Biology subjects

Millar, A. H.

Publications and source records attributed to Millar, A. H..

24 records · Page 2Linked to original sources

bioRxiv2021

The mitochondrial pyruvate carrier (MPC) complex is one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate (OAA) and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. 13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis were largely maintained except for alanine and glutamate, indicating that transamination contributes to restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases (AlaAT) when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

plant biology↗

bioRxiv2020

Increased wheat protein content via introgression of a HMW glutenin selectively reshapes the grain proteome

Introgression of a high molecular weight glutenin subunit (HMW-GS) gene, 1Ay21*, into commercial wheat cultivars increased overall grain protein content and bread-making quality by unknown mechanisms. As well as increased abundance of 1Ay HMW-GS, 115 differentially expressed proteins (DEPs) were discovered between three cultivars and corresponding introgressed near-isogenic lines (NILs). Functional category analysis showed that the DEPs were predominantly other storage proteins, and proteins involved in protein synthesis, protein folding, protein degradation, stress response and grain development. Nearly half the genes encoding the DEPs showed strong co-expression patterns during grain development. Promoters of these genes are enriched in elements associated with transcription initiation and light response, indicating a potential connection between these cis-elements and grain protein accumulation. A model of how this HMW-GS enhances the abundance of machinery for protein synthesis and maturation during grain filling is proposed. This analysis not only provides insights into how introgression of the 1Ay21* improves grain protein content, but also directs selection of protein candidates for future wheat quality breeding programmes. One sentence summaryIntrogression of the 1Ay21* HMW-GS increases wheat grain protein content and improves bread-making quality in association with a broad reshaping of the grain proteome network.

plant biology↗

bioRxiv2020

Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsis tz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mech-anism in planta. Unexpectedly, transcriptome datamining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in devel-oping grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thia-zole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain filling period. AbbreviationsDHAla, dehydroalanine; EST, expressed sequence tag; FDR, false discovery rate; IPTG, {beta}-D-1-thiogalactopyranoside

plant biology↗

bioRxiv2020

The Arabidopsis mitochondrial dicarboxylate carrier 2 maintains leaf metabolic homeostasis by uniting malate import and citrate export

Malate is the major substrate for respiratory oxidative phosphorylation in illuminated leaves. In the mitochondria malate is converted to citrate either for replenishing tricarboxylic acid (TCA) cycle with carbon, or to be exported as substrate for cytosolic biosynthetic pathways or for storage in the vacuole. In this study, we show that DIC2 functions as a mitochondrial malate/citrate carrier in vivo in Arabidopsis. DIC2 knockout (dic2-1) results in growth retardation that can only be restored by expressing DIC2 but not its closest homologs DIC1 or DIC3, indicating that their substrate preferences are not identical. Malate uptake by non-energised dic2-1 mitochondria is reduced but can be restored in fully energised mitochondria by altering fumarate and pyruvate/oxaloacetate transport. A reduced citrate export but an increased citrate accumulation in substrate-fed, energised dic2-1 mitochondria suggest that DIC2 facilitates the export of citrate from the matrix. Consistent with this, metabolic defects in response to a sudden dark shift or prolonged darkness could be observed in dic2-1 leaves, including altered malate, citrate and 2-oxoglutarate utilisation. There was no alteration in TCA cycle metabolite pools and NAD redox state at night; however, isotopic glucose tracing reveals a reduction in citrate labelling in dic2-1 which resulted in a diversion of flux towards glutamine, as well as the removal of excess malate via asparagine and threonine synthesis. Overall, these observations indicate that DIC2 is responsible in vivo for mitochondrial malate import and citrate export which coordinate carbon metabolism between the mitochondrial matrix and the other cell compartments. SIGNIFICANCE STATEMENTMitochondria are pivotal for plant metabolism. One of their central functions is to provide carbon intermediates for the synthesis of critical building blocks, such as amino acids. Malate import and citrate export are two of the most recognised and specialised features of the mitochondrial role in the plant cellular metabolic network, yet the possibility that a single carrier would unite both functions has not been considered. Here, we have demonstrated that DIC2 preferentially fulfils these two functions in Arabidopsis thaliana in vivo, making it a bifunctional gateway for two major metabolite fluxes into and out of the mitochondrial matrix in the plant cell. Our results highlight the significance of DIC2 in cooperation with other mitochondrial carriers in maintaining metabolic balance even under challenging environmental conditions.

plant biology↗

bioRxiv2019

Refining the composition of the Arabidopsis thaliana 80S cytosolic ribosome

The cytosolic 80S ribosome is composed of protein and RNA molecules and its function in protein synthesis is modulated through interaction with other cytosolic components. Defining the role of each of the proteins associated with ribosomes in plants is a major challenge which is hampered by difficulties in attribution of different proteins to roles in ribosome biogenesis, the mature cytosolic ribosome (r-proteins) or to the broader translatome associated with functioning ribosomes. Here we refined the core r-protein composition in plants by determining the abundance of proteins in low, partially and highly purified ribosomal samples from Arabidopsis thaliana cell cultures. To characterise this list of proteins further we determined their degradation (KD) and synthesis (KS) rate by progressive labelling with 15N combined with peptide mass spectrometry analysis. The turnover rates of 55 r-proteins, including 26 r-proteins from the 40S subunit and 29 r-proteins from the 60S subunit could be determined. Overall, ribosome proteins showed very similar KD and KS rates suggesting that half of the ribosome population is replaced every 3-4 days. Three proteins showed significantly shorter half-lives; ribosomal protein P0D (RPP0D) with a half-life of 0.5 days and RACK1b and c with half-lives of 1-1.4 days. The ribosomal RPP0D protein is a homolog 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 as a 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 in Arabidopsis. Of this set, 26 were enriched more than 10-fold during ribosome purification. A number have known roles in translation or ribosome-association while others are newly proposed ribosome-associated factors in plants. This analysis provides a more robust understanding of Arabidopsis ribosome content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and reveals how protein turnover can identify r-proteins involved in ribosome biogenesis or regulation in plants. Data are available via ProteomeXchange with identifier PXD012839.

plant biology↗

bioRxiv2016

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 optimised data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents more than 78% of the genome with a scaffold N50 of 88.8kbp that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNAseq and PacBio full-length cDNAs to identify 104,091 high confidence protein-coding genes and 10,156 non-coding 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. [Supplemental material is available for this article.]

genomics↗