Quantitative trait loci controlling leaf venation in Arabidopsis

Leaf veins provide the mechanical support and are responsible for the transport of nutrients and water to the plant. High vein density is a prerequisite for plants to have C4 photosynthesis. We investigated the genetic variation and genetic architecture of leaf venation traits within the species Arabidopsis thaliana using natural variation. Leaf venation traits, including leaf vein density (LVD) were analysed in 66 worldwide accessions and 399 lines of the multi‐parent advanced generation intercross population. It was shown that there is no correlation between LVD and photosynthesis parameters within A. thaliana. Association mapping was performed for LVD and identified 16 and 17 putative quantitative trait loci (QTLs) in the multi‐parent advanced generation intercross and worldwide sets, respectively. There was no overlap between the identified QTLs suggesting that many genes can affect the traits. In addition, linkage mapping was performed using two biparental recombinant inbred line populations. Combining linkage and association mapping revealed seven candidate genes. For one of the candidate genes, RCI2c, we demonstrated its function in leaf venation patterning.     

中国花楸属单叶类群叶脉序特征研究

中国花楸属单叶类群(24种)包含落萼组与冠萼组,亦有学者将其分为两个属(白花楸属和水榆属)或合并成为一个属(白花楸属).由于其花序和果实的高度相似性,长期以来分类地位具有争议,而叶脉序特征在花楸属单叶类群中的分类作用尚不明确.因此,该研究以国产花楸属落萼组的13个物种和冠萼组的7个物种作为研究对象,以清净法制作标本对叶...     

叶脉网络功能性状及其生态学意义

叶脉网络结构是叶脉系统在叶片里的分布和排列样式。早期叶脉网络结构研究主要集中在其分类学意义上; 近年来叶脉网络功能性状及其在植物水分利用上的意义已成为植物生态学研究的热点。该文介绍了叶脉网络功能性状的指标体系(包括叶脉密度、叶脉直径、叶脉之间的距离、叶脉闭合度等), 综述了叶脉网络功能性状与叶脉系统功能(包括水分、养分和光合产物等物质运输、机械支撑和虫害防御等)的关系, 叶脉网络功能性状与叶片其他功能性状(包括比叶重、叶寿命、光合速率、叶片大小、气孔密度等)的协同变异和权衡关系, 以及叶脉网络功能性状随环境因子(包括水分、温度、光照等)的变化规律等方面的最新研究进展。此外, 叶脉网络功能性状的研究成果也被应用于古环境重建、城市交通规划、流域规划及全球变化研究中。由于叶脉网络功能性状是环境因子与系统发育共同作用的结果, 未来开展分子—叶片—植物—生态系统等多尺度的叶脉网络功能性状研究, 理清叶脉网络功能性状与气孔失水—茎干导水—根系吸水等植物水分利用的关系, 将为预测植物及生态系统对全球变化的响应提供新的启示。     

Quantifying leaf venation patterns: two-dimensional maps

The leaf vasculature plays crucial roles in transport and mechanical support. Understanding how vein patterns develop and what underlies pattern variation between species has many implications from both physiological and evolutionary perspectives. We developed a method for extracting spatial vein pattern data from leaf images, such as vein densities and also the sizes and shapes of the vein reticulations. We used this method to quantify leaf venation patterns of the first rosette leaf of Arabidopsis thaliana throughout a series of developmental stages. In particular, we characterized the size and shape of vein network areoles (loops), which enlarge and are split by new veins as a leaf develops. Pattern parameters varied in time and space. In particular, we observed a distal to proximal gradient in loop shape (length/width ratio) which varied over time, and a margin-to-center gradient in loop sizes. Quantitative analyses of vein patterns at the tissue level provide a two-way link between theoretical models of patterning and molecular experimental work to further explore patterning mechanisms during development. Such analyses could also be used to investigate the effect of environmental factors on vein patterns, or to compare venation patterns from different species for evolutionary studies. The method also provides a framework for gathering and overlaying two-dimensional maps of point, line and surface morphological data.     

