Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues

Macromolecular trafficking within the sieve element-companion cell complex, phloem unloading, and post-phloem transport were studied using the jellyfish green fluorescent protein (GFP). The GFP gene was expressed in Arabidopsis and tobacco under the control of the AtSUC2 promoter. In wild-type Arabidopsis plants, this promoter regulates expression of the companion cell-specific AtSUC2 sucrose-H+ symporter gene. Analyses of the AtSUC2 promoter-GFP plants demonstrated that the 27-kD GFP protein can traffic through plasmodesmata from companion cells into sieve elements and migrate within the phloem. With the stream of assimilates, the GFP is partitioned between different sinks, such as petals, root tips, anthers, funiculi, or young rosette leaves. Eventually, the GFP can be unloaded symplastically from the phloem into sink tissues, such as the seed coat, the anther connective tissue, cells of the root tip, and sink leaf mesophyll cells. In all of these tissues, the GFP can traffic cell to cell by symplastic post-phloem transport. The presented data show that plasmodesmata of the sieve element-companion cell complex, as well as plasmodesmata into and within the analyzed sinks, allow trafficking of the 27-kD nonphloem GFP protein. The data also show that the size exclusion limit of plasmodesmata can change during organ development. The results are also discussed in terms of the phloem mobility of assimilates and of small, low molecular weight companion cell proteins.     

Functional characterization of the Arabidopsis AtSUC2 Sucrose/H+ symporter by tissue-specific complementation reveals an essential role in phloem loading but not in long-distance transport

AtSUC2 (At1g22710) encodes a phloem-localized sucrose (Suc)/H(+) symporter necessary for efficient Suc transport from source tissues to sink tissues in Arabidopsis (Arabidopsis thaliana). AtSUC2 is highly expressed in the collection phloem of mature leaves, and its function in phloem loading is well established. AtSUC2, however, is also expressed strongly in the transport phloem, where its role is more ambiguous, and it has been implicated in mediating both efflux and retrieval to and from flanking tissues via the apoplast. To characterize the role of AtSUC2 in controlling carbon partitioning along the phloem path, AtSUC2 cDNA was expressed from tissue-specific promoters in an Atsuc2 mutant background. Suc transport in this mutant is highly compromised, as indicated by stunted growth and the accumulation of large quantities of sugar and starch in vegetative tissues. Expression of AtSUC2 cDNA from the 2-kb AtSUC2 promoter was sufficient to restore growth and carbon partitioning to nearly wild-type levels. The GALACTINOL SYNTHASE promoter of Cucumis melo (CmGAS1p) confers expression only in the minor veins of mature leaves, not in the transport phloem of larger leaf veins and stems. Mutant plants expressing AtSUC2 cDNA from CmGAS1p had intermediate growth and accumulated sugar and starch, but otherwise they had normal morphology. These characteristics support a role for AtSUC2 in retrieval but not efflux along the transport phloem and show that the only vital function of AtSUC2 in photoassimilate distribution is phloem loading. In addition, Atsuc2 mutant plants, although debilitated, do grow, and AtSUC2-independent modes of phloem transport are discussed, including an entirely symplastic pathway from mesophyll cells to sink tissues.     

Macromolecular trafficking between Nicotiana tabacum and the holoparasite Cuscuta reflexa

Transgenic tobacco plants expressing green fluorescent protein (GFP) under the control of the companion cell-specific promoter, AtSUC2, were parasitized by the holoparasite Cuscuta reflexa (dodder). GFP, moving in the translocation stream of the host, was transferred to the Cuscuta phloem via the absorbing hyphae of the parasite. An identical pattern of transfer was observed for the phloem-mobile probe, carboxyfluorescein. Following uptake by the parasite, GFP was translocated and unloaded from the Cuscuta phloem in meristematic sink tissues. Contrary to published data, these observations suggest the presence of a functional symplastic pathway between Cuscuta and its hosts, and demonstrate a considerable capacity for macromolecular exchange between plant species.     

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.     

