Arabidopsis seedling growth, storage lipid mobilization, and photosynthetic gene expression are regulated by carbon:nitrogen availability

Abstract

The objective of the current work was to establish the degree to which the effects of carbon and nitrogen availability on Arabidopsis seedling growth and development are due to these nutrients acting independently or together. Growth of seedlings on low (0.1 mM) nitrogen results in a significant reduction of seedling and cotyledon size, fresh weight, chlorophyll, and anthocyanin content but a slight increase in endogenous sugars. The addition of 100 mM sucrose (Suc) to the nitrogen-depleted growth media results in a further reduction in cotyledon size and chlorophyll content and an overall increase in anthocyanins and endogenous sugars. Storage lipid breakdown is almost completely blocked in seedlings grown on low nitrogen and 100 mM Suc and is significantly inhibited when seedlings are grown on either low nitrogen or high Suc. Carbohydrate repression of photosynthetic gene expression can only be observed under low nitrogen conditions. Low (0.1 mM) nitrogen in the absence of exogenous carbohydrate results in a significant decrease in chlorophyll a/b-binding protein and ribulose bisphosphate carboxylase small subunit gene transcript levels. Thus, carbon to nitrogen ratio rather than carbohydrate status alone appears to play the predominant role in regulating various aspects of seedling growth including storage reserve mobilization and photosynthetic gene expression.

Figure 1

Phenotypes of Arabidopsis seedlings germinated and grown for 6 d on media containing different concentrations of nitrogen and Suc. 60 N/0 Suc, 60 mm nitrogen and 0 Suc; 0.1 N/0 Suc, 0.1 mm nitrogen and 0 Suc; 60 N/100 Suc, 60 mm nitrogen and 100 mm Suc; 0.1 N/100 Suc, 0.1 mm nitrogen and 100 mm Suc. Scale bar = 2 mm. The mean width of approximately 20 cotyledons ± the sd is shown in each case. 0.1 mm nitrogen/100 mm sorbitol treatment resulted in seedlings similar to those grown on 0.1N/0 Suc. Seedlings grown on 100 mm sorbitol did not show the decrease in cotyledon size and purple pigmentation present in seedlings grown on 100 mm Suc and 0.1 mm nitrogen but were similar to seedlings grown on 0.1 mm nitrogen alone (not shown).

Figure 2

Primary root length of 6-d-old Arabidopsis seedlings grown in the presence of various nitrogen concentrations (60–0.1 mm) and 100 mm Suc. sd bars are shown; n = 20.

Figure 3

Fresh weight of Arabidopsis seedlings germinated for 6 d in the presence of various nitrogen concentrations (60–0.1 mm) and in the absence or presence of different carbohydrate sources. Each bar presents the average fresh weight of Arabidopsis seedlings. The average fresh weight was estimated in three independent experiments using three to five batches of 20 seedlings in each experiment. Error bars show the sd of the average fresh weight over the three independent experiments.

Figure 4

Effect of carbohydrate to nitrogen balance on storage lipid breakdown. Seeds were germinated on media with either 60 mm or 0.1 mm nitrogen in the presence or absence of 100 mm Suc. Samples of 20 seeds or seedlings were taken at the start of the experiment (d 0) and after 3 and 6 d of germination. Total lipids were extracted and measured. All experiments were done in triplicate. The levels of eicosenoic acid (C20:1, n = 11) are shown as an indicator of TAG content of the seedlings. ses are shown. In all cases total fatty acid levels followed a similar pattern to that of eicosenoic acid (not shown).

Figure 5

Soluble sugar content (A), chlorophyll amount (B), and anthocyanin levels (C) in 6-d-old Arabidopsis seedlings. Seedlings were germinated either in the absence of a carbohydrate source or in the presence of 100 mm Suc, Glc, or Fru. Germination in the presence of 100 mm sorbitol was used as an osmotic control. The nitrogen concentrations in the media were as follows: 60 mm, 6 mm, 0.6 mm, or 0.1 mm. Sugars, chlorophyll, and anthocyanins were extracted and measured as described in “Material and Methods.” Error bars indicate the sd as described (“Materials and Methods”).

Figure 6

Northern analysis of CAB, RBCS, and CHS gene expression in seedlings germinated for 6 d on media containing 60 mm, 6 mm, 0.6 mm, or 0.1 mm nitrogen in either the absence or presence of 100 mm Suc. Photographs of RNA gels show the amount of 18S rRNA in each lane as a loading control.