The Phloem as an Arena for Plant Pathogens.

Although the phloem is a highly specialized tissue, certain pathogens, including phytoplasmas, spiroplasmas, and viruses, have evolved to access and live in this sequestered and protected environment, causing substantial economic harm. In particular, Candidatus Liberibacter spp. are devastating citrus in many parts of the world. Given that most phloem pathogens are vectored, they are not exposed to applied chemicals and are therefore difficult to control. Furthermore, pathogens use the phloem network to escape mounted defenses. Our review summarizes the current knowledge of phloem anatomy, physiology, and biochemistry relevant to phloem/pathogen interactions. We focus on aspects of anatomy specific to pathogen movement, including sieve plate structure and phloem-specific proteins. Phloem sampling techniques are discussed. Finally, pathogens that cause particular harm to the phloem of crop species are considered in detail.

Modeling 'Candidatus Liberibacter asiaticus' Movement Within Citrus Plants.

The phloem-limited 'Candidatus Liberibacter asiaticus' (Las) causes huanglongbing, a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in the greenhouse, and the impacts of temperature on plant colonization in growth-chamber experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by quantitative PCR (qPCR) or symptoms at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, maintained at from 8 to 20°C, from 18 to 30°C, or from 24 to 38°C daily range, and assessed by qPCR of samples taken from noninoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to analysis of variance and binomial generalized linear model and logistic regression analyses. Probabilities of detecting Las in greenhouse plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day-1 average speed of movement. In growth chambers, the temperature impacted plant colonization by Las, new shoot emission, and symptom expression. After a 7-month exposure time, Las was absent in all new shoots in the cooler environment (average three per plant), and present in 70% at the milder environment (six shoots, severe symptoms) and 25% in the warmer environment (eight shoots, no visible symptoms). Temperature of 25.7°C was the optimum condition for plant colonization. This explains the higher impact and incidence of huanglongbing disease during the winter months or regions of milder climates in Brazil.

Complexity untwined: The structure and function of cucumber (Cucumis sativus L.) shoot phloem.

Cucurbit phloem is complex, with large sieve tubes on both sides of the xylem (bicollateral phloem), and extrafascicular elements that form an intricate web linking the rest of the vasculature. Little is known of the physical interconnections between these networks or their functional specialization, largely because the extrafascicular phloem strands branch and turn at irregular angles. Here, export in the phloem from specific regions of the lamina of cucumber (Cucumis sativus L.) was mapped using carboxyfluorescein and 14 C as mobile tracers. We also mapped vascular architecture by conventional microscopy and X-ray computed tomography using optimized whole-tissue staining procedures. Differential gene expression in the internal (IP) and external phloem (EP) was analyzed by laser-capture microdissection followed by RNA-sequencing. The vascular bundles of the lamina form a nexus at the petiole junction, emerging in a predictable pattern, each bundle conducting photoassimilate from a specific region of the blade. The vascular bundles of the stem interconnect at the node, facilitating lateral transport around the stem. Elements of the extrafascicular phloem traverse the stem and petiole obliquely, joining the IP and EP of adjacent bundles. Using pairwise comparisons and weighted gene coexpression network analysis, we found differences in gene expression patterns between the petiole and stem and between IP and EP, and we identified hub genes of tissue-specific modules. Genes related to transport were expressed primarily in the EP while those involved in cell differentiation and development as well as amino acid transport and metabolism were expressed mainly in the IP.

Physiological and Proteomic Responses of Mulberry Trees (Morus alba. L.) to Combined Salt and Drought Stress.