Enhanced Sucrose Loading Improves Rice Yield by Increasing Grain Size

Yield in cereals is a function of grain number and size. Sucrose (Suc), the main carbohydrate product of photosynthesis in higher plants, is transported long distances from source leaves to sink organs such as seeds and roots. Here, we report that transgenic rice plants (Oryza sativa) expressing the Arabidopsis (Arabidopsis thaliana) phloem-specific Suc transporter (AtSUC2), which loads Suc into the phloem under control of the phloem protein2 promoter (pPP2), showed an increase in grain yield of up to 16% relative to wild-type plants in field trials. Compared with wild-type plants, pPP2::AtSUC2 plants had larger spikelet hulls and larger and heavier grains. Grain filling was accelerated in the transgenic plants, and more photoassimilate was transported from the leaves to the grain. In addition, microarray analyses revealed that carbohydrate, amino acid, and lipid metabolism was enhanced in the leaves and grain of pPP2::AtSUC2 plants. Thus, enhancing Suc loading represents a promising strategy to improve rice yield to feed the global population.Rice (Oryza sativa) is a staple food for nearly one-half of the global population. Given the rapid growth of the world’s population, there is an urgent need to increase rice yield. Rice yield is a complex trait that is directly associated with grain size, panicle number, and the number of grains per panicle (Xing and Zhang, 2010). Increasing grain size is a prime breeding target, and several genes known to control rice grain size, such as GRAIN SIZE3 (GS3), GS5, GW2 QTL for rice grain width and weight (GW2), GW8, and rice seed width5, have been identified (Fan et al., 2006; Song et al., 2007; Shomura et al., 2008; Li et al., 2011a; Wang et al., 2012). However, our knowledge of the mechanisms that control rice yield is limited. Thus, further improving rice yield remains a challenge for breeders (Sakamoto and Matsuoka, 2008). Identifying and characterizing unique genes or targets that regulate yield traits would improve our understanding of the molecular mechanisms that regulate yield traits and facilitate the breeding of new rice varieties with higher yields.The carbohydrates in rice grains originate from photosynthesis that is carried out predominantly in leaves (sources). Therefore, grain filling and rice yield depend on the efficient transport of carbohydrates from the leaves to seeds (sinks). In most plants, Suc is the main carbohydrate transported long distance in the veins to support the growth and development of roots, flowers, fruits, and seeds (Baker et al., 2012; Braun, 2012). Recently, the entire pathway for the export of Suc from leaves has been elucidated (Baker et al., 2012; Braun, 2012). Suc is synthesized in leaf mesophyll cells and diffuses from cell to cell through plasmodesmata until it reaches the phloem parenchyma cells (Slewinski and Braun, 2010). The SWEET transporters mediate Suc efflux from the phloem parenchyma cells into the apoplast, where Suc is subsequently loaded into the phloem sieve element-companion cell (SE/CC) complexes by Suc transporters (SUTs; Braun and Slewinski, 2009; Ayre, 2011; Chen et al., 2012). The resultant accumulation of Suc in sieve elements produces a hydrostatic pressure gradient that results in the bulk flow of Suc through a conduit of contiguous sieve elements, leading to its arrival and unloading in sink tissues (Lalonde et al., 2004; Baker et al., 2012).Genetic evidence has demonstrated that apoplastic Suc phloem loading is critical for growth, development, and reproduction in Arabidopsis (Arabidopsis thaliana). AtSWEET11 and AtSWEET12 are localized to the plasma membrane of the phloem and are expressed in a subset of phloem parenchyma cells in minor veins. These transporters mediate Suc efflux from phloem parenchyma cells into the apoplast prior to Suc uptake by SE/CC (Chen et al., 2012). The atsweet11 or atsweet12 single mutants exhibit no aberrant phenotypes, possibly due to genetic redundancy. However, atsweet11;12 double mutants are mildly chlorotic and display slower growth and higher levels of starch and sugar accumulation in the leaves than do wild-type plants (Chen et al., 2012). Arabidopsis phloem-specific sucrose transporter (AtSUC2) is a phloem-specific SUT that is expressed specifically in companion cells (Stadler and Sauer, 1996). AtSUC2 plays an essential role in phloem Suc loading and is necessary for efficient Suc transport from source to sink tissues in Arabidopsis (Stadler and Sauer, 1996; Gottwald et al., 2000; Srivastava et al., 2008). The atsuc2 mutants show stunted growth, retarded development, and sterility. Furthermore, these mutants accumulate excess starch in the leaves and fail to transport sugar efficiently to the roots and inflorescences (Gottwald et al., 2000).The proper control of carbohydrate partitioning is fundamental to crop yield (Braun, 2012). It has been reported that increasing sink grain strength by improving assimilate uptake capacity could be a promising approach toward obtaining higher yield. For example, seed-specific overexpression of a potato (Solanum tuberosum) SUT increased Suc uptake and growth rates of developing pea (Pisum sativum) cotyledons (Rosche et al., 2002). In addition, the Suc uptake capacity of grains and storage protein biosynthesis was increased in transgenic wheat (Triticum aestivum) plants expressing the barley (Hordeum vulgare) SUT HvSUT1 under the control of an endosperm-specific promoter (Weichert et al., 2010). Moreover, it was recently found that these transgenic wheat plants had a higher thousand grain weight and grain width and length, as well as a 28% increase in grain yield (Saalbach et al., 2014).Since the carbohydrates in rice grains originate from photosynthesis in source leaves, and carbohydrate partitioning from source leaves to heterotrophic sinks (e.g. seeds) is mediated by Suc transport in plants (Lalonde et al., 2004; Ayre, 2011), enhancing the capacity for Suc transport from leaves to seeds theoretically could increase crop yield. However, until now, enhancing Suc transport from leaves to seeds has not been shown to improve yield (Ainsworth and Bush, 2011).Here, we tested the hypothesis that enhancing Suc transport from leaves to seeds would increase rice yield. We expressed Arabidopsis SUC2 under control of the phloem protein2 promoter (pPP2) in rice and found that enhancing Suc loading did indeed increase rice yield. The pPP2::AtSUC2 plants produced larger grain than the wild type and showed grain yield increases of up to 16% in field trials. Our results suggest that manipulating phloem Suc transport is a useful strategy for increasing grain yield in rice and other cereal crops.     

