Ecophysiology of phloem loading in source leaves

The nature of phloem loading of photosynthesis products – either symplastic or apoplastic – has been a matter of debate over the last two decades. This controversy was reconciled by proposing a multiprogrammed loading mechanism. Different modes of phloem loading were distinguished on the basis of the variety of plasmodesmatal connectivity between the minor vein elements. Physiological evidence for at least two phloem loading mechanisms as well as recent support for coincidence between plasmodesmatal connectivity and the loading mechanism is shortly reviewed. The present paper attempts to correlate the plasmodesmatal connectivity between sieve element/companion cell complex and the adjacent cells (the minor vein configuration) – and implicitly the associate phloem loading mechanisms – with different types of climate. The minor vein configuration is a family characteristic. This enables one to relate vein configuration with ecosystem using the family distribution over the globe. The uneven distribution of vein types between terrestrial ecosystems indicates that apoplastic phloem loading predominates in cold and dry climate zones. Projection of the minor vein configuration on the Takhtajan system of flowering plants suggests evolution from apoplastic to symplastic phloem loading. Accordingly, the distribution of minor vein configurations suggests that drought and temperature stress have led to the transformation of the ancient symplastic mode into the more advanced apoplastic mode of loading.     

Phloem loading in the tulip tree. Mechanisms and evolutionary implications

Minor vein ultrastructure and phloem loading were studied in leaves of the tulip tree (Liriodendron tulipifera; Magnoliaceae). Plasmodesmatal frequencies leading into minor vein companion cells are higher than in species known to load via the apoplast. However, these companion cells are not specialized as "intermediary cells" as they are in species in which the best evidence for symplastic phloem loading has been documented. Mesophyll cells plasmolyzed in 600 mM sorbitol, whereas sieve elements and companion cells did not plasmolyze even in 1.2 M sorbitol, indicating that solute accumulates in the phloem against a steep concentration gradient. Both [(14)C]sucrose and (14)C-labeled photo-assimilate accumulated in the minor vein network, as demonstrated by autoradiography. [(14)C]sucrose accumulation was prevented by p-chloromercuribenzenesulfonic acid, an inhibitor of sucrose-proton cotransport from the apoplast. p-Chloromercuribenzenesulfonic acid largely, but not entirely, inhibited exudation of radiolabeled photoassimilate. The evidence is most consistent with the presence of an apoplastic component to phloem loading in this species, contrary to speculation that the more basal members of the angiosperms load by an entirely symplastic mechanism.     

Ultrastructural indications for coexistence of symplastic and apoplastic phloem loading in Commelina benghalensis leaves

The ultrastructural ontogeny of Commelina benghalensis minor-vein elements was followed. The mature minor vein has a restricted number of elements: a sheath of six to eight mestome cells encloses one xylem vessel, three to five vascular parenchyma cells, a companion cell, a thin-walled protophloem sieve-tube member and a thick-walled metaphloem sieve-tube member. The protophloem sieve-tube member (diameter 4–5 m; wall thickness 0.12 m) and the companion cell originated from a common mother cell. The metaphloem sieve-tube member (diameter 3 m; wall thickness 0.2 m) developed from the same precursor cell as the phloem parenchyma cells. Counting the plasmodesmatal frequencies demonstrated a symplastic continuum from mesophyll to the minor-vein phloem. The metaphloem sievetube member and the phloem parenchyma cells are the termini of this symplast. The protophloem sieve-tube member and companion cell constitute an insulated symplastic domain. The symplastic route, mesophyll to metaphloem sieve tube, appears to offer a path for symplastic loading; the protophloem sieve tube may be capable of accumulation from the apoplast. A similar two-way system of loading may exist in a number of plant families. Plasmodesmograms (a novel way to depict cell elements, plasmodesmatal frequencies and vein architecture) of some other species also displayed the anatomical requirements for two routes from mesophyll to sieve tube and indicate the potential coexistence of symplastic and apoplastic loading.     