Intensive investigations have been conducted on the effect of sole drought or salinity stress on the growth of plants. However, there is relatively little knowledge on how plants, particularly woody species, respond to a combination of these two stresses although these stresses can simultaneously occur in the field. In this study, mulberry, an economically important resource for traditional medicine, and the sole food of domesticated silkworms was subjected to a combination of salt and drought stress and analyzed by physiological methods and TMT-based proteomics. Stressed mulberry exhibited significant alteration in physiological parameters, including root/shoot ratio, chlorophyll fluorescence, total carbon, and ion reallocation. A total of 577 and 270 differentially expressed proteins (DEPs) were identified from the stressed leaves and roots, respectively. Through KEGG analysis, these DEPs were assigned to multiple pathways, including carbon metabolism, photosynthesis, redox, secondary metabolism, and hormone metabolism. Among these pathways, the sucrose related metabolic pathway was distinctly enriched in both stressed leaves and roots, indicating an important contribution in mulberry under stress condition. The results provide a comprehensive understanding of the adaptive mechanism of mulberry in response to salt and drought stress, which will facilitate further studies on innovations in terms of crop performance.

Environmental conditions, not sugar export efficiency, limit the length of conifer leaves.

Most conifer species have needle-shaped leaves that are only a few centimeters long. In general, variation in leaf size has been associated with environmental factors, such as cold or drought stress. However, it has recently been proposed that sugar export efficiency is the limiting factor for conifer needle length, based on the results obtained using a mathematical model of phloem transport. Here, phloem transport rates in long conifer needles were experimentally determined to test if the mathematical model accurately represents phloem transport. The validity of the model's assumptions was tested by anatomical analyses and sugar quantification. Furthermore, various environmental and physiological factors were tested for their correlation with needle length. The results indicate that needle length is not limited by sugar transport efficiency, but, instead, by winter temperatures and light availability. The identification of factors that influence needle size is instrumental for using this trait as a variable in breeding programs.

Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development.

The phloem of the Cucurbitaceae has long been a subject of interest due to its complex nature and the economic importance of the family. As in a limited number of other families, cucurbit phloem is bicollateral, i.e. with sieve tubes on both sides of the xylem. To date little is known about the specialized functions of the internal phloem (IP) and external phloem (EP). Here, a combination of microscopy, fluorescent dye transport analysis, micro-computed tomography, laser capture microdissection and RNA-sequencing (RNA-Seq) were used to study the functions of IP and EP in the vascular bundles (VBs) of cucumber fruit. There is one type of VB in the peduncle, but four in the fruit: peripheral (PeVB), main (MVB), carpel (CVB) and placental (PlVB). The VBs are bicollateral, except for the CVB and PlVB. Phloem mobile tracers and 14 C applied to leaves are transported primarily in the EP, and to a lesser extent in the IP. RNA-Seq data indicate preferential gene transcription in the IP related to differentiation/development, hormone transport, RNA or protein modification/processing/transport, and nitrogen compound metabolism and transport. The EP preferentially expresses genes for stimulus/stress, defense, ion transport and secondary metabolite biosynthesis. The MVB phloem is preferentially involved in photoassimilate transport, unloading and long-distance signaling, while the PeVB plays a more substantial role in morphogenesis and/or development and defense response. CVB and PlVB transcripts are biased toward development of reproductive organs. These findings provide an integrated view of the differentiated structure and function of the vascular tissue in cucumber fruit.

Elucidation of the Mechanisms of Long-Distance mRNA Movement in a Nicotiana benthamiana/Tomato Heterograft System.

Recent heterograft analyses showed that large-scale messenger RNA (mRNA) movement takes place in the phloem, but the number of mobile transcripts reported varies widely. However, our knowledge of the mechanisms underlying large-scale mRNA movement remains limited. In this study, using a Nicotiana benthamiana/tomato (Solanum lycopersicum) heterograft system and a transgenic approach involving potato (Solanum tuberosum), we found that: (1) the overall mRNA abundance in the leaf is not a good indicator of transcript mobility to the root; (2) increasing the expression levels of nonmobile mRNAs in the companion cells does not promote their mobility; (3) mobile mRNAs undergo degradation during their movement; and (4) some mRNAs arriving in roots move back to shoots. These results indicate that mRNA movement has both regulated and unregulated components. The cellular origins of mobile mRNAs may differ between herbaceous and woody species. Taken together, these findings suggest that the long-distance movement of mRNAs is a complex process and that elucidating the physiological roles associated with this movement is challenging but remains an important task for future research.

Mechanisms of phloem loading.

