The H+-sucrose cotransporter NtSUT1 is essential for sugar export from tobacco leaves

Abstract

In many species translocation of sucrose from the mesophyll to the phloem is carrier mediated. A sucrose/H+-symporter cDNA, NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown to be highly expressed in mature leaves and at low levels in other tissues, including floral organs. To study the in vivo function of NtSUT1, tobacco plants were transformed with a SUT1 antisense construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly affected plants developed chloroses and necroses that led to death. The leaves exhibited impaired ability to export recently fixed 14CO2 and were unable to export transient starch during extended periods of darkness. As a consequence, soluble carbohydrates accumulated and photosynthesis was reduced. Autoradiographs of leaves show a heterogenous pattern of CO2 fixation even after a 24-h chase. The 14C pattern does not change with time, suggesting that movement of photosynthate between mesophyll cells may also be impaired. The affected lines show a reduction in the development of the root system and delayed or impaired flowering. Taken together, the effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic route and possibly for intermesophyll transport as well.

Figure 1

Computer-aided homology analysis by PHYLIP (Felsenstein, 1993) of aligned SUTs from tobacco (NtSut1), potato (StSut1) (Riesmeier et al., 1993), spinach (SoSut1) (Riesmeier et al., 1992), Arabidopsis (AtSuc1 and AtSuc2) (Sauer and Stolz, 1994), Plantago major (PmSuc1 and PmSuc2) (Gahrtz et al., 1994, 1996), castor bean (RcSut1) (Weig and Komor, 1996), rice (OsSut1) (Hirose et al., 1997), and fava bean (VfSut1) (Weber et al., 1997). The comparison was restricted to the region in NtSUT1 from amino acid position 19 to 472. The numbers indicate the occurrence of a branch in 100 bootstrap replicates of a given data set. OsSut1 was used as the outgroup.

Figure 2

Northern-blot analysis of NtSUT1 expression in tobacco using stringent conditions (see Methods; probe, NtSut1, 1.2 kb). A, Expression in various parts of the plant (16.5 μg/lane). B, Expression in flower organs (5 μg/lane).

Figure 3

Structure of the chimeric gene and analysis of transgenic tobacco plants with reduced expression of NtSUT1. Analysis of NtSUT1 mRNA expression in source leaves of transgenic and control plants. Total RNA (25 μg/lane) was hybridized with a radiolabeled NtSUT1 1.2-kb probe under stringent conditions (see Methods). Transcript sizes are given on the right. The hybridization signals at 1500 nt represent the antisense NtSUT1 mRNA.

Figure 4

Development of symptoms in tobacco plants transformed with the SUT1 antisense construct. A, Transgenic tobacco plants after 7 weeks in the greenhouse (from left to right): wild type, αNtSUT1-35S₃₃, αNtSUT1-35S₃₀, and αNtSUT1-35S₁₂. B, Top view of wild type (left) and αNtSUT1-35S₅₀ (right). C, Starch accumulation as determined after 16 h of darkness for wild type (control), αNtSUT1-35S₃₀, and αNtSUT1-35S₁₂ by KI staining.

Figure 5

Growth response of three selected antisense plants with weak to intermediate phenotypes. A, Phenotype; B, dry weight; C, shoot-to-root ratio; and D, photosynthesis of tobacco plants transformed with the SUT1 antisense construct. Rate of net photosynthesis of the youngest fully expanded leaf was measured at a growth irradiance of 500 μmol photons m⁻² s⁻¹. wt, Wild type; 17, αNtSUT1-35S₁₇; 30, αNtSUT1-35S₃₀; 55*, αNtSUT1-35S_55*.

Figure 6

The export of ¹⁴C from leaves of tobacco. Wild-type plants (•) and the transgenic lines αNtSUT1-35S₁₇ (▵), αNtSUT1-35S₃₀ (○), and αNtSUT1-35S_55* (□) expressing antisense NtSUT1 mRNA. A, Data from young leaves; B, data from mature, fully expanded leaves. Leaves were fed with ¹⁴CO₂ at 11:00 am and the export of ¹⁴C was followed with a Geiger-Müller tube positioned under the fed area of the leaf. Data are expressed as the maximum amount of isotope incorporated. The black bar on the x axis represents the dark period.

Figure 7

Typical pattern of chlorosis observed in the interveinal regions of mature leaves from line αNtSUT1-35S₃₀. This phenotype developed progressively after leaves attained full expansion and only in lines exhibiting a marked inhibition of sucrose export.

Figure 8

Photographs (A and D) and autoradiographs (B, C, E, and F) of wild-type (A–C) and transgenic (D–F) plants. Leaves were allowed to fix ¹⁴CO₂ for 5 min and were then detached from the plant (B and E) or allowed to remain attached for another 24 h to permit export of incorporated radioactivity (C and F).

Figure 9

Concentration of soluble sugars and starch in leaves from tobacco plants transformed with antisense constructs of the SUT. Samples were taken 5 h into the photoperiod. WT, Wild type; 17a, αNtSUT1-35S₁₇; 30, αNtSUT1-35S₃₀; 55, αNtSUT1-35S_55*. Data are means ± se. Open bars, Regions distal to major veins; shaded bars, equal regions proximal to major veins.