Plant Physiology and Biochemistry

Cadmium is a very toxic heavy metal. We studied Cd-treated barley plants with especial focus on rare atypical plants with signs of chlorosis. Cd treatment decreased the maximal photochemical activities of both photosystems while the activity of photosystem I decreased more than activity of photosystem II. In photosystem II, Cd treatment inhibited non-photochemical quenching that increased portion of unquenched "closed" complexes of photosystem II. The latter effect increased balance of limitations between the acceptor side of photosystem II (qC) and the donor side of photosystem I (Y(ND)) and raised the ratio qC/Y(ND). All these effects were enhanced in the atypical more damaged plants. Cd treatment reduced K content in the first leaves; in atypical plants, K content decreased even more. Cd treatment changed a pattern of stomatal conductance possibly by means of reducing K content in leaves. The untreated barley plants kept different stomatal conductance at adaxial and abaxial sides of leaves and fulfilled a complicated diurnal dynamics with large ups and downs of stomatal conductance. The typical Cd-treated plants were less flexible and demonstrated medium values. Stomatal conductance in the untreated plants were higher or lower than in the typical Cd-treated plants depending on a particular time; average daytime stomatal conductance was equal in both variants. At 10.00, stomatal conductance in the atypical Cd-treated plants was smaller than in the typical ones. Levels of 13 chloroplast mRNAs remained unchanged, while psbD decreased in both types of Cd-treated plants. HighlightsO_LISeveral Cd effects were enhanced in more damaged (atypical) chlorotic plants C_LIO_LICd treatment decreased activity of photosystem I and non-photochemical quenching C_LIO_LIRatio of limitations between photosystems II and I [qC/Y(ND)] was rather constant C_LIO_LICd treatment reduced K content in the first leaves C_LIO_LICd treatment changed pattern of stomatal conductance C_LI

Photosynthesis accounts for most of the final grain yield in rice, making improvements in radiation use efficiency (RUE) a key strategy for enhancing productivity. Agronomically, RUE is defined as the biomass produced per unit of total solar radiation or photosynthetically active radiation intercepted by the canopy. However, the interaction between carbon and nitrogen metabolism plays a critical role in determining plant growth and grain yield. Assimilated nitrogen is required for the synthesis of photosynthetic pigments and enzymes, while the reduction of nitrate (NOLL) and nitrite (NOLL), as well as the assimilation of ammonium (NHLL), depend on the reducing power and carbon skeletons generated by photosynthesis. In this study, two high-yielding rice (Oryza sativa) cultivars--an indica-type (El Paso 144) and a japonica-type (INIA Parao) were subjected to two nitrogen treatments (3 mM and 9 mM NOLL/NHLL) and two light intensities (850 and 1500 mol mL{superscript 2} sL{superscript 1}). A strong interaction between light intensity and nitrogen metabolism was observed, with contrasting responses between subspecies. These differences reflect a coordinated regulation of carbon assimilation and primary nitrogen metabolism. The results provide new insights into the metabolic strategies underlying nitrogen compound accumulation under variable irradiance. Such knowledge is essential for improving nitrogen fertilizer use efficiency and yield performance in elite rice genotypes cultivated under commercial field conditions.

Non-structural carbohydrates (NSC) stored in the stem play a crucial role in supporting yield formation in rice. However, internode morphological determinants of NSC accumulation are unclear. This study aimed to clarify the relationship between internode morphology and NSC accumulation and to identify a robust morphological indicator for evaluating NSC accumulation capacity. Two years of field experiments were conducted using multiple cultivars. The NSC content was quantified for individual internodes and at the whole-plant culm level, and its relationships with internode morphological traits were analyzed. Since the upper internodes (UIN; first and second internodes) and lower internodes (LIN; third and subsequent internodes) exhibited contrasting roles in NSC accumulation, a novel index was introduced, the volume composition ratio (VCR) of UIN/LIN, which represents their relative volumetric contributions within a culm. The VCR of UIN/LIN showed the strongest correlation with culm NSC and high reproducibility across years, outperforming simple morphological traits. Manipulation of internode development using plant growth regulators demonstrated that altering VCR effectively modified culm NSC accumulation. Accordingly, the VCR of UIN/LIN serves as a robust morphological indicator of culm NSC accumulation capacity, providing a practical framework for improving NSC accumulation to achieve high and stable yield performance in rice. HighlightThis novel internode structural index robustly predicts the culm non-structural carbohydrate accumulation capacity, providing a practical morphological indicator for improving yield stability in rice.

