using the nonparametric Mann-Whitney U-test, since most data were found to lack the assumption of normal distribution and homogeneity of variance. All statistical analyses were performed with Analyze-it v2.03 beta.
We wish to thank Bernhard Claus (Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben) for skillful help with confocal laser scanning microscopy and Marc Strickert (Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben) for valuable advice on the statistical analysis. We also like to thank Norbert Sauer (Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany) for providing the confocal laser scanning microscope of his department. Received May 25, 2007; accepted September 4, 2007; published September 7, 2007.
1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (grant no. DFG SO 300/9/1).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Uwe Sonnewald (usonne{at}biologie.uni-erlangen.de). www.plantphysiol.org/cgi/doi/10.1104/pp.107.102806 * Corresponding author; e-mail hofius{at}my.molbio.ku.dk.
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-D-glucan deposition (Rinne et al., 2005