Venation networks and the origin of the leaf economics spectrum

The leaf economics spectrum describes biome-invariant scaling functions for leaf functional traits that relate to global primary productivity and nutrient cycling. Here, we develop a comprehensive framework for the origin of this leaf economics spectrum based on venation-mediated economic strategies. We define a standardized set of traits - density, distance and loopiness - that provides a common language for the study of venation. We develop a novel quantitative model that uses these venation traits to model leaf-level physiology, and show that selection to optimize the venation network predicts the mean global trait-trait scaling relationships across 2548 species. Furthermore, using empirical venation data for 25 plant species, we test our model by predicting four key leaf functional traits related to leaf economics: net carbon assimilation rate, life span, leaf mass per area ratio and nitrogen content. Together, these results indicate that selection on venation geometry is a fundamental basis for understanding the diversity of leaf form and function, and the carbon balance of leaves. The model and associated predictions have broad implications for integrating venation network geometry with pattern and process in ecophysiology, ecology and palaeobotany.     

贵州小檗属植物(小檗科)叶脉序研究

采用制作叶脉标本和透明叶标本的方法,对贵州产28种2变种小檗属植物叶脉特征进行比较研究。结果表明:贵州小檗属植物的脉序类型有5种:半达缘羽状脉、花环状半达缘羽状脉、简单弓形羽状脉、花环状弓形羽状脉和混合型。叶脉分支一般有五级:1一级脉构架均为羽状脉,粗度有很粗、粗、中等粗细和纤细四种类型,分支方式包括单轴分支和合轴分支;2粗二级脉构架中有分支达缘或分支均不达缘,与中脉夹角变化各异,内二级脉存在或缺失,细二级脉半达缘、真曲行或简单弓形,间二级脉类型复杂多变但频度种间有差异;3三级脉贯串型、结网型或分支型;4四、五级脉网状或自由分支且常混合在一起。脉间区从发育差到良好,小脉从不分支到不均等分支等各种类型均有,叶缘末级脉缺失、不完整、钉状和环状。大部分种类叶缘具齿,每1cm齿数目和齿内腺点的特性等特征在不同种类间有区别,具有鉴定价值,但齿其它特征复杂多变或种间区别较小,同时齿内脉性状也不稳定。此外,齿的有无会对脉序类型产生影响。小檗属植物叶脉类型存在种间差异,具有重要的分类学价值,叶脉类型的变化和复杂程度显示了该属植物的进化特点;叶齿的有无和齿特征具有分类学和系统学意义。基于叶脉特征的研究结果并结合重要的外部形态学特征编制了贵州小檗属植物的分种检索表。研究结果可为小檗属植物分类寻找新的依据并探讨其系统学意义。     

Linking leaf veins to growth and mortality rates: an example from a subtropical tree community

A fundamental goal in ecology is to link variation in species function to performance, but functional trait–performance investigations have had mixed success. This indicates that less commonly measured functional traits may more clearly elucidate trait–performance relationships. Despite the potential importance of leaf vein traits, which are expected to be related to resource delivery rates and photosynthetic capacity, there are few studies, which examine associations between these traits and demographic performance in communities. Here, we examined the associations between species traits including leaf venation traits and demographic rates (Relative Growth Rate, RGR and mortality) as well as the spatial distributions of traits along soil environment for 54 co‐occurring species in a subtropical forest. Size‐related changes in demographic rates were estimated using a hierarchical Bayesian approach. Next, Kendall's rank correlations were quantified between traits and estimated demographic rates at a given size and between traits and species‐average soil environment. Species with denser venation, smaller areoles, less succulent, or thinner leaves showed higher RGR for a wide range of size classes. Species with leaves of denser veins, larger area, cheaper construction costs or thinner, or low‐density wood were associated with high mortality rates only in small size classes. Lastly, contrary to our expectations, acquisitive traits were not related to resource‐rich edaphic conditions. This study shows that leaf vein traits are weakly, but significantly related to tree demographic performance together with other species traits. Because leaf traits associated with an acquisitive strategy such as denser venation, less succulence, and thinner leaves showed higher growth rate, but similar leaf traits were not associated with mortality, different pathways may shape species growth and survival. This study suggests that we are still not measuring some of key traits related to resource‐use strategies, which dictate the demography and distributions of species.     