Betalamic acid is the chromophore of betalains, which are pigments of chemotaxonomic and physiological importance. This study involves RACE-PCR-based gene cloning, heterologous expression, protein purification, and steady-state kinetics of B. alba L. var. Rubra L-DOPA/dopamine-4,5-dioxygenase 1 (BrDOD1). BrDOD1 is a unique high betalamic acid-forming LigB homolog in plants having comparable affinity for both L-DOPA and dopamine (KM < 50 M). Ascorbic acid (10 mM) shifted the steady-state kinetics from inhibitory to activator at a particular substrate concentration of both L-DOPA and dopamine. This increased both KM and Vmax by more than 6.5-fold, indicating that ascorbic acid acted as a molecular crowding agent in the enzyme assay. BrDOD1s physiological substrate is L-DOPA as the reaction rate for L-DOPA was 6.6-fold higher than dopamine, L-DOPA was present in higher concentration than that of dopamine in the same plant, and molecular dynamic simulations showed better stability of BrDOD1-L-DOPA complex than that of dopamine. Further, two more LigB homologs from the same plant have also been cloned. Based on the betalamic acid-forming activity, molecular phylogeny, conserved structural regions, and theoretical pI, betalainic plant LigB homologs have been classified into three groups to better understand the evolutionary trajectory of the LigB homologs in plants.

BackgroundExtensin peroxidases (EPs) are class III plant peroxidases and are responsible for intermolecular covalent crosslinking of extensin (EXT) monomers to create scaffolds within plant cell walls. The formation of these scaffolds impacts plant development, mechanical wounding, and response to pathogen attacks. Therefore, elucidating the molecular mechanism controlling covalent crosslinking of EXT monomers is crucial for understanding cell wall deposition and potentially improving plant growth and adaptation. The focus of this work is to use in silico analysis to determine the structural characteristics of an EP from tomato (TomEP) to elucidate its specificity for crosslinking of EXT monomers. ResultsIn this study the two-dimensional (2D) and three-dimensional (3D) structures of TomEP were determined using several advanced bioinformatics tools and compared to two other peroxidases: GvEP1 (a known EP) and HRP-C (having a low affinity for EXT substrates). The results revealed that TomEP is a stable and hydrophilic protein with high thermal stability. The heme binding pockets of TomEP and GvEP1 have more hydrophobic residues and larger volume and pocket area compared to HRP-C. Molecular docking at the active site, which includes a heme heteroatom, showed that the ligands consisting of the hydrophobic Tyrosine-X-Tyrosine [-Y-X-Y-] motifs (i.e., [-Y-K-Y-], [-Y-V-Y-], and [-Y-Y-Y-] found in EXTs, and their derivatives, Isodityrosine (IDT), Pulcherosine (Pul), Di-Isodytirosine (diIDT), bind perfectly to the active site of TomEP with dominant interactions of Val54, Ser94, Ala96 and Phe196 residues. Pulcherosine had the highest binding affinity, while [-Y-K-Y-] showed the lowest binding affinity. Molecular dynamics simulations showed that [-Y-X-Y-] motifs (and the derivative substrate ligands) remain bound to the active site of TomEP throughout the 100 ns long simulation. Furthermore, the binding of these substrates stabilized the protein structure. ConclusionThese results may explain why TomEP is particularly well-suited for EXT crosslinking and will have significant implications on biochemistry, biotechnology, and the potential use of these EPs in crops improvement.

Atmospheric nitrogen (N) deposition is increasingly affecting global ecosystems, with nitrate contributing a growing proportion alongside ammonium. However, the interaction between N forms and leaf developmental stage in shaping physiological and metabolic strategies in Chinese fir remains poorly understood. In this study, a field experiment was conducted to explore the physiological and metabolic responses of young and old leaves to ammonium and nitrate N addition. Our findings showed that N addition enhanced photosynthetic performance in young leaves, with a stronger effect from nitrate. In contrast, old leaves exhibited limited photosynthetic response but accumulated higher non-structural carbohydrates and showed elevated N assimilation enzyme activities, particularly under nitrate addition. Phytohormone profiles varied between leaf ages, with young leaves having higher auxin levels while old leaves exhibiting increased abscisic and salicylic acid contents under N addition. Additionally, N addition induced differential reprogramming of amino acid metabolism, with age-dependent accumulation patterns. Metabolomic analysis identified key amino acids involved in coordinating carbon-nitrogen metabolism. These results highlighted the complementary metabolic strategies by young and old leaves of Chinese fir under contrasting N forms addition and emphasized the importance of considering both N form and leaf age in optimizing N management for sustainable plantation practices. HighlightsO_LINitrate enhanced photosynthesis in young Chinese fir leaves more effectively than ammonium. C_LIO_LIOld leaves prioritized C storage and N assimilation under N addition, especially nitrate. C_LIO_LIComplementary metabolic strategies between leaf ages optimized resource use under different N forms addition. C_LI

