Plant Physiology

Light is essential for plant growth and development. Sustainably feeding a constantly-growing human population will likely involve adapting crop plants to intercropping and high planting density by rational manipulation of light signaling. Here, we edited the tomato (Solanum lycopersicum) genome to generate lines with a light-stable version of ELONGATED HYPOCOTYL 5 (HY5), a master transcription factor involved in the integration of light and hormone signaling. Removing the tomato HY5 N-terminal domain required for interaction with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) prevented light-dependent protein degradation and resulted in a gain-of-function phenotype of short seedlings. Elongation growth was also compromised under proximity shade conditions either simulated by enriching white light (W) with far-red light (W+FR) or achieved by growing plants at a higher density. Transcriptomic analysis of gene expression changes after exposure to W+FR for 24h revealed a reduced number of shade-responsive genes in edited lines compared to unedited, wild-type controls, many of which are related to growth and hormone (notably auxin) biosynthesis and signaling. The reduced elongation observed in edited lines correlated with enhanced resistance to infection by viral, bacterial and fungal pathogens, both under low and high density conditions. These results indicate that our editing approach allows the generation of gain-of-function tomato plants in which HY5 is camouflaged to avoid COP1 recognition and eventual degradation. Our findings therefore provide a biotechnological tool to create more compact and pathogen-resistant plants amenable to high planting densities.

Water availability in drying soils is most often spatially heterogeneous, requiring plants to adjust their root system architecture to sustain water uptake. This acclimation response to water deficit (WD) relies on the integration of local and systemic signals. The later and their mode of action remain poorly understood. Using hydroponic split-root systems in maize and Arabidopsis, we show that, with respect to control well-watered plants, a local WD does not affect shoot biomass but suppresses lateral root (LR) growth in the water-deprived compartment while promoting LR elongation in the well-watered compartment. Spatiotemporal analyses in maize revealed an early and transient (<2h) transcriptomic response of LRs triggered by both local and systemic WD signals. This response involved activation of jasmonate (JA) biosynthesis and signaling components, including MYC2 like regulators and JAZ repressors. It occurred in both the water-deprived and well-watered root halves of split roots, with no evidence of long-distance transport of JA. Furthermore, exogenous methyl jasmonate locally inhibited LR elongation, whereas the Arabidopsis JA insensitive jar1-1 mutant showed an enhanced systemic LR growth response to a local WD. Together, these findings demonstrate that JA acts as an early negative regulator that counterbalances the systemic enhancement of LR growth induced by a local WD, thereby fine-tuning root system architecture and water uptake during local soil drying.

Sporadically, all plastids produce dynamic tubular extensions in response to myriad stresses. Although the use of fluorescent proteins revealed these transient tubules to be stroma-filled and led to their being called stromules, the mechanism underlying tubule extension from a spheroidal plastid remained unknown. As plastids in viridiplantae have two bounding membranes the fluid stroma remains confined within the inner envelope membrane (IEM) while the outer envelope membrane (OEM) around the IEM forms the interface with the rest of the cytoplasm. Here, using a sucrose treatment for inducing tubules in the dark in transgenic Arabidopsis plants expressing different stroma and OEM-targeted fluorescent proteins we carried out comparative molecular-genetic and lipidomics analyses. Our data strongly suggest that plastid tubule formation occurs due to increase in bilayer-forming galactolipids such as digalactosyldiacylglycerol (DGDG) on the OEM. While the dynamic behaviour of stromules occurs within an external encasing OEM, stresses such as phosphate starvation and high light intensity cause the galactolipid-rich OEM-tubule projecting beyond the stromule to be shed into the cytoplasm. OEM shedding from long tubules with extended plastid outreach into the cell suggests their important role in dispersing plastid-derived, membrane-stabilizing, galactolipids to the rest of the cell to augment its resilience to stress.

