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1.  Leaf Length Tracker: a novel approach to analyse leaf elongation close to the thermal limit of growth in the field 
Journal of Experimental Botany  2016;67(6):1897-1906.
A novel weatherproof in situ method for high-precision leaf growth measurements in monocot plants was used to detect genotype-specific growth responses even at low temperature.
Leaf growth in monocot crops such as wheat and barley largely follows the daily temperature course, particularly under cold but humid springtime field conditions. Knowledge of the temperature response of leaf extension, particularly variations close to the thermal limit of growth, helps define physiological growth constraints and breeding-related genotypic differences among cultivars. Here, we present a novel method, called ‘Leaf Length Tracker’ (LLT), suitable for measuring leaf elongation rates (LERs) of cereals and other grasses with high precision and high temporal resolution under field conditions. The method is based on image sequence analysis, using a marker tracking approach to calculate LERs. We applied the LLT to several varieties of winter wheat (Triticum aestivum), summer barley (Hordeum vulgare), and ryegrass (Lolium perenne), grown in the field and in growth cabinets under controlled conditions. LLT is easy to use and we demonstrate its reliability and precision under changing weather conditions that include temperature, wind, and rain. We found that leaf growth stopped at a base temperature of 0°C for all studied species and we detected significant genotype-specific differences in LER with rising temperature. The data obtained were statistically robust and were reproducible in the tested environments. Using LLT, we were able to detect subtle differences (sub-millimeter) in leaf growth patterns. This method will allow the collection of leaf growth data in a wide range of future field experiments on different graminoid species or varieties under varying environmental or treatment conditions.
PMCID: PMC4783369  PMID: 26818912
Field conditions; leaf elongation; low temperature; marker tracking; monocotyledons; phenotyping; plant growth.
2.  Leaf mineral nutrient remobilization during leaf senescence and modulation by nutrient deficiency 
Higher plants have to cope with fluctuating mineral resource availability. However, strategies such as stimulation of root growth, increased transporter activities, and nutrient storage and remobilization have been mostly studied for only a few macronutrients. Leaves of cultivated crops (Zea mays, Brassica napus, Pisum sativum, Triticum aestivum, Hordeum vulgare) and tree species (Quercus robur, Populus nigra, Alnus glutinosa) grown under field conditions were harvested regularly during their life span and analyzed to evaluate the net mobilization of 13 nutrients during leaf senescence. While N was remobilized in all plant species with different efficiencies ranging from 40% (maize) to 90% (wheat), other macronutrients (K–P–S–Mg) were mobilized in most species. Ca and Mn, usually considered as having low phloem mobility were remobilized from leaves in wheat and barley. Leaf content of Cu–Mo–Ni–B–Fe–Zn decreased in some species, as a result of remobilization. Overall, wheat, barley and oak appeared to be the most efficient at remobilization while poplar and maize were the least efficient. Further experiments were performed with rapeseed plants subjected to individual nutrient deficiencies. Compared to field conditions, remobilization from leaves was similar (N–S–Cu) or increased by nutrient deficiency (K–P–Mg) while nutrient deficiency had no effect on Mo–Zn–B–Ca–Mn, which seemed to be non-mobile during leaf senescence under field conditions. However, Ca and Mn were largely mobilized from roots (-97 and -86% of their initial root contents, respectively) to shoots. Differences in remobilization between species and between nutrients are then discussed in relation to a range of putative mechanisms.
PMCID: PMC4429656  PMID: 26029223
remobilization; ionomic; nutrient deficiencies; senescence; crop species; Brassica napus; trees
3.  Individual Leaf Development in Arabidopsis thaliana: a Stable Thermal‐time‐based Programme 
Annals of Botany  2002;89(5):595-604.
In crop species, the impact of temperature on plant development is classically modelled using thermal time. We examined whether this method could be used in a non‐crop species, Arabidopsis thaliana, to analyse the response to temperature of leaf initiation rate and of the development of two leaves of the rosette. The results confirmed the large plant‐to‐plant variability in the studied isogenic line of the Columbia ecotype: 100‐fold differences in leaf area among plants sown on the same date were commonly observed at a given date. These differences disappeared in mature leaves, suggesting that they were due to a variability in plant developmental stage. The whole population could therefore be represented by any group of synchronous plants labelled at the two‐leaf stage and followed during their development. Leaf initiation rate, duration of leaf expansion and maximal relative leaf expansion rate varied considerably among experiments performed at different temperatures (from 6 to 26 °C) but they were linearly related to temperature in the range 6–26 °C, with a common x‐intercept of 3 °C. Expressing time in thermal time with a threshold temperature of 3 °C unified the time courses of leaf initiation and of individual leaf development for plants grown at different temperatures and experimental conditions. The two leaves studied (leaf 2 and leaf 6) had a two‐phase development, with an exponential phase followed by a phase with decreasing relative elongation rate. Both phases had constant durations for a given leaf position if expressed in thermal time. Changes in temperature caused changes in both the rate of development and in the expansion rate which mutually compensated such that they had no consequence on leaf area at a given thermal time. The resulting model of leaf development was applied to ten experiments carried out in a glasshouse or in a growth chamber, with plants grown in soil or hydroponically. Because it predicts accurately the stage of development and the relative expansion rate of any leaf of the rosette, this model facilitates precise planning of sampling procedures and the comparison of treatments in growth analyses.
PMCID: PMC4233892  PMID: 12099534
Arabidopsis thaliana; thermal time; leaf initiation; leaf expansion; model; temperature
4.  From Individual Leaf Elongation to Whole Shoot Leaf Area Expansion: a Comparison of Three Aegilops and Two Triticum Species 
Annals of Botany  2004;94(1):99-108.
