BACKGROUND: Glandular trichomes, often referred to as \"phytochemical factories\", plays a crucial role in plant growth and metabolism. As the site for secretion and storage, the development of glandular trichomes is related to the dynamic biosynthesis of specialised metabolites. The study aims to explore the relationship between spatial phenotype and dynamic metabolism of glandular trichomes, and establish a novel approach for the exploration and study of the regulatory mechanism governing the development of glandular trichomes.
RESULTS: In this study, we proposed a technical route based on the relative deviation value to distinguish the peltate glandular trichomes (PGTs) from the background tissues and extract their spatial phenotype. By defining glandular trichome developmental stages based on the leaf vein growth axis, we found that young PGTs were densely distributed near the proximal end of growth axis of the leaf veins, where perillaketone, a primary metabolite of PGTs, is predominantly accumulated. Conversely, mature PGTs are typically found near the distal end of the mid-vein growth axis and the lateral end of the secondary vein growth axis, where the accumulation rate of isoegomaketone and egomaketone exceeds that of perillaketone in PGTs. We further identified spatial phenotypic parameters, Lsum and d, as independent variables to construct a linear regression model that illustrates the relationship between the spatial phenotypes and metabolite content of PGTs, including perillaketone (R2 = 0.698), egomaketone (R2 = 0.593), isoegomaketone (R2 = 0.662) and the sum of the amount (R2 = 0.773).
CONCLUSIONS: This model proved that the development of PGTs was correlated with the growth of the entire leaf, and the development stage of PGTs can be identifined by spatial phenotypes based on the leaf veins. In conclusion, the findings of this study enhance our understanding of correlation between spatial phenotype and development of glandular trichomes and offer a new approach to explore and study the regulatory mechanism of glandular trichome development.

The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3-day-old cotyledons using single-cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3-day-old cotyledons based on single-cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.

The unique, hierarchical patterns of leaf veins have attracted extensive attention in recent years. However, it remains unclear how biological and mechanical factors influence the topology of leaf veins. In this paper, we investigate the optimization mechanisms of leaf veins through a combination of experimental measurements and numerical simulations. The topological details of three types of representative plant leaves are measured. The experimental results show that the vein patterns are insensitive to leaf shapes and curvature. The numbers of secondary veins are independent of the length of the main vein, and the total length of veins increases linearly with the leaf perimeter. By integrating biomechanical mechanisms into the topology optimization process, a transdisciplinary computational method is developed to optimize leaf structures. The numerical results show that improving the efficiency of nutrient transport plays a critical role in the morphogenesis of leaf veins. Contrary to the popular belief in the literature, this study shows that the structural performance is not a key factor in determining the venation patterns. The findings provide a deep understanding of the optimization mechanism of leaf veins, which is useful for the design of high-performance shell structures.

Beyond facilitating transport and providing mechanical support to the leaf, veins have important roles in the performance and productivity of plants and the ecosystem. In recent decades, computational image analysis has accelerated the extraction and quantification of vein traits, benefiting fields of research from agriculture to climatology. However, most of the existing leaf vein image analysis programs have been developed for the reticulate venation found in dicots. Despite the agroeconomic importance of cereal grass crops, like Oryza sativa (rice) and Zea mays (maize), a dedicated image analysis program for the parallel venation found in monocots has yet to be developed. To address the need for an image-based vein phenotyping tool for model and agronomic grass species, we developed the grass vein image quantification (grasviq) framework. Designed specifically for parallel venation, this framework automatically segments and quantifies vein patterns from images of cleared leaf pieces using classical computer vision techniques. Using image data sets from maize inbred lines and auxin biosynthesis and transport mutants in maize, we demonstrate the utility of grasviq for quantifying important vein traits, including vein density, vein width and interveinal distance. Furthermore, we show that the framework can resolve quantitative differences and identify vein patterning defects, which is advantageous for genetic experiments and mutant screens. We report that grasviq can perform high-throughput vein quantification, with precision on a par with that of manual quantification. Therefore, we envision that grasviq will be adopted for vein phenomics in maize and other grass species.

The identification of sites in leaves where transpiration water crosses cell membranes and enters the symplast has previously been made using freeze-substitution to locate concentrations of dye [e.g. sulphorhodamine (SR)] moving with the transpiration stream, and left outside the membranes where the water passes through. These concentrations were called sumps, and the sites of entry to the symplast were called flumes. A simple method of locating sumps, and therefore flumes, is described. Fresh leaves, fed SR solution through their cut petioles for pulse periods of 0.5 h or more, followed or not by a chase of water, were sectioned by hand under paraffin oil, and the sections mounted in the same fluid. Observation of the sections by simple bright-field microscopy revealed sumps of SR at the same sites, and of the same crystalline nature as found in the freeze-substituted preparations. The saving in preparation time is of the order of > 100-fold, at the sacrifice of resolution (5-10 μm compared with 0.2 μm). A limited survey of grass, sedge and dicotyledon leaves by this method confirmed in all essentials the results found by freeze-substitution, and in addition, revealed flumes at the fusoid cells on the flanks of the veins of bamboo leaves, and at the same position next to the water tissue of Cyperus leaves. The rate of accumulation of crystalline SR in the sumps inside tracheary elements suggests that the concentration of this non-permeating solute in the xylem sap increased by about 1000-fold in the finest veins during 1-2 h of transpiration in the dye solution.

