Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis.

Photoinhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional up-regulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light­induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation 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 the photosystem II (PSII) D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

The composition and turnover of the Arabidopsis thaliana 80S cytosolic ribosome.

Cytosolic 80S ribosomes contain proteins of the mature cytosolic ribosome (r-proteins) as well as proteins with roles in ribosome biogenesis, protein folding or modification. Here, we refined the core r-protein composition in Arabidopsis thaliana by determining the abundance of different proteins during enrichment of ribosomes from cell cultures using peptide mass spectrometry. The turnover rates of 26 40S subunit r-proteins and 29 60S subunit r-proteins were also determined, showing that half of the ribosome population is replaced every 3-4 days. Three enriched proteins showed significantly shorter half-lives; a protein annotated as a ribosomal protein uL10 (RPP0D, At1g25260) with a half-life of 0.5 days and RACK1b and c with half-lives of 1-1.4 days. The At1g25260 protein is a homologue of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role in the 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins; 26 were enriched more than 10-fold during ribosome enrichment. Some of these enriched proteins have known roles in translation, while others are newly proposed ribosome-associated factors in plants. This analysis provides an improved understanding of A. thaliana ribosome protein content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and how protein turnover can help identify r-proteins involved in ribosome biogenesis or regulation in plants.

Impact of oxidative stress on the function, abundance, and turnover of the Arabidopsis 80S cytosolic ribosome.

Abiotic stress in plants causes accumulation of reactive oxygen species (ROS) leading to the need for new protein synthesis to defend against ROS and to replace existing proteins that are damaged by oxidation. Functional plant ribosomes are critical for these activities, however we know little about the impact of oxidative stress on plant ribosome abundance, turnover, and function. Using Arabidopsis cell culture as a model system, we induced oxidative stress using 1 µm of H2 O2 or 5 µm menadione to more than halve cell growth rate and limit total protein content. We show that ribosome content on a total cell protein basis decreased in oxidatively stressed cells. However, overall protein synthesis rates on a ribosome abundance basis showed the resident ribosomes retained their function in oxidatively stressed cells. 15 N progressive labelling was used to calculate the rate of ribosome synthesis and degradation to track the fate of 62 r-proteins. The degradation rates and the synthesis rates of most r-proteins slowed following oxidative stress leading to an ageing population of ribosomes in stressed cells. However, there were exceptions to this trend; r-protein RPS14C doubled its degradation rate in both oxidative treatments. Overall, we show that ribosome abundance decreases and their age increases with oxidative stress in line with loss of cell growth rate and total cellular protein amount, but ribosome function of the ageing ribosomes appeared to be maintained concomittently with differences in the turnover rate and abundance of specific ribosomal proteins. Data are available via ProteomeXchange with identifier PXD012840.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development.

We applied 15N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome.

Changes in specific protein degradation rates in Arabidopsis thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis.

Mitochondrial Lon1 loss impairs oxidative phosphorylation complexes and TCA enzymes and causes accumulation of specific mitochondrial proteins. Analysis of over 400 mitochondrial protein degradation rates using 15 N labelling showed that 205 were significantly different between wild type (WT) and lon1-1. Those proteins included ribosomal proteins, electron transport chain subunits and TCA enzymes. For respiratory complexes I and V, decreased protein abundance correlated with higher degradation rate of subunits in total mitochondrial extracts. After blue native separation, however, the assembled complexes had slow degradation, while smaller subcomplexes displayed rapid degradation in lon1-1. In insoluble fractions, a number of TCA enzymes were more abundant but the proteins degraded slowly in lon1-1. In soluble protein fractions, TCA enzymes were less abundant but degraded more rapidly. These observations are consistent with the reported roles of Lon1 as a chaperone aiding the proper folding of newly synthesized/imported proteins to stabilise them and as a protease to degrade mitochondrial protein aggregates. HSP70, prohibitin and enzymes of photorespiration accumulated in lon1-1 and degraded slowly in all fractions, indicating an important role of Lon1 in their clearance from the proteome.

Resource: Mapping the Triticum aestivum proteome.

Yield and quality improvement of bread wheat (Triticum aestivum) is a focus in efforts to meet new demands from population growth and changing human diets. As the complexity of the wheat genome is unravelled, determining how it is used to build the protein machinery of wheat plants is a key next step in explaining detailed aspects of wheat growth and development. The specific functions of wheat organs during vegetative development and the role of metabolism, protein degradation and remobilisation in driving grain production are the foundations of crop performance and have recently become accessible through studies of the wheat proteome. We present a large scale, publicly accessible proteome mapping of wheat consisting of 24 organ and developmental samples. Tissue specific sub-proteomes and ubiquitously expressed markers of the wheat proteome are identified, alongside hierarchical assessment of protein functional classes, their presence in different tissues and correlations between the abundance of functional classes of proteins. Gene-specific identifications and protein family relationships are accounted for in the organisation of the data and 202 new protein-coding transcripts identified by proteogenomic mapping. The interactive database will serve as a vehicle to build, refine and deposit confirmed targeted proteomic assays for wheat proteins and protein families to assess function (www.wheatproteome.org).