• Background and Aims Rapid leaf area expansion is a desirable trait in the early growth stages of cereal crops grown in low‐rainfall areas. In this study, the traits associated with inherent variation in early leaf area expansion rates have been investigated in two wheat species (Triticum aestivum and T. durum) and three of its wild relatives (Aegilops umbellulata, A. caudata and A. tauschii) to find out whether the Aegilops species have a faster leaf area expansion in their early developmental stage than some of the current wheat species.
• Methods Growth of individual leaves, biomass allocation, and gas exchange were measured on hydroponically grown plants for 4 weeks.
• Key Results Leaf elongation rate (LER) was strongly and positively correlated with leaf width but not with leaf elongation duration (LED). The species with more rapidly elongating leaves showed a faster increase with leaf position in LER, leaf width and leaf area, higher relative leaf area expansion rates, and more biomass allocation to leaf sheaths and less to roots. No differences in leaf appearance rate were found amongst the species.
• Conclusions Aegilops tauschii was the only wild species with rapid leaf expansion rates similar to those of wheat, and it achieved the highest photosynthetic rates, making it an interesting species for further study.
PMCID: PMC4242366  PMID: 15155374
Aegilops; biomass allocation; leaf elongation duration; leaf elongation rate; leaf expansion; leaf width; photosynthesis; relative growth rate; Triticum; wheat; wild relatives
5.  Genetical genomics of Populus leaf shape variation 
BMC Plant Biology  2015;15:166.
Leaf morphology varies extensively among plant species and is under strong genetic control. Mutagenic screens in model systems have identified genes and established molecular mechanisms regulating leaf initiation, development, and shape. However, it is not known whether this diversity across plant species is related to naturally occurring variation at these genes. Quantitative trait locus (QTL) analysis has revealed a polygenic control for leaf shape variation in different species suggesting that loci discovered by mutagenesis may only explain part of the naturally occurring variation in leaf shape. Here we undertook a genetical genomics study in a poplar intersectional pseudo-backcross pedigree to identify genetic factors controlling leaf shape. The approach combined QTL discovery in a genetic linkage map anchored to the Populus trichocarpa reference genome sequence and transcriptome analysis.
A major QTL for leaf lamina width and length:width ratio was identified in multiple experiments that confirmed its stability. A transcriptome analysis of expanding leaf tissue contrasted gene expression between individuals with alternative QTL alleles, and identified an ADP-ribosylation factor (ARF) GTPase (PtARF1) as a candidate gene for regulating leaf morphology in this pedigree. ARF GTPases are critical elements in the vesicular trafficking machinery. Disruption of the vesicular trafficking function of ARF by the pharmacological agent Brefeldin A (BFA) altered leaf lateral growth in the narrow-leaf P. trichocarpa suggesting a molecular mechanism of leaf shape determination. Inhibition of the vesicular trafficking processes by BFA interferes with cycling of PIN proteins and causes their accumulation in intercellular compartments abolishing polar localization and disrupting normal auxin flux with potential effects on leaf expansion.
In other model systems, ARF proteins have been shown to control the localization of auxin efflux carriers, which function to establish auxin gradients and apical-basal cell polarity in developing plant organs. Our results support a model where PtARF1 transcript abundance changes the dynamics of endocytosis-mediated PIN localization in leaf cells, thus affecting lateral auxin flux and subsequently lamina leaf expansion. This suggests that evolution of differential cellular polarity plays a significant role in leaf morphological variation observed in subgenera of genus Populus.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0557-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4486686  PMID: 26122556
Leaf morphology; QTL analysis; Expression QTL; Genomics; Populus trichocarpa; ADP-ribosylation factor
6.  The Leaf Size–Twig Size Spectrum of Temperate Woody Species Along an Altitudinal Gradient: An Invariant Allometric Scaling Relationship 
Annals of Botany  2006;97(1):97-107.
• Background and Aims The leaf size–twig size spectrum is one of the leading dimensions of plant ecological variation, and now it is under development. The purpose of this study was to test whether the relationship between leaf size and twig size is isometric or allometric, and to examine the relationship between plant allometric growth and life history strategies in the spectrum.
• Methods Leaf and stem characters—including leaf and stem mass, total leaf area, individual leaf area, stem cross-sectional area, leaf number and stem length—at the twig level for 59 woody species were investigated along an altitudinal gradient on Changbaishan Mountain in the temperate zone of China. The environmental gradient ranges from temperate broad-leaved mixed forest at low altitude, to conifer forest at middle altitude, and to sub-alpine birch forest at high altitude. The scaling relationships between stem cross-sectional area and stem mass, stem mass and leaf mass, and leaf mass and leaf area at the twig level were simultaneously determined.
• Key Results Twig cross-sectional area was found to have invariant allometric scaling relationships with the stem mass, leaf mass, total leaf area and individual leaf area, all with common slopes being significantly larger than 1, for three altitudinal-zoned vegetation types under investigation. However, leaf mass was found to be isometrically related to stem mass and leaf area along the environmental gradient. Based on the predictions of previous models, the exponent value of the relationship between twig cross-sectional area and total leaf area can be inferred to be 1·5, which falls between the confidence intervals of the relationship at each altitude, and between the confidence intervals of the common slope value (1·17–1·56) of this study. This invariant scaling relationship is assumed to result from the fractural network and/or developmental constraints of plants. The allometric constants (y-intercepts) of the relationships between the stem cross-sectional area and leaf area (both total leaf area and individual leaf area) were found to decrease significantly along the altitudinal gradient. This suggests that the species would support less leaf area at a given twig cross-sectional area with increasing environmental stress.
• Conclusions This study demonstrated that plants respond to the environmental gradient by changing the y-intercepts of the relationship between leaf size–twig size, while keeping the exponent value of the allometric relationship as an invariant constant. The allometric growth in the twig size–leaf size spectrum is related to many other components of plant life history strategy, including the well established life history trade-off between efficiency and safety in the hydraulic transport of water.
