Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response

Abstract

Sucrose (Suc) transporters belong to a large gene family. The physiological role of SUT1 proteins has been intensively investigated in higher plants, whereas that of SUT4 proteins is so far unknown. All three known Suc transporters from potato (Solanum tuberosum), SUT1, SUT2, and SUT4, are colocalized and their RNA levels not only follow a diurnal rhythm, but also oscillate in constant light. Here, we examined the physiological effects of transgenic potato plants on RNA interference (RNAi)-inactivated StSUT4 expression. The phenotype of StSUT4-RNAi plants includes early flowering, higher tuber production, and reduced sensitivity toward light enriched in far-red wavelength (i.e. in canopy shade). Inhibition of StSUT4 led to tuber production of the strict photoperiodic potato subsp. andigena even under noninductive long-day conditions. Accumulation of soluble sugars and Suc efflux from leaves of transgenic plants are modified in StSUT4-RNAi plants, leading to modified Suc levels in sink organs. StSUT4 expression of wild-type plants is induced by gibberellins and ethephon, and external supply of gibberellic acid leads to even more pronounced differences between wild-type and StSUT4-RNAi plants regarding tuber yield and internode elongation, indicating a reciprocal regulation of StSUT4 and gibberellins.

Figure 1.

A, Expression pattern of StSUT4 in sink and source organs as determined by real-time PCR. StSUT4 expression increases during flower development and strongest expression is detected in young developing tubers and mature flowers. B, Western-blot analysis of StSUT4 in leaves of potato. The microsomal fraction (MF) has been loaded in the first two lanes. Plasma membranes (PM) and endosomal membranes (EF) have been separated by two-phase partitioning and loaded on SDS-PAGE. In each lane, 15 μg of membrane proteins are loaded. StSUT4-specific peptide antibodies (Weise et al., 2000) detected the StSUT4 protein in the correct size of 47 kD only in the plasma membrane fraction. C, D, F, and G, Expression of StSUT4-GFP fusion expressed under the cauliflower mosaic virus 35S promoter in a pCF203 derivative in A. tumefaciens-infiltrated tobacco leaves. E, The same StSUT4-GFP construct expressed in infiltrated potato leaves. C, F, and G, Single scans. D and E, Overlay projections of confocal z stacks. GFP is not only detectable at the plasma membrane, but also in a perinuclear ring as shown by propidium iodide staining. F, StSUT4-GFP fluorescence is detectable at the plasma membrane of tobacco cells as well as in perinuclear rings. G, Same cell shown in F with propidium iodide-specific filter settings. H, Yeast cells expressing a LeSUT4-GFP construct under control of the Adh1 promoter in the yeast expression vector 112A1NE. GFP fluorescence is detected at the plasma membrane and in ER stacks surrounding the nucleus. n, Nucleus.

Figure 2.

Quantification of Suc transporter mRNA accumulation by real-time PCR analysis in constant light and StSUT1 transcript quantification in constant darkness. All three known Suc transporters from potato are expressed diurnally with distinct maxima. StSUT2 shows peak levels at the beginning of the light period, whereas StSUT1 and StSUT4 show maximal transcript accumulation at the end of the light period. Oscillation of transcript amounts is continuous even under 72 h of constant light. The amplitude of StSUT1 oscillation strongly decreases in constant darkness. Relative quantification was performed with ubiquitin as internal standard. sd is given.

Figure 3.

Phenotype of StSUT4-RNAi plants. A, Plants with reduced StSUT4 expression show early flowering under LD conditions. ‘Désirée’ wild-type and StSUT4-RNAi₁₀ plants after 5 weeks in the greenhouse. B, Internode elongation of potato ‘Désirée’ wild-type and StSUT4-RNAi plants. Quantification of internode elongation is shown in Figure 9C. C, Internodes of transformed potato andigena plants are reduced in length in comparison with andigena wild-type plants. andigena plants transformed with a StSUT4-RNAi construct showing reduced StSUT4 transcript levels are able to produce tubers even under LD conditions (D), whereas andigena wild type does not (E). StSUT4-RNAi_2/5 shows early flowering compared to potato andigena wild-type plants grown under LD conditions (F).

Figure 4.

A, StSUT4-RNAi ‘Désirée’ plants flower on average 6 d earlier than wild-type plants when grown under LD conditions. Flowering was observed with StSUT4-RNAi lines 10 (n = 8), 81 (n = 9), and 38 (n = 9) and potato subsp. tuberosum plants (n = 23) grown in the greenhouse under LD conditions. B, StSUT4-RNAi ‘Désirée’ plants have significantly fewer leaves at flowering if grown under LD conditions in the greenhouse (n = 6 for each plant line). C, The tuber yield of StSUT4-RNAi potato plants is significantly increased under noninductive LD. All experiments are reproduced at least three times. One representative example is given (n = 5–6 for each line and each time point). sd is given.

Figure 5.