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

Increasing attention is being focused on the glycemic index (GI) of daily food for humans, and the resistant starch content (RSC) is an important indicator of GI for starch-rich staple foods. In recent years, some studies revealed that the loss function of single or multiple key enzymes in the primary pathway of starch synthesis substantially increases RSC in rice, such as starch branching enzyme IIb (BEIIb) and soluble starch synthase IIIa (OsSSIIIa). However, a noteworthy negative characteristic of these high RSC mutants is the substantially increased amylose content (AC). AC as a major determinator of rice eating quality, must not be higher than an acceptable limit for most consumers. To solve this problem, in this study, we adopted two promoter editing (PE) editing strategies to develop rice germplasms with a better balance of RSC and AC: one is to edit the promoter of BEIIb in a low AC rice variety, another is to edit the promoter of Waxy (Wx) gene in a BEIIb loss of function mutant. Using AC[&le;]20%, which is the range of premium quality rice in China as a criteria, we finally obtained 2 homozygous lines with significantly increased RSC ([&ge;]5%) in the NG46 background by promoter editing of BEIIb and 1 homozygous line in the YouTang2 (YT2, a BEIIb mutant) background by promoter editing of Wx gene. Further analysis revealed that AC and the amount of long-chain branches of amylopectin are positively correlated with RSC in the population of BEIIb PE lines. However, unexpectedly, the Wx PE-line with substantially decreased AC (17.7%) also showed significantly increased RSC (16.9%). Our study not only produces useful germplasms for the high RSC rice breeding in the future but also provides an insight into understanding the relationship between AC and RSC in defective BEIIb rice.

Neck blast (NB), caused by Magnaporthe oryzae, damages rice panicles and reduces yield. Knowledge of NB resistance remains limited due to the lack of reliable resistance evaluation methods. Here, we applied a newly established neck injection method and performed a GWAS on 335 diverse accessions from the 3K Rice Genomes Project to identify loci associated with NB resistance. We detected a significant association on chromosome 12, explaining 15-18% of the symptom variations caused by a highly virulent Philippine blast isolate (M64-1-3-9-1). Linkage disequilibrium analysis refined this region to a 42.3-kb interval containing Pita2, a known leaf blast resistance gene. We found that two Pita2 allelic variants, Pita2a and Pita2c, both harboring the variant A/G (Lys879) in the last exon (Chr12:10,833,400), are associated with NB resistance. IR64 and a CO39 near-isogenic line (NIL) IRBLta2-Pi[CO] harboring Pita2a were resistant, whereas CRISPR-Cas9 knock-out of Pita2a in IR64 caused susceptibility to M64-1-3-9-1 and IK81-25. These results indicate that Pita2a is required for NB resistance. Furthermore, the CO39 NIL, IRBLta2-Pi[CO], and Lijiangxintuanheigu monogenic line (IRBLta2-Pi) harboring the Pita2a allele exhibited broad-spectrum resistance to 75% and 80% of Philippine differential blast isolates, respectively. The superior haplotype of Pita2 contains two major SNPs (A/G and A/C at Chr12:10,833,400 and Chr12:10,845,095) occurs in 83% of IRRI elite breeding lines and can be used to select NB-resistant genotypes with an accuracy of 86%. Our findings identify Pita2a as a major gene for NB resistance and provide a valuable genetic resource for developing blast-resistant rice. PLAIN LANGUAGE SUMMARYRice blast, caused by the fungus Magnaporthe oryzae, is a major threat to global rice production. Neck blast (NB) is the most severe type of blast, however, the genetic basis of NB resistance remains poorly understood. In this study, we analyzed 335 rice accessions to identify genes underlying the resistance against a Philippine blast isolate. We found that allelic variants Pita2a and Pita2c are strongly-associated with NB resistance. Knock-out of Pita2a allele made resistant rice plants susceptible while introgression into susceptible rice lines enhanced resistance to multiple blast isolates, confirming its role in NB resistance. Importantly, the superior alleles of Pita2 are already present in 83% of elite breeding lines and can be used to select NB-resistant genotypes with an accuracy of 86%. Our findings clarify the genetic control of NB resistance and offer new tools for protecting rice yields in blast-endemic regions.

