Diameters of phloem sieve elements can predict stem growth rates of woody plants.

Understanding forest dynamics is crucial to addressing climate change and reforestation challenges. Plant anatomy can help predict growth rates of woody plants, contributing key information on forest dynamics. Although features of the water-transport system (xylem) have long been used to predict plant growth, the potential contribution of carbon-transporting tissue (phloem) remains virtually unexplored. Here, we use data from 347 woody plant species to investigate whether species-specific stem diameter growth rates can be predicted by the diameter of both the xylem and phloem conducting cells when corrected for phylogenetic relatedness. We found positive correlations between growth rate, phloem sieve element diameter and xylem vessel diameter in liana species sampled in the field. Moreover, we obtained similar results for data extracted from the Xylem Database, an online repository of functional, anatomical and image data for woody plant species. Information from this database confirmed the correlation of sieve element diameter and growth rate across woody plants of various growth forms. Furthermore, we used data subsets to explore potential influences of biomes, growth forms and botanical family association. Subsequently, we combined anatomical and geoclimatic data to train an artificial neural network to predict growth rates. Our results demonstrate that sugar transport architecture is associated with growth rate to a similar degree as water-transport architecture. Furthermore, our results illustrate the potential value of artificial neural networks for modeling plant growth under future climatic scenarios.

Phloem anatomy and function as shaped by the cell wall.

The partitioning of assimilated carbon is a complex process that involves the loading, long-distance transport, and subsequent unloading of carbohydrates from source to sink tissues. The network of plumbing that facilitates this coordinated process is the phloem tissue. Our understanding of the physiology of phloem transport has grown tremendously since the modern theory of mass flow was first put forward, aided by the concomitant progress of technology and experimental methodologies. Recent findings have put a renewed emphasis on the underlying anatomy of the phloem, and in particular the important role that cell walls play in enabling the high-pressure flow of photoassimilates through the sieve element. This review will briefly summarize the foundational work in phloem anatomy and highlight recent work exploring the physiology of phloem cell wall structure and mechanics.

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.).

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.

Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis.

KEY MESSAGE: Overexpression of CiNPR4 enhanced resistance of transgenic citrus plants to Huanglongbing by perceiving the salicylic acid and jasmonic acid signals and up-regulating the transcriptional activities of plant-pathogen interaction genes. Developing transgenic citrus plants with enhanced immunity is an efficient strategy to control citrus Huanglongbing (HLB). Here, a nonexpressor of pathogenesis-related gene 1 (NPR1) like gene from HLB-tolerant 'Jackson' grapefruit (Citrus paradisi Macf.), CiNPR4, was introduced into 'Wanjincheng' orange (Citrus sinensis Obseck). CiNPR4 expression was determined in transgenic citrus plants using quantitative real-time PCR analyses. The Candidatus Liberibacter asiaticus (CLas) pathogen of HLB was successfully transmitted to transgenic citrus plants by grafting infected buds. HLB symptoms developed in transgenic and wild-type (WT) plants by 9 months after inoculation. A CLas population analysis showed that 26.9% of transgenic lines exhibited significantly lower CLas titer levels compared with the CLas-infected WT plants at 21 months after inoculation. Lower starch contents and anatomical aberration levels in the phloem were observed in transgenic lines having enhanced resistance compared with CLas-infected WT plants. CiNPR4 overexpression changed the jasmonic acid, but not salicylic acid, level. Additionally, the jasmonic acid and salicylic acid levels increased after CLas infection. Transcriptome analyses revealed that the enhanced resistance of transgenic plants to HLB resulted from the up-regulated transcriptional activities of plant-pathogen interaction-related genes.

Characterization of a new natural cellulosic fiber extracted from Derris scandens stem.