PMCID: PMC2803375  PMID: 16254019
Allometric growth; altitudinal gradient; leaf area; stem mass; leaf mass; life history trade-off; scaling; twig cross-sectional area
7.  Canopy Apparent Photosynthetic Characteristics and Yield of Two Spike-Type Wheat Cultivars in Response to Row Spacing under High Plant Density 
PLoS ONE  2016;11(2):e0148582.
In northern China, large-spike wheat (Triticum aestivum L) is considered to have significant potential for increasing yields due to its greater single-plant productivity despite its lower percentage of effective tillers, and increasing the plant density is an effective means of achieving a higher grain yield. However, with increases in plant density, the amount of solar radiation intercepted by lower strata leaves is decreased and the rate of leaf senescence is accelerated. Row spacing can be manipulated to optimize the plant spatial distribution under high plant density, therefore improving light conditions within the canopy. Consequently, field experiments were conducted from 2010 to 2012 to investigate whether changes in row spacing under high plant density led to differences in canopy apparent photosynthesis (CAP), individual leaf photosynthesis and grain yield. Two different spike-type winter wheat cultivars, Jimai22 (a small-spike cultivar as a control cultivar) and Wennong6 (a large-spike cultivar), were grown at a constant plant density of 3,600,000 plants ha–1 (a relatively higher plant density) over a wide range of row spacing as follows: 5-cm row spacing (R0), 15-cm row spacing (R1), 25-cm conventional row spacing (R2), and 35-cm row spacing (R3). The two-year investigations revealed that increased row spacing exhibited a significantly higher light transmission ratio (LT), which improved light conditions within the canopy; however, excessive light leakage losses in R2 and R3 treatments were not favorable to improved irradiation energy utilization efficiency. Aboveground biomass accumulation was influenced by row spacing. Two spike-type wheat accumulated greater biomass under 15-cm row spacing compared to other row spacing treatments, although a markedly improved photosynthetic rate (PN), effective quantum yield of photosystem II (ΦPSII) and maximal efficiency of photosystem II photochemistry (Fv/Fm) in the penultimate and third leaves were observed in R2 and R3 treatments. At the same time, a longer duration of CAP and green leaf area was maintained in R1 during grain filling. Compared with conventional row spacing, Wennong6 in R1 treatment obtained 21.0% and 19.1% higher grain yield in 2011 and 2012, respectively, while for Jimai22 it increased by 11.3% and 11.4%, respectively. A close association of yield with CAP and LAI at mid-grain filling was observed. In conclusion, for the tested growing conditions, decreasing the row spacing to an optimal distance (15 cm) maintained a longer duration of LAI and CAP during grain filling, made a better coordination of group and individual leaf photosynthesis, and accumulated higher aboveground biomass, leading to a greater grain yield. In addition, Wennong6 had a more rational canopy architecture than Jimai22 (improved LT and higher LAI) and CAP under 15-cm row spacing, leading to a higher grain yield, which indicated that the large-spike type cultivar has the potential to obtain higher yields by increasing plant density through optimum row spacing allocation (15 cm).
PMCID: PMC4741391  PMID: 26845330
8.  Developmental Changes in Composition and Morphology of Cuticular Waxes on Leaves and Spikes of Glossy and Glaucous Wheat (Triticum aestivum L.) 
PLoS ONE  2015;10(10):e0141239.
The glossy varieties (A14 and Jing 2001) and glaucous varieties (Fanmai 5 and Shanken 99) of wheat (Triticum aestivum L.) were selected for evaluation of developmental changes in the composition and morphology of cuticular waxes on leaves and spikes. The results provide us with two different wax development patterns between leaf and spike. The general accumulation trend of the total wax load on leaf and spike surfaces is first to increase and then decrease during the development growth period, but these changes were caused by different compound classes between leaf and spike. Developmental changes of leaf waxes were mainly the result of variations in composition of alcohols and alkanes. In addition, diketones were the third important contributor to the leaf wax changes in the glaucous varieties. Alkanes and diketones were the two major compound classes that caused the developmental changes of spike waxes. For leaf waxes, β- and OH-β-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of β- and OH-β-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, β-diketone existed in all varieties, but OH-β-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-β-diketone. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C28 alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes was C24 or C26 depending on varieties. Epicuticular wax crystals on wheat leaf and glume were comprised of platelets and tubules, and the crystal morphology changed constantly throughout plant growth, especially the abaxial leaf crystals. Moreover, our results suggested that platelets and tubules on glume surfaces could be formed rapidly within a few days.
PMCID: PMC4624236  PMID: 26506246
9.  LEAF-E: a tool to analyze grass leaf growth using function fitting 
Plant Methods  2014;10:37.
In grasses, leaf growth is often monitored to gain insights in growth processes, biomass accumulation, regrowth after cutting, etc. To study the growth dynamics of the grass leaf, its length is measured at regular time intervals to derive the leaf elongation rate (LER) profile over time. From the LER profile, parameters such as maximal LER and leaf elongation duration (LED), which are essential for detecting inter-genotype growth differences and/or quantifying plant growth responses to changing environmental conditions, can be determined. As growth is influenced by the circadian clock and, especially in grasses, changes in environmental conditions such as temperature and evaporative demand, the LER profiles show considerable experimental variation and thus often do not follow a smooth curve. Hence it is difficult to quantify the duration and timing of growth. For these reasons, the measured data points should be fitted using a suitable mathematical function, such as the beta sigmoid function for leaf elongation.
In the context of high-throughput phenotyping, we implemented the fitting of leaf growth measurements into a user-friendly Microsoft Excel-based macro, a tool called LEAF-E. LEAF-E allows to perform non-linear regression modeling of leaf length measurements suitable for robust and automated extraction of leaf growth parameters such as LER and LED from large datasets. LEAF-E is particularly useful to quantify the timing of leaf growth, which forms an important added value for detecting differences in leaf growth development. We illustrate the broad application range of LEAF-E using published and unpublished data sets of maize, Miscanthus spp. and Brachypodium distachyon, generated in independent experiments and for different purposes. In addition, we show that LEAF-E could also be used to fit datasets of other growth-related processes that follow the sigmoidal profile, such as cell length measurements along the leaf axis.
