Extended darkness induces internal turnover of glucosinolates in Arabidopsis thaliana leaves

Abstract

Prolonged darkness leads to carbohydrate starvation, and as a consequence plants degrade proteins and lipids to oxidize amino acids and fatty acids as alternative substrates for mitochondrial ATP production. We investigated, whether the internal breakdown of glucosinolates, a major class of sulfur-containing secondary metabolites, might be an additional component of the carbohydrate starvation response in Arabidopsis thaliana (A. thaliana). The glucosinolate content of A. thaliana leaves was strongly reduced after seven days of darkness. We also detected a significant increase in the activity of myrosinase, the enzyme catalyzing the initial step in glucosinolate breakdown, coinciding with a strong induction of the main leaf myrosinase isoforms TGG1 and TGG2. In addition, nitrilase activity was increased suggesting a turnover via nitriles and carboxylic acids. Internal degradation of glucosinolates might also be involved in diurnal or developmental adaptations of the glucosinolate profile. We observed a diurnal rhythm for myrosinase activity in two-week-old plants. Furthermore, leaf myrosinase activity and protein abundance of TGG2 varied during plant development, whereas leaf protein abundance of TGG1 remained stable indicating regulation at the transcriptional as well as post-translational level.

Fig 1. Myrosinase activity in rosette leaves during development of A. thaliana.

Complete rosettes were harvested at the beginning of the light period after 2–5 weeks of growth under long-day conditions (16/8h light/dark), and 3–9 weeks of growth under short-day conditions (8/16 h light/dark). Error bars indicate the standard deviation of four to five biological replicates or pools. Significantly different means are indicated by different letters (Student Newman Keuls; P < 0.05).

Fig 2. Protein abundance of TGG1 and TGG2 during development of A. thaliana plants grown under long-day conditions.

Rosette leaves were harvested at the beginning of the light period and protein abundance was quantified after western-blotting and immunodetection using ImageJ. Asterics indicate significant differences (Student’s T-test; P < 0.01) compared to the protein abundance in two-week-old plants. Error bars indicate the standard deviation of two to three independent experiments with pools (2- and 3-week-old plants) and single plants (4- and 5-week-old plants).

Fig 3. Diurnal myrosinase activity and protein abundance in two-week-old A. thaliana plants grown under long-day conditions.

The plants were harvested at four different time points: 1 Beginning of the light period; 2 Middle of the day; 3 End of the light period; 4 Middle of the night. The myrosinase activity (A) was measured photometrically and normalized to the maximal activity. Error bars show the standard deviation of four to five biological replicates (pools). For protein abundance of TGG1 and TGG2 (B), immunoblotting and normalization with actin was performed. Error bars represent the standard deviation of three independent experiments with one pool of plants. Significantly different means are indicated by different letters (Student Newman Keuls; P < 0.05).

Fig 4. Myrosinase activity, myrosinase protein abundance and nitrilase activity in rosette leaves after extended darkness (ED).

The plants were transferred to complete darkness for 3 d (3d ED) or 7 d (7d ED) after six weeks of growth under short day conditions. Myrosinase (A) as well as nitrilase (C) activity was measured photometrically (error bars show the standard deviation of five to 19 biological replicates). For protein abundance of TGG1 and TGG2 (B), immunoblotting and normalization to the control was performed (error bars represent the standard deviation of three biological replicates from three independently performed Western blots). Asterics indicate significant differences (Student’s T-test; P < 0.05) compared to the control.

Fig 5. Glucosinolate content in rosette leaves after extended darkness (ED).

Plants were grown for six weeks under short day condition and transferred to darkness for 7 d. Glucosinolate content of rosette leaves was measured via LC-MS. Values are means of three to four biological replicates (error bars represent the standard deviation) and Student’s T-test shows *P < 0.05; ** P < 0.01; ***P < 0.001. 1: general structure of methylsulfinyl-GLS; 2: general structure of methylthio-GLS; 3: structure of I3M. 4MSOB (4-Methylsulfinylbutyl-GLS); 5MSOP (5-Methylsulfinylpentyl-GLS); 6MSOH (6-Methylsulfinylhexyl-GLS); 7MSOH (7-Methylsulfinylheptyl); 8MSOO (8-Methylsulfinyloctyl); 4MTB (4-Methylthiobutyl-GLS); 5MTP (5-Methylthiopentyl-GLS); 7MTH (7-Methylthioheptyl-GLS); 8MTO (8-Methylthiooctyl-GLS); I3M (Indol-3-ylmethyl-GLS); 4MI3M (4-Methoxy-indol-3-ylmethyl-GLS); 1MI3M (N-Methoxy-indol-3-ylmethyl-GLS).