Quantitative analysis of venation patterns of Arabidopsis leaves by supervised image analysis

The study of transgenic Arabidopsis lines with altered vascular patterns has revealed key players in the venation process, but details of the vascularization process are still unclear, partly because most lines have only been assessed qualitatively. Therefore, quantitative analyses are required to identify subtle perturbations in the pattern and to test dynamic modeling hypotheses using biological measurements. We developed an online framework, designated Leaf Image Analysis Interface (LIMANI), in which venation patterns are automatically segmented and measured on dark-field images. Image segmentation may be manually corrected through use of an interactive interface, allowing supervision and rectification steps in the automated image analysis pipeline and ensuring high-fidelity analysis. This online approach is advantageous for the user in terms of installation, software updates, computer load and data storage. The framework was used to study vascular differentiation during leaf development and to analyze the venation pattern in transgenic lines with contrasting cellular and leaf size traits. The results show the evolution of vascular traits during leaf development, suggest a self-organizing mechanism for leaf venation patterning, and reveal a tight balance between the number of end-points and branching points within the leaf vascular network that does not depend on the leaf developmental stage and cellular content, but on the leaf position on the rosette. These findings indicate that development of LIMANI improves understanding of the interaction between vascular patterning and leaf growth.     

Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild‐type and leaf vein mutants

Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (K_leaf), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col‐0) and four vein mutants (dot3‐111 and dot3‐134, and cvp1‐3 and cvp2‐1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean K_leaf. Across all genotypes, there was a strong correlation of K_leaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence K_leaf, indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing K_leaf.     

Decline of leaf hydraulic conductance with dehydration: relationship to leaf size and venation architecture

Across plant species, leaves vary enormously in their size and their venation architecture, of which one major function is to replace water lost to transpiration. The leaf hydraulic conductance (K(leaf)) represents the capacity of the transport system to deliver water, allowing stomata to remain open for photosynthesis. Previous studies showed that K(leaf) relates to vein density (vein length per area). Additionally, venation architecture determines the sensitivity of K(leaf) to damage; severing the midrib caused K(leaf) and gas exchange to decline, with lesser impacts in leaves with higher major vein density that provided more numerous water flow pathways around the damaged vein. Because xylem embolism during dehydration also reduces K(leaf), we hypothesized that higher major vein density would also reduce hydraulic vulnerability. Smaller leaves, which generally have higher major vein density, would thus have lower hydraulic vulnerability. Tests using simulations with a spatially explicit model confirmed that smaller leaves with higher major vein density were more tolerant of major vein embolism. Additionally, for 10 species ranging strongly in drought tolerance, hydraulic vulnerability, determined as the leaf water potential at 50% and 80% loss of K(leaf), was lower with greater major vein density and smaller leaf size (|r| = 0.85-0.90; P < 0.01). These relationships were independent of other aspects of physiological and morphological drought tolerance. These findings point to a new functional role of venation architecture and small leaf size in drought tolerance, potentially contributing to well-known biogeographic trends in leaf size.     

Topological Phenotypes Constitute a New Dimension in the Phenotypic Space of Leaf Venation Networks

The leaves of angiosperms contain highly complex venation networks consisting of recursively nested, hierarchically organized loops. We describe a new phenotypic trait of reticulate vascular networks based on the topology of the nested loops. This phenotypic trait encodes information orthogonal to widely used geometric phenotypic traits, and thus constitutes a new dimension in the leaf venation phenotypic space. We apply our metric to a database of 186 leaves and leaflets representing 137 species, predominantly from the Burseraceae family, revealing diverse topological network traits even within this single family. We show that topological information significantly improves identification of leaves from fragments by calculating a “leaf venation fingerprint” from topology and geometry. Further, we present a phenomenological model suggesting that the topological traits can be explained by noise effects unique to specimen during development of each leaf which leave their imprint on the final network. This work opens the path to new quantitative identification techniques for leaves which go beyond simple geometric traits such as vein density and is directly applicable to other planar or sub-planar networks such as blood vessels in the brain.     