<Emphasis Type="Italic">Amborella trichopoda</Emphasis>, plasmodesmata,and the evolution of phloem loading

Phloem loading is the process by which photoassimilates synthesized in the mesophyll cells of leaves enter the sieve elements and companion cells of minor veins in preparation for long distance transport to sink organs. Three loading strategies have been described: active loading from the apoplast, passive loading via the symplast, and passive symplastic transfer followed by polymer trapping of raffinose and stachyose. We studied phloem loading in Amborella trichopoda, a premontane shrub that may be sister to all other flowering plants. The minor veins of A. trichopoda contain intermediary cells, indicative of the polymer trap mechanism, forming an arc on the abaxial side and subtending a cluster of ordinary companion cells in the interior of the veins. Intermediary cells are linked to bundle sheath cells by highly abundant plasmodesmata whereas ordinary companion cells have few plasmodesmata, characteristic of phloem that loads from the apoplast. Intermediary cells, ordinary companion cells, and sieve elements form symplastically connected complexes. Leaves provided with ¹⁴CO₂ translocate radiolabeled sucrose, raffinose, and stachyose. Therefore, structural and physiological evidence suggests that both apoplastic and polymer trapping mechanisms of phloem loading operate in A. trichopoda. The evolution of phloem loading strategies is complex and may be difficult to resolve.     

Phloem loading. A reevaluation of the relationship between plasmodesmatal frequencies and loading strategies

The incidence of plasmodesmata in the minor vein phloem of leaves varies widely between species. On this basis, two pathways of phloem loading have been proposed: symplastic where frequencies are high, and apoplastic where they are low. However, putative symplastic-loading species fall into at least two categories. In one, the plants translocate raffinose-family oligosaccharides (RFOs). In the other, the primary sugar in the phloem sap is sucrose (Suc). While a thermodynamically feasible mechanism of symplastic loading has been postulated for species that transport RFOs, no such mechanism is known for Suc transporters. We used p-chloromercuribenzenesulfonic acid inhibition of apoplastic loading to distinguish between the two pathways in three species that have abundant minor vein plasmodesmata and are therefore putative symplastic loaders. Clethra barbinervis and Liquidambar styraciflua transport Suc, while Catalpa speciosa transports RFOs. The results indicate that, contrary to the hypothesis that all species with abundant minor vein plasmodesmata load symplastically, C. barbinervis and L. styraciflua load from the apoplast. C. speciosa, being an RFO transporter, loads from the symplast, as expected. Data from these three species, and from the literature, also indicate that plants with abundant plasmodesmata in the minor vein phloem have abundant plasmodesmata between mesophyll cells. Thus, plasmodesmatal frequencies in the minor veins may be a reflection of overall frequencies in the lamina and may have limited relevance to phloem loading. We suggest that symplastic loading is restricted to plants that translocate oligosaccharides larger than Suc, such as RFOs, and that other plants, no matter how many plasmodesmata they have in the minor vein phloem, load via the apoplast.     

AmSUT1, a sucrose transporter in collection and transport phloem of the putative symplastic phloem loader Alonsoa meridionalis

A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80-91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.     

A three-step screening procedure to identify the mode of phloem loading in intact leaves

A three-step screening method was developed to identify the mode of phloem loading in intact leaves. Phloem loading of ¹⁴CO₂-derived photosynthate was challenged by p-chloromercuribenzenesulfonic acid (PCMBS) in leaves of dicotyledons with either a symplasmic (type 1, with intermediary cells as companion cells) or apoplasmic (type 2b, with transfer cells as companion cells) minor-vein configuration. Firstly, photosynthate export as the result of phloem loading was measured by collection of phloem exudate from the petiole. The PCMBS had virtually no effect on photosynthate export in representatives of type-1 families (Lamiaceae, Lythraceae, Onagraceae, Saxifragaceae). In contrast, photosynthate export was strongly reduced by PCMBS in representatives of type-2b families (Asteraceae, Balsaminaceae, Dipsacaceae, Linaceae, Tropaeolaceae, Valerianaceae) and type-2b members of polytypical families (Fabaceae, Scrophulariaceae). Secondly, densitometric measurements of leaf autoradiographs demonstrated that the contrast between the mesophyll and the lower-order veins was hardly affected by PCMBS treatment in type-1 species, whereas PCMBS strongly reduced the contrast in type-2b species. Thirdly, separate ¹⁴C-radioassays of vein and mesophyll tissues confirmed this observation. The three-step procedure thus revealed a strong and consistent reduction of phloem loading by PCMBS in type-2b species which was absent in type-1 species. In conclusion, phloem loading in type-2b species occurs via the apoplast and type-1 species execute an alternative — most likely symplasmic — mode of phloem loading.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE/CC-complex sieve element/companion cell complex We gratefully acknowledge the expert help of Dr. Maarten Terlou, Department of Image Processing and Design, University of Utrecht, in carrying out the densitometric measurements.     