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

Rhamnogalacturonan I (RG-I) is a major pectin domain and an important wall component for cell-cell adhesion. Arabidopsis seed mucilage serves as a powerful model to identify and characterize pectin-related enzymes, since this gelatinous capsule is composed predominantly of RG-I. Although multiple glycosyltransferase (GT) families participate in pectin biosynthesis, only a limited number of their members have been functionally characterized in vivo or biochemically characterized in vitro. In this study, we characterized the biological functions of PEELING WALLS1 (PEEL1), a Golgi-localized GT106 protein that is related to known RG-I rhamnosyltransferases (RRTs). Knocking out PEEL1, but not its close paralog PEEL1-LIKE (PEEL1L), led to patchy mucilage release from seeds upon hydration. However, RG-I content was not compromised in mucilage extracted from peel1 or peel1 peel1L mutant seeds. Treatment of seeds with a cation chelator restored mucilage expansion but failed to rescue the underlying defect in epidermal cell adhesion. Histological experiments and transgene complementation demonstrated that optimal expression of PEEL1 is required for the adhesion of primary walls to the seed surface. Collectively, these findings identify PEEL1 as a previously unrecognized contributor to primary cell wall adhesion and suggest that different RRT-related proteins contribute to unique pectic structures or localizations in vivo, rather than total mucilage RG-I abundance.

Plants frequently experience recurrent drought events separated by periods of rehydration. Although drought imposes strong constraints on plant physiology, prior exposure may alter subsequent stress responsiveness through memory-based mechanisms. Here, we investigated whether recurrent drought at distinct developmental stages establishes stress memory in sugarcane and whether this response persists across vegetative propagation. Two genotypes contrasting in drought tolerance and productivity (IACCTC07-8008 and IACSP95-5000, respectively) were grown under greenhouse conditions and subjected to three drought cycles imposed either at tillering or maturation stage. Gas exchange, photochemical performance, leaf water status, primary metabolite profile, and growth traits were assessed across cycles, and vegetative propagules were subsequently evaluated under renewed drought. The first drought cycle imposed strong limitations on carbon assimilation and photochemistry in both genotypes and developmental stages. However, subsequent cycles resulted in attenuated reductions in A and g, improved intrinsic water use efficiency, and partial stabilization of PSII performance, indicating a modified stress-response trajectory. Young plants displayed earlier improvements (from the second cycle), whereas in mature plants this shift was evident mainly during the third cycle. Recurrent drought promoted sustained reorganization of amino acid, carbohydrate, organic acid and polyol metabolism alongside increased root biomass and higher relative water content during later cycles. Importantly, propagules derived from drought-conditioned plants exhibited faster recovery of photosynthetic performance and reduced cumulative physiological impairment under renewed drought, despite showing similar stress sensitivity at maximum water deficit. This persistence of enhanced recovery capacity across vegetative propagation indicates that drought-induced memory was maintained beyond the initially stressed plants. Together, our findings demonstrate that recurrent drought establishes a metabolically imprinted state in clonal sugarcane, integrating physiological adjustment, metabolic reprogramming, and whole-plant acclimation. These results highlight the potential of stress memory as a mechanism supporting resilience in perennial crops exposed to increasingly recurrent drought events.

Groundnut (Arachis hypogaea) is an important oilseed crop, and its production is severely affected by drought worldwide. Improving the drought-tolerance of groundnut without compromising its agronomic traits is a major challenge for researchers. Here, a meta-transcriptome analysis identified tandemly duplicated G{beta} subunits (AhG{beta}1 and AhG{beta}3) as early signaling genes under water deficit conditions. Heterotrimeric G-protein {beta}-subunit is a major signaling element that can regulate diverse responses in plants. Heterologous complementation studies in Arabidopsis G{beta} mutant (agb1-2) demonstrated evolutionary conservation of AhG{beta} function, while the transgenic overexpression and RNAi knockdown in groundnut established AhG{beta}1 as a positive regulator of plant growth, yield, and drought tolerance. Integrated transcriptomic, transcription factor regulatory network, and metabolomic analyses demonstrated that AhG{beta}1 coordinates drought-responsive transcriptional and metabolic reprogramming characterized by suppression of cell wall-associated processes and activation of phenylpropanoid, flavonoid, and isoflavonoid biosynthesis. This reprogramming resulted in enhanced accumulation of antioxidant flavonoids, including quercetin and rutin, reduced ROS accumulation, and improved drought tolerance. Furthermore, exogenous application of these flavonoids enhanced drought recovery, highlighting their role as protective downstream metabolites. Collectively, our findings establish AhG{beta}1 as a conserved regulator linking early G-protein signaling with transcriptional and metabolic reprogramming to maintain redox homeostasis during drought stress and identify promising molecular and metabolic targets for improving drought resilience in groundnut.

