Predominantly symplastic phloem unloading of photosynthates maintains efficient starch accumulation in the cassava storage roots (Manihot esculenta Crantz).

BACKGROUND: Cassava (Manihot esculenta Crantz) efficiently accumulates starch in its storage roots. However, how photosynthates are transported from the leaves to the phloem (especially how they are unloaded into parenchymal cells of storage roots) remains unclear. RESULTS: Here, we investigated the sucrose unloading pattern and its impact on cassava storage root development using microstructural and physiological analyses, namely, carboxyfluorescein (CF) and C14 isotope tracing. The expression profiling of genes involved in symplastic and apoplastic transport was performed, which included enzyme activity, protein gel blot analysis, and transcriptome sequencing analyses. These finding showed that carbohydrates are transported mainly in the form of sucrose, and more than 54.6% was present in the stem phloem. Sucrose was predominantly unloaded symplastically from the phloem into storage roots; in addition, there was a shift from apoplastic to symplastic unloading accompanied by the onset of root swelling. Statistical data on the microstructures indicated an enrichment of plasmodesmata within sieve, companion, and parenchyma cells in the developing storage roots of a cultivar but not in a wild ancestor. Tracing tests with CF verified the existence of a symplastic channel, and [14C] Suc demonstrated that sucrose could rapidly diffuse into root parenchyma cells from phloem cells. The relatively high expression of genes encoding sucrose synthase and associated proteins appeared in the middle and late stages of storage roots but not in primary fibrous roots, or secondary fibrous roots. The inverse expression pattern of sucrose transporters, cell wall acid invertase, and soluble acid invertase in these corresponding organs supported the presence of a symplastic sucrose unloading pathway. The transcription profile of genes involved in symplastic unloading and their significantly positive correlation with the starch yield at the population level confirmed that symplastic sucrose transport is vitally important in the development of cassava storage roots. CONCLUSIONS: In this study, we revealed that the cassava storage root phloem sucrose unloading pattern was predominantly a symplastic unloading pattern. This pattern is essential for efficient starch accumulation in high-yielding varieties compared with low-yielding wild ancestors.

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.

Involvement of SUT1 and SUT2 Sugar Transporters in the Impairment of Sugar Transport and Changes in Phloem Exudate Contents in Phytoplasma-Infected Plants.

Phytoplasmas inhabit phloem sieve elements and cause abnormal growth and altered sugar partitioning. However, how they interact with phloem functions is not clearly known. The phloem responses were investigated in tomatoes infected by "Candidatus Phytoplasma solani" at the beginning of the symptomatic stage, the first symptoms appearing in the newly emerged leaf at the stem apex. Antisense lines impaired in the phloem sucrose transporters SUT1 and SUT2 were included. In symptomatic sink leaves, leaf curling was associated with higher starch accumulation and the expression of defense genes. The analysis of leaf midribs of symptomatic leaves indicated that transcript levels for genes acting in the glycolysis and peroxisome metabolism differed from these in noninfected plants. The phytoplasma also multiplied in the three lower source leaves, even if it was not associated with the symptoms. In these leaves, the rate of phloem sucrose exudation was lower for infected plants. Metabolite profiling of phloem sap-enriched exudates revealed that glycolate and aspartate levels were affected by the infection. Their levels were also affected in the noninfected SUT1- and SUT2-antisense lines. The findings suggest the role of sugar transporters in the responses to infection and describe the consequences of impaired sugar transport on the primary metabolism.

Computational Tools for Serial Block Electron Microscopy Reveal Plasmodesmata Distributions and Wall Environments.

Plasmodesmata are small channels that connect plant cells. While recent technological advances have facilitated analysis of the ultrastructure of these channels, there are limitations to efficiently addressing their presence over an entire cellular interface. Here, we highlight the value of serial block electron microscopy for this purpose. We developed a computational pipeline to study plasmodesmata distributions and detect the presence/absence of plasmodesmata clusters, or pit fields, at the phloem unloading interfaces of Arabidopsis (Arabidopsis thaliana) roots. Pit fields were visualized and quantified. As the wall environment of plasmodesmata is highly specialized, we also designed a tool to extract the thickness of the extracellular matrix at and outside of plasmodesmata positions. We detected and quantified clear wall thinning around plasmodesmata with differences between genotypes, including the recently published plm-2 sphingolipid mutant. Our tools open avenues for quantitative approaches in the analysis of symplastic trafficking.

Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

Sieve elements rapidly develop 'nacreous walls' following injury - a common wounding response?

