γ-Aminobutyric acid (GABA), a nonproteinogenic amino acid first identified in biological systems over 70 years ago, has long been recognized as a metabolic intermediate. More recently, GABA has also been acknowledged as a signaling molecule that couples physiological responses to metabolic status. This review presents a conceptual framework for how metabolism sets GABA concentration and localization, which then modulate ion transport and membrane potential dynamics to influence plant growth, development, and adaptation to stress. We explore the emerging network of GABA's interactions with other signaling pathways, highlighting its involvement in environmental sensing and internal regulatory mechanisms via hormones and reactive oxygen species. These interactions influence key physiological processes including stomatal regulation, pathogen and herbivore defense, root growth, and even the modulation of flavor. Collectively, these findings position GABA as a metabolic signal integrator of plant physiological status and responses, with broad implications for enhancing crop stress resilience and food quality.

Abstract 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. Abstract Figure Graphical abstract: 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).

Enzyme protein turnover accounts for about half the maintenance energy budget in plants. Slowing turnover─i.e., extending the effective working life (Catalytic Cycles till Replacement, CCR)─of short-lived enzymes is thus a rational strategy to conserve energy and carbon and raise crop productivity. Arabidopsis histidinol dehydrogenase (HDH) is a short-lived enzyme that can sustain life-shortening damage from its aminoaldehyde reaction intermediate. We used the yeast OrthoRep continuous directed evolution system in a his4Δ strain to raise cumulative HDH function and, by proxy, lifespan as functional enzymes, by selecting for growth rate while tapering histidinol concentration and escalating that of the inhibitor histamine. Improved HDH variants carried diverse nonsynonymous mutations and ranged 20-fold in level. Improved HDH performance was associated with higher HDH abundance in some cases and with greater catalytic efficiency or histamine resistance in others. These findings indicate that OrthoRep-based directed evolution can extend enzyme working life in vivo in addition to, as expected, altering kinetic properties.

State transitions in algae and higher plants involve the phosphorylation and dephosphorylation of light-harvesting complex II, which are regulated by the kinase STN7 and phosphatase TAP38/PPH1. We systematically measured thylakoid protein turnover in stn7-1 and tap38-1 Arabidopsis (Arabidopsis thaliana) mutants, which are locked in permanent State I and State II, respectively. Although the abundance of photosynthetic proteins such as D1, LHCII, and PSAB remained unchanged, their turnover rates were antagonistically altered compared to wild-type plants. These changes correlated with differences in excitation energy distribution and electron transport rates. Our findings reveal that the thylakoid state minimally affects protein abundance but markedly impacts protein complex maintenance by altering protein turnover. This provides insights into the costs and benefits of thylakoid state transitions in plants, the impact of excitation energy distribution between photosynthetic electron transport pathways on their maintenance, and a potential method for quantifying long-term energy distribution changes.

Image-based plant phenotyping involves the quantitative determination of complex plant traits using image analysis. One important parameter to assess is the degree of greenness of photosynthetic tissues, as it may reflect plant health, development, or the pigment-depleting impact of stressful environments. Various attempts have been made to quantify leaf greenness scores, but each has shown restricted utility and efficacy. Often, these methods overlooked the precision needed to represent greenness differences. Here, we developed an improved method, the 'Green Index' (GI), to quantitatively score the greenness of photosynthetic tissues and track smooth transitions in seedling greening during de-etiolation. GI is open-source, uses widely available RGB values from image pixels, and does not require advanced computational skills (available at www.foodandplantbiology.com). We describe the conception of the GI formula and evaluate its superiority over existing methods using both literature-derived and new datasets. Furthermore, we demonstrated the utility of the GI in addressing common issues encountered in assessing plant phenotype in biology experiments, underscoring its potential as a reliable and accessible tool. Based on greenness, GI quantitatively discriminates leaf health, developmental stages, and stress sensitivity. We also report that GI significantly correlates with chlorophyll content, and can thus serve as a proxy for tracking chlorophyll trends.