Calcium (Ca2+) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca2+; signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani, affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani-infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca2+; sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca2+, and the Ca2+-binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca2+ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca2+ and undergo minimal secondary and tertiary structural changes upon Ca2+-binding, in contrast to several Ca2+-binding proteins. Of note, SlCBL1-2 binds Ca2+ with apparent KD values of 526 {+/-} 113 {micro}M and 28.7 {+/-} 9.47 {micro}M, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca2+-binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca2+ sensor in defense signalling.

{beta}-Cyclocitral ({beta}-cc), a {beta}-carotene oxidation product, has been proposed as an early retrograde signal that primes protection against photooxidative stress. Its temporal relationship with photosynthetic acclimation, however, remains unresolved. Exogenous {beta}-cc elicited cytosolic calcium changes within minutes and induced the {beta}-cc-responsive pAER::LUC detoxification reporter within 40 min. We exposed Arabidopsis thaliana plants to 1500 mol photons m-2 s-1 under ventilation creating a high-irradiance regime without concomitant heat stress and with a relatively mild photooxidative outcome (RT-EL). Non-photochemical quenching increased, whereas PSII maximum quantum yield progressively decreased during RT-EL, with both responses approaching a plateau by 16 h. Volatile {beta}-cc pretreatment did not improve photosynthetic performance or NPQ dynamics, whereas endogenous {beta}-cc and {beta}-cyclocitric acid accumulated only after prolonged RT-EL exposure (16 h). By contrast, hormone profiling and RNA-seq revealed extensive {beta}-cc-dependent reprogramming that was largely opposite to the RT-EL transcriptional response. Together, these results reveal a kinetic separation between rapid {beta}-cc signaling and its endogenous production. Under RT-EL major photosynthetic adjustments precede detectable {beta}-cc accumulation, whereas {beta}-cc triggers cellular responses within minutes once present. We therefore propose that the timing of {beta}-cc action during excess light is determined primarily by the onset of substantial {beta}-carotene oxidation.

Root nodule symbiosis allows legumes to meet their nitrogen requirements. However, the causes of symbiosis failure at high temperatures remain unknown. We demonstrated that elevated temperatures affect both the host and the microbial partner in Medicago truncatula and chickpea. Rhizobial viability and nod gene expression persisted under heat stress, but plant transcriptome analysis revealed a strong downregulation of the nodule organogenesis machinery. Nodule organogenesis was restored by constitutive overexpression of NIN-LBD16-NFYA1-NFYB16, miR2111, and CLE5 knockdown but failed to restore nitrogen fixation. In contrast, the genetic manipulation of the competence pathway involving CEP peptides significantly improved both nodulation and nitrogen fixation. Constitutive loss of the autoregulation of nodulation (AON) pathway in the SUNN alleles also rescued root nodulation and nitrogen fixation. Therefore, heat stress perturbs the balance between AON and the competence pathway, and genetic manipulation of these pathways is a promising approach to engineer thermotolerant nitrogen fixation in crop legumes.

Photosynthetic electron transport is tightly regulated to balance ATP and NADPH production and prevent over-reduction of the photosynthetic machinery. How this balance is maintained under severe ATP limitation, particularly under anoxia, is not fully resolved. Here, we used several deletion mutants of chloroplast ATP synthase subunits from Chlamydomonas reinhardtii to examine how electron flow is reorganized under anoxic conditions through the interplay between linear electron flow, cyclic electron flow, and alternative electron sinks. Using a combination of optical spectroscopy methods and membrane inlet mass spectrometry, we characterized changes in photosynthetic electron transport, proton motive force relaxation and gas exchange. Under oxic conditions, the {Delta}atpH mutant (lacking the c-subunit) showed strong acceptor-side limitation at photosystem I (PSI), whereas atpC1-2 (lacking the {gamma}-subunit) resembled the wild type. Under anoxia, this limitation was partially relieved, coinciding with sustained electron transport and prolonged hydrogen production. In particular, the {Delta}atpH mutant exhibited increased cyclic electron flow under oxic conditions and maintained PSI activity under anoxia despite impaired ATP synthesis. Prolonged hydrogen production was also observed in atpC1-2, which did not share the increased respiration of {Delta}atpH. Hydrogenase activity under anoxia is consistent with a role as an electron sink that may help alleviate PSI acceptor-side limitation and rebalance the redox state of the electron transport chain. These findings show that ATP synthase deficiency reorganizes photosynthetic electron flow and promotes prolonged hydrogen production, and point to hydrogenase as a candidate electron sink under energy-constrained conditions.

