Biology subjects

Millar, A. H.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Leaf movements as a quantitative metric for early stress detection

Early, precise, and non-destructive stress detection is essential for maintaining crop productivity, particularly in high-density plant growth systems like controlled environment agriculture (CEA), where manual monitoring is often impractical. Using plant motion as a proxy for growth and plant health, we demonstrate a method for early, non-invasive stress detection through quantitative leaf-movement analysis in lettuce and five other CEA relevant crops. Leaf-movement dynamics under stress were imaged with a low-cost, scalable Raspberry Pi imaging setup and quantified using a repurposed open-source motion estimation algorithm; Tracking Rhythms in Plants (TRiP). Our system detected stress-induced changes in leaf-movement within 1 hour of stress, with the timing dependent on the nature of the stress. Sustained reductions in leaf-movement coincide with decreased biomass accumulation. This approach offers a non-invasive, rapid, scalable, and cost-effective solution for continuous crop monitoring, with potential for application in both terrestrial and space farming CEA systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/732190v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@19ee20eorg.highwire.dtl.DTLVardef@b0804org.highwire.dtl.DTLVardef@3b3fa8org.highwire.dtl.DTLVardef@1d04026_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Quantification of leaf-movement dynamics as a high-throughput proxy for plant physiological status, enabling early stress detection and timely intervention to mitigate yield penalties in CEA settings (image made with biorender.org). C_FIG

plant biology↗

bioRxiv2025

Analysis of protein aging reveals rates of subcellular organelle renewal and selective post-translational modification in Arabidopsis

The cellular proteome represents a mixture of older and newer copies of each protein type and turnover of this mixture occurs by cycles of protein synthesis and degradation. There is considerable research on new protein synthesis, the nature of nascent proteins and cellular machinery of protein degradation. However, we have limited insights into older proteins or the protein aging process in plants at scale. In this study we use pulse chase biorthogonal non-canonical amino acid tagging (BONCAT) in Arabidopsis cells coupled to affinity purification to capture and analyse snapshots of the cellular proteome as it ages over a two-week period. Each snapshot was subjected to peptide mass spectrometry-based identification, quantitation and characterisation. We show that there are a broad range of lifespans among the 1688 proteins studied and that their subcellular location correlates strongly with protein longevity. Mitochondria, plastids and the extracellular environment contained the longest lived sub-proteomes while the vesicular pathway to ER, PM and peroxisomes contained the shortest-lived protein sets. Abundant primary metabolic enzymes have considerable longevity, while kinases and ubiquitination machinery do not. Through analysis of the aging profiles, we demonstrate that many proteins selectively accumulate posttranslational modifications (PTMs) as they age and that these are mostly oxidative in nature. We show by analysis of exemplar proteins that distinct PTM profiles and proportional changes with age exist between proteins, likely dictated by differences in subcellular environment and protein function. Implications of these insights for understanding cellular function and for biotechnological modification of the plant proteome are discussed.

plant biology↗

bioRxiv2025

Liquid-phase determination of Arabidopsis respiration and photosynthesis using Clark-type O2 electrodes

