Transgenic Arabidopsis plants with decreased activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase have altered carbon partitioning

Abstract

The role of fructose-2,6-bisphosphate (Fru-2,6-P(2)) as a regulatory metabolite in photosynthetic carbohydrate metabolism was studied in transgenic Arabidopsis plants with reduced activity of Fru-6-phosphate,2-kinase/Fru-2,6-bisphosphatase. A positive correlation was observed between the Fru-6-phosphate,2-kinase activity and the level of Fru-2,6-P(2) in the leaves. The partitioning of carbon was studied by (14)CO(2) labeling of photosynthetic products. Plant lines with Fru-2,6-P(2) levels down to 5% of the levels observed in wild-type (WT) plants had significantly altered partitioning of carbon between sucrose (Suc) versus starch. The ratio of (14)C incorporated into Suc and starch increased 2- to 3-fold in the plants with low levels of Fru-2,6-P(2) compared with WT. Transgenic plant lines with intermediate levels of Fru-2,6-P(2) compared with WT had a Suc-to-starch labeling ratio similar to the WT. Levels of sugars, starch, and phosphorylated intermediates in leaves were followed during the diurnal cycle. Plants with low levels of Fru-2,6-P(2) in leaves had high levels of Suc, glucose, and Fru and low levels of triose phosphates and glucose-1-P during the light period compared with WT. During the dark period these differences were eliminated. Our data provide direct evidence that Fru-2,6-P(2) affects photosynthetic carbon partitioning in Arabidopsis. Opposed to this, Fru-2,6-P(2) does not contribute significantly to regulation of metabolite levels in darkness.

Figure 1

Immunoblot analysis of antisense F2KP and WT Arabidopsis plants. Transgenic plants lines with decreased F6P,2-K/F26BPase activity were analyzed by western blot and the 93-kD F2KP protein was detected in the plant extracts using polyclonal antibodies against the AtF2KP-phosphatase region. Each lane contained 6.5 μg of protein.

Figure 2

Relationship between F6P,2-K activity and the Fru-2,6-P₂ levels. The specific F6P,2-K activities are plotted against the Fru-2,6-P₂ levels in seven independent plant lines. Each point represents one plant. The plant lines are AS1 (♦), AS2 (□), AS6 (▴), AS13 (▵), AS16 (▪), control (●), and WT (○). Control is a plant line only transformed with an empty vector construct. All samples were harvested during the last hour of the photoperiod.

Figure 3

F6P,2-K and F26BPase activities, Fru-2,6-P₂ levels, and sugars-to-starch ¹⁴C-labeling ratio in independent plant lines. Samples were collected and analyzed for activity of F6P,2-K (A), F26BPase (B), and levels of Fru-2,6-P₂ (C). Similar plants were radiolabeled with ¹⁴CO₂ for 10 min and the ratio between ¹⁴C incorporated into sugars and starch was determined (D). Each value represents the means ± sd of four different plants. ND, Not determined. All samples were harvested and radiolabeling was performed during the last hour of the photoperiod.

Figure 4

Photosynthetic carbon partitioning measured as ¹⁴C incorporation in WT and antisense plants. Plants with low levels of Fru-2,6-P₂ (AS1) and WT plants were photosynthetically labeled with ¹⁴CO₂ for 10 min from the beginning of the photoperiod (0) and after 10 min (10) and 60 min (60) of illumination. Each column represents the means ± sd of three individual plants. Black columns represent the sum of radioactivity incorporated into lipids, cationic, anionic, and insoluble compounds (excluding starch). Gray columns represent starch. White columns represent neutral compounds, mainly Suc.

Figure 5

The level of Fru-2,6-P_2, sugars, and starch in WT Arabidopsis and antisense plants with low Fru-2,6-P₂ (AS1) during the diurnal cycle. Each point represents the means ± sd of four different plants. The bars at the top of the graphs indicate light (white bars) or dark (black bars). A, Fru-2,6-P₂; B, Suc; C, Glc; D, Fru; E, starch. White symbols, AS1; black symbols, WT.

Figure 6

The hexose-phosphate and triose-phosphate content of WT plants and transgenic plants with low Fru-2,6-P₂. Each point represents the means ± sd of four different plants. A, Fru-6-P; B, Glc-6-P; C, Glc-1-P; D, GAP; E, DHAP. White symbols, AS1; black symbols, WT.

Figure 7

Light response curve for oxygen evolution in leaves of low Fru-2,6-P₂ plants (AS1) and for WT plants. Each point represents the means ± sd of five independent plants, with three independent determinations of each. White symbols, AS1; black symbols, WT.