Mitochondria of seeds face the challenge of remaining quiescent over long periods and rapidly resuming their respiratory function once environmental conditions become favorable. Despite the fundamental importance of mitochondrial functions for germination, the composition and functionality of seed mitochondria remain poorly understood. Previous work has suggested that dry seeds contain immature promitochondria largely devoid of cristae or respiratory complexes and that key mitochondrial functions need to be re-established by rebuilding a functional proteome to support germination. Here, we examined the onset of respiration in the germination of several plant species and investigated the composition of Arabidopsis seed mitochondria using cryo-preservation-based transmission electron microscopy, affinity- and centrifugation-based mitochondrial isolations, native gel electrophoresis, and advanced proteomic and protein localization analyses. Our data reveal pre-formed cristae, the full set of respiratory complexes in the mitochondria of dry seeds, as well as the presence of proteins for all key functions that mitochondria fulfill in vegetative tissues. While the overall protein composition of Arabidopsis seed mitochondria is similar to that in other developmental stages and dark-grown cell cultures, it deviates particularly strongly in selected proteoforms and yet-unassigned candidate mitochondrial or mitochondria-associated proteins. Our findings establish that Arabidopsis seed mitochondria are preserved in a structurally and functionally competent state, accounting for the immediate activation of respiration that occurs upon seed rehydration. They further expand our understanding of mitochondrial plasticity by assignment of novel proteins to the plant mitochondrial proteome, laying the foundation for future investigations of their potential significance in desiccation tolerance and metabolic regulation.

Abstract Seed longevity is a key determinant of crop establishment, productivity, and germplasm conservation. During storage and germination, reactive oxygen species accumulate and contribute to seed aging through oxidative damage and loss of viability. The maintenance of redox homeostasis therefore relies on NADPH-dependent antioxidant systems, which require a continuous supply of reducing power. NADP-dependent malic enzyme 1 (NADP-ME1), represents a source of NADPH supporting antioxidant defense during seed aging. Here, we show that enhanced expression of NADP-ME1 positively contributes to seed vigor and longevity in Arabidopsis thaliana . NADP-ME1 overexpression lines exhibited faster germination and higher overall germination after accelerated aging, whereas knockout mutants showed markedly reduced germination performance. Enhanced post-aging vigor in the overexpression lines was associated with reduced oxidative damage as indicated by lower malondialdehyde and hydrogen peroxide accumulation, along with preservation of specific polyunsaturated fatty acids, and increased γ-tocopherol levels in aged dry seeds. Enhanced expression of NADP-ME1 reshapes the transcriptome of germinated seeds under fresh conditions compared with the wild type, while only minimal differences between genotypes are detected in aged seeds. These results suggest that NADP-ME1 contributes to the establishment of a transcriptional state associated with enhanced seed vigor and improved post-aging germination. Finally, co-immunoprecipitation coupled to mass spectrometry and bimolecular fluorescence complementation identified aspartate aminotransferase 2 as a NADP-ME1 interactor, pointing to a link between malate metabolism and amino acid-related metabolic adjustment. Together, these results identify NADP-ME1 as a determinant of seed resilience to aging and a potential target for improving seed quality.

The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum-members of the same C4 lineage-underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis.

Plant structural biology is entering a new era. Advances in cryo-electron microscopy, tomography, and AI-based prediction are making it possible to study plant macromolecular machines at near-atomic resolution, including complexes that long resisted analysis by traditional approaches. Yet, despite these developments, plant proteins remain underrepresented in structural databases, reflecting persistent challenges in sample preparation, in situ imaging, and capturing dynamics. At the same time, plants present unique opportunities for structural biology, from the photosynthetic apparatus and cellulose synthase rosettes to receptor-like kinases, resistosomes, and plastid protein import machinery. Understanding these systems requires not only technical innovation but also conceptual shifts toward structural landscapes that capture molecular heterogeneity across time, space, and environmental conditions. Here, we outline the main frontiers for the field: improving sample preparation pipelines, advancing in situ and time-resolved methods, integrating structural biology with omics, and harnessing computational modeling. We highlight biological questions where structural insights are most urgently needed, including photosynthesis, hormone signaling, cell wall synthesis, organelle biology, and immunity. We argue that investment in infrastructure, training, and collaborative networks is essential if plant structural biology is to realize its potential. By revealing the molecular logic of the green world, the field can contribute solutions to urgent challenges in agriculture, sustainability, and climate resilience.