The present study aims to identify a potential substitute for the harmful synthetic fibers in the field of polymer composites. With this objective, a comprehensive characterization of Derris scandens stem fibers (DSSFs) was carried out. The presence of high strength gelatinous fibers with a traditional hierarchical cell structure was found in the anatomical study. The chemical compositional analysis estimated the cellulose, hemicellulose, and lignin contents of 63.3 wt%, 11.6 wt%, and 15.3 wt%, respectively. Further analysis with XRD confirmed the presence of crystalline cellulose having a size of 11.92 nm with a crystallinity index of 58.15%. SEM and AFM studies show that these fibers are porous, and the average roughness is 105.95 nm. Single fiber tensile tests revealed that the DSSFs exhibited the mean Young's modulus and tensile strength of 13.54 GPa and 633.87 MPa respectively. Furthermore, the extracted fibers were found to be thermally stable up to 230 °C, as confirmed by thermogravimetric analysis. The fibers extracted from the stem of medicinal plant Derris scandens have the properties comparable to that of existing natural fibers, thus, suggesting it to use as a highly promising reinforcing agent alternative to synthetic fibers in polymer matrix composites.

Raman Imaging of Plant Cell Walls.

Raman imaging is a microspectroscopic approach revealing the chemistry and structure of plant cell walls in situ on the micro- and nanoscale. The method is based on the Raman effect (inelastic scattering) that takes place when monochromatic laser light interacts with matter. The scattered light conveys a change in energy that is inherent of the involved molecule vibrations. The Raman spectra are thus characteristic for the chemical structure of the molecules and can be recorded spatially ordered with a lateral resolution of about 300 nm. Based on thousands of acquired Raman spectra, images can be assessed using univariate as well as multivariate data analysis approaches. One advantage compared to staining or labeling techniques is that not only one image is obtained as a result but different components and characteristics can be displayed in several images. Furthermore, as every pixel corresponds to a Raman spectrum, which is a kind of "molecular fingerprint," the imaging results should always be evaluated and further details revealed by analysis (e.g., band assignment) of extracted spectra. In this chapter, the basic theoretical background of the technique and instrumentation are described together with sample preparation requirements and tips for high-quality plant tissue sections and successful Raman measurements. Typical Raman spectra of the different plant cell wall components are shown as well as an exemplified analysis of Raman data acquired on the model plant Arabidopsis. Important preprocessing methods of the spectra are included as well as single component image generation (univariate) and spectral unmixing by means of multivariate approaches (e.g., vertex component analysis).

Optimal Preparation of Tissue Sections for Light-Microscopic Analysis of Phloem Anatomy.

In order to successfully analyze and describe any plant tissue, the first challenge is preparation of good anatomical slides. The challenge is even greater when the target tissue has heterogeneous characteristics, such as the phloem where soft and stiff tissues occur side by side. The goal of this chapter is to present a detailed protocol containing various techniques for optimal preparation of phloem tissue samples for light microscopic analysis. The process typically involves the steps of fixation, softening, embedding, sectioning, staining, and mounting. The protocol can be applied to make samples of phloem and surrounding tissues of stems and roots, from woody to herbaceous plants.

Scaling of phloem hydraulic resistance in stems and leaves of the understory angiosperm shrub Illicium parviflorum.

PREMISE OF THE STUDY: Recent studies in canopy-dominant trees revealed axial scaling of phloem structure. However, whether this pattern is found in woody plants of the understory, the environment of most angiosperms from the ANA grade (Amborellales-Nymphaeales-Austrobaileyales), is unknown. METHODS: We used seedlings and adult plants of the understory tropical shrub Illicium parviflorum, a member of the lineage Austrobaileyales, to explore the anatomy and physiology of the phloem in their aerial parts, including changes through ontogeny. KEY RESULTS: Adult plants maintain a similar proportion of phloem tissue across stem diameters, but larger conduit dimensions and number cause the hydraulic resistance of the phloem to decrease toward the base of the plant. Small sieve plate pores resulted in an overall higher sieve tube hydraulic resistance than has been reported in other woody angiosperms. Sieve elements increase in size from minor to major leaf veins, but were shorter and narrower in petioles. The low carbon assimilation rates of seedlings and mature plants contrasted with a 3-fold higher phloem sap velocity in seedlings, suggesting that phloem transport velocity is modulated through ontogeny. CONCLUSIONS: The overall architecture of the phloem tissue in this understory angiosperm shrub scales in a manner consistent with taller trees that make up the forest canopy. Thus, the evolution of larger sieve plate pores in canopy-dominant trees may have played a key role in allowing woody angiosperms to extend beyond their understory origins.