The complex form of higher plants requires continuous, balanced transport of nutrients in the phloem. The initial step of transferring sugars, amino acids, and other materials from photosynthetic cells to the conducting sieve tubes is known as phloem loading. Three phloem loading mechanisms have been described. The first involves release of sucrose into the apoplast and subsequent retrieval by the phloem. The initial release step in this process is now known to be mediated by a new class of transporters, the SWEET proteins. In the other two loading mechanisms, polymer trapping and diffusion, sucrose passes into the phloem through cytoplasmic channels, the plasmodesmata. Recent models have shed additional light on these mechanisms and their ability to sustain the growth of even the tallest trees.

FLOWERING LOCUS T mRNA is synthesized in specialized companion cells in Arabidopsis and Maryland Mammoth tobacco leaf veins.

Flowering is triggered by the transmission of a mobile protein, FLOWERING LOCUS T (FT), from leaves to the shoot apex. FT originates in the phloem of leaf veins. However, the identity of the FT-synthesizing cells in the phloem is not known. As a result, it has not been possible to determine whether the complex regulatory networks that control FT synthesis involve intercellular communication, as is the case in many aspects of plant development. We demonstrate here that FT in Arabidopsis thaliana and FT orthologs in Maryland Mammoth tobacco (Nicotiana tabacum) are produced in two unique files of phloem companion cells. These FT-activating cells, visualized by fluorescent proteins, also activate the GALACTINOL SYNTHASE (CmGAS1) promoter from melon (Cucumis melo). Ablating the cells by expression of the diphtheria toxin gene driven by the CmGAS1 promoter delays flowering in both Arabidopsis and Maryland Mammoth tobacco. In Arabidopsis, toxin expression reduces expression of FT and flowering-associated genes downstream, but not upstream, of FT Our results indicate that specific companion cells mediate the essential flowering function. Since the identified cells are present in the minor veins of two unrelated dicotyledonous species, this may be a widespread phenomenon.

Phloem Loading through Plasmodesmata: A Biophysical Analysis.

In many species, Suc en route out of the leaf migrates from photosynthetically active mesophyll cells into the phloem down its concentration gradient via plasmodesmata, i.e. symplastically. In some of these plants, the process is entirely passive, but in others phloem Suc is actively converted into larger sugars, raffinose and stachyose, and segregated (trapped), thus raising total phloem sugar concentration to a level higher than in the mesophyll. Questions remain regarding the mechanisms and selective advantages conferred by both of these symplastic-loading processes. Here, we present an integrated model-including local and global transport and kinetics of polymerization-for passive and active symplastic loading. We also propose a physical model of transport through the plasmodesmata. With these models, we predict that (1) relative to passive loading, polymerization of Suc in the phloem, even in the absence of segregation, lowers the sugar content in the leaf required to achieve a given export rate and accelerates export for a given concentration of Suc in the mesophyll and (2) segregation of oligomers and the inverted gradient of total sugar content can be achieved for physiologically reasonable parameter values, but even higher export rates can be accessed in scenarios in which polymers are allowed to diffuse back into the mesophyll. We discuss these predictions in relation to further studies aimed at the clarification of loading mechanisms, fitness of active and passive symplastic loading, and potential targets for engineering improved rates of export.

The complex character of photosynthesis in cucumber fruit.

The surface area of a mature green cucumber (Cucumis sativa L.) fruit is comparable with that of a functional leaf, but the characteristics of fruit photosynthesis and its contribution to growth are poorly understood. Here, the photosynthetic properties of two genotypes of cucumber (dark green and light green fruits) were studied using a combination of electron microscopy, immunogold enzyme localization, chlorophyll fluorescence imaging, isotope tracer, and fruit darkening techniques. Chlorophyll content of the exocarp is similar to that of leaves, but there are no distinctive palisade and spongy tissues. The efficiency of PSII is similar to that in leaves, but with lower non-photochemical quenching (NPQ). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is found mainly in the exocarp, while phosphoenolpyruvate carboxylase (PEPC) is primarily localized to vascular bundles and placenta tissue. Rubisco and PEPC expression at both transcriptional and translational levels increases concurrently during fruit growth. The contribution of fruit photosynthesis in exocarp to its own C accumulation is 9.4%, while ~88% of respiratory CO2 in fruit was captured and re-fixed. Photosynthesis by cucumber fruits, through direct fixation of atmospheric CO2 and recapture of respired CO2, as verified by 14CO2 uptake and gas exchange, makes an important contribution to fruit growth.