A morpho-anatomical characterisation of Myrothamnus moschatus (Myrothamnaceae) under the aspect of desiccation tolerance

Morpho-anatomical traits of the rarely studied dicotyledonous desiccation-tolerant shrub Myrothamnus moschatus were examined and compared for the first time to Myrothamnus flabellifolius under the aspect of desiccation tolerance. Both species almost exclusively occur on rock outcrops and differ mainly in their geographic range and leaf morphology (fan-shaped in M. flabellifolius, lanceolate in M. moschatus) but have a very similar leaf and wood anatomy, except for the lack of hydathodes in M. moschatus. Both species adopt the parallel leaf venation of monocots, although this is more pronounced in M. moschatus. This provides a mechanical and protective advantage over the net venation pattern of most dicots and facilitates the reversible, drought-induced, accordion-like leaf contraction. The sclerenchyma, as a stabilising tissue, is mainly confined to vascular bundles in leaves of both species. Here, mechanical support seems to be less crucial for survival in long periods of drought than other morpho-anatomical traits (e.g. parallel leaf venation).     

植物叶片水力与经济性状权衡关系的研究进展

叶片既是植物光合产物形成的主要场所, 又是整株植物的水力瓶颈、应对灾难性水力失调的安全阀门, 是植物碳水耦合权衡的重要器官。叶经济型谱反映了叶片经济性状“投资-收益”的权衡, 为验证植物进化过程中形成的物种对策提供了适用的理论框架。叶片水力性状变化会影响叶片经济性状及植物存活和生长。因此, 探索植物叶片水力与经济性状的权衡关系, 对建立植物碳-水耦合模型、揭示植物水-碳投资机理、扩展植物性状型谱等均有重要意义。该文首先综述了叶片水力性状、经济性状及两者之间的权衡关系, 分析了叶片导水率与水力脆弱性、失膨点水势、水容、安全阈值等水力性状以及与叶片的形态、结构和气体交换功能性状之间的关系。然后, 从叶片形态、解剖和叶脉网络结构以及气孔功能方面探讨了叶片水力性状与经济性状的调节机制。最后, 提出今后应加强三方面的研究: (1)探索建立植物根-茎-叶水力输导系统的碳-氮-水资源的整株经济型谱, 以揭示植物功能结构耦合、高效固碳用水的生理生态学机制; (2)探索叶片水力安全、水力效率和固碳效率之间的普适性权衡关系, 以深入理解抗旱植物叶片构建的生物物理结构与生理代谢的关系; (3)探索个体水平碳水代谢关系、水分运输与生长速率的耦合, 为代谢推演理论和植物群落尺度预测提供基础。     

Trade-offs between plant leaf hydraulic and economic traits

Leaf is the most important organ for carbon-water coupling of a plant because it is the primary medium for photosynthesis. It also acts as the hydraulic bottleneck and safety valve against hydraulic catastrophic dysfunctions. The leaf economics spectrum, which reflects the balance between investments and returns of leaf economic traits, provides a useful framework for examining species strategies as shaped by their evolutionary history. Changes in leaf hydraulic traits will influence leaf economic traits as well as plant survival and growth. Exploring trade-offs between leaf hydraulic and economic traits is thus of significance for modeling carbon-water relations, understanding the mechanisms of water/carbon investments, and extending the leaf economic spectrum. In this review, we first examined the trade-offs between leaf hydraulic and economic traits. Specially, we analyzed the relationships between leaf hydraulic conductivity and hydraulic vulnerability, water potential at the turgor loss point, water capacitance, safety margin, and leaf morphological, structural and functional traits. We then discussed potential mechanisms regulating leaf hydraulic and economic traits from leaf morphology, anatomy, venation, and stomatal functions. Finally, we proposed future research to: (1) develop an integrated whole-plant economics spectrum, including carbon-nitrogen-water resources and root-stem-leaf hydraulic transport system that will help revealing ecophysiological mechanisms of plant structure-functional coupling, carbon sequestration and water use; (2) explore a generalized trade-offs among leaf hydraulic safety, hydraulic efficiency and carbon fixation efficiency to advance our understanding of the relationships between biophysical structure and physiological metabolism in plant leaf construction under drought stress; and (3) explore the carbon-water metabolic relationship and coupling of water transport and growth rate for the metabolic theory and predictions at community scale.     