Thick glistening cell walls occur in sieve tubes of all major land plant taxa. Historically, these 'nacreous walls' have been considered a diagnostic feature of sieve elements; they represent a conundrum, though, in the context of the widely accepted pressure-flow theory as they severely constrict sieve tubes. We employed the cucurbit Gerrardanthus macrorhizus as a model to study nacreous walls in sieve elements by standard and in situ confocal microscopy and electron microscopy, focusing on changes in functional sieve tubes that occur when prepared for microscopic observation. Over 90% of sieve elements in tissue sections processed for microscopy by standard methods exhibit nacreous walls. Sieve elements in whole, live plants that were actively transporting as shown by phloem-mobile tracers, lacked nacreous walls and exhibited open lumina of circular cross-sections instead, an appropriate structure for Münch-type mass flow of the cell contents. Puncturing of transporting sieve elements with micropipettes triggered the rapid (<1 min) development of nacreous walls that occluded the cell lumen almost completely. We conclude that nacreous walls are preparation artefacts rather than structural features of transporting sieve elements. Nacreous walls in land plants resemble the reversibly swellable walls found in various algae, suggesting that they may function in turgor buffering, the amelioration of osmotic stress, wounding-induced sieve tube occlusion, and possibly local defence responses of the phloem.

Scanning Electron Microscopy of the Phloem.

In vascular plants, sugars are transported through the phloem tissue from areas of production, the leaves, to heterotrophic organs, where they are needed for growth and storage. Inside the phloem, transport takes place in specialized cells called sieve elements. Sieve elements are connected end-to-end by sieve plates to form a sieve tube. Sieve plates have small perforations called sieve pores. Transport of sugars is pushed through the tubes, plates, and pores by osmotic potential differences in the plant. Physical constraints govern the speed and volume of sugar flow through this tube system. Understanding the phloem requires precise anatomical measurements to model the effect of sieve element physical parameters on flow. Presented is a detailed method to prepare phloem tissue for scanning electron microscopy to obtain large quantities of high-resolution data of the plants sugar transport tissue.

Transmission Electron Microscopy of the Phloem with Minimal Artifacts.

It is a universal feature of seed plants that their phloem consists of a continuous sieve-tube system throughout the plant that is highly pressurized by its sugar contents. Cellular continuity and the pressure flow, osmotically generated in the source leaves, allow the assimilates to reach all sinks organs. However, both phloem features, the cellular continuity and the high pressure, are challenges when fixing the phloem for transmission electron microscopy. With very few exceptions, the tissue preparation necessary for the fixation evokes rapid wound responses that eventually result in artifacts.This chapter describes the steps necessary to minimize development of artifacts in the phloem and includes preparation of fixatives, a dissection procedure that optimizes penetration of the fixatives and application to axial and lateral plant organs. Moreover, as alternative to the established fixation of fresh hand sections, we suggest a xylem-assisted perfusion fixation method for herbaceous plants. After the initial fixation, the subsequent dehydration, embedding, and ultrathin sectioning of the material follow routine procedures, which are briefly discussed, as is the orientation of samples for obtaining transverse and longitudinal phloem sections.

Noninvasive Investigation of Phloem Structure by 3D Synchrotron X-Ray Microtomography.

X-ray microtomography (µCT) is a three-dimensional imaging technique, which has, over the past decade, established itself as a go-to method for nondestructive visualization of plant tissue with submicrometer resolution. µCT is closely related to medical computed tomography, in that a measurement consists of acquiring a series of radiographs from different directions around the sample. Especially with synchrotron X-ray sources, these radiographs exhibit significant phase contrast. This greatly enhances soft tissue contrast, making it well suited for plant imaging. Tomographic reconstruction techniques are then employed to convert the stack of radiographs into a 3D volumetric image. Compared with the laboratory X-ray tube-based systems, synchrotron tomography beamlines also offer high throughput, with tens of samples scanned over the course of a typical 24-h beam time.Synchrotrons are typically operated as user facilities, with a staff member assisting users in aligning the beamline and all instrumentation-related matters. From the user's point of view, success of a synchrotron µCT experiment is often dependent on secure sample mounting, choice of appropriate beam parameters, and post-processing the data, i.e., extracting scientifically meaningful results from the 3D image. In this chapter, we review the issues to consider in preparation of a µCT experiment from the point of view of a phloem researcher, emphasizing those aspects which are directly under the user's control rather than technical specifics, which vary from one beamline to another.

Quantification of Leaf Phloem Anatomical Features with Microscopy.