Estimation of phloem carbon translocation belowground at stand level in a hinoki cypress stand.

At stand level, carbon translocation in tree stems has to match canopy photosynthesis and carbohydrate requirements to sustain growth and the physiological activities of belowground sinks. This study applied the Hagen-Poiseuille equation to the pressure-flow hypothesis to estimate phloem carbon translocation and evaluate what percentage of canopy photosynthate can be transported belowground in a hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) stand. An anatomical study revealed that, in contrast to sieve cell density, conductive phloem thickness and sieve cell hydraulic diameter at 1.3 m in height increased with increasing tree diameter, as did the concentration of soluble sugars in the phloem sap. At tree level, hydraulic conductivity increased by two orders of magnitude from the smallest to the largest trees in the stand, resulting in a stand-level hydraulic conductance of 1.7 × 10-15 m Pa-1 s-1. The osmotic potential of the sap extracted from the inner bark was -0.75 MPa. Assuming that phloem water potential equalled foliage water potential at predawn, the turgor pressure in the phloem at 1.3 m in height was estimated at 0.22 MPa, 0.59 MPa lower than values estimated in the foliage. With this maximal turgor pressure gradient, which would be lower during day-time when foliage water potential drops, the estimated stand-level rate of carbon translocation was 2.0 gC m-2 day-1 (30% of daily gross canopy photosynthesis), at a time of the year when aboveground growth and related respiration is thought to consume a large fraction of photosynthate, at the expense of belowground activity. Despite relying on some assumptions and approximations, this approach, when coupled with measurements of canopy photosynthesis, may further be used to provide qualitative insight into the seasonal dynamics of belowground carbon allocation.

Similarities and differences in the balances between leaf, xylem and phloem structures in Fraxinus ornus along an environmental gradient.

The plant carbon balance depends on the coordination between photosynthesis and the long-distance transport of water and sugars. How plants modify the allocation to the different structures affecting this coordination under different environmental conditions has been poorly investigated. In this study, we evaluated the effect of soil water availability on the allocation to leaf, xylem and phloem structures in Fraxinus ornus L. We selected small individuals of F. ornus (height ~2 m) from sites contrasting in soil water availability (wet vs dry). We measured how the leaf (LM) and stem + branch biomass (SBM) are cumulated along the stem. Moreover, we assessed the axial variation in xylem (XA) and phloem tissue area (PA), and in lumen area of xylem vessels (CAxy) and phloem sieve elements (CAph). We found a higher ratio of LM:SBM in the trees growing under drier conditions. The long-distance transport tissues of xylem and phloem followed axial patterns with scaling exponents (b) independent of site conditions. PA scaled isometrically with XA (b ~ 1). While CAxy was only marginally higher at the wet sites, CAph was significantly higher at the drier sites. Our results showed that under reduced soil water availability, F. ornus trees allocate relatively more to the leaf biomass and produce more conductive phloem, which is likely to compensate for the drought-related hydraulic limitations to the leaf gas exchanges and the phloem sap viscosity.

The impact of prolonged drought on phloem anatomy and phloem transport in young beech trees.