Photosynthesis and respiration are fundamental metabolic processes in plants, tightly connected through shared substrates, energy dynamics, and redox balance. Arabidopsis is the key genetic model for plants but monitoring these sorts of physiological processes presents significant challenges using traditional gas-exchange or fluorescence-based techniques due to the small size of intact Arabidopsis thaliana (arabidopsis) seedlings. Here, we validate and characterize the use of Clark-type oxygen electrodes, specifically the Hansatech Oxytherm+P system, to quantify both photosynthetic and respiratory activity in intact arabidopsis seedlings. By monitoring oxygen evolution in dark and light phases, we demonstrate that oxygen consumption and production correspond to mitochondrial respiration and photosynthesis, respectively. These processes were modulated by tissue biomass, light intensity, developmental stage, and stress conditions. Specific inhibitors such as potassium cyanide and paraquat confirmed that the recorded changes in oxygen concentrations reflected mitochondrial cytochrome oxidase activity and photosystem electron transport-dependent oxygen production, respectively. Moreover, oxygen evolution increased significantly with bicarbonate supplementation, validating the systems sensitivity to carbon fixation. We further showed that photosynthetic activity measured with this method correlates with a quantitative green index and responds dynamically to de-etiolation, abiotic stress (salt, osmotic, oxidative), and temperature shifts. Our study lays the groundwork for measuring photosynthesis based on oxygen evolution and respiration in arabidopsis knockout mutants, CRISPR lines, overexpression lines and ecotypes using Clark-type oxygen electrodes and highlights key considerations and limitations to consider when applying this approach. This platform could also be adapted for many other small tissue plant samples.

plant biology↗

bioRxiv2023

Green Index: a widely accessible method to quantify greenness of photosynthetic organisms

Plant phenotyping involves the quantitative determination of complex plant traits using image analysis. One important parameter is how green plant tissues appear to the observer, which is indicative of their health and developmental stage. Various formulas have been developed to quantify this by calculating leaf greenness scores. We have developed a revised formula called "Green Index" (GI) devised out of the need to quantitatively assess how the apparent greenness of seedlings changes during de-etiolation. The GI calculation is simple, uses widely available RGB values of pixels in images as input, and does not require commercial software platforms or advanced computational skills. In this study we describe the conception of the GI formula, compare it with other widely used greenness formulas, and test its wider application in plant phenotyping using the open source free software platform RawTherapee. We demonstrate the utility of the GI in addressing common issues encountered in assessing plant biology experiments, underscoring its potential as a reliable and accessible tool. Finally, we explore the correlation between GI and chlorophyll content, assess its reliance on different types of photography, and summarizes the key steps for its effective utilization.

plant biology↗

bioRxiv2023

Metabolic adaptations leading to lignification in wheat roots under salinity stress

Analysis of salinity tolerance processes in wheat has focused on salt exclusion from shoots while root phenotypes have received limited attention. Here we consider the varying phenotypic response of four bread wheat varieties that differ in their type and degree of salt tolerance and consider in detail their molecular responses to salinity and changes in root cell wall lignification. These varieties were Westonia introgressed with Nax1 and Nax2 root sodium transporters (HKT1;5-A) that reduce Na+ accumulation in leaves, as well as the tissue tolerant Portugese landrace Mocho de Espiga Branca that has a mutation in the homologous gene HKT1;5-D and has high Na+ concentration in leaves. These three varieties were compared with the more salt-sensitive cultivar Gladius. Through the use of root structural analysis, ion concentrations, as well as differential proteomics and targeted metabolomics we provide an integrated view of the wheat root response to salinity. We show different metabolic re-arrangements in energy conversion, primary metabolic machinery and phenylpropanoid pathway leading to monolignol production in a genotype and genotype by treatment dependent manner that alters the extent and localisation of root lignification which correlated with an improved capacity of wheat roots to cope better under salinity stress.