Plasmodesmatal frequency in relation to short-distance transport and phloem loading in leaves of barley (Hordeum vulgare). Phloem is not loaded directly from the symplast

We investigated the phloem loading pathway in barley, by determining plasmodesmatal frequencies at the electron microscope level for both intermediate and small blade bundles of mature barley leaves. Lucifer yellow was injected intercellularly into bundle sheath, vascular parenchyma, and thin-walled sieve tubes. Passage of this symplastically transported dye was monitored with an epifluorescence microscope under blue light. Low plasmodesmatal frequencies endarch to the bundle sheath cells are relatively low for most interfaces terminating at the thin- and thick-walled sieve tubes within this C₃ species. Lack of connections between vascular parenchyma and sieve tubes, and low frequencies (0.5% plasmodesmata per μm cell wall interface) of connections between vascular parenchyma and companion cells, as well as the very low frequency of pore-plasmodesmatal connections between companion cells and sieve tubes in small bundles (0.2% plasmodesmata per μm cell wall interface), suggest that the companion cell-sieve tube complex is symplastically isolated from other vascular parenchyma cells in small bundles. The degree of cellular connectivity and the potential isolation of the companion cell-sieve tube complex was determined electrophysiologically, using an electrometer coupled to microcapillary electrodes. The less negative cell potential (average –52 mV) from mesophyll to the vascular parenchyma cells contrasted sharply with the more negative potential (–122.5 mV) recorded for the companion cell-thin-walled sieve tube complex. Although intercellular injection of lucifer yellow clearly demonstrated rapid (0.75 μm s^-1) longitudinal and radial transport in the bundle sheath-vascular parenchyma complex, as well as from the bundle sheath through transverse veins to adjacent longitudinal veins, we were neither able to detect nor present unequivocal evidence in support of the symplastic connectivity of the sieve tubes to the vascular parenchyma. Injection of the companion cell-sieve tube complex, did not demonstrate backward connectivity to the bundle sheath. We conclude that the low plasmodesmatal frequencies, coupled with a two-domain electropotential zonation configuration, and the negative transport experiments using lucifer yellow, precludes symplastic phloem loading in barley leaves.     

Ultrastructure of minor-vein phloem and assimilate export in summer and winter leaves of the symplasmically loading evergreens Ajuga reptans L., Aucuba japonica Thunb., and Hedera helix L.

Minor-vein ultrastructure and sugar export were studied in mature summer and winter leaves of the three broadleaf-evergreen species Ajuga reptans var. artropurpurescens L., Aucuba japonica Thunb. and Hedera helix L. to assess temperature effects on phloem loading. Leaves of the perennial herb Ajuga exported substantial amounts of assimilates in form of raffinose-family oligosaccharides (RFOs). Its minor-vein companion cells represent typical intermediary cells (ICs), with numerous small vacuoles and abundant plasmodesmal connectivity to the bundle sheath. The woody plants Hedera and Aucuba translocated sucrose as the dominant sugar species, and only traces of RFOs. Their minor-vein phloem possessed a layer of highly vacuolated cells (VCs) intervening between mesophyll and sieve elements. Depending on their location and ontogeny, VCs were classified either as companion or parenchyma cells. Both cell types showed symplasmic continuity to the adjacent mesophyll tissue although at a lower plasmodesmal frequency compared to the Ajuga ICs. p-Chloromercuribenzenesulfonic acid did not reduce leaf sugar export in any of the plants, indicating a symplasmic mode of phloem loading. Winter leaves did not show symptoms of frost injury, and the vacuolar pattern in ICs and VCs was equally prominent in both seasons. Starch accumulation as a result of reduced phloem loading was not observed to be triggered by low temperature. In contrast, high amounts of starch were found in mesophyll and bundle-sheath cells of summer leaves. Physiological data on season-dependent leaf exudation showed the maintenance of sugar export in cold-acclimated winter leaves.     