Given its user-friendliness, ability to quantify duration and timing of leaf growth and broad application range, LEAF-E is a tool that could be routinely used to study growth processes following the sigmoidal profile.
Electronic supplementary material
The online version of this article (doi:10.1186/1746-4811-10-37) contains supplementary material, which is available to authorized users.
PMCID: PMC4246515  PMID: 25435898
Leaf elongation rate; Non-linear regression; Leaf length; Cell length; Growth zone
10.  Latent developmental and evolutionary shapes embedded within the grapevine leaf 
The New Phytologist  2015;210(1):343-355.
Across plants, leaves exhibit profound diversity in shape. As a single leaf expands, its shape is in constant flux. Plants may also produce leaves with different shapes at successive nodes. In addition, leaf shape varies among individuals, populations and species as a result of evolutionary processes and environmental influences.Because leaf shape can vary in many different ways, theoretically, the effects of distinct developmental and evolutionary processes are separable, even within the shape of a single leaf. Here, we measured the shapes of > 3200 leaves representing > 270 vines from wild relatives of domesticated grape (Vitis spp.) to determine whether leaf shapes attributable to genetics and development are separable from each other.We isolated latent shapes (multivariate signatures that vary independently from each other) embedded within the overall shape of leaves. These latent shapes can predict developmental stages independent from species identity and vice versa. Shapes predictive of development were then used to stage leaves from 1200 varieties of domesticated grape (Vitis vinifera), revealing that changes in timing underlie leaf shape diversity.Our results indicate that distinct latent shapes combine to produce a composite morphology in leaves, and that developmental and evolutionary contributions to shape vary independently from each other.
PMCID: PMC5063178  PMID: 26580864
development; grape (Vitis vinifera); leaf morphology; leaf shape; phenotype
11.  Increasing leaf hydraulic conductance with transpiration rate minimizes the water potential drawdown from stem to leaf 
Journal of Experimental Botany  2014;66(5):1303-1315.
Under well-watered conditions leaf hydraulic conductance increases with transpiration rate. This reduces the water potential gradient in leaves and potentially improves productivity under daily variation in evaporative demand.
Leaf hydraulic conductance (k leaf) is a central element in the regulation of leaf water balance but the properties of k leaf remain uncertain. Here, the evidence for the following two models for k leaf in well-hydrated plants is evaluated: (i) k leaf is constant or (ii) k leaf increases as transpiration rate (E) increases. The difference between stem and leaf water potential (ΔΨstem–leaf), stomatal conductance (g s), k leaf, and E over a diurnal cycle for three angiosperm and gymnosperm tree species growing in a common garden, and for Helianthus annuus plants grown under sub-ambient, ambient, and elevated atmospheric CO2 concentration were evaluated. Results show that for well-watered plants k leaf is positively dependent on E. Here, this property is termed the dynamic conductance, k leaf(E), which incorporates the inherent k leaf at zero E, which is distinguished as the static conductance, k leaf(0). Growth under different CO2 concentrations maintained the same relationship between k leaf and E, resulting in similar k leaf(0), while operating along different regions of the curve owing to the influence of CO2 on g s. The positive relationship between k leaf and E minimized variation in ΔΨstem–leaf. This enables leaves to minimize variation in Ψleaf and maximize g s and CO2 assimilation rate over the diurnal course of evaporative demand.
PMCID: PMC4339593  PMID: 25547915
Leaf hydraulic conductance; leaf water potential; stem water potential; stomatal conductance; transpiration; water relations.
12.  Identification of Nitrogen, Phosphorus, and Potassium Deficiencies in Rice Based on Static Scanning Technology and Hierarchical Identification Method 
PLoS ONE  2014;9(11):e113200.
Establishing an accurate, fast, and operable method for diagnosing crop nutrition is very important for crop nutrient management. In this study, static scanning technology was used to collect images of a rice sample's fully expanded top three leaves and corresponding sheathes. From these images, 32 spectral and shape characteristic parameters were extracted using an RGB mean value function and using the Regionprops function in MATLAB. Hierarchical identification was used to identify NPK deficiencies. First, the normal samples and non-normal (NPK deficiencies) samples were identified. Then, N deficiency and PK deficiencies were identified. Finally, P deficiency and K deficiency were identified. In the identification of every hierarchy, SVFS was used to select the optimal characteristic set for different deficiencies in a targeted manner, and Fisher discriminant analysis was used to build the diagnosis model. In the first hierarchy, the selected characteristics were the leaf sheath R, leaf sheath G, leaf sheath B, leaf sheath length, leaf tip R, leaf tip G, leaf area and leaf G. In the second hierarchy, the selected characteristics were the leaf sheath G, leaf sheath B, white region of the leaf sheath, leaf B, and leaf G. In the third hierarchy the selected characteristics were the leaf G, leaf sheath length, leaf area/leaf length, leaf tip G, difference between the 2nd and 3rd leaf lengths, leaf sheath G, and leaf lightness. The results showed that the overall identification accuracies of NPK deficiencies were 86.15, 87.69, 90.00 and 89.23% for the four growth stages. Data from multiple years were used for validation, and the identification accuracies were 83.08, 83.08, 89.23 and 90.77%.
PMCID: PMC4245116  PMID: 25426712
13.  Polyploidy and Cellular Mechanisms Changing Leaf Size: Comparison of Diploid and Autotetraploid Populations in Two Species of Lolium 
Annals of Botany  2005;96(5):931-938.
• Background and Aims Growth and development of plant organs, including leaves, depend on cell division and expansion. Leaf size is increased by greater cell ploidy, but the mechanism of this effect is poorly understood. Therefore, in this study, the role of cell division and expansion in the increase of leaf size caused by polyploidy was examined by comparing various cell parameters of the mesophyll layer of developing leaves of diploid and autotetraploid cultivars of two grass species, Lolium perenne and L. multiflorum.