Abstract Malate is a central metabolite in plant energy metabolism and biosynthesis and serves as a major carrier of carbon and reducing equivalents between chloroplasts, the cytosol, and mitochondria. However, how individual malate-converting systems contribute to physiology in specific subcellular compartments remains incompletely understood. Here, we investigated the impact of combined loss of mitochondrial malate dehydrogenase (MDH) and NAD-dependent malic enzyme (NAD-ME) activity in Arabidopsis thaliana by integrating reverse genetics, physiological analyses, transcriptomics, quantitative proteomics, and metabolite profiling. Specifically, we generated triple mutants ( mdh1xme1xme2 ) lacking the predominant mitochondrial isoform MDH1 together with both NAD-ME subunits, thereby reducing overall mitochondrial malate conversion capacity. By growing plants under contrasting photoperiod and irradiance regimes to vary photosynthetic demand on malate-linked fluxes, we uncovered a conditional phenotype that was most pronounced under short-day/low-light conditions. Under these conditions, mdh1xme1xme2 exhibited impaired growth and photosynthetic performance, accompanied by cytosolic redox imbalance and altered chloroplast ultrastructure. Transcriptomic profiling revealed that low light unmasks a dawn-phase bottleneck in establishing photosynthetic and redox homeostasis. Consistent with this, the low-light plastid proteome revealed a reallocation away from chloroplast translation and photosynthetic capacity toward proteome maintenance, photoprotection/repair, and iron/ROS management, consistent with a protective acclimation state that nevertheless constrains carbon gain under energy limitation. Low light also triggered C/N imbalance and ammonium accumulation in the mutants. In contrast, increasing irradiance or extending the photoperiod largely alleviated these defects. Together, our results identified mitochondrial malate conversion capacity as a key control point coupling respiratory energy supply and redox homeostasis to photosynthetic metabolism when photosynthetic energy input is limiting.

NADP-dependent malic enzyme (NADP-ME) has been repeatedly co-opted into distinct metabolic roles across plants, most prominently as the decarboxylase of NADP-ME-type C4 photosynthesis. In maize, the plastidic C4- and nonC4-NADP-ME isoforms are closely related in sequence yet display strikingly different catalytic properties, suggesting that small changes in ligand recognition can re-tune reaction chemistry. However, mechanistic interpretation has been hampered by the scarcity of plant NADP-ME structures captured in catalytically informative, ligand-bound states. Here, we integrate X-ray crystallography with structure-guided docking and atomistic molecular dynamics (MD) to resolve ligand-site interaction networks across reaction states. We determined a 2.55 Å structure of maize plastidic nonC4-NADP-ME bound to NADP+, pyruvate, and Mg2+, revealing a conserved NADP-ME fold with localized active-site flexibility. Comparison with maize C4-NADP-ME uncovers isoform-specific rewiring of NADP+ and pyruvate contacts, with the nonC4 enzyme forming a denser product-cofactor interaction network. To access substrate-bound states, we reconstructed malate-NADP+-Mg2+ complexes by docking followed by MD, identifying distinct malate-Mg2+ coordination geometries and alternative NADP+ positioning between isoforms. Together, these structures and simulations provide a network-level framework for plastidic NADP-ME functional diversification and generate testable hypotheses for how ligand coordination drives isoform-specific catalysis.

Photosynthesis provides energy and organic substrates to most life. In plants, photosynthesis dominates chloroplast physiology but represents only a fraction of the tightly interconnected metabolic network that spans the entire cell. Here, we explore how photosynthetic activity affects energy physiology within and beyond the chloroplast. We developed a new standard for the live-monitoring of subcellular energy physiology by combining confocal imaging of genetically encoded fluorescent protein biosensors with advanced on-stage illumination technology to investigate pH, MgATP[2][1]- and NADH/NAD+ dynamics at dark-light transitions in Arabidopsis mesophyll cells. Our findings reveal a stromal alkalinization signature induced by photosynthetic proton pumping, extending to the cytosol and mitochondria as an ’alkalinization wave’. Photosynthesis leads to increased MgATP[2][1]- levels in both the stroma and cytosol. Additionally, we observed reduction of the NAD pool driven by photosynthesis-derived electron export. Arabidopsis lines defective in chloroplast NADP- and mitochondrial NAD-dependent malate dehydrogenases show more reduced cytosolic NAD redox status even in darkness, highlighting the involvement of chloroplasts and mitochondria in shaping cytosolic redox metabolism via malate metabolism. Our study sets a novel methodological standard for precision live-monitoring of photosynthetic cell physiology. Applying this technology reveals signatures of photosynthetic physiology within and beyond the chloroplast with unprecedented resolution. Those signatures link photosynthetic activity and the fundamental biochemical functions of phototrophic cells. Significance statement By applying novel live microscopy monitoring using fluorescent protein biosensors in plant cells, we reveal that dark-light transitions trigger profound re-orchestration of subcellular pH, ATP and NAD redox physiology not limited to chloroplasts but extending into the cytosol and the mitochondria. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 386512654, 507704013, 289357231 China Scholarship Council, 202006910019 [1]: #ref-2