The Unfolded Protein Response (UPR) maintains endoplasmic reticulum (ER) homeostasis during proteotoxic stress while also contributing to plant immunity. However, the functional relationship between UPR signaling, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and salicylic acid (SA) remains poorly understood. Here, we investigated this interplay using Arabidopsis mutants impaired in the two major UPR branches. Mutants defective in the INOSITOL-REQUIRING ENZYME 1 (IRE1)-bZIP60 branch displayed enhanced resistance to Pseudomonas syringae pv. tomato DC3000 and to the type III secretion-deficient strain hrcC-, indicating potentiation of PTI. Consistently, the ire1a ire1b double mutant exhibited increased flg22-induced callose deposition, whereas reactive oxygen species production was unaffected. Despite enhanced basal resistance, all UPR mutants retained full responsiveness to benzothiadiazole, demonstrating that downstream SA signaling remains functional independently of UPR integrity. Conversely, acute ER stress induced by tunicamycin promoted SA accumulation and extensive leaf necrosis. Reverse genetics revealed that SA-deficient mutants were largely protected from ER stress-induced cell death, whereas mutants over-accumulating SA exhibited enhanced susceptibility, demonstrating that endogenous SA is required for the full execution of this response. Analysis of an independent transcriptomic dataset further showed that loss of IRE1 de-represses genes involved in SA biosynthesis, PTI and defense under ER stress. Together, our results identify IRE1 as a key regulator coordinating ER homeostasis with immune signaling by limiting SA-dependent defense activation during ER stress, while simultaneously preventing excessive cell death.

- C2 photosynthesis reduces photorespiratory carbon loss by shuttling glycine from mesophyll (M) to bundle sheath (BS) tissues, where glycine decarboxylase (GDC) releases CO2 that accumulates around rubisco. C2 photosynthesis has been considered a constitutive character, but as a metabolic trait, it may acclimate to the rate of photorespiratory glycine production. - We grew eleven species from two eudicot and two grass lineages spanning C3 to C2 phenotypes at 32 {degrees}C days and 200 or 800 {micro}mol mol-1 CO2. Acclimation was assessed using leaf gas exchange, GDC P-protein immunolocalization, M and BS ultrastructure, photosynthetic enzyme activity, and photosynthesis modelling. - Low relative to high growth CO2 enhanced C2-related phenotypes in sub-C2 and C2 species but not C3 or proto-Kranz species. Sub-C2 species positioned chloroplasts closer to mitochondria and had lower modelled BS conductance. C2 species increased rubisco capacity and GDC allocation to the BS. Aspartate aminotransferase activity increased in both sub-C2 and C2 species. - Acclimation to greater photorespiratory conditions strengthens C2 photosynthesis, but only in species whose BS has crossed a threshold of organelle and GDC investment. We hypothesize that acclimation could expose C2-related traits to selection and facilitate the origin of C4 photosynthesis.

Although iron (Fe) is abundant in soils, its low solubility, particularly under alkaline conditions, often limits plant Fe acquisition. Under these conditions, coumarin-mediated Fe mobilization becomes increasingly important. Arabidopsis thaliana FE DEFICIENCY-INDUCED KELCH REPEAT PROTEIN 1 (FDK1, AT3G07720) is strongly induced by Fe deficiency and regulated by the central transcription factor FIT, but its function is unknown. We investigated FDK1 using genetic, physiological, metabolite, and transcriptomic analyses. Loss of FDK1 altered root growth, whereas this phenotype was restored in the FDK1-Ox overexpression line. Under alkaline Fe deficiency, growth medium from FDK1-Ox contained strongly increased levels of the Fe-mobilizing coumarins fraxetin and esculetin compared with wild type, whereas their induction was attenuated in fdk1-1. RNA-seq analysis linked FDK1 abundance to coumarin biosynthesis and iron-homeostasis gene expression. FIT and many FIT-associated genes were highly expressed in FDK1-Ox, whereas several upstream iron-homeostasis regulators, including bHLH Ib factors, were highest in fdk1-1. These findings identify FDK1 as a component of the alkaline Fe-deficiency response that promotes iron-mobilizing coumarin accumulation and is associated with coordinated regulation of iron-homeostasis genes.