Two forward genetic screens for vein density mutants in sorghum converge on a cytochrome P450 gene in the brassinosteroid pathway

The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C₄ traits into C₃ plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C₄ plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C₄ species from C₃ species. To identify genetic factors that specify C₄ leaf anatomy, we generated ethyl methanesulfonate‐ and γ‐ray‐mutagenized populations of the C₄ species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F₂ populations as homozygous recessive alleles. Bulk segregant analysis using next‐generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C₄ and C₃ leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.     

闽楠叶形态与叶脉网络性状关系对城市生长环境的响应

植物叶形态与叶脉网络功能性状的协同变异与权衡关系,对深入理解植物叶脉网络功能性状对环境变异的生态适应,以及预测植物物种生活习性对城市化过程的响应具有重要意义。闽楠作为珍贵的常绿阔叶树种,正在城市绿化中逐步推广。针对不同生长环境中（行道与植物园混交林）的闽楠,开展了叶形态与叶脉网络功能性状关系对城市生长环境的响应研究。研究结果表明：闽楠叶性状值基本满足正态分布,各性状变异系数保持在10%-20%之间,群体内性状变异较为丰富,单因素方差分析表明两种环境对叶形态性状的影响比叶脉网络系统的影响更明显;两种生长环境下闽楠叶形态性状组与叶脉网络功能性状组都具有极显著相关性,行道和植物园混交林典型性相关系数分别为0.804和0.795,叶形态性状与叶脉直径呈显著正相关,形态性状、叶脉直径与初级脉密度呈显著负相关;闽楠在响应城市生长环境的过程中呈现出相应的经济权衡机制,行道环境中闽楠以较大的初级脉密度和较小叶面积来确保水分获取和光合之间的平衡,植物园闽楠则采用较低初级叶脉密度、较高叶面积和叶脉直径的叶形态和叶脉网络构建模式。在选择闽楠作为城市绿化树种时,可将叶片形态性状组与叶脉网络功能性状组的协同变化和权衡关系作为选种依据,以提高闽楠在城市环境中的成活率和适应性。     

