Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic ¹³CO₂ Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein

Abstract

ORCID IDs: 0000-0002-7658-0473 (H.I.); 0000-0001-8931-7722 (T.O.); 0000-0002-6113-9570 (R.S.).Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied 13 CO 2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d 21), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark.

Cellulose Synthesis and Cell Expansion Are Regulated by Different Mechanisms in Growing Arabidopsis Hypocotyls

“…techniques show that the incorporation of glucose units into cell wall polymers decreases during the night in wild-type Arabidopsis plants and is abolished from ;4 h into the night in the starchless pgm mutant, showing a close temporal relationship to the depletion of sucrose in the mutant (Boex-Fontvieille et al, 2014;Ishihara et al, 2015). These labeling experiments are in close agreement with our results on diurnal changes in cellulose synthesis estimated from CSC dynamics.…”

“…These labeling experiments are in close agreement with our results on diurnal changes in cellulose synthesis estimated from CSC dynamics. Based on our data and the report from Ishihara et al (2015), it is tempting to speculate that the production and secretion of other cell wall components, like pectin and hemicelluloses, may be regulated in a similar manner. This would perhaps not be surprising as they are also abundant components of cell walls, their synthesis is a significant resourceconsuming flux in growing cells, and their deposition likewise depends on vesicle trafficking and exocytosis (McFarlane et al, 2014;Kim and Brandizzi, 2016).…”

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

“…We note here that although the ambient temperature in our experiment was 37°C, aerial tissues reached a peak temperature of only approximately 31°C (measured by infrared thermometer at the end of the stress period just prior to harvest), likely owing to evaporative cooling effects. Notably, Ishihara et al (2015) report higher protein synthesis rates at 28°C than at 21°C in Arabidopsis Figure 2. A, Probes used in this study.…”

Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day

“…13 CO 2 labeling was carried out as described by Ishihara et al (2015). Threeweek-old plants were placed in advance of the experiment in a Plexiglas chamber inside a controlled environment cabinet under the conditions described in the text and legends.…”