Leaf senescence is a programmed plant developmental process that can also be regulated by environmental factors, like nutrient availability. Although phosphorus is an essential element determining plants growth and productivity, mechanisms underlying adaptation to phosphorus availability in plants are not well understood. In this study, we combined physiological, biochemical and molecular approaches to investigate the effect of phosphate supply on leaf senescence in rice. We show that short-term treatment of rice seedlings with low phosphate increases photosynthetic pigments content, confers tolerance to methyl viologen-induced oxidative stress in chloroplasts, and increases antioxidant enzyme activities. Leaves from low-Pi-treated plants also showed a reduction in membrane lipid peroxidation and electrolyte leakage. Opposite trends were observed in seedlings under high Pi supply, in which accelerated leaf senescence occurs. Further analyses indicated that CRISPR/Cas9-mediated editing of MIR827, and subsequent reduction in Pi content, promotes delayed leaf senescence, while Pi accumulation in MIR827 or MIR399 overexpressing plants accelerates senescence. These findings strongly support that short treatment with low phosphate delays rice leaf senescence. Transcriptomic analysis demonstrated multiple biological processes underlying adaptation of rice plants to low phosphate, including senescence-associated and metabolic processes. These findings provide novel insights into leaf senescence potentially contributing to sustainable rice production.

Glutathione peroxidases (GPXs) are widely recognized as key antioxidants that mitigate oxidative stress by detoxifying reactive oxygen species (ROS). However, GPXs are largely uncharacterized in citrus. Here, we demonstrated that Citrus sinensis contains four GPX proteins (CsGPX1-4). Unexpectedly, overexpression of CsGPX4, a homolog of AtGPX8 in Arabidopsis, in citrus resulted in typical oxidative stress phenotypes including severe growth inhibition, chlorosis, and elevated intracellular ROS accumulation. Transmission electron microscopy (TEM) analysis further revealed stress responses at cellular level. Whole genome shot gun sequencing analysis showed that T-DNA insertion occurs in the UTR of SWEET2 gene, which is unlikely to be responsible for the oxidative stress phenotypes. Immunoblotting revealed that CsGPX4 accumulates as a truncated protein in citrus, in contrast to the full-length version expressed in Nicotiana benthamiana. MALDI-TOF assays further confirmed the truncation of CsGPX4 in the transgenic line with the predicted cleavage site between L115-K117. This truncation was associated with altered subcellular localization, shifting from cytoplasmic and nuclear distribution in N. benthamiana to membrane association in citrus. Proteomic profiling further indicated extensive reprogramming of pathways involved in detoxification, cytoskeletal stability, hormone signaling, and cell wall modification. Our data suggests that de facto overexpression of truncated CsGPX4 may have dominant-negative effects on proteins interacting with CsGPX4, thus interfering with their normal functions. In conclusion, our study demonstrates CsGPX4 as a critical regulator of redox homeostasis and ROS homeostasis in citrus and reveals selective truncation of CsGPX4 as a unique proteolytic or regulatory strategies in such processes.