Phloem failure has recently been recognized as one of the mechanisms causing tree mortality under drought, though direct evidence is still lacking. We combined 13C pulse-labelling of 8-year-old beech trees (Fagus sylvatica L.) growing outdoors in a nursery with an anatomical study of the phloem tissue in their stems to examine how drought alters carbon transport and phloem transport capacity. For the six trees under drought, predawn leaf water potential ranged from -0.7 to -2.4 MPa, compared with an average of -0.2 MPa in five control trees with no water stress. We also observed a longer residence time of excess 13C in the foliage and the phloem sap in trees under drought compared with controls. Compared with controls, excess 13C in trunk respiration peaked later in trees under moderate drought conditions and showed no decline even after 4 days under more severe drought conditions. We estimated higher phloem sap viscosity in trees under drought. We also observed much smaller sieve-tube radii in all drought-stressed trees, which led to lower sieve-tube conductivity and lower phloem conductance in the tree stem. We concluded that prolonged drought affected phloem transport capacity through a change in anatomy and that the slowdown of phloem transport under drought likely resulted from a reduced driving force due to lower hydrostatic pressure between the source and sink organs.

Similar photosynthetic response to elevated carbon dioxide concentration in species with different phloem loading strategies.

Species have different strategies for loading sugars into the phloem, which vary in the route that sugars take to enter the phloem and the energetics of sugar accumulation. Species with passive phloem loading are hypothesized to have less flexibility in response to changes in some environmental conditions because sucrose export from mesophyll cells is dependent on fixed anatomical plasmodesmatal connections. Passive phloem loaders also have high mesophyll sugar content, and may be less likely to exhibit sugar-mediated down-regulation of photosynthetic capacity at elevated CO2 concentrations. To date, the effect of phloem loading strategy on the response of plant carbon metabolism to rising atmospheric CO2 concentrations is unclear, despite the widespread impacts of rising CO2 on plants. Over three field seasons, five species with apoplastic loading, passive loading, or polymer-trapping were grown at ambient and elevated CO2 concentration in free air concentration enrichment plots. Light-saturated rate of photosynthesis, photosynthetic capacity, leaf carbohydrate content, and anatomy were measured and compared among the species. All five species showed significant stimulation in midday photosynthetic CO2 uptake by elevated CO2 even though the two passive loading species showed significant down-regulation of maximum Rubisco carboxylation capacity at elevated CO2. There was a trend toward greater starch accumulation at elevated CO2 in all species, and was most pronounced in passive loaders. From this study, we cannot conclude that phloem loading strategy is a key determinant of plant response to elevated CO2, but compelling differences in response counter to our hypothesis were observed. A phylogenetically controlled experiment with more species may be needed to fully test the hypothesis.

Scaling relationships and vessel packing in petioles.

PREMISE OF THE STUDY: While tradeoffs among mechanical and conductive functions have been well investigated in woody stems, these tradeoffs are relatively unexplored in petioles, the structural link between stems and laminas. We investigated size-independent scaling relationships between cross-sectional areas of structural and vascular tissues, relationships between tissue areas of xylem and phloem, vessel packing within xylem, and scaling of vascular and structural tissues with lamina traits. METHODS: We examined allometric relationships among petiole tissues and as a function of lamina and petiole size variation on eleven species of Pelargonium. From transverse sections of methacrylate-embedded tissue, we measured the cross-sectional areas of all tissues within the petiole and vessel lumen, and cell wall areas of each vessel. Allometric scaling relationships were analyzed using standardized major axis regressions. KEY RESULTS: Pelargonium petiole vessels were packed as predicted by Sperry's packing rule for woody stems. In contrast to woody stems, there was no evidence of a tradeoff between vessel area and fiber area. Within cross-sections, more xylem was produced than phloem. Among bundles, xylem and phloem scaling relationships varied with bundle position. Except for lamina dry mass and petiole fiber cross-sectional area, petiole and lamina traits were independent. CONCLUSIONS: Petioles share vascular tissue traits with stems despite derivation from leaf primordia. We did not find evidence for a tradeoff between structural and vascular tissues, in part because fibers occur outside the xylem. We propose this separation of conduction and support underlies observed developmental and evolutionary plasticity in petioles.

Phloem physics: mechanisms, constraints, and perspectives.