phenoVein—A Tool for Leaf Vein Segmentation and Analysis

Precise measurements of leaf vein traits are an important aspect of plant phenotyping for ecological and genetic research. Here, we present a powerful and user-friendly image analysis tool named phenoVein. It is dedicated to automated segmenting and analyzing of leaf veins in images acquired with different imaging modalities (microscope, macrophotography, etc.), including options for comfortable manual correction. Advanced image filtering emphasizes veins from the background and compensates for local brightness inhomogeneities. The most important traits being calculated are total vein length, vein density, piecewise vein lengths and widths, areole area, and skeleton graph statistics, like the number of branching or ending points. For the determination of vein widths, a model-based vein edge estimation approach has been implemented. Validation was performed for the measurement of vein length, vein width, and vein density of Arabidopsis (Arabidopsis thaliana), proving the reliability of phenoVein. We demonstrate the power of phenoVein on a set of previously described vein structure mutants of Arabidopsis (hemivenata, ondulata3, and asymmetric leaves2-101) compared with wild-type accessions Columbia-0 and Landsberg erecta-0. phenoVein is freely available as open-source software.Leaf veins are an important aspect of leaf structure and responsible for both the mechanical support of leaves and the long-distance transport of water, nutrients, and photoassimilates (Onoda et al., 2011; Malinowski, 2013). The molecular mechanisms by which vascular tissues acquire their identities are yet largely unknown (Roschzttardtz et al., 2014), and there is high interest in analyzing and evaluating traits of veins or leaf venation networks and their genetic regulation. The impact of vein density on photosynthesis is a major investigated topic (Sack and Scoffoni, 2013). During the last decade, a positive correlation between leaf venation and photosynthesis has been observed (Sack and Holbrook, 2006; Brodribb et al., 2007). An optimization of photosynthetic rates was shown to occur by spatial coordination between leaf vein and stomatal densities (Zhang et al., 2012; Carins Murphy et al., 2014; Fiorin et al., 2015). Additionally, there is interest in the impact of vein density on interveinal distances (Dengler et al., 1994; McKown and Dengler, 2009) and the effect of climate, habitat, or growth form on vein density (Sack and Scoffoni, 2013; Scoffoni et al., 2015) or vein width with respect to leaf hydraulic conductance (Feild and Brodribb, 2013; Xiong et al., 2015). Other researchers are particularly interested in the evolution from C3 to C4 plants, which requires higher vein density (Gowik and Westhoff, 2011) and led to selecting for variation of vein density within species (e.g. in a mutant collection by Feldman et al., 2014).Leaf venation studies analyzing traits of veins and venation networks are generally performed on microscopic images of leaves that are properly cleared after harvest. For very small leaves, e.g. the cotyledons or the first leaves (leaves 2–5) of Arabidopsis (Arabidopsis thaliana), basic traits, such as total vein length or vein density (vein length per leaf area), can be achieved manually. However, for larger leaves, manual vein segmentation may become tedious, and at least partially automated analysis is needed for studies on large series of leaf collections. Furthermore, the quantification of vein widths and in particular mean values of vein width of certain vein pieces of interest can hardly be achieved manually. Dedicated image processing tools are, therefore, needed to support researchers for fast and reliable data analysis.A number of software tools have been published that are either specifically made or adapted to analyze leaf veins. These programs have some common properties, like image processing functionalities for vein/areole segmentation and trait extraction. However, they differ in handling strategies or vein parameter analysis methods. A general overview on plant image analysis tools is collected in an online database at http://www.plant-image-analysis.org (Lobet et al., 2013). Programs allowing automated or semiautomated analysis of leaf venation parameters are, for example, a method to extract leaf venation patterns (Rolland-Lagan et al., 2009), the leaf extraction and analysis framework graphical user interface LeafGUI (Price et al., 2011), the leaf image analysis interface LIMANI (Dhondt et al., 2012), the user-interactive vessel generation analysis tool VESGEN (Vickerman et al., 2009; Parsons-Wingerter et al., 2014), and the software network extraction from images NEFI (Dirnberger et al., 2015). Nevertheless, for the analysis of large-scale leaf vein phenotyping experiments, there are certain needs that are only partly covered by each of the approaches and programs mentioned above. Specifically, the following properties are needed: (1) automated vein segmentation with optional manual correction; (2) invariance of the segmentation procedure to inhomogeneous illumination or brightness variations in the leaf image; (3) automated determination of total vein length and projected leaf area; (4) a well-defined and automated determination of vein widths, which is, as far as possible, independent of user chosen thresholds; (5) ability to process large high-resolution images of whole leafs; and (6) full transparency of the source code as well as offline availability of the tool. To provide these functionalities, we developed the user-friendly analysis tool phenoVein. It features automated leaf vein segmentation based on advanced image filtering techniques and includes determination of various vein traits, particularly a model-based vein width estimation. phenoVein allows easy and fast visual control and manual correction on the automatically achieved skeleton of the veins enabled by a real-time overlay of the segmented leaf vein structures on the original image. The length measurement algorithm of phenoVein was validated against complete manual segmentation. We evaluated the impact of image resolution on the results, which has recently been discussed (Price et al., 2014; Sack et al., 2014), and tested whether the orientation (angle) of a leaf on an image may affect the results as suspected from image analysis theory on binary skeleton length measurements (Russ, 2011). To show the powerful phenotyping capabilities of phenoVein, we analyzed the venation traits of leaves of Arabidopsis at different developmental stages (cotyledons, pooled leaves 1 + 2, and leaf 6) harvested from previously described venation mutants and corresponding wild-type lines: asymmetric leaves2-101 (as2-101), ondulata3 (ond3), and hemivenata2 (hve-2) versus Columbia-0 (Col-0) and Landsberg erecta-0 (Ler-0; Semiarti et al., 2001; Alonso-Peral et al., 2006; Robles et al., 2010; Pérez-Pérez et al., 2011). We offer the source code of phenoVein to the public as open-source software that can be further adapted or improved (for details, see “Materials and Methods”).     