Light wavelengths modulate plant growth, metabolism, and physiology. Amaranthus, a C4 underutilized climate resilient crop with promising nutritional properties remained unexplored in terms of metabolite enrichment under monochromatic light wavelengths of visible spectrum. In current study, two cultivars of Amaranthus tricolor (green and red) were exposed to seven light regimes of photosynthetically active radiation (PAR; 400-700 nm): deep blue, blue, green, amber, red, deep red, far red, and their metabolic responses were captured using Gas Chromatography-Mass Spectrometry. The metabolic analysis revealed wavelength-specific reprogramming in the levels of organic acids, sugars, amino acids, fatty acids as well as phenolics. In both the green and red Amaranthus, branched-chain amino acids and phenylalanine, which are nutritionally essential, were significantly elevated under far-red light. While the phenolics such as caffeic acid and ferulic acid were elevated under green and deep blue light respectively in green Amaranthus, amber light wavelengths enhanced these phenolics in red Amaranthus. The study highlighted cultivar-specific metabolic rewiring triggered by specific wavelengths. Altogether, these findings provides insights into metabolic adaptation and demonstrate the ability of light wavelength to specifically enrich the targeted metabolite of nutritional relevance in Amaranthus. It offers strategies to improve the nutritional value of crops in controlled agriculture systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/714947v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1a4477dorg.highwire.dtl.DTLVardef@518550org.highwire.dtl.DTLVardef@7682dorg.highwire.dtl.DTLVardef@4876e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and AimsL-DOPA is an important pharmaceutical that accumulates to high levels in the legume faba bean (Vicia faba). L-DOPA is likely derived from L-tyrosine but the responsible enzyme (L-tyrosine oxidase) remains unknown. Availability of L-tyrosine may be a key factor controlling L-DOPA accumulation. In legumes, L-tyrosine is supplied via either a plastidial TyrA enzyme (ADH) or a deregulated cytosolic homolog (PDH). This study aimed at identifying L-tyrosine oxidase and TyrA genes from faba bean. MethodsWe used gene-to-metabolite correlations and homology-based searches to select fifteen L-tyrosine oxidase candidates, which were tested in yeast and in the model plant Nicotiana benthamiana. We also used isotopically labeled L-tyrosine to measure biosynthetic activity in different faba bean tissues and to test an alternative biosynthetic hypothesis. Three faba bean TyrA genes were inferred by homology and assayed in N. benthamiana by co-expression with a known L-tyrosine oxidase, CYP76AD6. Key ResultsNone of the L-tyrosine oxidase candidates produced L-DOPA upon heterologous expression. Feeding experiments showed a lack of correlation between L-DOPA accumulation and biosynthetic capacity. Feeding studies also disproved an alternative route to L-DOPA by oxidation of 4-hydroxyphenylpyruvate. Of the TyrA genes, two were able to increase L-tyrosine levels in N. benthamiana 2-3-fold (VfADH and VfPDH), and one of them was able to boost the levels of L-DOPA derivatives up to 6-fold (VfADH). ConclusionsThe faba bean L-tyrosine oxidase remains unidentified, with a possible transport of L-DOPA across tissues likely having confounded our correlation-based selection strategies. In N. benthamiana, both VfADH and VfPDH can increase the levels of L-tyrosine, while VfADH can further boost the levels of L-DOPA derivatives. Our work delivers a strategy to boost the provision of L-tyrosine in N. benthamiana and provides valuable insights in the search for the elusive L-tyrosine oxidase from faba bean.

Acidic stress severely restricts crop growth by disrupting nutrient uptake, redox homeostasis, and membrane stability, yet mitigation strategies remain limited. Here, we investigated the role of melatonin (MT) in regulating growth, photosynthesis, oxidative stress, antioxidant defense and proton transport in peanut seedlings under controlled hydroponics acidic (pH 4.0) and near-optimal (pH 6.5) conditions, and validated these findings in naturally acidic field soil (pH 4.3-4.5). Acid stress markedly reduced biomass accumulation, chlorophyll content, and redox balance, while enhancing ROS (H2O2) and lipid peroxidation (MDA). Exogenous MT application, particularly at 50-100 {micro}M, significantly improved shoot and root biomass, restored chlorophyll pigments and reduced H2O2 and MDA accumulation, with more pronounced effects under pH 4.0 than pH 6.5. MT strongly activated antioxidant enzymes (SOD, CAT, APX), while POD activity declined, reflecting melatonins dual role as both a direct ROS scavenger and a regulator of enzymatic redox networks. Notably, MT induced strong, dose-dependent upregulation of HL-ATPase genes (AH1 and AH2) in both leaves and roots under acidic conditions, suggesting enhanced proton extrusion, intracellular pH homeostasis, and stress adaptation. The soil validation experiment confirmed the agronomic relevance of these findings, where MT dose-dependent concentrations improved germination, vegetative growth, chlorophyll fluorescence (Fv/Fm), and yield-related traits under natural acidic conditions. Although MT also conferred benefits at pH 6.5, responses were generally moderate compared with acid stress. Collectively, these results demonstrate that MT enhances peanut tolerance to acid stress across both controlled and natural field-relevant environments, highlighting its potential application for sustainable crop production on low-pH soils.

