Abstract Reactive oxygen species (ROS) produced by plant NADPH oxidases (RBOHs) must be precisely controlled in their concentration and spatial distribution to support diverse developmental and stress responses. RBOHs are activated by Ca 2+ binding and phosphorylation, yet how internal regulatory domains within RBOHs have evolved to translate these inputs into precise levels of ROS production remains unclear. To address this, we performed phylogenetic analyses to define RBOH subfamilies and identify protein regions underlying functional diversification. This analysis revealed that the most variable regions across land-plant RBOHs are two unstructured regions in the N-terminus, UR1 and UR2, which flank the EF-hand Ca 2+ -binding domain (EFD). We dissected the roles of these regions in Arabidopsis RBOHD, which is central to plant immunity, and in RBOHH, which drives pollen tube elongation and exhibits high Ca 2+ -induced ROS production. Our analyses revealed that UR1 plays opposing roles in these RBOHs: in RBOHD, UR1 functions as an autoinhibitory module that restrains Ca 2+ -mediated activation, whereas in RBOHH, UR1 is essential for Ca 2+ -dependent activation and has coevolved with the EFD to maximize Ca 2+ -induced ROS production. We further uncovered divergent regulatory roles for UR2. In RBOHD, but not in RBOHH, phosphorylation of UR2 stabilizes an α-helical conformation that promotes interaction with the catalytic domain required for enzymatic activation. Furthermore, unlike in RBOHH, the EFD of RBOHD has coevolved with UR2 to maximize phosphorylation-induced activity. Together, our results show how evolution of unstructured regulatory regions adapts a conserved enzymatic core to distinct demands of immune signaling and polarized growth.

Mitochondria are the centres of carbon and energy metabolism in cells and are functionally integrated with other organelles. Under environmental stress, disturbances in organellar functions trigger stress signals that activate the necessary metabolic responses and maintain cell redox homeostasis. The tricarboxylic acid cycle enzyme aconitase has emerged as a key component in stress-induced organellar signalling and a regulator of metabolic and redox balance in photosynthetic organisms. Aconitase mediates mitochondrial and chloroplast retrograde signalling and contributes to the activation of the alternative oxidase (AOX) pathway in mitochondria. Aconitase-driven citrate metabolism plays a crucial role in providing reducing equivalents and metabolic precursors for cytosolic nitrogen metabolism and biosynthetic pathways relevant for stress acclimation. Besides its enzymatic activity, aconitase has a non-canonical function as it is a post-transcriptional regulator of specific gene transcripts. The varied functions of aconitase under stress are facilitated by the regulation of specific aconitase isoforms at multiple levels. This review discusses the emerging role of aconitase as a central regulator of stress responses and signalling in photosynthetic organisms.

Modified atmosphere packaging (MAP) combined with low temperatures is a proven method for maintaining the quality of fresh-cut lettuce (Lactuca sativa), particularly in relation to cut surface discoloration. However, even though fresh-cut products in retail environments can be influenced by even low levels of photosynthetic light, few studies have focused on the impact of light on MAP products. In this study, we found that constant low light illumination undermines the effectiveness of MAP for fresh-cut lettuce. This is associated with a slight temperature increase in the storage cabinet and photosynthetic gas exchange restoring normal atmospheric gas compositions, accelerating quality loss. These light-induced changes are correlated with transcriptional hallmarks of senescence. In contrast, even with extended storage beyond typical retail standards, these hallmarks were absent in darkness. Instead, there were transcriptional and metabolite profile signatures related to low oxygen and oxylipin metabolism. Furthermore, the phenylpropanoid pathway, which is central to cut-surface discoloration, appeared to be dynamically responsive to lighting and increasing storage time throughout the storage period. In conclusion, this study reveals temporal transcriptomic and metabolomic responses that are highly conditioned by lighting during MAP-storage of fresh-cut lettuce.