Passive phloem loading and long-distance transport in a synthetic tree-on-a-chip.

Vascular plants rely on differences in osmotic pressure to export sugars from regions of synthesis (mature leaves) to sugar sinks (roots, fruits). In this process, known as Münch pressure flow, the loading of sugars from photosynthetic cells to the export conduit (the phloem) is crucial, as it sets the pressure head necessary to power long-distance transport. Whereas most herbaceous plants use active mechanisms to increase phloem sugar concentration above that of the photosynthetic cells, in most tree species, for which transport distances are largest, loading seems, counterintuitively, to occur by means of passive symplastic diffusion from the mesophyll to the phloem. Here, we use a synthetic microfluidic model of a passive loader to explore the non-linear dynamics that arise during export and determine the ability of passive loading to drive long-distance transport. We first demonstrate that in our device, the phloem concentration is set by the balance between the resistances to diffusive loading from the source and convective export through the phloem. Convection-limited export corresponds to classical models of Münch transport, where the phloem concentration is close to that of the source; in contrast, diffusion-limited export leads to small phloem concentrations and weak scaling of flow rates with hydraulic resistance. We then show that the effective regime of convection-limited export is predominant in plants with large transport resistances and low xylem pressures. Moreover, hydrostatic pressures developed in our synthetic passive loader can reach botanically relevant values as high as 10 bars. We conclude that passive loading is sufficient to drive long-distance transport in large plants, and that trees are well suited to take full advantage of passive phloem loading strategies.

The scaling of the hydraulic architecture in poplar leaves.

Although much is known about the hydraulics of xylem, the hydraulic interconnectivity and dimensional scaling of phloem with respect to xylem in leaves has not been adequately studied to test alternative hydraulic architectural rules such as da Vinci's rule or Murray's rule, or physiological models such as Münch's Pressure Flow hypothesis. Using confocal and electron microscopy as well as mathematical analyses, we examined the hydraulic architecture of the mature leaves of the model species Populus tremula × alba across all seven hierarchical orders of the vascular branching. We show that: phloem and xylem conductive areas increase from minor to major veins; the sum of the conductive areas for each vein order increases exponentially from major to minor veins; the volume of individual sieve tube and vessel members increases from minor to major veins; and phloem conductive area scales isometrically with respect to xylem area across all vein orders. The application of first principles to our data shows that conductive areas scale according to da Vinci's rule and not according to Murray's rule, and that the phloem network in poplar leaves can generate the pressure gradient envisioned in Münch's hypothesis.

The hydraulic architecture of Ginkgo leaves.

PREMISE OF THE STUDY: The hydraulics of xylem has been widely studied in numerous species and organ types. However, comparatively little is known about how phloem and xylem are hydraulically coupled or about many of the basic structural properties of phloem (such as conducting cell numbers and conductive areas), which nevertheless have direct bearing on understanding phloem loading and unloading. METHODS: Using a combination of light, epifluorescence, confocal, and transmission electron microscopy, we quantified the hydraulic architecture of Ginkgo biloba leaf laminae and examined the scaling relationships between phloem and xylem in five fully mature leaves. KEY RESULTS: The conductive areas and lengths of sieve cells and tracheids increase basipetally toward the petiole in a manner that is consistent with Münch's pressure flow hypothesis for phloem transport. This trend holds true for individual veins, the sum of conductive areas across all veins at any distance from the petiole, and for individual sieve cells and tracheids. Further, the conductive areas of phloem and xylem are isometrically correlated across the entire vasculature of the leaf lamina. The data for conducting cell areas do not conform with the predictions of the hydraulic models of da Vinci and Murray. CONCLUSIONS: The scaling of Ginkgo lamina hydraulics complies with that observed in leaves of other gymnosperms and most angiosperms and is inconsistent with theoretical models that assume that the volume of transported incompressible fluids is conserved.