• Methods Three cultivars of each ploidy level of both species were grown under pot conditions in a controlled growth chamber, and leaf elongation rate and the cell length profile at the leaf base were measured on six plants in each cultivar. Cell parameters related to division and elongation activities were calculated by a kinematic method.
• Key Results Tetraploid cultivars had faster leaf elongation rates than did diploid cultivars in both species, resulting in longer leaves, mainly due to their longer mature cells. Epidermal and mesophyll cells differed 20-fold in length, but were both greater in the tetraploid cultivars of both species. The increase in cell length of the tetraploid cultivars was caused by a faster cell elongation rate, not by a longer period of cell elongation. There were no significant differences between cell division parameters, such as cell production rate and cell cycle time, in the diploid and tetraploid cultivars.
• Conclusion The results demonstrated clearly that polyploidy increases leaf size mainly by increasing the cell elongation rate, but not the duration of the period of elongation, and thus increases final cell size.
PMCID: PMC4247059  PMID: 16100224
Cell division; cell elongation; kinematic method; leaf elongation rate; leaf size; Lolium; mesophyll cells; tetraploid
14.  Multi-trait interactions, not phylogeny, fine-tune leaf size reduction with increasing altitude 
Annals of Botany  2011;107(3):455-465.
Background and Aims
Despite long-held interest, knowledge on why leaf size varies widely among species is still incomplete. This study was conducted to assess whether abiotic factors, phylogenetic histories and multi-trait interactions act together to shape leaf size.
Fifty-seven pairs of altitudinal vicariant species were selected in northern Spain, and leaf area and a number of functionally related leaf, shoot and whole plant traits were measured for each pair. Structural equation modelling helped unravel trait interactions affecting leaf size, and Mantel tests weighed the relative relevance of phylogeny, environment and trait interactions to explain leaf size reduction with altitude.
Key Results
Leaves of highland vicariants were generally smaller than those of lowlands. However, the extent of leaf size reduction with increasing altitude was widely variable among genera: from approx. 700 cm2 reduction (96 % in Polystichum) to approx. 30 cm2 increase (37 % in Sorbus). This was partially explained by shifts in leaf, shoot and whole plant traits (35–64 % of explained variance, depending on models), with size/number trade-offs more influential than shifts in leaf form and leaf economics. Shifts in traits were more important than phylogenetic distances or site-specific environmental variation in explaining the degree of leaf size reduction with altitude.
Ecological filters, constraints due to phylogenetic history (albeit modest in the study system), and phenotypic integration contribute jointly to shape single-trait evolution. Here, it was found that phenotypic change was far more important than shared ancestry to explaine leaf size differences of closely related species segregated along altitudes.
PMCID: PMC3043936  PMID: 21199835
Leaf size evolution; leaf economics; phylogeny; traits; altitude; indirect selection; morphological correlates; structural equation models
15.  Profiling of spatial metabolite distributions in wheat leaves under normal and nitrate limiting conditions 
Phytochemistry  2015;115:99-111.
•Nitrogen and carbon assimilation interaction is essential in leaf metabolism.•Along the developing wheat leaf there is a switch from hetero- to auto-trophy.•GC–MS metabolite profiling was combined with Bayesian network (BN) correlation analysis.•Amino acid, organic acid and carbohydrate distribution changed with nitrate levels.
Graphical abstract
Classical phenotyping and metabolite profiling were combined to study the spatial development of photosynthetic capacity within the developing wheat leaf, revealing bonafide metabolic changes associated with hetero- and auto-trophy.
The control and interaction between nitrogen and carbon assimilatory pathways is essential in both photosynthetic and non-photosynthetic tissue in order to support metabolic processes without compromising growth. Physiological differences between the basal and mature region of wheat (Triticum aestivum) primary leaves confirmed that there was a change from heterotrophic to autotrophic metabolism. Fourier Transform Infrared (FT-IR) spectroscopy confirmed the suitability and phenotypic reproducibility of the leaf growth conditions. Principal Component–Discriminant Function Analysis (PC–DFA) revealed distinct clustering between base, and tip sections of the developing wheat leaf, and from plants grown in the presence or absence of nitrate. Gas Chromatography-Time of Flight/Mass Spectrometry (GC-TOF/MS) combined with multivariate and univariate analyses, and Bayesian network (BN) analysis, distinguished different tissues and confirmed the physiological switch from high rates of respiration to photosynthesis along the leaf. The operation of nitrogen metabolism impacted on the levels and distribution of amino acids, organic acids and carbohydrates within the wheat leaf. In plants grown in the presence of nitrate there was reduced levels of a number of sugar metabolites in the leaf base and an increase in maltose levels, possibly reflecting an increase in starch turnover. The value of using this combined metabolomics analysis for further functional investigations in the future are discussed.
PMCID: PMC4518043  PMID: 25680480
Triticum aestivum; Wheat; Leaves; Metabolite fingerprinting; Metabolite profiling; Bayesian network analysis; Nitrate
16.  Metabolic responses to salt stress of barley (Hordeum vulgare L.) cultivars, Sahara and Clipper, which differ in salinity tolerance 
Journal of Experimental Botany  2009;60(14):4089-4103.