Ultraviolet-B radiation (UV-B) is the most-energetic region of the solar spectrum received by plants and its absorption by leaves requires specialised adaptations. Natural light is essentially dynamic, yet the effects of fluctuating UV-B radiation on plant ecophysiology remain poorly understood. We investigated how two grapevine (Vitis vinifera) cultivars, Tempranillo (red) and Viura (white), respond to fluctuating and steady UV-B treatments in a controlled environment, and explored the underlying processes mediating acclimation using photoreceptor mutants of Arabidopsis thaliana. Plants were grown in a greenhouse under the same daily dose of fluctuating or steady UV-B radiation, paired with their respective attenuated UV-B controls. Leaf chlorophyll epidermal flavonols and photosynthetic capacity were monitored in both cultivars as well as photoassimilates in Arabidopsis. Acclimation was cultivar-specific: whereby flavonol accumulation in response to UV-B was greater in Viura (a 28 % increase), although in Tempranillo within-treatment flavonol accumulation was associated with less inhibition of operating efficiency of photosystem II (ɸPSII). In Arabidopsis, responses to both fluctuating and steady UV-B regimes were primarily mediated by the photoreceptor UV RESISTANCE LOCUS 8, with cryptochromes also contributing to flavonol regulation and playing a greater role in photoassimilate accumulation under steady UV-B. Knowledge of these processes across cultivars and conditions can assist in grapevine selection, to guide photoreceptor-centric approaches on the basis of differences in UV-B-radiation acclimation capacity, and to support vineyard management under naturally variable UV-B conditions. These aspects are increasingly important under current global climate change scenarios for such a widespread crop as grapevine.

Kales (Brassica oleracea convar. acephala) are fast-growing, nutritious leafy vegetables ideal for year-round indoor farming. However, selection of the best cultivars for growth under artificial lighting necessitates a deeper understanding of leaf metabolism in different kale types. Here we examined a curly-leaved cultivar, Half Tall, and a lacinato-type cultivar, Black Magic, under moderate light (130 µmol photons m-2 s-1/22 °C) and high light (800 µmol photons m-2 s-1/26 °C) conditions. These conditions induced genotype-dependent differences in nutritionally important metabolites, especially anthocyanins and glucosinolates (GSLs), in kale cultivars. In the pale green Half Tall, growth under high light conditions did not induce changes in either pigmentation or total GSL content. In contrast, the purple pigmentation of Black Magic intensified due to increased anthocyanin accumulation. Black Magic showed reduced contents of indole GSLs and increased contents of aliphatic GSLs under high light conditions, with notable cultivar-specific adjustments in individual GSL species. Correlation analysis of metabolite profiles suggested cultivar-specific metabolic interplay between serine biosynthesis and the production of indole GSLs. RNA sequencing identified candidate genes encoding metabolic enzymes and regulatory components behind anthocyanin and GSL biosynthesis. These findings improve our understanding of leaf metabolism and its effects on the nutritional quality of kale cultivars.

Increasing the availability of fresh vegetables and reducing food waste are essential for healthy and sustainable production. However, fresh-cut vegetables such as lettuce (Lactuca sativa L.) often experience rapid quality loss after harvest and processing. To maintain freshness in retail, modified atmosphere packaging (MAP) with low oxygen (O2) and high carbon dioxide (CO2) concentrations, combined with refrigeration, is commonly used. Packaged leaves are then displayed under dark or low-light conditions. The postharvest physiology of lettuce under these conditions (including changes in energy metabolism, photosynthesis, senescence progression, and light-dependent metabolic alterations) remains poorly understood, limiting the development of strategies to extend shelf life. Using spectroscopic and biochemical approaches, we investigated the physiological changes in fresh-cut lettuce stored in MAP under refrigeration in either darkness or constant low light. Our analysis revealed distinct light-dependent and light-independent adjustments in photosynthesis. MAP triggered rapid and dramatic changes in photosynthetic light reactions, detectable within 1 h of packaging, as observed by pulse amplitude modulated (PAM) and OJIP kinetics assays. These changes were likely associated with compromised electron sink strength in the photosynthetic electron transfer chain and altered dynamics of the energy component of nonphotochemical quenching (qE-NPQ). Notably, these functional alterations coincided with only minor modifications in photosynthetic supercomplexes, as determined by blue native gel electrophoresis. The MAP-induced changes in photosynthesis deteriorated during storage in darkness but were reversed by storage under low light, likely due to photosynthetic gas exchange. Our findings provide new insights into photosynthesis of packaged lettuce and highlight promising physiological readouts for assessing lettuce quality in retail settings.