Measurements of vein density and foliar minor vein phloem cell numbers, minor vein phloem cell sizes, and transfer cell wall ingrowths provide quantitative proxies for the leaf's capacities to load and export photosynthates. While overall infrastructural capacity for sugar loading and sugar export correlated positively and closely with photosynthetic capacity, the specific targets of the adjustment of minor vein organization varied with phloem-loading mechanism, plant life-cycle characteristics, and environmental growth conditions. Among apoplastic loaders, for which sugar loading into the phloem depends on cell membrane-spanning transport proteins, variation in minor vein density, phloem cell number, and level of cell wall ingrowth (when present) were consistently associated with photosynthetic capacity. Among active symplastic loaders, for which sugar loading into the phloem depends on cytosolic enzymes, variation in vein density and phloem cell size were consistently associated with photosynthetic capacity. All of these anatomical features were also subject to acclimatory adjustment depending on species and environmental conditions, with increased levels of these features supporting higher rates of photosynthesis. We present a procedure for the preparation of leaf tissue for minor vein analysis, using both light and transmission electron microscopy, that facilitates quantification of not only phloem features but also xylem features that provide proxies for foliar water import capacity.

Methods of Phloem Visualization: A Clear Future in Sight?

There have been exciting new results in phloem research in recent years, at least in part made possible by the rapid advancement of microscopic techniques. Several methods for visualizing phloem cells are available. The suitability of each method depends on the organ and species being studied, and on the scientific question being addressed. This review will briefly explain the specific challenges associated with phloem cell visualization. It will then focus on common methods currently being used for studying phloem anatomy, development, and function. Emphasis will be placed on the most recent improvements in imaging techniques which had, or most certainly will have, an impact on phloem research.

Down-regulation of the Sucrose Transporter CsSUT1 Causes Male Sterility by Altering Carbohydrate Supply.

In plants, male sterility is an important agronomic trait, especially in hybrid crop production. Many factors are known to affect crop male sterility, but it remains unclear whether Suc transporters (SUTs) participate directly in this process. Here, we identified and functionally characterized the cucumber (Cucumis sativus) CsSUT1, a typical plasma membrane-localized energy-dependent high-affinity Suc-H+ symporter. CsSUT1 is expressed in male flowers and encodes a protein that is localized primarily in the tapetum, pollen, and companion cells of the phloem of sepals, petals, filaments, and pedicel. The male flowers of CsSUT1-RNA interference (RNAi) lines exhibited a decrease in Suc, hexose, and starch content, relative to those of the wild type, during the later stages of male flower development, a finding that was highly associated with male sterility. Transcriptomic analysis revealed that numerous genes associated with sugar metabolism, transport, and signaling, as well as with auxin signaling, were down-regulated, whereas most myeloblastosis (MYB) transcription factor genes were up-regulated in these CsSUT1-RNAi lines relative to wild type. Our findings demonstrate that male sterility can be induced by RNAi-mediated down-regulation of CsSUT1 expression, through the resultant perturbation in carbohydrate delivery and subsequent alteration in sugar and hormone signaling and up-regulation of specific MYB transcription factors. This knowledge provides a new approach for bioengineering male sterility in crop plants.

Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells.

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants.

Intra-annual dynamics of phloem formation and ultrastructural changes in sieve tubes in Fagus sylvatica.

Despite increased interest in the timing and dynamics of phloem formation, seasonal changes in the structure of phloem sieve elements remain largely unexplored. To understand better the dynamics of phloem formation and the functioning of sieve tubes in the youngest phloem in Fagus sylvatica L., we investigated repeatedly taken phloem samples during the growing season of 2017 by means of light microscopy, and transmission and scanning electron microscopy. Phloem formation started with the expansion of the overwintered early phloem sieve tubes adjacent to the cambium and concurrent cambial cell production. The highest phloem growth rate was observed in general 1 week after the onset of cambial cell production, whereas the transition from early to late phloem occurred at the end of May. Cambial cell production ceased at the end of July. The final width of the phloem increment was 184 ± 10 µm, with an early phloem proportion of 59%. Collapse of older phloem tissue is a progressive process, which continuously occurred during the sampling period. Collapse of early phloem sieve tubes started shortly after the cessation of cambial cell production. Prior to the onset of radial growth, late phloem from the previous year represented 80% of the total non-collapsed part; during the growth period, this percentage decreased to 20%. Differences were observed in both sieve tube ultrastructure and sieve plate geometry between the youngest and older phloem. However, sieve plates were never completely occluded by callose, suggesting that processes affecting the functionality of sieve tubes may differ in the case of regular collapse or injury. The youngest parts of the phloem increment from the previous year (i.e., previous late phloem) continue functioning for some time in the current growing season, but the two-step development of overwintered phloem cells also ensures a sufficient translocation pathway for photosynthates to the actively growing tissues.