Plants show varied cellular responses to salinity that are partly associated with maintaining low cytosolic Na+ levels and a high K+/Na+ ratio. Plant metabolites change with elevated Na+, some changes are likely to help restore osmotic balance while others protect Na+-sensitive proteins. Metabolic responses to salt stress are described for two barley (Hordeum vulgare L.) cultivars, Sahara and Clipper, which differed in salinity tolerance under the experimental conditions used. After 3 weeks of salt treatment, Clipper ceased growing whereas Sahara resumed growth similar to the control plants. Compared with Clipper, Sahara had significantly higher leaf Na+ levels and less leaf necrosis, suggesting they are more tolerant to accumulated Na+. Metabolite changes in response to the salt treatment also differed between the two cultivars. Clipper plants had elevated levels of amino acids, including proline and GABA, and the polyamine putrescine, consistent with earlier suggestions that such accumulation may be correlated with slower growth and/or leaf necrosis rather than being an adaptive response to salinity. It is suggested that these metabolites may be an indicator of general cellular damage in plants. By contrast, in the more tolerant Sahara plants, the levels of the hexose phosphates, TCA cycle intermediates, and metabolites involved in cellular protection increased in response to salt. These solutes remain unchanged in the more sensitive Clipper plants. It is proposed that these responses in the more tolerant Sahara are involved in cellular protection in the leaves and are involved in the tolerance of Sahara leaves to high Na+.
PMCID: PMC2755029  PMID: 19666960
Barley; GC-MS; metabolomics; salt stress; tissue tolerance
17.  Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM) 
Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (Ψleaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol • m-2 • s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- Δψleaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.
PMCID: PMC3577864  PMID: 23299126
Plant Biology; Issue 70; Molecular Biology; Physiology; Ecology; Biology; Botany; Leaf traits; hydraulics; stomata; transpiration; xylem; conductance; leaf hydraulic conductance; resistance; evaporative flux method; whole plant
18.  Leaf hydraulic conductance is coordinated with leaf morpho-anatomical traits and nitrogen status in the genus Oryza  
Journal of Experimental Botany  2014;66(3):741-748.
The range of leaf hydraulic conductance across the genus Oryza is caused by leaf morpho-anatomical traits and leaf N status.
Leaf hydraulic conductance (K leaf) is a major determinant of photosynthetic rate in plants. Previous work has assessed the relationships between leaf morpho-anatomical traits and K leaf with woody species, but there has been very little focus on cereal crops. The genus Oryza, which includes rice (Oryza sativa) and wild species (such as O. rufipogon cv. Griff), is ideal material for identifying leaf features associated with K leaf and gas exchange. Leaf morpho-anatomical traits, K leaf, leaf N content per leaf area, and CO2 diffusion efficiency were investigated in 11 Oryza cultivars. K leaf was positively correlated with leaf thickness and related traits, and therefore positively correlated with leaf mass per area and leaf N content per leaf area, and negatively with inter-veinal distance. K leaf was also positively correlated with leaf area and its related traits, and therefore negatively correlated with the proportion of minor vein length per area. In addition, coordination between K leaf and CO2 diffusion conductance in leaves was observed. We conclude that leaf morpho-anatomical traits and N content per leaf area strongly influence K leaf. Our results suggest that more detailed anatomical and structural studies are needed to elucidate the impacts of leaf feature traits on K leaf and gas exchange in grasses.
PMCID: PMC4321541  PMID: 25429002
CO2 diffusion conductance; leaf anatomy; leaf hydraulic conductance; leaf N content per leaf area; photosynthesis; rice.
19.  Defending the leaf surface: intra- and inter-specific differences in silicon deposition in grasses in response to damage and silicon supply 
Understanding interactions between grasses and their herbivores is central to the conservation of species-rich grasslands and the protection of our most important crops against pests. Grasses employ a range of defenses against their natural enemies; silicon-based defenses have been shown to be one of the most effective. Silicon (Si) is laid down on the leaf surface as spines and other sharp bodies, known as phytoliths, making grasses abrasive and their foliage indigestible to herbivores. Previous studies on Si defenses found that closely related species may have similar levels of Si in the leaves but differ markedly in abrasiveness. Here we show how the number, shape and distribution of Si-rich phytoliths and spines differ within and between different grass species and demonstrate that species also differ in their ability to change the deposition and distribution of these defenses in response to damage or increases in Si supply. Specifically, we tested the response of two genotypes of Festuca arundinacea known to differ in their surface texture and three different grass species (F. ovina, F. rubra, and Deschampsia cespitosa) differing in their abrasiveness to combined manipulation of leaf damage and Si supply. F. arundinacea plants with a harsh leaf surface had higher Si content and more spines on their leaf surface than soft varieties. F. ovina and D. cespitosa plants increased their leaf Si concentration and produced an increase in the number of leaf spines and phytoliths on the leaf surface in response to Si addition. F rubra also increased leaf Si content in response to treatments, particularly in damaged leaves, but did not deposit this in the form of spines or increased densities of phytoliths. We discuss how the form in which grasses deposit Si may affect their anti-herbivore characteristics and consider the ecological and agricultural implications of the differences in allocation to Si-based defenses between grass species.
PMCID: PMC4324063  PMID: 25717331
plant defense; silicon; abrasion; grasses; leaf surface; phytoliths; spines
20.  Delivering high-resolution landmarks using inkjet micropatterning for spatial monitoring of leaf expansion 
Plant Methods  2011;7:1.
Inkjet micropatterning is a versatile deposition technique with broad applications in numerous fields. However, its application in plant science is largely unexplored. Leaf expansion is one of the most important parameters in the field of plant science and many methods have been developed to examine differential expansion rates of different parts of the leaf lamina. Among them, methods based on the tracking of natural landmarks through digital imaging require a complicated setup in which the leaf must remain fixed and under tension. Furthermore, the resolution is limited to that of the natural landmarks, which are often difficult to find, particularly in young leaves. To study the fine scale expansion dynamics of the leaf lamina using artificial landmarks it is necessary to place small, noninvasive marks on a leaf surface and then recover the location of those marks after a period of time.
To monitor leaf expansion in two dimensions, at very fine scales, we used a custom designed inkjet micropatterning system to print a grid composed of c. 0.19 mm2 cells on small developing leaves of ivy (Hedera helix) using 40 μm dots at a spacing of c. 91 μm. The leaves in different growing stages were imaged under magnification to extract the coordinates of the marks which were then used in subsequent computer-assisted leaf expansion analyses. As an example we obtained quantified global and local expansion information and created expansion maps over the entire leaf surface. The results reveal a striking pattern of fine-scale expansion differences over short periods of time. In these experiments, the base of the leaf is a "cold spot" for expansion, while the leaf sinuses are "hot spots" for expansion. We have also measured a strong shading effect on leaf expansion. We discuss the features required to build an inkjet printing apparatus optimized for use in plant science, which will further maximize the range of tissues that can be printed at these scales.
