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Accumulation of cadmium (Cd) shows a serious problem for the environment and poses a threat to plants. Plants employing various cellular and molecular mechanisms to limit Cd toxicity and alterations of the cell wall structure were observed upon Cd exposure. This study focuses on changes in the cell wall protein-enriched subproteome of alfalfa (Medicago sativa) leaves during long-term Cd exposure. Plants grew on Cd-contaminated soil (10 mg/kg dry weight (DW)) for an entire season. A targeted approach was used to sequentially extract cell wall protein-enriched fractions from the leaves and quantitative analyses were conducted with two-dimensional difference gel electrophoresis (2D DIGE) followed by protein identification with matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time of flight (TOF/TOF) mass spectrometry. In 212 spots that showed a significant change in intensity upon Cd exposure a single protein was identified. Of these, 163 proteins are predicted to be secreted and involved in various physiological processes. Proteins of other subcellular localization were mainly chloroplastic and decreased in response to Cd, which confirms the Cd-induced disturbance of the photosynthesis. The observed changes indicate an active defence response against a Cd-induced oxidative burst and a restructuring of the cell wall, which is, however, different to what is observed in M. sativa stems and will be discussed.

• Leaf functional traits and their covariation underlie plant ecological adaptations along environmental gradients, but there is limited information on the global covariation patterns of key leaf construction traits. • To explore how leaf construction traits co-vary across diverse climate and soil environmental conditions, we compiled a global dataset including cell wall mass per unit leaf mass (CWmass), leaf carbon (C) and calcium (Ca) concentrations, and specific leaf area (SLA) for 2348 angiosperm species from 340 sites world-wide. • Our results demonstrated negative correlations between leaf C and Ca concentrations and between leaf C and SLA across diverse nongraminoid angiosperms. Leaf C concentration increased with increasing mean annual temperature (MAT) and mean annual precipitation (MAP) and with decreasing soil pH and calcium carbonate (CaCO₃) concentration, whereas leaf Ca concentration and SLA exhibited the opposite responses to these environmental variables. The covariations of leaf Ca–C and of leaf SLA–C were stronger in habitats with lower MAT and MAP, and/or higher soil CaCO₃ content. • This global-scale analysis demonstrates that the leaf C and Ca concentrations and SLA together govern the C and biomass investment strategies in leaves of nongraminoids. We conclude that environmental conditions strongly shape leaf construction traits and their covariation patterns.

Single-cell RNA-seq (scRNA-seq) has been highlighted as a powerful tool for the description of human cell transcriptome, but the technology has not been broadly applied in plant cells. Herein, we describe the successful development of a robust protoplast cell isolation system in the peanut leaf. A total of 6,815 single cells were divided into eight cell clusters based on reported marker genes by applying scRNA-seq. Further, a pseudo-time analysis was used to describe the developmental trajectory and interaction network of transcription factors (TFs) of distinct cell types during leaf growth. The trajectory enabled re-investigation of the primordium-driven development processes of the mesophyll and epidermis. These results suggest that palisade cells likely differentiate into spongy cells, while the epidermal cells originated earlier than the primordium. Subsequently, the developed method integrated multiple technologies to efficiently validate the scRNA-seq result in a homogenous cell population. The expression levels of several TFs were strongly correlated with epidermal ontogeny in accordance with obtained scRNA-seq values. Additionally, peanut AHL23 (AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 23), which is localized in nucleus, promoted leaf growth when ectopically expressed in Arabidopsis by modulating the phytohormone pathway. Together, our study displays that application of scRNA-seq can provide new hypotheses regarding cell differentiation in the leaf blade of Arachis hypogaea. We believe that this approach will enable significant advances in the functional study of leaf blade cells in the allotetraploid peanut and other plant species.

The effects of nitrogen (N) availability on cell number and cell size, and the contribution of these determinants to the final area of fully expanded leaves of sunflower (Helianthus annuus L.) were investigated in glasshouse experiments. Plants were given a high (N =315 ppm) or low (N=21 ppm) N supply and were transferred between N levels at different developmental stages (5 to 60% of final size) of target leaves. The dynamics of cell number in unemerged (< 0.01 m in length) leaves of plants growing at high and low levels of N supply were also followed. Maximum leaf area (LAmax) was strongly (up to two-fold) and significantly modified by N availability and the timing of transfer between N supplies, through effects on leaf expansion rate. Rate of cell production was significantly (P<0.05) reduced in unemerged target leaves under N stress, but there was no evidence of a change in primordium size or in the duration of the leaf differentiation–emergence phase. In fully expanded leaves, number of cells per leaf (Ncell), leaf area per cell (LAcell) and cell area (Acell) were significantly reduced by N stress. WhileLAcell and Acellresponded to changeover treatments irrespective of leaf size, significant (P<0.05) changes in Ncellonly occurred when the changeover occurred before the leaf reached approx. 10% of LAmax. There were no differential effects of N on numbers of epidermal vs. mesophyll cells. The results show that the effects of N on leaf size are largely due to effects on cell production in the unemerged leaf and on both cell production and expansion during the first phase of expansion of the emerged leaf. During the rest of the expansion period N mainly affects the expansion of existing cells. Cell area plasticity permitted a response to changes in N supply even at advanced stages of leaf expansion. Increased cell expansion can compensate for low Ncellif N stress is relieved early in the expansion of emerged leaves, but in later phases Ncellsets a limit to this response.