Sucrose transporters and plasmodesmal regulation in passive phloem loading

An essential step for the distribution of carbon throughout the whole plant is the loading of sugars into the phloem in source organs. In many plants,accumulation of sugars in the sieve element-companion cell(SE-CC)complex is mediated and regulated by active processes.However,for poplar and many other tree species,a passive symplasmic mechanism of phloem loading has been proposed,characterized by symplasmic continuity along the pre-phloem pathway and the absence of active sugar accumulation in the SE-CC complex. A high overall leaf sugar concentration is thought to enable diffusion of sucrose into the phloem. In this review,we critically evaluate current evidence regarding the mechanism of passive symplasmic phloem loading,with a focus on the potential influence of active sugar transport and plasmodesmal regulation. The limited experimental data,combined with theoretical considerations,suggest that a concomitant operation of passive symplasmic and active phloem loading in the same minor vein is unlikely.However,active sugar transport could well play an important role in how passively loading plants might modulate the rate of sugar export from leaves. Insights into the operation of this mechanism has direct implications for our understanding of how these plants utilize assimilated carbon.     

Identification of phloem involved in assimilate loading in leaves by the activity of the galactinol synthase promoter

The definition of "minor" veins in leaves is arbitrary and of uncertain biological significance. Generally, the term refers to the smallest vein classes in the leaf, believed to function in phloem loading. We found that a galactinol synthase promoter, cloned from melon (Cucumis melo), directs expression of the gusA gene to the smallest veins of mature Arabidopsis and cultivated tobacco (Nicotiana tabacum) leaves. This expression pattern is consistent with the role of galactinol synthase in sugar synthesis and phloem loading in cucurbits. The expression pattern in tobacco is especially noteworthy since galactinol is not synthesized in the leaves of this plant. Also, we unexpectedly found that expression in tobacco is limited to two of three companion cells in class-V veins, which are the most extensive in the leaf. Thus, the "minor" vein system is defined and regulated at the genetic level, and there is heterogeneity of response to this system by different companion cells of the same vein.     

Structure and function of leaf minor veins in trees and herbs

Summary The structure of leaf minor veins in 700 species from 140 families of dicotyledons, monocotyledons and conifers has been studied by light and electron microscopy. The presence of several structural types of minor veins has been shown. The main types are open and closed veins characteristic of trees and herbs, respectively. These vein types differ by the structure of intermediate cells, and by the mechanisms of phloem loading and sugar transport. Most woody plants have intermediate cells with numerous plasmodesmal fields, symplastic transport as the main phloem loading mechanism, as well as oligosaccharides and other complex sugars as the main phloem transport substances. By contrast, the majority of herbs have intermediate cells without plasmodesmal connections, and apoplastic loading of sucrose occurs only by membrane proton cotransport. The closed type is divided into three subtypes, differing in the degree of development of the structures used for sugar uptake from the apoplast. A list of the plants investigated with their vein types is given. The evolution of the minor vein structure and phloem loading mechanism are discussed in relation to the evolution of life forms of higher plants.     

Phloem loading in two Scrophulariaceae species. What can drive symplastic flow via plasmodesmata?

To determine the driving forces for symplastic sugar flux between mesophyll and phloem, gradients of sugar concentrations and osmotic pressure were studied in leaf tissues of two Scrophulariaceae species, Alonsoa meridionalis and Asarina barclaiana. A. meridionalis has a typical symplastic configuration of minor-vein phloem, i.e. intermediary companion cells with highly developed plasmodesmal connections to bundle-sheath cells. In A. barclaiana, two types of companion cells, modified intermediary cells and transfer cells, were found in minor-vein phloem, giving this species the potential to have a complex phloem-loading mode. We identified all phloem-transported carbohydrates in both species and analyzed the levels of carbohydrates in chloroplasts, vacuoles, and cytoplasm of mesophyll cells by nonaqueous fractionation. Osmotic pressure was measured in single epidermal and mesophyll cells and in whole leaves and compared with calculated values for phloem sap. In A. meridionalis, a 2-fold concentration gradient for sucrose between mesophyll and phloem was found. In A. barclaiana, the major transported carbohydrates, sucrose and antirrhinoside, were present in the phloem in 22- and 6-fold higher concentrations, respectively, than in the cytoplasm of mesophyll cells. The data show that diffusion of sugars along their concentration gradients is unlikely to be the major mechanism for symplastic phloem loading if this were to occur in these species. We conclude that in both A. meridionalis and A. barclaiana, apoplastic phloem loading is an indispensable mechanism and that symplastic entrance of solutes into the phloem may occur by mass flow. The conditions favoring symplastic mass flow into the phloem are discussed.     