Studies have uncovered delicate mechanisms that enable plant acclimation to fluctuating light. Translating the knowledge to controlled environment agriculture could advance the development of cost-effective dynamic lighting strategies where the light intensity is purposely alternated, mirroring the spot electricity price, but its effects on vegetable crops remain poorly understood. Here, we recorded photosynthetic parameters, metabolic responses, and growth of lettuce (Lactuca sativa L.) cv. "Katusa" under dynamic lighting. The light intensity was altered at different times of the photoperiod with uniform daily light integral. Three different setups, including a plant phenotyping facility, a small-scale vertical farm testbed and a larger-scale vertical farm, were utilized to address the physiological responses and scalability of lighting strategies. The lettuces readily adjusted their photosynthetic light reactions and carbon metabolism according to the changing light intensities. However, the overall metabolic composition of lettuce leaves did not respond to dynamic lighting. Upon simulation of commercial production in the larger-scale vertical farm, constant and dynamic lighting regimes yielded lettuce heads with equal saleable sizes of 87-89 g, even under artificial "Split-Night" regimes where the photoperiod was interrupted by two periods of darkness. These findings suggest that dynamic lighting strategies enable cost-effective lighting via optimization of electricity use in indoor cultivation.

Environmental stress induces mitochondrial retrograde signals that prompt protective responses in plants. The elusive mitochondrial signal has now been uncovered in a new study, which identifies formation of reactive oxygen species inside mitochondria as the key trigger of stress signals.

Crop productivity depends on the ability of plants to thrive across different growth environments. In nature, light conditions fluctuate due to diurnal and seasonal changes in direction, duration, intensity, and spectrum. Laboratory studies, predominantly conducted with arabidopsis (Arabidopsis thaliana), have provided valuable insights into the metabolic and regulatory strategies that plants employ to cope with varying light intensities. However, there has been less focus on how horticultural crops tolerate dynamically changing light conditions during the photoperiod. In this review we connect insights from photobiology in model plants to the application of dynamic lighting in indoor horticulture. We explore how model species respond to fluctuating light intensities and discuss how this knowledge could be translated for new lighting solutions in controlled environment agriculture.

Plants live in a world of changing environments, where they are continuously challenged by alternating biotic and abiotic stresses. To transfer information from the environment to appropriate protective responses, plants use many different signaling molecules and pathways. Reactive oxygen species (ROS) are critical signaling molecules in the regulation of plant stress responses, both inside and between cells. In natural environments, plants can experience multiple stresses simultaneously. Laboratory studies on stress interaction and crosstalk at regulation of gene expression, imply that plant responses to multiple stresses are distinctly different from single treatments. We analyzed the expression of selected marker genes and reassessed publicly available datasets to find signaling pathways regulated by ozone, which produces apoplastic ROS, and high light treatment, which produces chloroplastic ROS. Genes related to cell death regulation were differentially regulated by ozone versus high light. In a combined ozone + high light treatment, the light treatment enhanced ozone-induced cell death in leaves. The distinct responses from ozone versus high light treatments show that plants can activate stress signaling pathways in a highly precise manner.

Recent developments in targeted mass spectrometry-based proteomics have provided new methodological solutions for accurate and quantitative analysis of proteins and their posttranslational control, which has significantly advanced our understanding of stress responses in different plant species. Instrumentation allowing high-resolution, accurate-mass (HR/AM) analysis has provided new acquisition strategies for targeted quantitative proteomic analysis by targeted selected ion monitoring (tSIM) and parallel reaction monitoring (PRM). Here we report a sensitive and accurate method for targeted analysis of protein phosphorylation by tSIM coupled to PRM (tSIM/PRM). The tSIM/PRM method takes advantage of HR/AM mass spectrometers and benefits from the combination of highly sensitive precursor ion quantification by tSIM and highly confident peptide identification by spectral library matching in PRM. The detailed protocol describes tSIM/PRM analysis of Arabidopsis thaliana foliar proteins, from the building of a spectral library to sample preparation, mass spectrometry, and data analysis, and provides a methodological approach for specifying the molecular mechanisms of interest.