To apply inkjet micropatterning to plant studies, we have successfully delivered landmarks on ivy leaf surfaces and achieved high-resolution, two-dimensional monitoring of leaf expansion at different growing stages. The measurement is capable of reliably identifying the fine scale changes during plant growth. As well as delivering landmarks, this technology may be used to deliver microscale targeted biological components such as growth hormones, and possibly be used to pattern sensors directly on the leaves.
PMCID: PMC3035580  PMID: 21266074
21.  Investigations of the Host Range of the Corn Cyst Nematode, Heterodera zeae, from Maryland 
Journal of Nematology  1987;19(Annals 1):97-106.
The host range of the corn cyst nematode, Heterodera zeae, recently detected in Maryland, was investigated. A total of 269 plant entries, representing 68 families, 172 genera, and 204 species, was inoculated with cysts or a mixture of eggs and second-stage juveniles of H. zeae. The host range of the Maryland population of H. zeae was limited to plants of the Gramineae and included 11 tribes, 33 genera, 42 species, and 77 entries. All 22 corn (Zea mays) cultivars tested were hosts. Other economic hosts included certain cultivars of barley (Hordeum vulgare), oat (Arena sativa), rice (Oryza sativa), sorghum (Sorghum bicolor), sugar cane (Saccharum interspecific hybrid), and wheat (Triticum aestivum). Fall panicum (Panicum dichotomiflorum), a weed species common to cultivated fields in Maryland, was also a host for H. zeae. Other hosts included meadow foxtail (Alopecurus pratensis), Calamagrostis eipgeios, Job's tears (Coix Lachryma-Jobi), green sprangletop (Leptochloa dubia), witchgrass (Panicum capillare), broomcorn (Panicum miliaceum), fountain grass (Pennisetum rueppeli), reed canary grass (Phalaris arundinacea), common reed (Phragmites australis), eastern gamagrass (Tripsacum dactyloides), corn (Zea mays), and teosinte (Zea mexicana).
PMCID: PMC2618679  PMID: 19290286
corn; corn cyst nematode; Heterodera zeae; host range; maize; Zea mays
22.  Comparing salt tolerance of beet cultivars and their halophytic ancestor: consequences of domestication and breeding programmes 
AoB Plants  2014;7:plu083.
Our results indicate that the salt tolerance of sugarbeet cultivars is only slightly less than that of their sea beet ancestor and that domestication and selection among sugar beet cultivars have not improved salt tolerance. While the yield of many traditional crops is reduced in salinized soils, sugar beet cultivars are tolerant to increased salinity. It is expected that salt tolerant sugar beet will be productive under seawater and brackish water irrigation in saline agriculture. The use of brackish and saline water for saline agriculture helps to prevent depletion of fresh water on earth.
Salt tolerance of higher plants is determined by a complex set of traits, the timing and rate of evolution of which are largely unknown. We compared the salt tolerance of cultivars of sugar beet and their ancestor, sea beet, in hydroponic studies and evaluated whether traditional domestication and more recent breeding have changed salt tolerance of the cultivars relative to their ancestor. Our comparison of salt tolerance of crop cultivars is based on values of the relative growth rate (RGR) of the entire plant at various salinity levels. We found considerable salt tolerance of the sea beet and slightly, but significantly, reduced salt tolerance of the sugar beet cultivars. This indicates that traditional domestication by selection for morphological traits such as leaf size, beet shape and size, enhanced productivity, sugar content and palatability slightly affected salt tolerance of sugar beet cultivars. Salt tolerance among four sugar beet cultivars, three of which have been claimed to be salt tolerant, did not differ. We analysed the components of RGR to understand the mechanism of salt tolerance at the whole-plant level. The growth rate reduction at higher salinity was linked with reduced leaf area at the whole-plant level (leaf area ratio) and at the individual leaf level (specific leaf area). The leaf weight fraction was not affected by increased salinity. On the other hand, succulence and leaf thickness and the net assimilation per unit of leaf area (unit leaf rate) increased in response to salt treatment, thus partially counteracting reduced capture of light by lower leaf area. This compensatory mechanism may form part of the salt tolerance mechanism of sea beet and the four studied sugar beet cultivars. Together, our results indicate that domestication of the halophytic ancestor sea beet slightly reduced salt tolerance and that breeding for improved salt tolerance of sugar beet cultivars has not been effective.
PMCID: PMC4381740  PMID: 25492122
Beta; breeding; cultivar; domestication; growth analysis; saline agriculture; salt tolerance; sea beet; sugar beet.
23.  Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C 
eLife  2013;2:e00260.
Nutrients shape the growth, maturation, and aging of plants and animals. In plants, the juvenile to adult transition (vegetative phase change) is initiated by a decrease in miR156. In Arabidopsis, we found that exogenous sugar decreased the abundance of miR156, whereas reduced photosynthesis increased the level of this miRNA. This effect was correlated with a change in the timing of vegetative phase change, and was primarily attributable to a change in the expression of two genes, MIR156A and MIR156C, which were found to play dominant roles in this transition. The glucose-induced repression of miR156 was dependent on the signaling activity of HEXOKINASE1. We also show that the defoliation-induced increase in miR156 levels can be suppressed by exogenous glucose. These results provide a molecular link between nutrient availability and developmental timing in plants, and suggest that sugar is a component of the leaf signal that mediates vegetative phase change.
eLife digest
Like animals, plants go through several stages of development before they reach maturity, and it has long been thought that some of the transitions between these stages are triggered by changes in the nutritional status of the plant. Now, based on experiments with the plant Arabidopsis thaliana, Yang et al. and, independently, Yu et al. have provided fresh insights into the role of sugar in ‘vegetative phase change'—the transition from the juvenile form of a plant to the adult plant.