Sieve Elements: The Favourite Habitat of Phytoplasmas.

The sieve elements are the only plant compartments, where phytoplasmas can survive and propagate. Therefore, this chapter is focussed on the specific molecular and cell-biological properties of the sieve element. Sieve element-companion cell complexes arise from (pro)cambial mother cells induced by key genes known to be decisive for sieve-element differentiation. The special anatomy, cell biology, and plasma-membrane outfit of sieve elements allows them to act collectively as a tube system that is able to drive a mass flow against the flow induced by transpiration. Plasmodesmal corridors are vital for the translocation of photoassimilates and systemic signals and for survival of the enucleate sieve elements. Of paramount importance is the Ca2+-dependent gating of plasmodesmata by callose and proteins. Hence, some of the complex, regulatory mechanisms to maintain Ca2+ homoeostasis in sieve elements are presented. Finally, the peculiarities of the chemical and physical sieve-element environment offered to phytoplasmas are discussed.

DAPI and Confocal Laser-Scanning Microscopy for In Vivo Imaging of Phytoplasmas.

As phytoplasmas are located inside the phloem tissue, always surrounded by numerous layers of other cells, they can result difficult candidates for microscopical investigations. Moreover, the necessity to kill the plant tissues for microscopy observations causes instantaneous and irreversible modifications in the sieve elements, leading to misleading information and erroneous interpretations. Phytoplasmas were here investigated in intact Vicia faba host plants using DAPI as fluorescent probe and confocal laser scanning microscopy. The described nondestructive technique may be applied for the imaging of phytoplasmas and of different pathogen-related responses in planta.

Computational models evaluating the impact of sieve plates and radial water exchange on phloem pressure gradients.

The sugar conducting phloem in angiosperms is a high resistance pathway made up of sieve elements bounded by sieve plates. The high resistance generated by sieve plates may be a trade-off for promoting quick sealing in the event of injury. However, previous modeling efforts have demonstrated a wide variation in the contribution of sieve plates towards total sieve tube resistance. In the current study, we generated high resolution scanning electron microscope images of sieve plates from balsam poplar and integrated them into a mathematical model using Comsol Multiphysics software. We found that sieve plates contribute upwards of 85% towards total sieve tube resistance. Utilizing the Navier-Stokes equations, we found that oblong pores may create over 50% more resistance in comparison with round pores of the same area. Although radial water flows in phloem sieve tubes have been previously considered, their impact on alleviating pressure gradients has not been fully studied. Our novel simulations find that radial water flow can reduce pressure requirements by half in comparison with modeled sieve tubes with no radial permeability. We discuss the implication that sieve tubes may alleviate pressure requirements to overcome high resistances by regulating their membrane permeability along the entire transport pathway.

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.

Sugar transport played a more important role than sugar biosynthesis in fruit sugar accumulation during Chinese jujube domestication.

MAIN CONCLUSION: Sugar transport, including the symplasmic pathway in plasmodesmata and apoplasmic pathway mediated by sugar transporters, accelerated sugar accumulation in cultivated jujube, while sugar metabolism-related genes played weak roles in jujube domestication. The fruit of Chinese jujube (Ziziphus jujuba Mill.) is high in sugar concentration. By contrast, wild type-sour jujube (Z. jujuba Mill. var. spinosa Hu) contains markedly less sugar. It is unknown whether sugar transport or sugar metabolism drove sugar accumulation during jujube domestication. Using a combination of ultrastructural observations, phylogenetic analysis, testing for soluble sugars, and transcriptional analysis, the sugar accumulation mechanism was studied in the developmental stages of cultivated jujube and sour jujube. Our results indicate that the symplasmic transport pathway in plasmodesmata is present in cultivated jujube, but not in sour jujube. Sugar transporter genes have higher frequencies of duplication than sugar metabolism-related genes. Gene expression patterns indicate that sugar transporter genes, especially ZjSUT2, ZjSWEET1, ZjSWEET7, ZjSWEET11, ZjSTP3, and ZjSTP13a, rather than sugar metabolism-related genes showed higher expression levels in cultivated jujube versus sour jujube during fruit sugar accumulation. These findings suggest that sugar transport, including apoplasmic and symplasmic transport, rather than sugar biosynthesis, is associated with the difference in sugar accumulation between jujube and sour jujube, and that it may drive jujube domestication. This study provides valuable genetic information for jujube improvement, and offers new insights into fruit tree domestication related to sugar accumulation.