plant biology↗

bioRxiv2023

PLANT UNCOUPLING MITOCHONDRIAL PROTEIN 2 localizes to the Golgi

Mitochondria act as cellular hubs of energy transformation and metabolite conversion in most eukaryotes. Plant mitochondrial electron transport chains are particularly flexible, featuring alternative components, such as ALTERNATIVE NAD(P)H DEHYDROGENASES and ALTERNATIVE OXIDASES (AOXs), that can bypass proton translocation steps. PLANT UNCOUPLING MITOCHONDRIAL PROTEINS (named PUMPs or plant UCPs) have been identified in plants as homologues of mammalian Uncoupling Proteins (UCPs), and their biochemical and physiological roles have been investigated in the context of mitochondrial energy metabolism. To dissect UCP function in Arabidopsis, the two most conserved (UCP1 and UCP2) have been targeted in recent work by combining mutant lines to circumvent potential functional redundancy in vivo. Such approaches rely on the assumption that both proteins reside in the inner mitochondrial membrane as a prerequisite for functional redundancy. Yet, contradicting results have been reported on UCP2 localization in plants. Here we provide evidence that, conversely to UCP1, which is an abundant inner mitochondrial membrane protein, UCP2 localizes to the Golgi rather than to mitochondria. Based on multiple lines of new and prior evidence, we summarize the consensus view that we have reached and provide an example of how open, critical exchange within the research community is able to constructively address ambiguities. Our observations and considerations provide direction to the ongoing discussion about the functions of UCP proteins. They further offer new perspectives for the study of Golgi membrane transport and subcellular targeting principles of membrane proteins. Since 20 to 30 % of genes in plant genomes are predicted to encode transmembrane proteins and the function of most of those proteins has not been experimentally investigated, we highlight the importance of using independent evidence for localization as a prerequisite for understanding physiological function of membrane proteins.

plant biology↗

bioRxiv2023

ENOD93 interacts with cytochrome c oxidase altering respiratory ATP production and root growth in plants

The early nodulin 93 (ENOD93) gene family in plants can regulate biological nitrogen fixation in legumes and nitrogen use efficiency in cereals but its molecular function is unknown. We show profile hidden Markov models define ENOD93 as a distant homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 is reported to regulate cytochrome oxidase (CIV) influencing the generation of a mitochondria proton motive force in yeast. Knockout of enod93 in Arabidopsis leads to a short root phenotype. ENOD93 is associated with a protein complex the size of CIV in isolated mitochondria but neither CIV abundance nor its activity in ruptured organelles changed in enod93. However, a progressive loss of ADP-dependent respiration rate was observed in enod93 mitochondria which could be fully recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 but ATP synthesis and ADP depletion rates progressively decreased. Respiration rate of whole enod93 seedlings was elevated and root ADP content was nearly double that in WT without a change in ATP content. These altered energetic states correlated with elevated respiratory substrate levels in roots of enod93 compared to WT and complemented lines. Overexpression of ENOD93 lowered ATP content in roots and increased the abundance of a range of amino acids in both roots and leaves. We propose that two previously unconnected gene families in plants, ENOD93 and HYPOXIA INDUCED GENE DOMAIN, are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded in two separate genes. Highlight significanceWe identify the enigmatic early nodulin ENOD93 gene family as the plant homolog of the N-terminal regulatory domain of the yeast RESPIRATORY SUPERCOMPLEX 2 (RCF2) of the mitochondrial oxidative phosphorylation system and provide biochemical and physiological evidence of its role in plant ATP production, broadly explaining the role of ENOD93 in plants.

plant biology↗

bioRxiv2022

Knockdown of mitochondrial atp1 mRNA by a custom-designed pentatricopeptide repeat protein alters F1Fo ATP synthase

We show that a custom-designed RNA-binding protein binds and specifically induces cleavage of atp1 RNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis thaliana. The transformed plants are characterized by delayed vegetative growth and reduced fertility. Five-fold depletion of Atp1 level was accompanied by a decrease in abundance of other ATP synthase subunits, lowered ATP synthesis rate of isolated mitochondria, but no change to mitochondrial electron transport chain complexes, adenylates or energy charge in planta. Transcripts for amino acid transport and a variety of stress response processes were differentially expressed in lines containing the PPR protein, indicating changes to achieve cellular homeostasis when ATP synthase was highly depleted. Leaves of ATP-synthase-depleted lines showed higher respiratory rates and elevated levels of most amino acids at night, most notably serine family amino acids. The results show the value of using custom-designed PPR proteins to influence expression of specific mitochondrial transcripts to carry out reverse genetics studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis. One sentence SummaryKnockdown of mitochondrial atp1 mRNA by a custom-designed pentatricopeptide repeat protein alters F1Fo ATP synthase, plant growth and amino acid metabolism and ATP synthesis in Arabidopsis thaliana