Plant glycolysis and the tricarboxylic acid (TCA) cycle are key pathways of central carbon metabolism. They facilitate energy transformation, provide redox balance, and supply the building blocks for biosynthetic processes that underpin plant survival, growth, and productivity. Yet, rather than acting as static pathways, the fluxes that are mediated by the enzymes involved form a branched network. Flux modes can change flexibly to match cellular demands and environmental fluctuations. Several of the enzymes involved in glycolysis and the TCA cycle have been identified as targets of posttranslational modifications (PTMs). PTMs can act as regulators to facilitate changes in flux by rapidly and reversibly altering enzyme organization and function. Consequently, PTMs enable plants to rapidly adjust their metabolic flux landscape, match energy and precursor provision with the changeable needs, and enhance overall metabolic flexibility. Here, we review the impact of different PTMs on glycolytic and TCA cycle enzymes, focusing on modifications that induce functional changes rather than the mere occurrence of PTMs at specific sites. By synthesizing recent findings, we provide a foundation for a system-level understanding of how PTMs choreograph the remarkable flexibility of plant central carbon metabolism.

Reactive carbonyl species (RCS) are toxic byproducts of normal metabolism that become more prevalent under oxidative stress. Here, we show that Arabidopsis thaliana ecotypes exhibit natural variation in their ability to detoxify glucose-derived RCS. We identified the IP-Pal-0 ecotype showing enhanced tolerance to glucose-derived RCS via upregulation of the glyoxalase system. In particular, the Viridiplantae-specific isoforms GLXI;2, GLXII;4, and GLXII;5 are highly expressed in IP-Pal-0 when plants are grown in the presence of 2-keto-D-glucose (KDG; glucosone) or methylglyoxal, and protein extracts from these plants display enhanced GLXI activity on KDG than other ecotypes. We identified specific motif/cis-regulatory elements in the GLXI;2 promoter regions of Col-0 and IP-Pal-0 that may underlie the differential expression of GLXI;2 associated with KDG detoxification. IP-Pal-0 GLXI;2 contains two different amino acids compared to Col-0, but these do not affect the basic kinetics of the protein. Interestingly, we found that the simultaneous change of these amino acids also occurs together in the GLXI proteins of some other organisms, suggesting a convergence in the simultaneous change of both amino acid residues. Our findings underscore the importance of Viridiplantae-specific glyoxalase isoforms in detoxifying glucose-derived RCS, particularly KDG, and highlight the promise of harnessing natural genetic diversity in the glyoxalase pathway to enhance plant stress tolerance.

Advancements in structural biology have significantly deepened our understanding of plant proteins, which are central to critical biological functions such as photosynthesis, metabolism, signal transduction, and structural architechture. Gaining insights into their structures is crucial for unraveling their functions and mechanisms, which in turn has profound implications for agriculture, biotechnology, and environmental sustainability. Traditional methods in protein structural biology often fall short in addressing large protein assemblies and membrane proteins, and, in particular the dynamics and structural features of proteins in the native cellular context. This paper explores how next-generation technologies are transforming the field of plant protein structural biology, offering powerful tools to overcome longstanding obstacles and enabling remarkable scientific breakthroughs. Key technologies discussed include advanced X-ray crystallography, Cryo-Electron microscopy, Nuclear Magnetic Resonance spectroscopy, Cross-linking mass spectrometry, and Artificial Intelligence-driven approaches. These technologies are examined in terms of their challenges, innovations, and application with particular emphasis on their relevance to plant systems. Future directions in plant protein structural biology are also discussed. Although technical details are not covered in depth, readers are referred to the primary literature for more comprehensive information.