Salinity stress in wheat affects physiological and biochemical parameters in tissues that alter plant development and ultimately lower crop yield. Shoot tissues can accumulate high concentrations of sodium over time through the transpiration stream coming from the roots. This imposes physiological responses that align salt effects with the basipetal developmental gradient of the monocot leaf. The role of metabolic processes in generating and responding to these increases in sodium concentration over time was explored by linking changes in ion distributions to those of enzyme abundance from the base to the tip of leaves under salt stress. We found that enzymes for methionine synthesis and lipid degradation pathways increase, concomitantly with proteins in jasmonate synthesis, which are key players in plant stress-induced responses. Combining the use of Differential Abundance of Protein analysis and Weighted Correlation Network Analysis we have focused on identifying key protein hubs associated with responses to salt stress or salt susceptibility, shedding light on potential sites of salt sensitivity as targets for enhancing salt tolerance in wheat. We found chloroplast protein synthesis machinery, including the 30S and 50S ribosomal proteins, and plastid localised protein synthesis elongation factors, were significantly reduced in abundance and correlated with the altered K+/Na+ ratio along salt-stressed wheat leaves. Additionally, the plastid protease system including ATP-dependent caseinolytic protease and filamentous temperature-sensitive H proteases involved in chloroplast protein homeostasis, show decreased abundance with salt. The complex interplay of these processes in and across the leaf affects overall plant viability under salt stress mainly affecting the energy homeostasis in wheat shoot. Data are available via ProteomeXchange with identifier PXD059765. SIGNIFICANCE: Soil salinity is a major agricultural challenge that cause significant reduction in wheat yields, a staple crop vital for global food security. Despite extensive breeding efforts, developing salt-tolerant wheat remains challenging due to the complex, multi-genic nature of salinity tolerance. While numerous studies have explored molecular responses to salt stress making salt to control comparisons, there is little consensus on the primary points of metabolic disruptions that would determine the salt response in wheat. Our study addresses this gap by integrating proteomics with Weighted Correlation Network Analysis to examine metabolic responses along the developmental gradient of wheat leaves. By exploiting the natural base-to-tip progression of leaf maturation under salt stress, we identify key protein groups linked to salt response. These findings provide new insights into potential metabolic targets for enhancing wheat's resilience to salinity stress.

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 DCMU 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 system's 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.

Target of rapamycin (TOR) kinase is the hub of a eukaryotic master signaling network that integrates nutritional and hormonal signals into cellular activities. Most studies on TOR in plants have focused on seedlings, where TOR is most responsive to light and sucrose. Here, we observed differences in nutrient regulation of TOR across plant tissues. Biochemical analyses highlighted the predominance of Gln-TOR signaling in mature Arabidopsis leaves and developing pea seeds, and its integration with hormone signaling and amino acid metabolism. Phosphoproteomic and transcriptomic analysis of developing pea seeds identified established and novel components of TOR signaling, which were enriched for proteins/genes regulating gene expression and autophagy. Unexpectedly, Gln-TOR signaling in pea embryos inhibited or delayed growth and protein accumulation during seed filling. A developmental profile was evident wherein high TOR activity and Gln levels during pea cotyledon cellularization reduced sharply as embryos progressed to seed filling. We observed strong interactions between TOR and abscisic acid (ABA) signaling such that TOR-inhibited embryos were hypersensitive to ABA-induced protein accumulation. We propose that legume seed storage protein biosynthesis displays atypical regulatory properties because it occurs in the face of increasing desiccation stress and is promoted by ABA signaling rather than TOR signaling.

Continuous directed evolution is a powerful Synthetic Biology tool to engineer proteins with desired functions in vivo. Mimicking natural evolution, it involves repeated cycles of high-frequency mutagenesis, selection, and replication within platform cells, where the function of the target gene is tightly linked to the host cell's fitness. However, cells might escape the selection pressure due to the inherent flexibility of their metabolism, which allows for adaptation. Whole-proteome analysis as well as targeted proteomics offer valuable insights into global and specific cellular changes. They can identify modifications in the target protein and its interactors to help understand its evolution and network integration. Using the continuous evolution of the Arabidopsis thaliana methionine synthases AtMS1 and AtMS2 as an example, we show how mass spectrometry-based proteomics was able to assess the abundance of target enzymes, identify flaws in population construction, measure methionine metabolic adaptation, and allow informed decision-making in the evolution campaign.

Plant protein production systems are scalable and sustainable platforms capable of meeting the growing demand for functional proteins in nutrition, pharmaceuticals, and industry. Recent advances in essential amino acid (EAA) biosynthesis, gene regulation, and subcellular targeting have enhanced protein yields and stability, but are yet to be integrated into holistic engineering approaches. Metabolic engineering can improve amino acid (AA) metabolism and energy efficiency, while genetic engineering enables finetuned, spatiotemporal expression of target proteins. Coupled with in silico tools for protein design, novel proteins with enhanced stability and functionality can be developed. Integrating these strategies would enable the fine-tuning of protein synthesis while balancing cellular energy costs, offering context-dependent opportunities to advance protein production in plant systems.