Plants have evolved specialized vascular tissues for the distribution of energy, water, nutrients, and for communication. The phloem transports sugars from photosynthetic source regions (e.g. mature leaves) to sugar sinks (e.g. developing tissues such as buds, flowers, roots). Moreover, chemical signals such as hormones, RNAs and proteins also move in the phloem. Basic physical processes strongly limit phloem anatomy and function. This paper provides an overview of recent research and perspectives on phloem biomechanics and the physical constraints relevant to sugar transport in plants.

Is desiccation tolerance and avoidance reflected in xylem and phloem anatomy of two coexisting arid-zone coniferous trees?

Plants close their stomata during drought to avoid excessive water loss, but species differ in respect to the drought severity at which stomata close. The stomatal closure point is related to xylem anatomy and vulnerability to embolism, but it also has implications for phloem transport and possibly phloem anatomy to allow sugar transport at low water potentials. Desiccation-tolerant plants that close their stomata at severe drought should have smaller xylem conduits and/or fewer and smaller interconduit pits to reduce vulnerability to embolism but more phloem tissue and larger phloem conduits compared with plants that avoid desiccation. These anatomical differences could be expected to increase in response to long-term reduction in precipitation. To test these hypotheses, we used tridimensional synchroton X-ray microtomograph and light microscope imaging of combined xylem and phloem tissues of 2 coniferous species: one-seed juniper (Juniperus monosperma) and piñon pine (Pinus edulis) subjected to precipitation manipulation treatments. These species show different xylem vulnerability to embolism, contrasting desiccation tolerance, and stomatal closure points. Our results support the hypothesis that desiccation tolerant plants require higher phloem transport capacity than desiccation avoiding plants, but this can be gained through various anatomical adaptations in addition to changing conduit or tissue size.

Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots.

The OLIGOPEPTIDE TRANSPORTER 3 (OPT3) has recently been identified as a component of the systemic network mediating iron (Fe) deficiency responses in Arabidopsis. Reduced expression of OPT3 induces an over accumulation of Fe in roots and leaves, due in part by an elevated expression of the IRON-REGULATED TRANSPORTER 1. Here we show however, that opt3 leaves display a transcriptional program consistent with an Fe overload, suggesting that Fe excess is properly sensed in opt3 leaves and that the OPT3-mediated shoot-to-root signaling is critical to prevent a systemic Fe overload. We also took advantage of the tissue-specific localization of OPT3, together with other Fe-responsive genes, to determine the timing and location of early transcriptional events during Fe limitation and resupply. Our results show that the leaf vasculature responds more rapidly than roots to both Fe deprivation and resupply, suggesting that the leaf vasculature is within the first tissues that sense and respond to changes in Fe availability. Our data highlight the importance of the leaf vasculature in Fe homeostasis by sensing changes in apoplastic levels of Fe coming through the xylem and relaying this information back to roots via the phloem to regulate Fe uptake at the root level.

Mechanistic modelling of coupled phloem/xylem transport for L-systems: combining analytical and computational methods.

Background and Aims: Transport of carbohydrates and water are essential aspects of plant function. The aim of this study was to develop and test the methods for mechanistic modelling of quasi-stationary coupled phloem/xylem transport in the context of functional-structural plant modelling. Methods: The novelty of this approach is in combining analytical and computational methods. The plant structure is modelled at a metamer level with the internodes represented by conduit elements and the lateral organs represented by sources and sinks. Transport equations are solved analytically for each internode and then the solutions are adjusted and 'sewn' together using an iterative computational procedure taking into account concentration-dependent sinks and sources. The model is implemented in L-studio and uses the aspect-oriented modelling approach for phloem/xylem coupling. Key Results: To our knowledge, this is the first transport model that provides continuous distributions of the system variables in a complex developing structure. The model takes into account non-linear dependence of phloem resistance and osmotic potential on the local carbohydrate concentration. The model solutions show excellent agreement with the existing results of other analytical and numerical models. These comparisons confirm the validity of the approximations made in the model. Combining analytical and computational methods made it possible to take into account continuous sink/source distribution within internodes without much increase in the complexity of the computational procedure, because the necessary changes in the model were mostly in the analytical part. The results emphasize sensitivity of phloem flux and lateral xylem flux to the presence of distributed sinks and sources along the transport system. Conclusions: The presented approach provides a new insight into mechanistic modelling of phloem/xylem transport in growing plants. It will be useful for both fine-scale modelling of carbohydrate dynamics and for creating simpler models at a growth unit level.