Heat-tolerant rice cultivars are essential for mitigating global warming impacts. Basal anther dehiscence length (BDL) is a promising visible morphological marker for heat tolerance through stable pollination. We investigated the effects of sowing date on anther morphology, pollination, and fertility under controlled high-temperature conditions (35, 37, or 39 {degrees}C at flowering). Three japonica cultivars-- Akitakomachi (early heading), Koshihikari (medium), and Hatsushimo (late)--were sown monthly over 3 months and grown in pots. At heading, the plants were exposed to the temperature treatments for 3 days, and the proportion of florets with [&ge;]10 germinated pollen grains on the stigma (GP10) and seed set were assessed. Among anther traits, BDL showed the greatest variation, with all cultivars from the second sowing exhibiting the shortest BDL. Analysis of variance revealed significant effects of genotype, sowing date, and their interaction on anther traits and fertility. Regression analysis indicated that fertility was associated with GP10, with BDL contributing significantly to GP10 in the late-heading Hatsushimo, together with maximum temperature at flowering. Thus, both genotype and environment shape anther morphology, pollination, and fertility, indicating that BDL plasticity and genotype-specific environmental responses must be carefully considered when using BDL as a breeding marker for heat tolerance. HighlightVariation in sowing date significantly affects anther morphology and heat tolerance in rice. Genotype-specific responses to the growing environment require careful consideration for reliable breeding assessments.

Nitrogen is an important element for all living organisms. Photoautotrophic organisms need to assimilate nitrogen from the environment, therefore changes in nitrogen availability have a strong influence on their growth and metabolism. Many microalgae have been known to contain crystalline inclusions, and recently, it has been shown that many of these consist of purines like guanine and thus must be linked to the cellular nitrogen metabolisms. The alveolate alga Chromera velia contains such guanine crystals, and during its lifecycle, the alga is thought to be subjected to strong changes in external nitrogen availability. Here, we investigated the formation or decline of crystalline guanine in dependence of the availability of inorganic nitrogen in the growth medium. Cells were examined using polarised light microscopy, Raman micro-spectroscopy, chromatography (HPLC), transmission and scanning electron microscopy. The cellular guanine crystal content decreased during nitrogen starvation and increased upon transfer of the cells back to standard growth medium containing nitrate. Raman micro-spectroscopy showed that the crystals were composed of anhydrous guanine in beta-polytype. They appear in unspecific positions throughout the cell, and staining with the green dye Lysotracker DND-26 suggests that they are within vacuoles. Stacks of crystals could be observed in cells via freeze fracture and freeze etching electron microscopy, which unambiguously showed a membrane around the crystal aggregates, in a similar arrangement as has been shown for guanine storage vacuoles (GSV) in Chlamydomonas reinhardtii. We developed a method to isolate the guanine crystals from whole cells, and were able to obtain crystals which retained their flat, plate-like structure, matching the electron microscopic observations from whole cells. The isolated crystals were shown to consist of nitrogen rich compounds via energy-dispersive X-ray (EDX) analysis, and Raman micro-spectroscopy confirmed that they consist of guanine.

Light is essential for photosynthetic organisms, but excess light can generate toxic levels of reactive oxygen species (ROS). To neutralize these ROS, plants and algae produce a variety of antioxidants like carotenoids, tocopherols, and glutathione. However, the role of alternative ROS scavengers, such as ovothiols, has not been studied in the context of oxidative stress in photosynthetic organisms. Here, we report that many algal groups have the potential for the biosynthesis of ovothiols, a group of thiohistidines. We discovered that the model green microalga Chlamydomonas reinhardtii produces millimolar concentrations of ovothiol A, whose biosynthesis is mediated by the ovothiol synthase OVOA1. Using CRISPR-generated ovoa1 knockout mutants, we found that ovothiol production is essential for resistance and acclimation to singlet oxygen, a prominent ROS in photosynthetic organisms. Finally, we demonstrated that OVOA1 expression is activated by singlet oxygen and light signaling pathways in which we identified the major regulatory factors. Overall, our results show that ovothiol A is a major, previously overlooked antioxidant in Chlamydomonas. This work broadens our understanding of cellular mechanisms that combat the damaging effects of oxidative stress. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/702910v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@cddb9corg.highwire.dtl.DTLVardef@10d0a43org.highwire.dtl.DTLVardef@11cc087org.highwire.dtl.DTLVardef@a40cc5_HPS_FORMAT_FIGEXP M_FIG C_FIG