An approach to improving radiation use efficiency (RUE) in wheat is to screen for variability in rates of leaf respiration in darkness (R-dark). We used a high-throughput system to quantify variation in R-dark among a diverse range of spring wheat genotypes (301 lines) grown in two countries (Mexico and Australia) and two seasons (2017 and 2018), and in doing so quantify the relative importance of genotype (G) and environment (E) in influencing variations in leaf R-dark. Through careful design, residual (unexplained) variation represented <10% of the total observed. Up to a third of the variation in R-dark (and related traits) was under genetic control. This suggests opportunities for breeders to use R-dark as a novel selection tool. In addition, E accounted for more than half of the total variation in area-based rates of R-dark. Here, the day of measurement was crucial, suggesting that day-to-day variations in the environment influence rates of R-dark measured at a common temperature. Overall, this study provides new insights into the role G and E play in determining variation in rates of leaf R-dark of one of the most important cereal crops, with implications for future improvements in carbon use efficiency and yield.

Enzyme protein turnover accounts for about half the maintenance energy budget in plants. Slowing turnover – i.e., extending lifespan – of short-lived enzymes is thus a rational strategy to conserve energy and carbon, and raise crop productivity. Arabidopsis histidinol dehydrogenase (HDH) is a short-lived enzyme that can sustain life-shortening damage from its aminoaldehyde reaction intermediate. We used the yeast OrthoRep continuous directed evolution system in a his4 Δ strain to raise HDH protein abundance (a proxy for lifespan) by selecting for growth rate while tapering histidinol concentration and escalating that of the inhibitor histamine. Improved HDHs carried diverse nonsynonymous mutations and ranged 20-fold in level. Improved HDH performance was associated with higher HDH abundance in some cases and with greater catalytic efficiency or histamine resistance in others. These findings indicate that OrthoRep-based directed evolution can extend enzyme lifespan in vivo in addition to, as expected, altering kinetic properties. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Food and Agriculture, https://ror.org/05qx3fv49, FLA-HOS-005796 C.V. Griffin, Sr. Foundation Australian Research Council, https://ror.org/05mmh0f86, FL200100057 Bioplatforms Australia, https://ror.org/042gz1a70

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. ### Competing Interest Statement The authors have declared no competing interest. Australian Research Council, https://ror.org/05mmh0f86, FL200100057

Wheat is a staple crop crucial for global food security, but its production is significantly affected by salt stress. Exploring natural genetic diversity in wheat can identify ways to improve salt tolerance. We subjected five wheat genotypes: Mocho de Espiga Branca (enhanced tissue tolerance), Fretes (tissue tolerance), Wyalkatchem and Westonia (salt exclusion) and Westonia Nax1 (enhanced salt exclusion), to 150 mM NaCl for 8 days. We measured changes in biomass, photosynthesis, chlorophyll content, Na+/K+ ratios and protein abundance. Mocho maintained growth despite high tissue Na+, showing physiological tolerance supported by differential regulation of mitochondrial proteins, central carbon metabolism, the GABA shunt and compatible solutes. Mitochondrial complexome profiling revealed salt-induced instability of 2-oxoglutarate dehydrogenase complex (OGDC) and a hydroxyglutarate synthase orthologue (HglS). In vitro assays confirmed subtle but significant OGDC activity and stability differences in Mocho, which also retained higher TCA cycle enzyme levels in vivo. Whole-plant treatment with the OGDC inhibitor succinyl phosphonate reproduced salt-like reductions in chlorophyll and biomass, particularly in Mocho. These findings highlight distinct strategies of tissue tolerance and salt exclusion in wheat, emphasising OGDC's role in Mocho's salt tolerance and pointing to metabolic pathways that could improve tissue tolerance traits and support sustainable agriculture.

Loss of Lon1 led to stunted plant growth and accumulation of nuclear-encoded mitochondrial proteins including Lon1 substrates. However, an in-depth label-free proteomics quantification of mitochondrial proteins in lon1 revealed that the majority of mitochondrial-encoded proteins decreased in abundance. Additionally, we found that lon1 mutants contained protein aggregates in the mitochondrial that were enriched in metabolic enzymes, ribosomal subunits and PPR-containing proteins of the translation apparatus. These mutants exhibited reduced general mitochondrial translation as well as deficiencies in RNA splicing and editing. These findings support the role of Lon1 in maintaining a functional translational apparatus for mitochondrial-encoded gene translation. Transcriptome analysis of lon1 revealed a mitochondrial unfolded protein response reminiscent of the mitochondrial retrograde signalling dependent on the transcription factor ANAC017. Notably, lon1 mutants exhibited transiently elevated ethylene production, and the shortened hypocotyl observed in lon1 mutants during skotomorphogenesis was partially alleviated by ethylene inhibitors. Furthermore, the short root phenotype was partially ameliorated by introducing a mutation in the ethylene receptor ETR1. Interestingly, the upregulation of only a select few target genes was linked to ETR1-mediated ethylene signalling. Together this provides multiple steps in the link between loss of Lon1 and signalling responses to restore mitochondrial protein homoeostasis in plants.