Lipid droplets (LDs) play a central role in cellular carbon and energy homeostasis, in particular through the roles played by numerous LD-associated proteins. A long-standing question concerns how these proteins are molecularly organized at the seemingly overcrowded LD surface. Delayed in TAG Hydrolysis 1 (DTH1) has been identified as a key protein for efficient LD degradation in Chlamydomonas reinhardtii, however the underlying molecular mechanism remains unknown. DTH1 contains several structural features including a coiled-coil domain, a phosphatidylethanolamine (PE) specific lipid-binding domain, intrinsically disordered regions, and an alpha-helical C-terminal domain, that make it a compelling candidate for acting as a molecular scaffold in protein recruitment and/or organelle tethering. In this study, we used the Chlamydomonas knock-out mutant dth1 as an entry point to begin dissecting the molecular organization of proteins at the LD surface. Comparative lipidomic and proteomic analyses of isolated LDs from both WT and dth1 mutants revealed several key features associated with LD degradation during nutrient recovery. First, PE emerged as an important lipid species involved in LD degradation. Second, active communication occurs among multiple subcellular organelles particularly between LDs, peroxisomes and mitochondria. Third, DTH1 was found to play a critical role in remodeling both the LD proteome and lipidome. Furthermore, detailed analyses of LD degradation during nitrogen recovery uncovered the formation of micro-LDs, a phenomenon not yet reported in microalgae. Collectively, these results highlight the tight coordination of metabolic activities across subcellular compartments and emphasize the importance of intracellular carbon trafficking during stress recovery.

Progression of leaf senescence is regulated by internal factors like the plant hormone cytokinin (CK) that acts to delay senescence and retain photosynthesis by maintaining chloroplast-functionality. While initial CK-signaling components controlling senescence have been described, downstream targets regulating physiological performance have remained less characterized. Here we present the CHLOROPLAST IMPORT APARATUS 2 (CIA2) and CIA2-like (CIL) transcription factors (TFs) as mediators of CK senescence activity, enabling chloroplast-function. Utilizing loss-of-function mutants, we show CIA2 and CIL negatively regulate senescence progression and alter physiological performance in Arabidopsis thaliana. Integrated transcriptomic-metabolomic analysis shows that CIA2 and CIL action is distinct from other known chloroplast-related TFs (like GLKs/GATAs) and can boost antioxidant response over senescence. We find that CIA2 and CIL are essential in relaying CK activity during chloroplast biogenesis, photosynthesis, rubisco function, and metabolite accumulation. In turn, ARR10/12 CK signaling components were needed for CK induction of CIA2 and CIL themselves - a cascade that induced chloroplast- and photosynthesis-associated nuclear genes, to boost chloroplast-function and metabolism. We also show that CK acts to repress senescence through accumulation of the polyamine, spermidine, in a CIA2/CIL dependent manner. This work presents a CIA2/CIL-cascade as a new anterograde signal for CK antisenescence effects, indicating a mechanism through which CK prolongs leaf function. This fundamental understanding of CK action has implications towards engineering plants with elevated photosynthesis performance and delayed senescence.

Plastidial phosphorylase (Pho1) regulates starch metabolism and also modulates photosystem I (PSI) activity. In rice, Pho1 contains an 80 amino acid intrinsically disordered region (IDR), L80, that acts as a negative regulator. While total removal of the IDR boosts seedling growth, flowering time, overall biomass, and grain yield and alters PSI redox properties, the precise underlying regulatory basis remains unclear. To pinpoint IDR regulatory elements, four Pho1 partial-IDR deletion variants ({delta}N, {delta}C, {delta}M, and {delta}N{delta}C) were evaluated. Lines carrying {delta}M and {delta}C exhibited enhanced starch metabolism, yielding larger, heavier grains, and flowered 5 to10 days earlier than wild type. Both d{delta}M and {delta}C share an acidic motif, (E/V)SEEV, identifying it as a primary negative regulator of starch synthesis and floral timing. By contrast, no partial deletion fully mirrored the PSI redox modifications of Pho1{delta}L80. These results demonstrate that starch biosynthesis and flowering are governed by one or more discrete sequence motifs within the middle and C-terminal regions, whereas PSI modulation likely requires the structural integrity of the intact IDR.