The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions. ### Competing Interest Statement The authors have declared no competing interest.

Cytokinins (CKs) are phytohormones structurally similar to purines that play important roles in various aspects of plant physiology and development. The local and long-distance distribution of CKs is very important to control their action throughout the plant body. Over the past decade, several novel CK transporters have been described, many of which have been linked to a physiological function rather than simply their ability to transport the hormone in vitro. Purine permeases, equilibrative nucleotide transporters and ATP-binding cassette transporters are involved in the local and long-range distribution of CK. In addition, members of the Arabidopsis AZA-GUANINE RESISTANT (AZG) protein family, AZG1 and AZG2, have recently been shown to mediate CK uptake at the plasma membrane and endoplasmic reticulum. Despite sharing ∼50% homology, AZG1 and AZG2 have unique transport mechanisms, tissue-specific expression patterns, and subcellular localizations that underlie their distinct physiological functions. AZG2 is expressed in a small group of cells in the overlying tissue around the lateral root primordia, where its expression is induced by auxins and it is involved in the regulation of lateral root growth. AZG1 is ubiquitously expressed, with high levels in the division zone of the root apical meristem. Here, it binds and stabilises the auxin efflux carrier PIN1, thereby shaping root architecture, particularly under salt stress. This review highlights the latest findings on the protein properties, transport mechanisms and cellular functions of this new family of CK transporters and discusses perspectives for future research in this field.

Photosynthesis-the conversion of energy from sunlight into chemical energy-is essential for life on Earth. Yet there is much we do not understand about photosynthetic energy conversion on a fundamental level: how it evolved and the extent of its diversity, its dynamics, and all the components and connections involved in its regulation. In this commentary, researchers working on fundamental aspects of photosynthesis including the light-dependent reactions, photorespiration, and C4 photosynthetic metabolism pose and discuss what they view as the most compelling open questions in their areas of research.

Drought is a major threat to food security. Water loss through stomata is an inevitable consequence of CO2 uptake, and water deficit inhibits plant growth, making it challenging to develop drought-resistant strategies without compromising yield. Here, we generated tobacco plants expressing a maize NADP-dependent malate decarboxylating enzyme in stomata and vascular cells (ME plants), which show higher seed yield and faster maturation compared to wild-type (WT) plants under normal irrigation and after drought. While WT plants die after 45 days of drought, ME plants survive without any adverse effects on seed production. In addition, ME plants exhibit improved photosynthetic efficiency despite reduced stomatal conductance and changes in stem morphology, which are likely related to their ability to withstand drought. We propose that enhanced C4-like biochemistry in cells surrounding the vascular system and increased sugar export likely compensated for the reduced stomatal conductance in ME plants. The study demonstrates that cell-targeted metabolic modifications can avoid pleiotropic effects and facilitate the stacking of beneficial traits to improve crop design. Significance Statement Drought is one of the biggest threats to global food security, and its impact on crop yield is expected to worsen due to climate change. Traditionally, drought resistance has often come at the expense of yield, creating a negative trade-off. However, we present here a promising solution to this challenge. We have developed a novel approach that successfully uncouples the negative balance between drought resistance and yield. By introducing a maize enzyme into specific tobacco cells, we have created drought-resistant plants with faster growth and higher seed yield. Most importantly, after prolonged drought, while the wild type dies, the modified plants maintain their high yield. This technology paves the way for greater food security and resilience to climate change. ### Competing Interest Statement The authors have declared no competing interest.

The flexibility of plant growth, development and stress responses is choreographed by an intricate network of signaling cascades and genetic programs. However, it is metabolism that ultimately executes these programs through the selective delivery of specific building blocks and energy. Photosynthetic carbon fixation is the central pillar of the plant metabolic network, the functioning of which is conditioned by environmental fluctuations. Hence, regulation of carbon assimilation metabolism must be particularly versatile and rapid to maintain efficiency and avoid dysfunction. While changes in gene expression can adjust the global inventory and abundance of relevant proteins, their specific characteristics are dynamically altered at the post-translational level. Here we highlight studies that show the extent of the regulatory impact by post-translational modification (PTM) on carbon assimilation metabolism. We focus on examples for which there has been empirical evidence of functional changes associated with a PTM, rather than just the occurrence of PTMs at specific sites in proteins, as regularly detected in proteomic studies. The examples indicate that we are only at the beginning of deciphering the PTM-based regulatory network that operates in plant cells. However, it is becoming increasingly clear that targeted exploitation of PTM engineering has the potential to control the metabolic flux landscape as a prerequisite for increasing crop yields, modifying metabolite composition, optimizing stress tolerance, and even executing novel growth and developmental programs.