Flooding causes severe crop losses in many parts of the world. Genetic variation in flooding tolerance exists in many species; however, there are few examples for the identification of tolerance genes and their underlying function. We conducted a genome-wide association study (GWAS) in 387 Arabidopsis (Arabidopsis thaliana) accessions. Plants were subjected to prolonged submergence followed by desubmergence, and seven traits (score, water content, Fv/Fm, and concentrations of nitrate, chlorophyll, protein, and starch) were quantified to characterize their acclimation responses. These traits showed substantial variation across the range of accessions. A total of 35 highly significant single-nucleotide polymorphisms (SNPs) were identified across the 20 GWA datasets, pointing to 22 candidate genes, with functions in TCA cycle, DNA modification, and cell division. Detailed functional characterization of one candidate gene, ACONITASE3 (ACO3), was performed. Chromatin immunoprecipitation followed by sequencing showed that a single nucleotide polymorphism in the ACO3 promoter co-located with the binding site of the master regulator of retrograde signaling ANAC017, while subcellular localization of an ACO3-YFP fusion protein confirmed a mitochondrial localization during submergence. Analysis of mutant and overexpression lines determined changes in trait parameters that correlated with altered submergence tolerance and were consistent with the GWAS results. Subsequent RNA-seq experiments suggested that impairing ACO3 function increases the sensitivity to submergence by altering ethylene signaling, whereas ACO3 overexpression leads to tolerance by metabolic priming. These results indicate that ACO3 impacts submergence tolerance through integration of carbon and nitrogen metabolism via the mitochondrial TCA cycle and impacts stress signaling during acclimation to stress.

Mitochondria are tightly embedded within metabolic and regulatory networks that optimize plant performance in response to environmental challenges. The best-known mitochondrial retrograde signaling pathway involves stress-induced activation of the transcription factor NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), which initiates protective responses to stress-induced mitochondrial dysfunction in Arabidopsis (Arabidopsis thaliana). Posttranslational control of the elicited responses, however, remains poorly understood. Previous studies linked protein phosphatase 2A subunit PP2A-B'γ, a key negative regulator of stress responses, with reversible phosphorylation of ACONITASE 3 (ACO3). Here we report on ACO3 and its phosphorylation at Ser91 as key components of stress regulation that are induced by mitochondrial dysfunction. Targeted mass spectrometry-based proteomics revealed that the abundance and phosphorylation of ACO3 increased under stress, which required signaling through ANAC017. Phosphomimetic mutation at ACO3-Ser91 and accumulation of ACO3S91D-YFP promoted the expression of genes related to mitochondrial dysfunction. Furthermore, ACO3 contributed to plant tolerance against ultraviolet B (UV-B) or antimycin A-induced mitochondrial dysfunction. These findings demonstrate that ACO3 is both a target and mediator of mitochondrial dysfunction signaling, and critical for achieving stress tolerance in Arabidopsis leaves.

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.

This article is a Commentary on Jacquot et al. (2020), 228: 1038–1054.

In the version of this article initially published, there was a mistake in the calculation of the nucleotide mutation rate per site per generation: 1 × 10 −9 mutations per site per generation was used, whereas 9.5 × 10 −9 was correct. This error affects the interpretation of population-size changes over time and their possible correspondence with known geological events, as shown in the original Fig. 4 and supporting discussion in the text, as well as details in the Supplementary Note. Neither the data themselves nor any other results are affected. Figure 4 has been revised accordingly. Images of the original and corrected figure panels are shown in the correction notice.

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'gamma is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'gamma regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'gamma to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'gamma is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'gamma depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'gamma age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.