SUMOylation is an essential and evolutionarily conserved post-translational modification that enables rapid cellular adaptation to environmental stress. Despite its importance, how the SUMOylation machinery controls its dynamics remains poorly understood. Here, we integrated a mathematical model of the plant SUMO cycle with quantitative imaging and biochemical data to decode the regulatory logic of SUMOylation dynamics. The model reveals a fundamental principle: steady-state SUMOylation levels are determined not by the full complement of SUMO-cycle components, but by the abundance of SUMO modifier proteins and E1 enzymes, together with SUMO protease abundance and activity. Strikingly, when we integrated fluorescence measurements from reporters for SUMO-cycle components in Arabidopsis roots after three hours of abiotic stress, the model predicted decreased SUMOylation levels across several tissues, contrary to prevailing evidence. Motivated by this unexpected prediction, we performed time-resolved imaging to better characterize the dynamics of key SUMO-cycle components after salt and osmotic stress. These data revealed highly dynamic component levels within the first three hours of stress that were not captured in the previous measurements. Incorporating this temporal data into the model uncovered a twin-peaked SUMOylation response to salt stress: an early surge underpinned by protease dynamics, followed by a decline and a second peak driven by elevated SUMO modifier and E2 levels. Western blot analysis revealed a corresponding twin-peaked pattern of SUMOylation. Together, our findings demonstrate that the plant SUMO cycle is a highly dynamic system that achieves tissue- and stress-specific control of SUMOylation through the coordinated interplay of multiple SUMO-cycle components.

Pigment composition and regulation are critical for efficient photosynthesis in the ocean, where light intensity decreases and the spectrum narrows with depth. Fucoxanthin (Fx), the main carotenoid of several microalgal lineages, harvests the blue-green light prevailing in the deep euphotic zone. In pelagophytes, the acylated derivative 19'-butanoyloxyfucoxanthin (19'-BFx) is abundant, yet its function and regulation remain unclear. Here, we investigated how irradiance and spectral quality shape photoacclimation in Pelagomonas calceolata, an abundant and cosmopolitan low-light pelagophyte. Cultures were grown under blue or white light across 2-60 umol photons m^-2 s^-1. We analysed growth, PSII photophysiology, pigment composition, gene expression levels, and the impact of spectral shifts on the 19'-BFx/Fx ratio. Pelagomonas calceolata grew optimally under dim blue light and displayed low non-photochemical quenching (NPQ) under assay conditions. Transcriptomes revealed broad remodelling driven mainly by irradiance and modulated by light colour, including carotenoid-related processes. Dim blue light progressively increased the 19'-BFx/Fx ratio, whereas white light kept it low and reversed the response after a blue-to-white shift. These results identify reversible, spectrum-dependent 19'-BFx accumulation as a key component of photoacclimation in P. calceolata and separate irradiance-driven from spectrum-dependent cellular responses. This plasticity may explain the ecological success of P. calceolata in low-light oceans.

Agrobacterium-mediated transformation is a cornerstone of plant genetic engineering, yet many crops remain recalcitrant to transformation and its efficiency is highly species- and genotype-dependent. The limited diversity of disarmed agrobacterial strains available for biotechnology, and the technically challenging workflows typically required for strain modification, constrain efforts to develop improved plant transformation platforms. Here, we describe the development of AGROtools-a versatile, expandable, and user-friendly modular cloning plasmid collection comprising more than 75 individual components, that is designed to simplify genetic engineering of agrobacteria. Constructs assembled with this toolkit couple visual reporters with selectable and counterselectable markers to facilitate identification of recombinant clones, thereby reducing reliance on PCR-based screening. We constructed and tested reporter cassettes that are functional across diverse agrobacterial strains and genomic contexts. By linking a visual reporter combinatorially with various antibiotic resistance markers and orthogonal site-specific recombinase recognition sequences, AGROtools can enable marker removal and recycling for up to four successive genomic modifications. We demonstrate the utility of AGROtools by disarming 10 novel wild-type strains, and accomplishing serial modifications within individual strain backgrounds. In addition to conventional recA-mediated homologous recombination, the collection incorporates components for applying contemporary strain engineering approaches, including CRISPR/dCas9 base editing, INTEGRATE-mediated targeted insertion, and phage-derived recombineering systems. Constructs can be assembled and deployed rapidly, enabling isolation of strains carrying an intended modification in less than two weeks. AGROtools provides a broadly applicable resource for accelerating strain development and genetic studies in agrobacteria, with potential to advance plant biotechnology by aiding improvement of disarmed strains.