plant biology↗

bioRxiv2021

Metabolic evidence for distinct pyruvate pools inside plant mitochondria

The majority of the pyruvate inside plant mitochondria is either transported into the matrix from the cytosol via the mitochondria pyruvate carrier (MPC) or synthesised in the matrix by alanine aminotransferase (AlaAT) or NAD-malic enzyme (NAD-ME). Pyruvate from these origins could mix into a single pool in the matrix and contribute indistinguishably to respiration, or they could maintain a degree of independence in metabolic regulation. Here, we demonstrated that feeding isolated mitochondria with U-13C-pyruvate and unlabelled malate enables the assessment of pyruvate contribution from different sources to TCA cycle intermediate production. Imported pyruvate is the preferred source for citrate production even when the synthesis of NAD-ME-derived pyruvate was optimised. Genetic or pharmacological elimination of MPC activity removed this preference and allowed an equivalent amount of citrate to be generated from the pyruvate produced by NAD-ME. Increasing mitochondrial pyruvate pool size by exogenous addition only affected metabolites from pyruvate transported by MPC whereas depleting pyruvate pool size by transamination to alanine only affected metabolic products derived from NAD-ME. Together, these data reveal respiratory substrate supply in plants involves distinct pyruvate pools inside the matrix that can be flexibly mixed based on the rate of pyruvate transport from the cytosol. These pools are independently regulated and contribute differentially to organic acids export from plant mitochondria. Significance statementPyruvate is the primary respiratory substrate for energy production to support plant growth and development. However, it is also the starting material of many other pathways. Prioritisation of respiratory use over other competing pathways would enable a level of control when pyruvate is delivered to mitochondria via the mitochondrial pyruvate transporter. We demonstrated the existence of two distinct pyruvate pools in plant mitochondria suggesting inner mitochondrial organisation allows metabolic heterogeneity, hence metabolic specialisation. This explains why NAD-ME flux into plant respiration is low and confirms the prominent link between imported pyruvate and energy production. This compartmentation also reveals how NAD-ME supplies substrate to the mitochondrial pyruvate exporter in plants, especially during C4 metabolism.

plant biology↗

bioRxiv2021

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

Photo-inhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional upregulation 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 light-induced 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 PSII D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

plant biology↗

bioRxiv2021

Alternative oxidase1a and 1d limit proline-dependent oxidative stress and aid salinity recovery in Arabidopsis

A link between Pro catabolism and mitochondrial reactive oxygen species production has been established across eukaryotes and in plants increases in leaf respiration rates have been reported following Pro exposure. Here we investigated how alternative oxidases (AOXs) of the mitochondrial electron transport chain accommodate the large, atypical flux resulting from Pro catabolism and limit oxidative stress during Pro breakdown in mature Arabidopsis leaves. Following Pro treatment, AOX1a and AOX1d accumulate at transcript and protein levels, with AOX1d approaching the level of the typically dominant AOX1a isoform. We therefore sought to determine the function of both AOX isoforms under Pro respiring conditions. Oxygen consumption rate measurements in aox1a and aox1d leaves suggested these AOXs can functionally compensate for each other to establish enhanced AOX catalytic capacity in response to Pro. Generation of aox1a.aox1d lines showed complete loss of AOX proteins and activity upon Pro treatment, yet full respiratory induction in response to Pro remained possible via the cytochrome pathway. However, aox1a.aox1d leaves suffered increased levels of oxidative stress and damage during Pro metabolism compared to WT or the single mutants. During recovery from salt stress, when high rates of Pro catabolism occur naturally, photosynthetic rates in aox1a.aox1d recovered slower than WT or the single aox lines, showing that both AOX1a and AOX1d are beneficial for cellular metabolism during Pro drawdown following osmotic stress. This work provides physiological evidence of a beneficial role for AOX1a but also the less studied AOX1d isoform in allowing safe catabolism of alternative respiratory substrates like Pro. One sentence summaryThe alternative oxidase of plant mitochondria contributes to Pro catabolism by preventing oxidative stress in the electron transport chain and this aids recovery of leaf metabolic rates following salinity stress.