Phloem unloading of potato virus X movement proteins is regulated by virus and host factors

To determine the requirements for viral proteins exiting the phloem, transgenic plants expressing green fluorescent protein (GFP) fused to the Potato virus X (PVX) triple gene block (TGB)p1 and coat protein (CP) genes were prepared. The fused genes were transgenically expressed from the companion cell (CC)-specific Commelina yellow mottle virus (CoYMV) promoter. Transgenic plants were selected for evidence of GFP fluorescence in CC and sieve elements (SE) and proteins were determined to be phloem mobile based on their ability to translocate across a graft union into nontransgenic scions. Petioles and leaves were analyzed to determine the requirements for phloem unloading of the fluorescence proteins. In petioles, fluorescence spread throughout the photosynthetic vascular cells (chlorenchyma) but did not move into the cortex, indicating a specific barrier to proteins exiting the vasculature. In leaves, fluorescence was mainly restricted to the veins. However, in virus-infected plants or leaves treated with a cocktail of proteasome inhibitors, fluorescence spread into leaf mesophyll cells. These data indicate that PVX contributes factors which enable specific unloading of cognate viral proteins and that proteolysis may play a role in limiting proteins in the phloem and surrounding chlorenchyma.     

Movement of protein and macromolecules between host plants and the parasitic weed Phelipanche aegyptiaca Pers.

Little is known about the translocation of proteins and other macromolecules from a host plant to the parasitic weed Phelipanche spp. Long-distance movement of proteins between host and parasite was explored using transgenic tomato plants expressing green fluorescent protein (GFP) in their companion cells. We further used fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite. Accumulation of GFP was observed in the central vascular bundle of leaves and in the root phloem of transgenic tomato plants expressing GFP under the regulation of AtSUC2 promoter. When transgenic tomato plants expressing GFP were parasitized with P. aegyptiaca, extensive GFP was translocated from the host phloem to the parasite phloem and accumulated in both Phelipanche tubercles and shoots. No movement of GFP to the parasite was observed when tobacco plants expressing GFP targeted to the ER were parasitized with P. aegyptiaca. Experiments using fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite demonstrated that Phelipanche absorbs dextrans up to 70 kDa in size from the host and that this movement can be bi-directional. In the present study, we prove for the first time delivery of proteins from host to the parasitic weed P. aegyptiaca via phloem connections, providing information for developing parasite resistance strategies.     

Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots

Transgenic Arabidopsis plants were constructed to express a range of GFP-fusion proteins (36-67 kDa) under the companion cell (CC)-specific AtSUC2 promoter. These plants were used to monitor the trafficking of these GFP-fusion proteins from the CCs into the sieve elements (SEs) and their subsequent translocation within and out of the phloem. The results revealed a large size exclusion limit (SEL) (>67 kDa) for the plasmodesmata connecting SEs and CCs in the loading phloem. Membrane-anchored GFP-fusions and a GFP variant targeted to the endoplasmic reticulum (ER) remained inside the CCs and were used as 'zero trafficking' controls. In contrast, free GFP and all soluble GFP-fusions, moved from the CCs into the SEs and were subsequently translocated through the phloem. Phloem unloading and post-phloem transport of these mobile GFP-fusions were studied in root tips, where post-phloem transport occurred only for the free form of GFP. All of the other soluble GFP-fusion variants were unloaded and restricted to a narrow zone of cells immediately adjacent to the mature protophloem. It appears that this domain of cells, which has a peripheral SEL of about 27-36 kDa, allows protein exchange between protophloem SEs and surrounding cells, but restricts general access of large proteins into the root tip. The presented data provide additional information on phloem development in Arabidopsis in relation to the formation of symplasmic domains.     

Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues

The Arabidopsis AtSUC3 gene encodes a sucrose (Suc) transporter that differs in size and intron number from all other Arabidopsis Suc transport proteins. Each plant species analyzed so far possesses one transporter of this special type, and several functions have been discussed for these proteins, including the catalysis of transmembrane Suc transport, and also Suc sensing and regulation of other Suc transporters. Here, we show that the AtSUC3 protein is localized in the sieve elements of the Arabidopsis phloem and is not colocalized with the companion cell-specific AtSUC2 phloem loader. Even stronger AtSUC3 expression is observed in numerous sink cells and tissues, such as guard cells, trichomes, germinating pollen, root tips, the developing seed coat, or stipules. Moreover, AtSUC3 expression is strongly induced upon wounding of Arabidopsis tissue. The physiological role of AtSUC3 in these different cells and tissues is discussed.     

The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development

The sucrose (Suc) H(+)-cotransporter StSUT1 from potato (Solanum tuberosum), which is essential for long-distance transport of Suc and assumed to play a role in phloem loading in mature leaves, was found to be expressed in sink tubers. To answer the question of whether SUT1 serves a function in phloem unloading in tubers, the promoter was fused to gusA and expression was analyzed in transgenic potato. SUT1 expression was unexpectedly detected not in tuber parenchyma but in the phloem of sink tubers. Immunolocalization demonstrated that StSUT1 protein was present only in sieve elements of sink tubers, cells normally involved in export of Suc from the phloem to supply developing tubers, raising the question of the role of SUT1 in tubers. SUT1 expression was inhibited by antisense in transgenic potato plants using a class I patatin promoter B33, which is primarily expressed in the phloem of developing tubers. Reduced SUT1 expression in tubers did not affect aboveground organs but led to reduced fresh weight accumulation during early stages of tuber development, indicating that in this phase SUT1 plays an important role for sugar transport. Changes in Suc- and starch-modifying enzyme activities and metabolite profiles are consistent with the developmental switch in unloading mechanisms. Altogether, the findings may suggest a role of SUT1 in retrieval of Suc from the apoplasm, thereby regulating the osmotic potential in the extracellular space, or a direct role in phloem unloading acting as a phloem exporter transferring Suc from the sieve elements into the apoplasm.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

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.     

Immunolocalization of solanaceous SUT1 proteins in companion cells and xylem parenchyma: new perspectives for phloem loading and transport

Leaf sucrose (Suc) transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Suc loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 Suc transporters from tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) were immunolocalized in plasma membranes of enucleate sieve elements. Here, we present data that identify solanaceous SUT1 proteins with high specificity in phloem companion cells. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species.     

Carbon partitioning and export in transgenic Arabidopsis thaliana with altered capacity for sucrose synthesis grown at low temperature: a role for metabolite transporters

We investigated the role of metabolite transporters in cold acclimation by comparing the responses of wild-type (WT) Arabidopsis thaliana (Heynh.) with that of transgenic plants over-expressing sucrose-phosphate synthase (SPSox) or with that of antisense repression of cytosolic fructose-1,6-bisphosphatase (FBPas). Plants were grown at 23 degrees C and then shifted to 5 degrees C. We compared the leaves shifted to 5 degrees C for 3 and 10 d with new leaves that developed at 5 degrees C with control leaves on plants at 23 degrees C. At 23 degrees C, ectopic expression of SPS resulted in 30% more carbon being fixed per day and an increase in sucrose export from source leaves. This increase in fixation and export was supported by increased expression of the plastidic triose-phosphate transporter AtTPT and, to a lesser extent, the high-affinity Suc transporter AtSUC1. The improved photosynthetic performance of the SPSox plants was maintained after they were shifted to 5 degrees C and this was associated with further increases in AtSUC1 expression but with a strong repression of AtTPT mRNA abundance. Similar responses were shown by WT plants during acclimation to low temperature and this response was attenuated in the low sucrose producing FBPas plants. These data suggest that a key element in recovering flux through carbohydrate metabolism in the cold is to control the partitioning of metabolites between the chloroplast and the cytosol, and Arabidopsis modulates the expression of AtTPT to maintain balanced carbon flow. Arabidopsis also up-regulates the expression of AtSUC1, and to lesser extent AtSUC2, as down-stream components facilitate sucrose transport in leaves that develop at low temperatures.     