Assessing Rates of Long-distance Carbon Transport in Arabidopsis by Collecting Phloem Exudations into EDTA Solutions after Photosynthetic Labeling with [14C]CO2.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves ( Yadav et al., 2017a ), (2) the relative rates of carbon loading and transport through the phloem (this protocol), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport ( Yadav et al., 2017b ), We propose that conducting all three protocols on experimental and control plants provides a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). In this protocol, [14C]CO2 is photoassimilated in source leaves and phloem loading and transport of photoassimilate is quantified by collecting phloem exudates into an EDTA solution followed by scintillation counting.

Assessing Long-distance Transport from Photosynthetic Source Leaves to Heterotrophic Sink Organs with [14C]CO2.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves ( Yadav et al., 2017a ), (2) the relative rates of carbon loading and transport through the phloem ( Yadav et al., 2017b ), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport (this protocol). We propose that conducting all three protocols on experimental and control plants provides a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). In this protocol, [14C]CO2 is photoassimilated in source leaves and phloem loading and transport of the 14C label to heterotrophic sink organs, particularly roots, is quantified by scintillation counting. Using this protocol, we demonstrated that overexpression of sucrose transporters and a vacuolar proton pumping pyrophosphatase in the companion cells of Arabidopsis enhanced transport of 14C label photoassimilates to sink organs ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). This method can be adapted to quantify long-distance transport in other plant species.

Quantifying the Capacity of Phloem Loading in Leaf Disks with [14C]Sucrose.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves (this protocol), (2) the relative rates of carbon loading and transport through the phloem ( Yadav et al., 2017a ), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport ( Yadav et al., 2017b ). We propose that conducting all three protocols on experimental and control plants provides a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). In this protocol, Arabidopsis leaf disks isolated from mature rosette leaves are infiltrated with a buffered solution containing [14C]Suc. Suc transporters (SUCs or SUTs) load Suc into the phloem and excess, unloaded Suc in the leaf disk is then washed away. Loading of labeled Suc into the veins is visualized by autoradiography of lyophilized leaf disks and quantified by scintillation counting. Results are expressed as disintegration per minute per unit of leaf disk fresh weight or area.

Allocation, stress tolerance and carbon transport in plants: how does phloem physiology affect plant ecology?

Despite the crucial role of carbon transport in whole plant physiology and its impact on plant-environment interactions and ecosystem function, relatively little research has tried to examine how phloem physiology impacts plant ecology. In this review, we highlight several areas of active research where inquiry into phloem physiology has increased our understanding of whole plant function and ecological processes. We consider how xylem-phloem interactions impact plant drought tolerance and reproduction, how phloem transport influences carbon allocation in trees and carbon cycling in ecosystems and how phloem function mediates plant relations with insects, pests, microbes and symbiotes. We argue that in spite of challenges that exist in studying phloem physiology, it is critical that we consider the role of this dynamic vascular system when examining the relationship between plants and their biotic and abiotic environment.

Comparative analyses of C4 and C3 photosynthesis in developing leaves of maize and rice.

C4 and C3 photosynthesis differ in the efficiency with which they consume water and nitrogen. Engineering traits of the more efficient C4 photosynthesis into C3 crops could substantially increase crop yields in hot, arid conditions. To identify differences between C4 and C3 photosynthetic mechanisms, we profiled metabolites and gene expression in the developing leaves of Zea mays (maize), a C4 plant, and Oryza sativa (rice), a C3 plant, using a statistical method named the unified developmental model (UDM). Candidate cis-regulatory elements and transcription factors that might regulate photosynthesis were identified, together with differences between C4 and C3 nitrogen and carbon metabolism. The UDM algorithms could be applied to analyze and compare development in other species. These data sets together with community viewers to access and mine them provide a resource for photosynthetic research that will inform efforts to engineer improvements in carbon fixation in economically valuable grass crops.