EARLY NODULIN 93 (ENOD93) has been genetically associated with biological nitrogen fixation in legumes and nitrogen use efficiency in cereals, but its precise function is unknown. We show that hidden Markov models define ENOD93 as a homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 regulates cytochrome oxidase (CIV), influencing the generation of a mitochondrial proton motive force in yeast (Saccharomyces cerevisiae). Knockout of ENOD93 in Arabidopsis (Arabidopsis thaliana) causes a short root phenotype and early flowering. ENOD93 is associated with a protein complex the size of CIV in mitochondria, but neither CIV abundance nor its activity changed in ruptured organelles of enod93. However, a progressive loss of ADP-dependent respiration rate was observed in intact enod93 mitochondria, which could be recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 in a CIV-dependent manner, but ATP synthesis and ADP depletion rates progressively decreased. The respiration rate of whole enod93 seedlings was elevated, and root ADP content was nearly double that in wild type without a change in ATP content. We propose that ENOD93 and HYPOXIA-INDUCED GENE DOMAIN 2 (HIGD2) are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded by 2 distinct genes. The plant homolog of a yeast component of the mitochondrial oxidative phosphorylation system plays a role in plant ATP production.

In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (gbM). gbM is correlated with ubiquitously expressed genes; however, its function, or absence thereof, is highly debated. The different outputs that mC can have raise questions as to how it is interpreted—or read—differently in these sequence and genomic contexts. To screen for potential mC-binding proteins, we performed an unbiased DNA affinity pull-down assay combined with quantitative mass spectrometry using methylated DNA probes for each DNA sequence context. All mC readers known to date preferentially bind to the methylated probes, along with a range of new mC-binding protein candidates. Functional characterization of these mC readers, focused on the MBD and SUVH families, was undertaken by ChIP-seq mapping of genome-wide binding sites, their protein interactors, and the impact of high-order mutations on transcriptomic and epigenomic profiles. Together, these results highlight specific context preferences for these proteins, and in particular the ability of MBD2 to bind predominantly to gbM. This comprehensive analysis ofArabidopsismC readers emphasizes the complexity and interconnectivity between DNA methylation and chromatin remodeling processes in plants.

In contrast to its close homolog PLANT UNCOUPLING MITOCHONDRIAL PROTEIN 1 (UCP1), which is an abundant carrier protein in the mitochondria, UCP2 localizes to the Golgi.

Chickpea (Cicer arietinum L.), a rainfed crop in semi-arid regions, experiences frequent intermittent drought stress, hindering growth. This study explored morpho-physiological and molecular responses of six chickpea genotypes (ICC 4958, BG 4005, Pusa 362, BGM 10218, RSG 888, ICC 1882) under terminal drought to enhance yield and adaptability. Drought stress was imposed during early podding in a controlled glasshouse experiment. Various physiological, root growth, fertility, and yield parameters were measured. Proteomics analysis was conducted on leaf, pod, and root tissue samples, collected at similar to 0.3 fraction of transpirable soil water (FTSW). Significant differences occurred for various traits, including phenologies, water relations, pollen viability and germination, flower and pod numbers, abortion rates, photosynthetic apparatus and pigments, osmotic adjustments, canopy temperatures, root length, and densities, and overall yield and biomass. FTSW dynamics can aid genotype screening during early drought. Characterizing stress-responsive proteins can provide insights into cellular processes and adaptations. Terminal drought affected leaf central shoot metabolism, reducing photosynthetic capacity, while roots and pods reconfigured enzyme abundances to promote root growth and pod development. The extensive genotypic variation in proteome data, morpho-physiological traits, and water-use patterns presents opportunities for developing drought-adapted cultivars.

Spontaneous mutations are rare in mitochondria and the lack of mitochondrial transformation methods has hindered genetic analyses. We show that a custom-designed RNA-binding pentatricopeptide repeat (PPR) protein binds and specifically induces cleavage of ATP synthase subunit1 (atp1) mRNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis (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 and 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 steady-state levels of numerous amino acids, most notably of the serine family. The results show the value of using custom-designed PPR proteins to influence the expression of specific mitochondrial transcripts to carry out reverse genetic studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis.