GSE131545
Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis
Photo-inhibitory high-light stress induces damage to photosystems and changes a myriad of metabolic processes in plants. In Arabidopsis it leads to increased abundance of markers of protein degradation and transcriptional upregulation of proteases and proteolytic machinery, but proteostasis is largely maintained. To identify specific enzymes degrading at a faster rate under high light conditions, we developed a 13C partial labelling approach to measure rates of turnover of specific proteins in Arabidopsis rosettes. We identified 73 proteins with rates of protein degradation enhanced by high-light. In addition to the expected increase in degradation rate of the photosystem II (PSII) D1 subunit, we discovered significant increases in degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase. Significant increases in degradation of 65 other plastid, mitochondrial, peroxisomal, and cytosolic enzymes were also found, including factors involved in redox shuttles within and between organelles and the cytosol. Coupled analysis of protein degradation rate, transcriptional rate, and protein abundance revealed that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light-induced transcriptional responses, ensuring proteostasis. In contrast, plastid-encoded proteins with enhanced turnover rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of PSII D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high-light stress. Overall design: Arabidopsis thaliana accession Columbia-0 plants were grown under 16/8-h light/dark conditions with cool white T8 tubular fluorescent lamps 4000K 3350 lm (Osram, Germany) with intensity of 100–125 µmol m-2 s-1 at 22 °C. Arabidopsis plants were grown in soil pots for 21 days until they reached leaf production stage 1.10 (Boyes et al., 2001). Shoots of Arabidopsis at the leaf production stage 1.10 were positioned into the sealed growth chamber with the soil pots kept underneath. Six tandem growth chambers were supplied with air at a continuous flow rate 6 L/min and kept overnight before the labelling experiment (T0). A homemade water column was connected to the air hose to keep the air humidity inside the growth chamber. A commercial LED (Heliospectra) were used as the light source for the labelling experiment and the light spectra was set as (420nm-250, 450nm-638, 530nm-750, 630nm-1000, 660nm-250 and 735nm-25). Normal and high light intensity at 100 and 500 µE was achieved by adjusting the distance between the growth chamber and the light source. Shoot tissues were harvested in biological triplicate after differing light transitions from dark: T0D - end of night (dark control), T2H - 2 hours high light, T2L - 2 hours standard light, T5H - 5 hours high light, T5L - 5 hours standard light, T8H - 8 hours high light, and T8L - 8 hours standard light. Total RNA was isolated using TRI reagent based on an adapted protocol (Crisp et al., 2017). Full details are available at protocols.io: dx.doi.org/10.17504/protocols.io.bt8wnrxe. Total RNA-sequencing libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Plant kit (RS-122-2402, Illumina, CA, USA) as per manufacturer's instructions but with input RNA and reaction volumes adjusted by one-third. PCR amplified libraries were pooled equal-molar and sequenced (75 bp, single-end) on one lane of the NextSeq500.