The peltate glandular trichomes (PGTs) on Nepeta tenuifolia leaves can secrete and store bioactive essential oils. ScRNA-seq is a powerful tool for uncovering heterogeneous cells and exploring the development and differentiation of specific cells. Due to leaves rich in PGTs, the young leaves were used to isolated protoplasts and successfully captured 33,254 protoplasts for sequencing purposes. After cell type annotation, all the cells were partitioned into six broad populations with 19 clusters. Cells from PGTs were identified based on the expression patterns of trichome-specific genes, monoterpene biosynthetic genes, and metabolic analysis of PGT secretions. The developmental trajectories of PGTs were delineated by pseudotime analysis. Integrative analysis of scRNA-seq data from N. tenuifolia leaves and Arabidopsis thaliana shoot revealed that PGTs were specific to N. tenuifolia . Thus, our results provide a promising basis for exploring cell development and differentiation in plants, especially glandular trichome initiation and development.

Plant bZIP group I transcription factors have been reported mainly for their role during vascular development and osmosensory responses. Interestingly, bZIP29 has been identified in a cell cycle interactome, indicating additional functions of bZIP29 in plant development. Here, bZIP29 was functionally characterized to study its role during plant development. It is not present in vascular tissue but is specifically expressed in proliferative tissues. Genome-wide mapping of bZIP29 target genes confirmed its role in stress and osmosensory responses, but also identified specific binding to several core cell cycle genes and to genes involved in cell wall organization. bZIP29 protein complex analyses validated interaction with other bZIP group I members and provided insight into regulatory mechanisms acting on bZIP dimers. In agreement with bZIP29 expression in proliferative tissues and with its binding to promoters of cell cycle regulators, dominant-negative repression of bZIP29 altered the cell number in leaves and in the root meristem. A transcriptome analysis on the root meristem, however, indicated that bZIP29 might regulate cell number through control of cell wall organization. Finally, ectopic dominant-negative repression of bZIP29 and redundant factors led to a seedling-lethal phenotype, pointing to essential roles for bZIP group I factors early in plant development.

Background As an essential trace element for plants, manganese (Mn) poses significant threats to ecosystems and human health when it accumulates excessively in soils and aquatic environments. Celosia argentea Linn. is a promising Mn hyperaccumulator. However, the regulatory mechanisms underlying leaf responses to Mn stress remain poorly understood, particularly regarding the interplay between stomatal dynamics and detoxification strategies. This study investigated the physiological and biochemical adaptive mechanisms of C. argentea leaves under Mn toxicity, with a focus on elucidating the roles of antioxidant defence systems, stomatal behaviour modulation, and Mn chemical transformation in detoxification processes. Results The experimental findings demonstrate three outcomes. (1) The antioxidant system mitigates Mn-induced oxidative damage through synergistic interactions between enzymatic and nonenzymatic components, although the defensive capacity becomes limited under extreme Mn concentrations. (2) The presence of Mn stress significantly reduces gas exchange parameters, specifically the transpiration rate (Tr) and stomatal conductance (Gs), in leaves, with more pronounced decreases observed in leaf tip parts, revealing the adaptive mechanisms in leaves under such stress conditions. (3) The chemical forms of Mn undergo dynamic transformations along concentration gradients, shifting from water-soluble fractions to protein-bound forms, which indicates the existence of an adaptive detoxification strategy. Conclusions These findings provide theoretical insights and scientific evidence for understanding the heavy metal tolerance strategies of hyperaccumulators and advancing the application of phytoremediation technologies.

