Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis

Maria Piques¹, Waltraud X Schulze¹, Melanie Höhne¹, Björn Usadel¹, Yves Gibon^1,a, Johann Rohwer^1,b & Mark Stitt¹

^aPresent address: INRA Bordeaux, University of Bordeaux 1&2, UMR619 Fruit Biology, F-33883 Villenave d'Ornon, France

^bPermanent address: Triple-J Group for Molecular Cell Physiology, Department of Biochemistry, Stellenbosch University, Private Bag X1, 7602 Matieland, South Africa

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Synopsis

In the following article, we quantify ribosome and transcript concentrations, and polysome composition in Arabidopsis rosettes. These data are used to predict translation rates, globally, and for individual enzymes in central metabolism. We then explore the consequences of these molecular events for growth. First, we compare the rates of synthesis with protein abundance to predict which enzymes are likely to be subject to rapid turnover and which are synthesized at rates similar to those required for growth. Second, we estimate the costs of protein synthesis and relate them to the whole plant carbon budget.

Plants experience continual changes in the environment. One of the most striking is the daily alternation between light and darkness. This leads to a repeated alternation between two states; a large positive balance of energy and carbon in the light period, and a deficit in the dark period. It is buffered by storing some newly fixed C as starch, and remobilizing it during the night (Smith and Stitt, 2007). We do not know how energetically expensive processes like protein synthesis are coordinated with these dramatic diurnal changes in the energy budget.

Plants also experience changes in the environment over a time range of days or weeks due to changing weather patterns and seasons, raising the question how they integrate their response over a wide range of time spans. Although there are large diurnal changes for thousands of transcripts (Usadel et al, 2008), most enzymes show only small diurnal changes in their maximum activity, and require days to adjust when plants are transferred to new conditions. Gibon et al (2004b) proposed that translation is so slow that several days are required to produce a major change in protein abundance. This would buffer enzymatic capacities against recurring changes caused by the light–dark cycle, while allowing them to adjust to sustained changes in the surroundings. To test this hypothesis, it was necessary to obtain information about rates of protein synthesis.

Protein synthesis occurs by the recruitment of transcripts to ribosomes, to form polysomes. The rate of translation of a given transcript species depends upon transcript abundance, the proportion present in polysomes, the number of ribosomes present on the transcript, and their speed of progression along the transcript. Real time RT–PCR combined with spiked external RNA standards was used to quantify the abundance of cytosolic, plastidic and mitochondrial ribosomal RNA and 98 transcripts, including 84 for enzymes involved in central metabolism. This was done in whole rosettes, and in fractions from polysome density gradients. The estimated rates of synthesis of proteins varied by >1000-fold. The measurements were carried out in the dark and the light periods. Ribosomal and transcript loading into polysomes and the estimated rates of synthesis of most proteins decreased by up to twofold in the dark.

Protein abundance was estimated in two ways; from maximum enzyme activities, corrected by literature values for specific activity, and from MS analyses using the emPAI index. Comparison of the resulting estimates for protein abundance revealed a highly significant agreement (Pearson's R²=0.590).

The estimated rates of protein synthesis were compared with protein abundance to calculate how many days it would require to synthesize all the protein in an Arabidopsis rosette (T_P, this term is used instead of T_0.5, because part of the newly synthesized protein represents the flux to growth). For some enzymes like nitrate reductase and ADP glucose pyrophosphorylase, the estimated rate of synthesis was high compared with their abundance. These enzymes also show marked changes of their maximum activity during diurnal cycles. However, estimated rates of synthesis of most enzymes were low compared with the amount present in the leaf, resulting in a T_P of four or more days. This resembles that needed for growth. An independent estimate of the global rate of protein synthesis was obtained using information about the ribosome concentration (about 0.1 nmol ribosomes g^-1 FW). This would support a maximum rate of protein synthesis of approximately 3 mg protein g^-1 FW day^-1, allowing synthesis of all proteins in the rosette (15 mg g^-1 FW) in about 5 days, resembling the rate required for the observed rate of growth.

There are considerable energetic costs associated with protein synthesis; conversion of one molecule of nitrate to amino acids requires 5 ATP molecules, and addition of an amino acid to a growing peptide chain requires >5 ATP molecules. ATP is supplied by photophosphorylation in the light period, and by respiration in the dark period. The latter is thermodynamically less efficient, and requires transient storage of large amounts of starch. When Arabidopsis is grown in a 12-h light–dark cycle, the rosette contains about 40 mol glucose g^-1 FW as starch at the end of the light period, and has a rate of respiration of about 8 mol CO₂ g^-1 FW h^-1 in the dark period. If protein synthesis continued at maximal rates in the dark period, about 65 mol hexose g^-1 FW would be required each night to supply amino acids, and another 7 mol hexose g^-1 FW for respiration to deliver ATP. Thus, insufficient starch is stored to support high rates of protein synthesis during the night. A deficit is avoided in two ways. First, up to 30 mol amino acids g^-1 FW is accumulated in the light period for use at night (Gibon et al, 2009). Second, (see above) protein synthesis is about twofold slower in the dark period than in the light period. These diurnal adjustments abolish the nocturnal requirement for carbon for amino acid synthesis, and decrease the respiratory cost by about 75%.

In conclusion, quantitative measurements of transcripts, ribosomes, polysomes and protein levels provide information that can be used to estimate protein synthesis and turnover rates. This allows proteins to be identified that are subject to rapid turnover, and provides an integrative framework in which the metabolic and energetic costs of protein synthesis and turnover can be related to central molecular components of cellular growth.