An efficient method for estimating vein density of Glossopteris and its application

Glossopteris-type leaves are the most abundant floristic element from the Gondwanan continent and are recorded throughout the Permian, which was a period of extreme icehouse-to-hothouse climatic global change. Fossil leaf traits can be useful for the reconstruction of palaeoenvironments and identification of climatic changes throughout geological time, but the conservative morphology of Glossopteris leaves has thus far made them difficult to use for this purpose. If the characters of Glossopteris can be better quantified then it should make them useful for tracking environmental changes over a wide geographical area and over a long time interval. Venation density is a highly variable leaf trait that might be useful for this purpose. This trait can be calculated, usually as vein length per centimetre squared, but this can be a time-consuming procedure. In this paper we propose a new rapid method to estimate venation density in a conical sector of Glossopteris leaf lamina using an accurate linear model whose predictors are three linear venation densities, measured as veins per centimetre. In addition to substantially reducing the data collection time, it is less biased and more reproducible than methods applied previously with this leaf type. Using this more robust method, preliminary results significantly distinguish the venation densities of leaves produced in wet and drier ecosystems, matching a pattern similar to modern plants. This is the first survey using a large sample size to reveal that environmental stress controlled the vein architecture of Palaeozoic plants, in a manner similar to plants in modern ecosystems.     

Leaf architecture of the genus Didymaea Hook. f. (Rubiaceae)

We examined 532 specimens representing eight species of Didymaea (Rubiaceae) to describe their leaf architecture. Characters observed in the herbarium specimens and cleared leaves were leaf base, apex, and margin; trichome type and distribution; vein orders from primary to higher order veins, veinlets, and ultimate marginal vein. Six morphometric variables were analyzed using variance and principal component analysis to identify which characters are useful to distinguish species. Most of the species present brochidodromous venation pattern, except D. hispidula, which has eucamptodromous venation pattern; Didymaea floribunda is unique in having cylindrical and bicellular trichomes. Principal component analysis revealed that 78% of the total variation is mainly related to leaf maximum width. Variance analysis showed significant differences between D. microflosculosa and the other species. Architectural features were useful for differentiating all the species, and an identification key for the species of Didymaea based only on leaf characters is presented.     

Reviewing models of auxin canalization in the context of leaf vein pattern formation in Arabidopsis

In both plants and animals vein networks play an essential role in transporting nutrients. In plants veins may also provide mechanical support. The mechanism by which vein patterns are formed in a developing leaf remains largely unresolved. According to the canalization hypothesis, a signal inducing vein differentiation is transported in a polar manner and is channeled into narrow strands. Since inhibition of auxin transport affects venation patterns, auxin is likely to be part of the signal involved. However, it is not clear whether the canalization hypothesis, initially formulated over 25 years ago, is compatible with recent experimental data. In this paper we focus on three aspects of this question, and show that: (i) canalization models can account for an acropetal development of the midvein if vein formation is sink-driven; (ii) canalization models are in agreement with venation patterns resulting from inhibited auxin transport and (iii) loops and discontinuous venation patterns can be obtained assuming proper spacing of discrete auxin sources.