The genetic basis of plant response to light and heat stresses had been unveiled, and different molecular mechanisms of leaf cell homeostasis to keep high physiological performances were recognized in grapevine varieties. However, the ability to develop heat stress tolerance strategies must be further elucidated since the morpho-anatomical and physiological traits involved may vary with genotype × environment combination, stress intensity, and duration. A 3-year experiment was conducted on potted plants of Sardinian red grapevine cultivars Cannonau (syn. Grenache) and Carignano (syn. Carignan), exposed to prolonged heat stress inside a UV-blocking greenhouse, either submitted to low daily UV-B doses of 4.63 kJ m −2 d −1 (+UV) or to 0 kJ m −2 d −1 (−UV), and compared to a control (C) exposed to solar radiation (4.05 kJ m −2 d −1 average UV-B dose). Irrigation was supplied to avoid water stress, and canopy light and thermal microclimate were monitored continuously. Heat stress exceeded one-third of the duration inside the greenhouse and 6% in C. In vivo spectroscopy, including leaf reflectance and fluorescence, allowed for characterizing different patterns of leaf traits and metabolites involved in oxidative stress protection. Cannonau showed lower stomatal conductance under C (200 mmol m −2 s −1 ) but more than twice the values inside the greenhouse (400 to 900 mmol m −2 s −1 ), where water use efficiency was reduced similarly in both varieties. Under severe heat stress and −UV, Cannonau showed a sharper decrease in primary photochemical activity and higher leaf pigment reflectance indexes and leaf mass area. UV-B increased the leaf pigments, especially in Carignano, and different leaf cell regulatory traits to prevent oxidative damage were observed in leaf cross-sections. Heat stress induced chloroplast swelling, plastoglobule diffusion, and the accumulation of secretion deposits in both varieties, aggravated in Cannonau −UV by cell vacuolation, membrane dilation, and diffused leaf blade spot swelling. Conversely, in Carignano UV-B, cell wall barriers and calcium oxalate crystals proliferated in mesophyll cells. These responses suggest an adaptive divergence among cultivars to prolonged heat stress and UV-B light. Further research on grapevine biodiversity, heat, and UV-B light interactions may give new insights on the extent of stress tolerance to improve viticulture adaptive strategies in climate change hotspots.

• Plants grow and transpire during the night. The aim of the present work was to assess the relative flows of carbon, water and solutes, and the energy involved, in sustaining night-time transpiration and leaf expansive growth under control and salt-stress conditions. • Published and unpublished data were used, for barley plants grown in presence of 0.5–1 mM NaCl (control) and 100 mM NaCl. • Night-time leaf growth presents a more efficient use of taken-up water compared with day-time growth. This efficiency increases several-fold with salt stress. Night-time transpiration cannot be supported entirely through osmotically driven uptake of water through roots under salt stress. Using a simple three- (root medium/cytosol/vacuole) compartment approach, the energy required to support cell expansion during the night is in the lower percentage region (0.03–5.5%) of the energy available through respiration, under both, control and salt-stress conditions. Use of organic (e.g. hexose equivalents) rather than inorganic (e.g. Na⁺, Cl⁻, K⁺) solutes for generation of osmotic pressure in growing cells, increases the energy demand by orders of magnitude, yet requires only a small portion of carbon assimilated during the day. • Night-time transpiration and leaf expansive growth should be considered as a potential acclimation mechanism to salinity.

Main conclusionΔClnps6 induced iron redistribution in maize B73 leaf cells and resulted in reactive oxygen species (ROS) burst to enhance plant resistance against Curvularia lunata.Iron is an indispensable co-factor of various crucial enzymes that are involved in cellular metabolic processes and energy metabolism in eukaryotes. For this reason, plants and pathogens compete for iron to maintain their iron homeostasis, respectively. In our previous study, ΔClnps6, the extracellular siderophore biosynthesis deletion mutant of Curvularia lunata, was sensitive to exogenous hydrogen peroxide and virulence reduction. However, the mechanism was not studied. Here, we report that maize B73 displayed highly resistance to ΔClnps6. The plants recruited more iron at cell wall appositions (CWAs) to cause ROS bursts. Intracellular iron deficiency induced by iron redistribution originated form up-regulated expression of genes involved in intracellular iron consumption in leaves and absorption in roots. The RNA-sequencing data also showed that the expression of respiratory burst oxidase homologue (ZmRBOH4) and NADP-dependent malic enzyme 4 (ZmNADP-ME4) involved in ROS production was up-regulated in maize B73 after ΔClnps6 infection. Simultaneously, jasmonic acid (JA) biosynthesis genes lipoxygenase (ZmLOX), allene oxide synthase (ZmAOS), GA degradation gene gibberellin 2-beta-dioxygenase (ZmGA2OX6) and ABA degradation genes abscisic acid hydroxylase (ZmABH1, ZmABH2) involved in iron homeostasis were up-regulated expression. Ferritin1 (ZmFER1) positive regulated maize resistance against C. lunata via ROS burst under Fe-limiting conditions. Overall, our results showed that iron played vital roles in activating maize resistance in B73-C. lunata interaction.