The intermediary cell: Minor-vein anatomy and raffinose oligosaccharide synthesis in the Scrophulariaceae

Minor-vein anatomy, sugar content, sugar synthesis, and translocation were studied in mature leaves of nine members of the Scrophulariaceae to determine if there is a correlation between companion-cell type and class of sugar translocated. Three types of companion cell were found: intermediary cells with extensive plasmodesmatal connections to the bundle sheath; transfer cells with wall ingrowths and few plasmodesmata; and ordinary companion cells with few plasmodesmata and no wall ingrowths. Alonsoa warscewiczii Regal., Verbascum chaixi Vill., and Mimulus cardinalis Dougl. ex. Benth. have intermediary cells and ordinary companion cells in the minor veins. These plants synthesize large amounts of raffinose and stachyose as well as sucrose. Nemesia strumosa Benth., and Rhodochiton atrosanguineum Zucc. have both intermediary cells and transfer cells and make proportionately less raffinose oligosaccharide than the species above. In N. strumosa, a single sieve element may abut both an intermediary cell and a transfer cell. The minor veins of Asarina scandens (Cav.) Penn. have transfer cells and what appear to be modified intermediary cells that have fewer plasmodesmata than other species, and occasional wall ingrowths. Asarina scandens synthesizes little raffinose or stachyose. Cymbalaria muralis P. Gaertn et al. and Linaria maroccana Hook.f. have only transfer cells and Digitalis grandiflora Mill. has only ordinary companion cells; these species make a trace of galactinol and raffinose, but no stachyose. Translocation experiments indicate that there is long-distance movement of raffinose oligosaccharide in these plants, even when it is synthesized in very small quantities in the leaves. We conclude that intermediary cells are as distinct a cell type as the transfer cell. In contrast to transfer cells, which are specialized for uptake of solute from the apoplast, intermediary cells are specialized for symplastic transfer of photoassimilate from the mesophyll and for synthesis of raffinose oligosaccharide. This supports our contention that raffinose oligosaccharide synthesis and symplastic phloem loading are mechanistically linked (Turgeon and Gowan 1990, Plant Physiol. 94, 1244–1249). Minor-vein anatomy and sugar synthesis may be useful characters in determining the phylogenetic relationships of plants in this family.We thank Andrea Wolfe and Wayne Elisens for helpful discussions on the taxonomy of the Scrophulariaceae. This research was supported by National Science Foundation grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 92-37306-7819, and Hatch funds.     

西瓜小叶脉超微结构研究

用透射电子显微技术研究了西瓜叶片小叶脉,结果表明,小叶脉是由大型维管束鞘细胞包围的维管束,维管束呈现大的头部和线形的柄部,柄部是单列细胞的木质部,由维管薄壁细胞和导管分子组成;头部是韧皮部,由维管薄壁细胞、伴胞和筛管分子组成。同一小叶脉内常见有超微结构特征显著不同的两种伴胞:一种伴胞体积小,与维管束鞘细胞接触面较小或不接触,细胞内有大液泡,细胞壁上没有胞间连丝或只有少数不分枝的胞间连丝,这种伴胞为2a型;另一种伴胞体积大,通常位于韧皮部两翼,不含大液泡而含大量小泡,与维管束鞘细胞接触面较大,接触面上有大量具分枝的胞间连丝,分枝部分比未分枝部分直径小,这种伴胞为中间细胞类型。显然,西瓜是小叶脉内兼具两种类型伴胞的植物。     

西瓜小叶脉超微结构研究

用透射电子显微技术研究了西瓜叶片小叶脉,结果表明,小叶脉是由大型维管束鞘细胞包围的维管束,维管束呈现大的头部和线形的柄部,柄部是单列细胞的木质部,由维管薄壁细胞和导管分子组成;头部是韧皮部,由维管薄壁细胞、伴胞和筛管分子组成。同一小叶脉内常见有超微结构特征显著不同的两种伴胞：一种伴胞体积小,与维管束鞘细胞接触面较小或不接触,细胞内有大液泡,细胞壁上没有胞间连丝或只有少数不分枝的胞间连丝,这种伴胞为2a型;另一种伴胞体积大,通常位于韧皮部两翼,不含大液泡而含大量小泡,与维管束鞘细胞接触面较大,接触面上有大量具分枝的胞间连丝,分枝部分比未分枝部分直径小,这种伴胞为中间细胞类型。显然,西瓜是小叶脉内兼具两种类型伴胞的植物。     