plant biology↗

bioRxiv2021

Protein turnover in the developing Triticum aestivum grain

Protein abundance in cereal grains is determined by the relative rates of protein synthesis and protein degradation during grain development. Through combining in vivo stable isotope labelling and in-depth quantitative proteomics, we have measured the turnover of 1400 different types of proteins during wheat grain development. We demonstrate that there is a spatiotemporal pattern to protein turnover rates which explain part of the variation in protein abundances that is not attributable to differences in wheat gene expression. We show that approximately 20% of total grain ATP production is used for grain proteome biogenesis and maintenance, and nearly half of this budget is invested exclusively in storage protein synthesis. We calculate that 25% of newly synthesized storage proteins are turned over during grain development rather than stored. This approach to measure protein turnover rates at proteome scale reveals how different functional categories of grain proteins accumulate, calculates the costs of protein turnover during wheat grain development and identifies the most and the least stable proteins in the developing wheat grain.

plant biology↗

bioRxiv2021

Distinct changes in mature root and growing root tip proteomes underlie physiological responses of bread wheat to salinity stress

The impact of salinity on wheat plants is often studied by analysis of shoot responses, even though the main mechanism of tolerance is shoot Na+ exclusion. There is a need to understand the molecular responses of root tissues that directly experience rising NaCl concentrations. We have combined analysis of root growth, ion content and respiration with proteome responses in wheat root tip and mature root tissues under saline conditions. We find significant changes in translation and protein synthesis, energy metabolism and amino acid metabolism in a root tissue specific manner. Translation and protein synthesis related proteins showed significant decreases in abundance only in root tips, as did most of the glycolytic enzymes and selected TCA cycle enzymes and ATP synthase subunits. This selective root tip proteome response indicates protein synthesis capacity and energy production were impaired under salt stress, correlating with the anatomical response of roots and reduced root tip respiration rate. Wheat roots respond directly to soil salinity, therefore shoot responses such as reduction in shoot growth and photosynthetic capacity need to be considered in light of these effects.

plant biology↗

bioRxiv2021

ePlant in 2021: New Species, Viewers, Data Sets, and Widgets

ePlant was introduced in 2017 for exploring large Arabidopsis thaliana data sets from the kilometre to nanometre scales. In the past four years we have used the ePlant framework to develop ePlants for 15 agronomically-important species: maize, poplar, tomato, Camelina sativa, soybean, potato, barley, Medicago truncatula, eucalyptus, rice, willow, sunflower, Cannabis sativa, wheat and sugarcane. We also updated the interface to improve performance and accessibility, and added two new views to the Arabidopsis ePlant - the Navigator and Pathways viewers. The former shows phylogenetic relationships between homologs in other species and their expression pattern similarities, with links to view data for those genes in the respective ePlants. The latter shows Plant Reactome metabolic reactions. We also describe new Arabidopsis data sets including single cell RNA-seq data from roots, and how to embed ePlant eFP expression pictographs into any web page.