Phloem networks in leaves.

The survival of all vascular plants depends on phloem and xylem, which comprise a hydraulically coupled tissue system that transports photosynthates, water, and a variety of other molecules and ions. Although xylem hydraulics has been extensively studied, until recently, comparatively little is known quantitatively about the phloem hydraulic network and how it is functionally coupled to the xylem network, particularly in photosynthetic leaves. Here, we summarize recent advances in quantifying phloem hydraulics in fully expanded mature leaves with different vascular architectures and show that (1) the size of phloem conducting cells across phylogenetically different taxa scales isometrically with respect to xylem conducting cell size, (2) cell transport areas and lengths increase along phloem transport pathways in a manner that can be used to model Münch's pressure-flow hypothesis, and (3) report observations that invalidate da Vinci's and Murray's hydraulic models as plausible constructs for understanding photosynthate transport in the leaf lamina.

Maintenance of carbohydrate transport in tall trees.

Trees present a critical challenge to long-distance transport because as a tree grows in height and the transport pathway increases in length, the hydraulic resistance of the vascular tissue should increase. This has led many to question whether trees can rely on a passive transport mechanism to move carbohydrates from their leaves to their roots. Although species that actively load sugars into their phloem, such as vines and herbs, can increase the driving force for transport as they elongate, it is possible that many trees cannot generate high turgor pressures because they do not use transporters to load sugar into the phloem. Here, we examine how trees can maintain efficient carbohydrate transport as they grow taller by analysing sieve tube anatomy, including sieve plate geometry, using recently developed preparation and imaging techniques, and by measuring the turgor pressures in the leaves of a tall tree in situ. Across nine deciduous species, we find that hydraulic resistance in the phloem scales inversely with plant height because of a shift in sieve element structure along the length of individual trees. This scaling relationship seems robust across multiple species despite large differences in plate anatomy. The importance of this scaling becomes clear when phloem transport is modelled using turgor pressures measured in the leaves of a mature red oak tree. These pressures are of sufficient magnitude to drive phloem transport only in concert with structural changes in the phloem that reduce transport resistance. As a result, the key to the long-standing mystery of how trees maintain phloem transport as they increase in size lies in the structure of the phloem and its ability to change hydraulic properties with plant height.

Cell-type- and tissue-specific transcriptomes of the white spruce (Picea glauca) bark unmask fine-scale spatial patterns of constitutive and induced conifer defense.

Plant defenses often involve specialized cells and tissues. In conifers, specialized cells of the bark are important for defense against insects and pathogens. Using laser microdissection, we characterized the transcriptomes of cortical resin duct cells, phenolic cells and phloem of white spruce (Picea glauca) bark under constitutive and methyl jasmonate (MeJa)-induced conditions, and we compared these transcriptomes with the transcriptome of the bark tissue complex. Overall, ~3700 bark transcripts were differentially expressed in response to MeJa. Approximately 25% of transcripts were expressed in only one cell type, revealing cell specialization at the transcriptome level. MeJa caused cell-type-specific transcriptome responses and changed the overall patterns of cell-type-specific transcript accumulation. Comparison of transcriptomes of the conifer bark tissue complex and specialized cells resolved a masking effect inherent to transcriptome analysis of complex tissues, and showed the actual cell-type-specific transcriptome signatures. Characterization of cell-type-specific transcriptomes is critical to reveal the dynamic patterns of spatial and temporal display of constitutive and induced defense systems in a complex plant tissue or organ. This was demonstrated with the improved resolution of spatially restricted expression of sets of genes of secondary metabolism in the specialized cell types.