Nematode infection triggers the de novo formation of unloading phloem that allows macromolecular trafficking of green fluorescent protein into syncytia

Syncytial feeding complexes induced by the cyst nematode Heterodera schachtii represent strong metabolic sinks for photoassimilates. These newly formed structures were described to be symplastically isolated from the surrounding root tissue and their mechanism of carbohydrate import has repeatedly been under investigation. Here, we present analyses of the symplastic connectivity between the root phloem and these syncytia in nematode-infected Arabidopsis (Arabidopsis thaliana) plants expressing the gene of the green fluorescent protein (GFP) or of different GFP fusions under the control of the companion cell (CC)-specific AtSUC2 promoter. In the same plants, phloem differentiation during syncytium formation was monitored using cell-specific antibodies for CCs or sieve elements (SEs). Our results demonstrate that free, CC-derived GFP moved freely from the phloem into the syncytial domain. No or only marginal cell-to-cell passage of GFP was observed into other root cells adjacent to these syncytia. In contrast, membrane-anchored GFP variants as well as soluble GFP fusions with increased molecular masses were restricted to the SE-CC complex. The presented data also show that nematode infection triggers the de novo formation of phloem containing an approximately 3-fold excess of SEs over CCs. This newly formed phloem exhibits typical properties of unloading phloem previously described in other sink tissues. Our results reveal the existence of a symplastic pathway between phloem CCs and nematode-induced syncytia. The plasmodesmata responsible for this symplastic connectivity allow the cell-to-cell movement of macromolecules up to 30 kD and are likely to represent the major or exclusive path for the supply of assimilates from the phloem into the syncytial complex.     

Arabidopsis sucrose transporter AtSUC9. High-affinity transport activity, intragenic control of expression, and early flowering mutant phenotype

AtSUC9 (At5g06170), a sucrose (Suc) transporter from Arabidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ultrahigh affinity for Suc (K(0.5) = 0.066 +/- 0.025 mm). AtSUC9 showed low substrate specificity, similar to AtSUC2 (At1g22710), and transported a wide range of glucosides, including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and alpha-methyl-d-glucose. The ability of AtSUC9 to transport 10 glucosides was compared directly with that of AtSUC2, HvSUT1 (from barley [Hordeum vulgare]), and ShSUT1 (from sugarcane [Saccharum hybrid]), and results indicate that type I and type II Suc transporters have different substrate specificities. AtSUC9 protein was localized to the plasma membrane by transient expression in onion (Allium cepa) epidermis. Using a whole-gene translational fusion to beta-glucuronidase, AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression in Arabidopsis was dependent on intragenic sequence, and this was found to also be true for AtSUC1 (At1g71880) but not AtSUC2. Plants containing mutations in Suc transporter gene AtSUC9 were found to have an early flowering phenotype under short-day conditions. The transport properties of AtSUC9 indicate that it is uniquely suited to provide cellular uptake of Suc at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition.     

Termination of nutrient import and development of vein loading capacity in albino tobacco leaves

The sink-source conversion in developing leaves of tobacco (Nicotiana tabacum L.) was studied to determine whether import termination is caused by the onset of export or is related to achievement of positive carbon balance. Albino shoots were grown in vitro and grafted to detopped stems of green tobacco plants. Termination of import was studied by providing mature leaves of the stock plant with ¹⁴CO₂ and detecting the presence of labeled nutrient in developing albino leaves by whole-leaf autoradiography. In albino leaves, import terminated progressively in the basipetal direction at the same stage of development as in leaves of green shoots. Starch was not present in the plastids of mesophyll cells of mature albino leaves but starch was synthesized when discs were cut from these leaves and incubated on 3 millimolar sucrose. Import ceased progressively in developing green leaves even when photosynthesis was prevented by darkening. It was concluded that cessation of import does not require achievement of positive carbon balance and is not the direct result of export initiation.

To determine whether vein loading capacity develops in albino leaves, discs were cut from mature leaves and floated on [¹⁴C]sucrose solution. Uptake of label into the veins was detected by autoradiography and this uptake was sensitive to the phloem loading inhibitor p-chloromercuribenzenesulfonic acid. However, the amount of label taken up by veins in albino leaves was less than that taken up by veins of mature green leaves.