Changes of view on the course of the transpiration stream beyond the veins in leaves are followed from the imbibition theory of Sachs, through the (symplastic) endosmotic theory of Pfeffer (which prevailed almost unquestioned until the late 1930s), to Strugger\'s experiments with fluorescent dye tracers and the epifluorescence microscope. This latter work persuaded many to return to the apoplastic-(wall)-path viewpoint, which, despite early and late criticisms that were never rebutted, is still widely held. Tracer experiments of the same kind are still frequently published without consideration of the evidence that they do not reveal the paths of water movement. Experiments on rehydration kinetics of leaves have not produced unequivocal evidence for either path. The detailed destinies of the solutes that reach the leaf in the transpiration stream have received little attention. Consideration of physical principles governing flow and evaporation in a transpiring leaf emphasizes that: (1) Diffusion over interveinal distances at the rates in water will account for substantial solute movement in a few minutes, even in the absence of flow. (2) Diffusion can occur also against opposing now. (3) Volume fluxes in veins are determined by the diameter of the largest leaves examined contain high conductance supply veins which are tapped into by low-conductance distributing veins. (4) Edges and teeth of leaves will be places of especially rapid evaporation, and they often have high-conductance veins leading to them. (5) Solutes in the stream will tend to accumulate at leaf margins. On the basis of recent work, the view is maintained that the water of the stream enters the symplast through cell membranes very close to tracheary elements. Also, that this occurs locally over a small area of membrane. Many solutes in the stream are left outside in the apoplast. This produces regions of high solute concentration in the apoplast and an enrichment of solutes in the stream as it perfuses the leaf. Solutes that enter the symplast are not so easily tracked. Suggestions about where some of them may go can be gained from a fluorescent probe that identifies particular cells (scavenging cells) as having H+ -ATPase porter systems to scrub selected solutes from the stream. Unpublished case-histories are presented which illustrate many aspects of these processes and principles. These are: (1) Maize leaf veins, where the symplastic water path starts at the parenchyma sheath; (2) Lupin veins, where the symplastic path starts at the bundle sheath and where solutes are concentrated in blind terminations; (3) The edges of maize leaves where flow is enhanced by a large vein (open to the apoplast), and solutes are deposited in the apoplast by evaporation; (4) Poplar leaf teeth, which receive strong flows, and where the epithem cells are scavenging cells; (5) Mimosa leaf marginal hairs, which have scavenging cells at their base; (6) Active hydathodes, whose epithem cells are scavenging cells; (7) Pine needle transfusion tissue, which is a site of both solute enrichment (in the tracheids), and scavenging (in the parenchyma); (8) Estimates are made of diffusion coefficients of a solute both along and at right angles to the major diffusive pathway in wheat leaves. The first is 1000 times the second, but is 1/100 of free diffusion in water. Five general themes of the behaviour and organization of the transpiration stream are induced from the facts reviewed. These are: (1) The stream is channelled into courses of graded intensities by the interplay of the physical forces with the anatomical features, each course with a distinct contribution to the processing of the stream. (2) Water enters the symplast at precise locations as close as possible to the tracheary elements. (3) As the stream moves through the leaf its solute concentration is enriched many-fold at predictable sites. (4) Solutes excluded from the symplast diffuse from these sources of high concentration in specially formed wall paths, in precise patterns, at rates which can be measured, and which are low compared with diffusion in water. (5) Other solutes permeate the symplast, often over the surfaces of groups of cells which are organized into recognized structural features. CONTENTS Summary 341 I. What becomes of the transpiration stream ? 342 II. Review 343 III. Preview 355 IV. Overview 361 Acknowledgements 365 References 365.

Little is known about the physiological and molecular mechanisms underlying magnesium (Mg)-deficiency-induced enlargement, cracking and lignification of midribs and main lateral veins of Citrus leaves. Citrus sinensis (L.) Osbeck seedlings were irrigated with nutrient solution at a concentration of 0 (Mg-deficiency) or 2 (Mg-sufficiency) mM Mg(NO3)2 for 16 weeks. Enlargement, cracking and lignification of veins occurred only in lower leaves, but not in upper leaves. Total soluble sugars (glucose + fructose + sucrose), starch and cellulose concentrations were less in Mg-deficiency veins of lower leaves (MDVLL) than those in Mg-sufficiency veins of lower leaves (MSVLL), but lignin concentration was higher in MDVLL than that in MSVLL. However, all four parameters were similar between Mg-deficiency veins of upper leaves (MDVUL) and Mg-sufficiency veins of upper leaves (MSVUL). Using label-free, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we identified 1229 and 492 differentially abundant proteins (DAPs) in MDVLL vs MSVLL and MDVUL vs MSVUL, respectively. Magnesium-deficiency-induced alterations of Mg, nonstructural carbohydrates, cell wall components, and protein profiles were greater in veins of lower leaves than those in veins of upper leaves. The increased concentration of lignin in MDVLL vs MSVLL might be caused by the following factors: (i) repression of cellulose and starch accumulation promoted lignin biosynthesis; (ii) abundances of proteins involved in phenylpropanoid biosynthesis pathway, hormone biosynthesis and glutathione metabolism were increased; and (iii) the abundances of the other DAPs [viz., copper/zinc-superoxide dismutase, ascorbate oxidase (AO) and ABC transporters] involved in lignin biosynthesis were elevated. Also, the abundances of several proteins involved in cell wall metabolism (viz., expansins, Rho GTPase-activating protein gacA, AO, monocopper oxidase-like protein and xyloglucan endotransglucosylase/hydrolase) were increased in MDVLL vs MSVLL, which might be responsible for the enlargement and cracking of leaf veins.