The new work takes advantage of the fact that vegetative phase change is controlled by two genes that encode microRNAs (MIRNAs). Arabidopsis has eight MIR156 genes and both groups confirmed that supplying plants with sugar reduces the expression of two of these—MIR156A and MIR156C—whereas sugar deprivation increases their expression. Removing leaves also leads to upregulation of both genes, and delays the juvenile to adult transition. Given that this effect can be partially reversed by providing the plant with sugar, it is likely that sugar produced in the leaves—or one of its metabolites—is the signal that triggers the juvenile to adult transition through the reduction of miR156 levels.
Consistent with this idea, Yang and co-workers revealed that mutant plants that are deficient in chlorophyll show elevated levels of miR156 and a delayed transition to the adult form. In addition, they showed that a gene called HXK1, which encodes a glucose signaling protein, helps to keep plants in the juvenile form under conditions of low sugar availability. HXK1 also contributes to the glucose-induced decrease in miR156 levels and does so, at least in part, by regulating the transcription of MIR156A and MIR156C genes into messenger mRNA. HXK1 is not solely responsible for the juvenile to adult transition, however, because plants that lack this protein are only slightly precocious in their transition to the adult form.
The works of Yang et al. and Yu et al. have thus provided key insights into the mechanisms by which a leaf-derived signal controls a key developmental change in plants. Just as fruit flies use their nutritional status to regulate the onset of metamorphosis, and mammals use it to control the onset of puberty, so plants use the level of sugar in their leaves to trigger the transition from juvenile to adult forms.
PMCID: PMC3608266  PMID: 23538384
phase change; heteroblasty; Nicotiana benthamiana; heterochrony; miRNAs; nutrition; Arabidopsis
24.  Quantitative Analysis of the Phenotypic Variability of Shoot Architecture in Two Grapevine (Vitis vinifera) Cultivars 
Annals of Botany  2007;99(3):425-437.
Background and Aims
Plant architecture and its interaction with agronomic practices and environmental constraints are determinants of the structure of the canopy, which is involved in carbon acquisition and fruit quality development. A framework for the quantitative analysis of grapevine (Vitis vinifera) shoot architecture, based on a set of topological and geometrical parameters, was developed for the identification of differences between cultivars and the origins of phenotypic variability.
Two commercial cultivars (‘Grenache N’, ‘Syrah’) with different shoot architectures were grown in pots, in well-irrigated conditions. Shoot topology was analysed, using a hidden semi-Markov chain and variable-order Markov chains to identify deviations from the normal pattern of succession of phytomer types (P0–P1–P2), together with kinematic analysis of shoot axis development. Shoot geometry was characterized by final internode and individual leaf area measurements.
Key Results
Shoot architecture differed significantly between cultivars. Secondary leaf area and axis length were greater for ‘Syrah’. Secondary leaf area distribution along the main axis also differed between cultivars, with secondary leaves preferentially located towards the basal part of the shoot in ‘Syrah’. The main factors leading to differences in leaf area between the cultivars were: (a) slight differences in main shoot structure, with the supplementary P0 phytomer on the lower part of the shoot in ‘Grenache N’, which bears a short branch; and (b) an higher rate and duration of development of branches bearing by P1–P2 phytomers related to P0 ones at the bottom of the shoot in ‘Syrah’. Differences in axis length were accounted for principally by differences in individual internode morphology, with ‘Syrah’ having significantly longer internodes. This trait, together with a smaller shoot diameter, may account for the characteristic ‘droopy’ habit of ‘Syrah’ shoots.
This study highlights the architectural parameters involved in the phenotypic variability of shoot architecture in two grapevine cultivars. Differences in primary shoot structure and in branch development potential accounted for the main differences in leaf area distribution between the two cultivars. By contrast, shoot shape seemed to be controlled by differences in axis length due principally to differences in internode length.
PMCID: PMC2802955  PMID: 17204533
Architecture; shoot; organogenesis; morphogenesis; branching; leaf area; genotypic variability; Vitis vinifera
25.  Native Environment Modulates Leaf Size and Response to Simulated Foliar Shade across Wild Tomato Species 
PLoS ONE  2012;7(1):e29570.
The laminae of leaves optimize photosynthetic rates by serving as a platform for both light capture and gas exchange, while minimizing water losses associated with thermoregulation and transpiration. Many have speculated that plants maximize photosynthetic output and minimize associated costs through leaf size, complexity, and shape, but a unifying theory linking the plethora of observed leaf forms with the environment remains elusive. Additionally, the leaf itself is a plastic structure, responsive to its surroundings, further complicating the relationship. Despite extensive knowledge of the genetic mechanisms underlying angiosperm leaf development, little is known about how phenotypic plasticity and selective pressures converge to create the diversity of leaf shapes and sizes across lineages. Here, we use wild tomato accessions, collected from locales with diverse levels of foliar shade, temperature, and precipitation, as a model to assay the extent of shade avoidance in leaf traits and the degree to which these leaf traits correlate with environmental factors. We find that leaf size is correlated with measures of foliar shade across the wild tomato species sampled and that leaf size and serration correlate in a species-dependent fashion with temperature and precipitation. We use far-red induced changes in leaf length as a proxy measure of the shade avoidance response, and find that shade avoidance in leaves negatively correlates with the level of foliar shade recorded at the point of origin of an accession. The direction and magnitude of these correlations varies across the leaf series, suggesting that heterochronic and/or ontogenic programs are a mechanism by which selective pressures can alter leaf size and form. This study highlights the value of wild tomato accessions for studies of both morphological and light-regulated development of compound leaves, and promises to be useful in the future identification of genes regulating potentially adaptive plastic leaf traits.
PMCID: PMC3257252  PMID: 22253737

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