A, Flowering behavior of grafted potato wild-type plants under LD conditions when grafted with StSUT4-RNAi plants. Early flowering is also observed in wild-type plants if grafted with StSUT4-RNAi plants, depending on the presence of source leaves at the rootstock. B, Schematic representation of graft experiments. Plants were regenerated from tubers and grafted after development of the first six leaves. Graft experiments were repeated twice with potato ‘Désirée’ with six reciprocal grafts from each transgenic line per experiment. C, Tuber yield of grafted StSUT4-RNAi potato plants (subsp. tuberosum) grafted on potato wild-type plants (subsp. andigena), which do not tuberize under LD conditions. Plants were grafted when they have four to five leaves and were kept under LD conditions. As a control, transgenic plants were grafted on their own rootstock (right, StSUT4-RNAi₈₁ grafted on StSUT4-RNAi₈₁ and StSUT4-RNAi₁₀ grafted on StSUT4-RNAi₁₀) and potato subsp. tuberosum wild type was grafted on potato subsp. andigena, which did not lead to tuber production (left, wild-type tuberosum grafted on wild-type andigena). sd is given. andig, andigena; tuberos, tuberosum.

Figure 6.

A to C, Content of soluble sugars in source leaves of StSUT4-RNAi plants compared to potato wild-type plants determined enzymatically. At the end of the light period, transgenic plants show significantly increased Glc (A), Fru (B), and Suc (C) content per gram fresh weight. D, Efflux of Suc from leaves of wild-type and StSUT4-RNAi plants was determined by exudation in the presence of EDTA. Suc exudation was determined enzymatically in intervals of 3 h during the light period from plants kept under LD conditions in the greenhouse. Suc efflux from wild-type leaves shows maxima at the end of the light period, whereas in StSUT4-RNAi plants Suc efflux remains high even in darkness. E, Suc and starch content of in vitro-grown microtubers (n = 4 for each plant line). Tubers were harvested 20 d after tuber induction in darkness. sd is given. F, Suc content in the shoot apical meristem of potato wild-type and StSUT4-RNAi plants. Samples were taken at the end of the light period (9 pm). Fresh weight of samples was between 20 and 60 mg. Error bars indicate sd. Experiments were performed under LD conditions. Note that floral buds of StSUT4-RNAi plants were first detected when plants had five to six mature leaves, whereas wild-type potato plants started transition from the vegetative to the generative phase when they had >10 leaves.

Figure 7.

A, Transcript levels of StSUT4, the GA biosynthetic enzyme GA20ox1, and the ethylene biosynthetic enzyme StACO3 in StSUT4-RNAi plants as determined by quantitative real-time PCR. A, StSUT4 expression in potato leaves treated with phytohormones and phytohormone inhibitors paclobutrazol (inhibitor of GA biosynthesis) and silver nitrate (inhibitor of the ethylene receptor). StSUT4 expression is inducible by GA₃ at the end of the light period and by ethephon treatment over the whole light period. Potato wild-type plants were treated with 20 μm GA₃, 350 μm paclobutrazol, 350 μm ethephon, or 1 mm silver nitrate, and StSUT4 mRNA was determined by real-time PCR analysis relative to the level of ubiquitin transcripts. B, Quantification of transcripts of the GA biosynthetic enzyme GA20ox1 in wild-type and StSUT4-RNAi plants showing reduced levels of GA20ox1 in source leaves of StSUT4-RNAi plants at the end of the light period if compared to wild-type levels. C, Quantification of the transcripts of the ethylene biosynthetic enzyme StACO3 in wild-type and StSUT4-RNAi plants showing significantly reduced levels of StACO3 mRNA levels in StSUT4-RNAi plants at any time. Ubiquitin transcript levels were used as internal standard for relative quantification in all experiments. Experiments were performed with greenhouse plants grown under LD conditions. sd is given.

Figure 8.

A, To reduce the red to far-red ratio for greenhouse-grown plants, they were planted at a density of 21 plants m⁻². Wild-type plants show shade avoidance response under canopy shade, showing elongated internodes and hyponastic leaf movement to capture light under crowded conditions (left side). StSUT4-RNAi plants do not show shade avoidance under canopy shade. Neither internode elongation nor leaf angle adaptation was observed (right side). B, Internode elongation of wild-type and StSUT4-RNAi potato plants grown under LD and SD conditions or in high-density populations under LD conditions. The experiment was reproduced in the greenhouse and in the growth chamber under LD and SD conditions showing the same results in each case. The length of the five upper internodes was measured as described by Martinez-Garcia et al. (2002). C, Internode elongation of potato plants grown under canopy shade in the greenhouse. Shaded plants were grown at high plant density (21 plants m⁻²), whereas control plants were grown at low density (7 plants m⁻²). D, Internode elongation of potato plants grown under artificial light conditions in the phytochamber. Internode length was measured after 3 weeks of growth under white light or under white light with additional far-red light. sd is given.

Figure 9.

GA₃ treatment of potato wild-type and StSUT4-RNAi plants grown under LD conditions in the greenhouse. A, Source leaves were treated with 20 μm GA₃ solution every 2 d over a period of 2 weeks. Flowering was analyzed after the indicated period of time. B, Tubers were harvested after 2 months of growth in the greenhouse. Water-treated StSUT4-RNAi plants show higher tuber yield than water-treated wild-type plants due to increased tuber size, whereas GA₃-treated StSUT4-RNAi plants show higher tuber number and tuber size than the wild-type control. C, Internode elongation of potato wild-type and StSUT4 plants treated with GA₃ or paclobutrazol. Error bars indicate sd.

Figure 10.

Hypothetical model of StSUT4-mediated interconnection of the photoreceptor and the GA₃ signaling pathway triggering tuberization, flowering, and shade avoidance response. The model is partially adapted from Rodriguez-Falcon et al. (2006).