bioinformatics↗

bioRxiv2021

Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis

Identification of autophagic protein cargo in plants by their abundance in autophagy related genes (ATG) mutants is complicated by changes in both protein synthesis and protein degradation. To detect autophagic cargo, we measured protein degradation rate in shoots and roots of Arabidopsis atg5 and atg11 mutant plants. These data show that less than a quarter of proteins changing in abundance are probable cargo and revealed roles of ATG11 and ATG5 in degradation of specific cytosol, chloroplast and ER-resident proteins, and a specialized role for ATG11 in degradation of proteins from mitochondria and chloroplasts. Our data support a role for autophagy in degrading glycolytic enzymes and the chaperonin containing T-complex polypeptide-1 complex. Autophagy induction by Pi limitation changed metabolic profiles and the protein synthesis and degradation rates of atg5 and atg11 plants. A general decrease in the abundance of amino acids and increase in several secondary metabolites in autophagy mutants was consistent with altered catabolism and changes in energy conversion caused by reduced degradation rate of specific proteins. Combining measures of changes in protein abundance and degradation rates, we also identify ATG11 and ATG5 associated protein cargo of low Pi induced autophagy in chloroplasts and ER-resident proteins involved in secondary metabolism. Single Sentence SummaryProtein cargo of autophagy in plants can be discovered by identifying proteins that increase in abundance and decrease in degradation rate in mutants deficient in autophagy machinery

plant biology↗

bioRxiv2021

Autophagy promotes photomorphogenesis during seedling development in Arabidopsis in carbon limiting conditions

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation condition and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12h post illumination. We investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in photomorphogenesis. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected photomorphogenesis. Moreover, we showed that lower levels of carbon resources exacerbated the delay in photomorphogenesis whereas higher levels of carbon resources had an opposite effect. This work provides evidence that autophagic process operate during de-etiolation in a manner that contributes to photomorphogenesis through increasing lipid turnover to physically or energetically sustain photomorphogenesis.

plant biology↗

bioRxiv2021

Large scale measurement of protein synthesis rates over the diurnal cycle reveal pathway specific regulation of translation

Plants have a diurnal separation of metabolic fluxes and a need for differential maintenance of protein machinery in the day and night. To directly assess aggregate protein translation and degradation for specific proteins and to estimate the ATP investment involved, the individual rates of synthesis and degradation of hundreds of different proteins were measured in Arabidopsis thaliana rosettes. This quantification of translation control through incorporation of heavy hydrogen into newly synthesised protein confirmed that most protein synthesis occurs during the day hours (~3:1 day:night), but revealed it was highly divergent across functional categories. Proteins involved in photosynthesis, especially components of the light harvesting complexes, were synthesized much faster in the day (~10:1), while the protein components of carbon metabolism and vesicle trafficking were translated at similar rates day or night. Comparison of aggregate translation with a range of comparable studies using polysomal loading of transcripts or transcript abundance suggests that translational control is a major contributor to effective gene expression over the diurnal cycle. Diurnal protein degradation rate was observed to be tightly coordinated with protein synthesis rate as few leaf proteins changed in abundance despite reduced translation rates during the night. The direct, quantitative and aggregate analysis of protein synthesis provides an integration of transcriptional, post-transcriptional and translational control of leaf proteome homeostasis and an opportunity to assess the ATP investments involved. The data reveals how the pausing of photosystem synthesis and degradation at night allows the redirection of a decreased energy budget to a selective night-time maintenance schedule. One sentence summaryAdvances in collection and processing of stable isotope incorporation data has allowed characterisation of differential protein synthesis rates over the diurnal cycle which reveal both global and specific translational regulation programs of core biochemical pathways.

plant biology↗

bioRxiv2021

In vivo homopropargylglycine incorporation enables nascent protein tagging, isolation and characterisation from Arabidopsis thaliana

Determining which proteins are actively synthesised at a given point in time and extracting them for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables BONCAT (Bio-Orthogonal Non-Canonical Amino acid Tagging) of proteins being synthesised in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met-sites throughout protein AA sequences and correlation with independent studies of protein labelling with 15N verified the data. We provide evidence that HPG-based BONCAT tags nascent plant proteins more efficiently than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that AHAs induction of Met metabolism and greater inhibition of cell growth rate than HPG likely limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for determining which plant proteins are being synthesised at a given time point and enriches new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of PTMs in plants.

plant biology↗