Reactive carbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are highly reactive, unwanted side-products of cellular metabolism maintained at harmless intracellular levels by specific scavenging mechanisms.MGO and GO are metabolized through the glyoxalase (GLX) system, which consists of two enzymes acting in sequence, GLXI and GLXII. While plant genomes encode a number of different GLX isoforms, their specific functions and how they arose during evolution are unclear. Here, we used Arabidopsis (Arabidopsis thaliana) as a model species to investigate the evolutionary history of GLXI and GLXII in plants and whether the GLX system can protect plant cells from the toxicity of RCS other than MGO and GO. We show that plants possess two GLX systems of different evolutionary origins and with distinct structural and functional properties. The first system is shared by all eukaryotes, scavenges MGO and GO, especially during seedling establishment, and features Zn2+-type GLXI proteins with a metal cofactor preference that were present in the last eukaryotic common ancestor. GLXI and GLXII of the second system, featuring Ni2+-type GLXI, were acquired by the last common ancestor of Viridiplantae through horizontal gene transfer from proteobacteria and can together metabolize keto-D-glucose (KDG, glucosone), a glucose-derived RCS, to D-gluconate. When plants displaying loss-of-function of a Viridiplantae-specific GLXI were grown in KDG, D-gluconate levels were reduced to 10%-15% of those in the wild type, while KDG levels showed an increase of 48%-67%. In contrast to bacterial GLXI homologs, which are active as dimers, plant Ni2+-type GLXI proteins contain a domain duplication, are active as monomers, and have a modified second active site. The acquisition and neofunctionalization of a structurally, biochemically, and functionally distinct GLX system indicates that Viridiplantae are under strong selection to detoxify diverse RCS.

Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light-dependent post-translational modifications (PTM) and have been studied at depth at the level of individual proteins. In contrast, a global picture of the light-dependent PTMome dynamics is lacking, leaving the response of a large proportion of cellular function undefined. Here, we investigated the light-dependent metabolome and proteome changes in Arabidopsis rosettes in a time resolved manner to dissect their kinetic interplay, focusing on phosphorylation, lysine acetylation, and cysteine-based redox switches. Of over 24 000 PTM sites that were detected, more than 1700 were changed during the transition from dark to light. While the first changes, as measured 5 min after onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin-Benson cycle and the synthesis of sugars at later timepoints. Our data reveal connections between metabolism and PTM-based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insight into the extent by which photosynthesis reprograms global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function.

Reactive carbonyl species (RCS) are highly toxic molecules produced during normal metabolism and increased under conditions of oxidative stress. Here we show that Arabidopsis thaliana ecotypes exhibit natural variation in the degree of detoxification of RCS. We have isolated the ecotype IP-Pal-0 as an ecotype that has evolved a higher resistance to the toxic effects of glucose-derived RCS by acquiring a higher activity of the glyoxalase system through increased expression of some of its components. In particular, Viridiplantae-specific GLXI;2, GLXII;4 and GLXII;5 isoforms are highly expressed when plants are grown in the presence of 2-keto-D-glucose (KDG; glucosone) or methylglyoxal. We found that specific motif/ cis -regulatory elements of the Col-0 and IP-Pal-0 GLXI;2 promoter regions may be involved in the differences in GLXI;2 gene expression associated with KDG detoxification. IP-Pal-0 GLXI;2 contains two different amino acids compared to Col-0, but these do not affect the basic kinetics of the protein. Interestingly, we found that the simultaneous change of these amino acids also occurs together in the GLXI proteins of some other organisms, suggesting a convergence in the simultaneous change of both amino acid residues. Our study of natural variants of A. thaliana suggests that the Viridiplantae-specific isoforms of the glyoxalase system are involved in the detoxification of glucose-derived RCS, particularly KDG.### Competing Interest StatementThe authors have declared no competing interest.