During drought, numerous compounds accumulate in plant tissues, but their physiological roles remain unclear - they may function as osmolytes, osmoprotectants, or merely arise as by-products of stress-induced metabolic shifts. We developed an experimental approach to link accumulation patterns with specific functions, using Scots pine (Pinus sylvestris L.) saplings subjected to water deprivation and subsequent rewatering as a model system. We monitored changes in relative water content (RWC) and osmotic adjustment dynamics, employed untargeted primary metabolite profiling for preliminary screening of compounds correlated with water status, and performed quantitative GC-MS and LC-MS analyses of selected metabolites. Major inorganic cations (K, Ca{superscript 2}, Mg{superscript 2}) were also quantified to assess their potential roles. Our results revealed that tryptophan, valine, and lysine - though generally present in low abundance - exhibited selective accumulation under severely reduced RWC ([&le;] 70%), suggesting their involvement as osmoprotectants. Major organic acids, particularly shikimic acid, showed trends consistent with osmotic adjustment. Notably, neither sucrose nor inorganic cations appeared to function as primary osmolytes in this context. The proposed approach offers a viable strategy for identifying compounds involved in plant adaptation to water deficit, with potential applications in breeding programs aimed at improving drought tolerance. HighlightsAn approach to identify osmolytes and osmoprotectants was implemented Accumulation of Trp, Val and Lys was consistent with their role in osmoprotection Osmotic adjustment relied predominantly on organic acids than on inorganic ions Monosaccharides but not sucrose correlates with changes in needle water status

Salinity and sodicity stresses adversely affect rice growth and yield. To overcome yield losses, suitable tolerant rice cultivars can be developed through a marker-assisted breeding (MAB) program. In the present study, genomic regions associated with sodicity stress tolerance at the reproductive stage were identified using a high-density 50kSNP array in a recombinant inbred line (RIL) population derived from the contrasting rice genotypes CSR11 and MI48. A total of 50 QTLs were detected for various yield-related traits; further, 19 QTLs with [&ge;]15% of phenotypic variance were selected for integrated (omics) analysis. RNA sequencing of leaves and panicles at the reproductive stage under sodic stress conditions was employed to find differentially expressed genes. A total of 1368 and 1410 SNPs; 104 and 144 indels were found for MI48 and CSR11, respectively, within the QTL regions from resequencing. At chromosomes 1 and 6, colocalized QTLs (qPH1-1/qGP1-1 and qGP6-2/qSSI6-2) were discovered. Differentially expressed genes (DEGs) were mapped over the QTL regions selected, and SNP variations and indels were screened for colocalized QTLs. Potential candidate genes, namely Os-pGlcT1 (Os01g0133400), OsHKT2;1 (Os06g0701600) and OsHKT2;4 (Os06g0701700), OsANTH12 (Os06g0699800), and OsPTR2 (Os06g0706400), were identified as being responsible for glucose transport, ion homeostasis, pollen germination, and nitrogen use efficiency, respectively, under salt stress. Finally, our study provides important insights into the genes and potential mechanisms affecting grain yield under sodic stress in rice, which will contribute to the development of molecular markers for rice breeding programs.

Published studies have reported species-variance between profiles of Calvin-Benson cycle (CBC) intermediates, not only between C4 species and C3 species, but also within C3 species (Arrivault et al., 2019, Borghi et al. 2019). It was proposed that this variance reflects lineage-dependent changes in the balance between different reactions, or poising, of the CBC. These earlier studies investigated phylogenetically-unrelated C3 species. In the current study, CBC intermediates were profiled in five closely-related species from Solanum sect. lycopersicon subsect. Lycopersicum. The levels of individual CBC intermediates showed many significant differences. In a principal component analysis, whilst three species (Solanum lycopersicum, Solanum cheesmaniae, Solanum neorickii) overlapped, Solanum pimpinellifolium and especially Solanum pennellii grouped separately, and were at opposing ends of the distribution. When combined with published data, whilst the separation between Solanum species was retained, they formed a group that was separated from five other C3 species, as well as two C4 species. It is discussed that the observed variation in CBC metabolites profiles within Solanum, together with their separation from other C3 species, supports the idea that CBC evolution is shaped both by phylogenetic relatedness and lineage-specific adaptation. HighlightVariance of intermediate levels points to poising of the Calvin-Benson cycle varying between closely-related species in the tomato clade Solanum sect. lycopersicon subsect. Lycopersicum