Effects of temperature on the conformation of the endoplasmic reticulum and on starch accumulation in leaves with the symplasmic minor-vein configuration

The phloem-loading-related effects of temperature on leaf ultrastructure were studied in seven species having numerous plasmodesmatal connections between the mesophyll and phloem (symplasmic minor-vein configuration). The response to temperature (between 5 and 30 °C) was characterized by drastic changes in the endoplasmic-reticulum labyrinth (ER labyrinth) of intermediary cells, in the position of the vacuole in bundle-sheath cells, and in the starch content in the chloroplasts of bundle-sheath cells and mesophyll cells. At temperatures above 20 °C, the ER system in the intermediary cells reached its maximal volume, while the vacuole in bundlesheath cells was positioned centripetally (proximal to the intermediary cell). With decreasing temperature, the ER labyrinth in intermediary cells gradually contracted till the ER was fully collapsed at 10 °C and the vacuole in bundle-sheath cells moved to a more centrifugal position. The apparent elimination of photosynthate transport via the ER and plasmodesmata at temperatures lower than 10 °C led to starch accumulation in the chloroplasts of bundle-sheath cells and mesophyll cells. All of these changes were fully temperature-reversible and probably reflect changes in the balance between photosynthate transport and storage. The ultrastructural shifts appear to be correlated with the passage of photosynthate through the intermediary cells and, as a consequence, with the rate of phloem loading at various temperatures. A contraction of the ER/plasmodesmata system imposed by cytoskeletal reorganisation is discussed as the reason for the blockage of phloem loading at low temperatures in association with the general chilling sensitivity of these species.Abbreviations BSC bundle-sheath cell - IC intermediary cell - MC mesophyll cell - PD plasmodesmata - PFD photon flux density - SE/CC-complex sieve element/companion cell complex The authors gratefully acknowledge the financial support by NWO (Dutch Organization for Scientific Research).     

Evidence for phloem loading via the abaxial bundle sheath cells in maize leaves

Leaves are asymmetric, with different functions for adaxial and abaxial tissue. The bundle sheath (BS) of C₃ barley (Hordeum vulgare) is dorsoventrally differentiated into three types of cells: adaxial structural, lateral S-type, and abaxial L-type BS cells. Based on plasmodesmatal connections between S-type cells and mestome sheath (parenchymatous cell layer below bundle sheath), S-type cells likely transfer assimilates toward the phloem. Here, we used single-cell RNA sequencing to investigate BS differentiation in C₄ maize (Zea mays L.) plants. Abaxial BS (^abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c mRNAs were also detected in the phloem parenchyma (PP). We show that maize has acquired a mechanism for phloem loading in which ^abBS cells provide the main route for apoplasmic sucrose transfer toward the phloem. This putative route predominates in veins responsible for phloem loading (rank-2 intermediate), whereas rank-1 intermediate and major veins export sucrose from the PP adjacent to the sieve element companion cell complex, as in Arabidopsis thaliana. We surmise that ^abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide insights into the unique transport-specific properties of ^abBS cells and support a modification to the canonical phloem loading pathway in maize.     

PLASMODESMATAL FREQUENCY AND OTHER STRUCTURAL ASPECTS OF ASSIMILATE COLLECTION AND PHLOEM LOADING IN LEAVES OF SONCHUS OLERACEUS (ASTERACEAE), A SPECIES WITH MINOR VEIN TRANSFER CELLS

Leaves of Sonchus oleraceus (Asteraceae) were examined with the electron microscope to determine plasmodesmatal frequencies and other structural features relating to the collection of photoassimilate and its subsequent loading into minor veins. Few plasmodesmata occur between mesophyll cells, which contain chloroplasts that are sometimes connected to both the plasmalemma and the tonoplast by membranous tubules. The minor veins consist of tracheary elements, sieve-tube members, vascular parenchyma cells, and companion cells. The latter two cell types are transfer cells, with some of the fingerlike wall ingrowths in companion cells being traversed lengthwise by plasmodesmata. The frequencies of plasmodesmata at the mesophyllbundle sheath boundary and within are higher at some interfaces than at corresponding interfaces in nine other species, including some that previously had been characterized as loading assimilate via the symplast. It is thus premature to designate all species containing transfer cells in their minor veins as loading assimilate only via the apoplast.