Effects of low light on phloem ultrastructure and subcellular localization of sucrose synthase in <Emphasis Type="Italic">Prunus persica</Emphasis> L. var. <Emphasis Type="Italic">nectarina</Emphasis> Ait. fruit

Using electron microscopy, the ultrastructure of phloem unloading zone was examined in the Prunus persica L. var. nectarina Ait. fruit. Our study showed that, in the SE/CC (sieve element/companion cell) complexes, CC developing under low light had a thin cytoplasm layer with few mitochondria and numerous small vacuoles, and not clearly seen nuclei. The cytoplasm vacuolation indicated that the cytoskeleton was destroyed at low light. The effects of low light on CC development suggest that unloading evidently linked to the low accumulation of soluble sugars by fruit. At the young fruit stage, flesh parenchyma around the phloem tissue had no starch grains in the plastids in fruit developing under low light. This is a further indication that less photoassimilates was translocated from source leaves to fruit sinks under low light during the young fruit developmental stage. The activity of sucrose synthase (SuSy), the key enzyme of sucrose metabolism in fruit, increased dramatically during fruit maturation. The highest SuSy activity during the rapid fruit growth phase suggests that sink strength could be correlated with the SuSy activity. The high SuSy activity under normal light possibly indicates that fruit had a capacity to utilize sucrose irrespective of their site of phloem unloading. Immunogold electron microscopy showed that SuSy was localized mainly in the vacuole of flesh parenchyma cells. The vacuole-localized SuSy can hydrolyze sucrose imported from the phloem, which may explain the apparent correlation between SuSy activity and phloem unloading. The double sieve element (SE/SE) complexes occurred in a greater number and had thicker cell walls under normal light intensity than under low light intensity. These data demonstrate clearly that low light decreased SuSy activity in the control of phloem unloading. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 509–517. This text was submitted by the authors in English.     

Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.     

Expression analysis of genes associated with sucrose accumulation in sugarcane under normal and GA<Subscript>3</Subscript>-induced source–sink perturbed conditions

Sugarcane accumulates high amount of sucrose, thus making it one of the important cash crops worldwide. The final destination of sucrose accumulation in sugarcane is sink tissue, i.e., stalk, supplied by the source, i.e., leaf, to fulfill the need of plant growth, respiration, storage, and other metabolic activities. Signals between sink and source tissues regulate sucrose accumulation in sink and possibly the negative feedback from the sink restrains further accumulation in the stalk. However, perturbation of this negative feedback may help to improve sugar yield. This can be achieved by the application of GA₃ (Gibberellic acid), a plant growth regulator, known to excite physiological responses and modify the source–sink metabolism through their effect on photosynthesis, which in turn improves sink strength by redistribution of the photoassimilates. In the present study, GA₃ applied canes showed prominent increase in invertase activity, at early stage of the application, to provide hexoses. This in turn helped increase the internodal length and cane capacity for additional accumulation of sucrose, thereby increasing sink strength. At maturity, sucrose% and brix% were found higher in middle and top portions of the GA₃-applied canes. Expression analysis of various sucrose metabolising genes viz., sucrose phosphate synthase (SPS), sucrose synthase (SuSy), soluble acid invertase, neutral invertase, and cell wall invertase (CWI) was carried out at different growth stages, using quantitative RT-PCR. CWI, which plays key role in phloem unloading in sink tissues, exhibited higher expression in GA₃ samples at the elongation stage which decreased with maturity, whereas both SuSy and SPS, involved in regulation of sucrose accumulation, showed a variable level of expression. Thus, GA₃ application on cane may improve the sucrose content in stalk and thus assuage maneuvering source–sink dynamics in sugarcane.     

Sucrose synthase levels do not limit or regulate carbon transfer in the arbuscular mycorrhizal symbiosis

Abstract

Sucrose synthase (SuSy) is the main sucrose breakdown enzyme in plant sink tissues, including nodules, and is a possible candidate for the diversion of plant carbon to arbuscular mycorrhizal (AM) fungi in roots. We tested the involvement of SuSy in AM symbiosis of Glomus intraradices and Pisum sativum (pea). We observed that peas deficient in the predominant root isoform of SuSy were colonized successfully by AM fungi similar to wild-type roots. SuSy protein levels did not increase in roots as AM symbiosis developed, although SuSy protein levels did increase in nodules as the rhizobium symbiosis developed. Our results lead us to conclude that, unlike nodule symbiosis, SuSy protein does not limit or regulate carbon transfer in the AM symbiosis.     

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.     

白及蔗糖合成酶基因的克隆及表达分析

为揭示白及蔗糖合成酶基因与生长发育的关系,该研究以白及为材料,利用RT-PCR技术同源克隆白及蔗糖合成酶的关键基因SuSy,对SuSy基因的生物学特性及表达特征进行了分析,并利用实时荧光定量PCR检测SuSy基因在不同组织中的表达规律。结果表明:(1)白及SuSy基因长度为2 215 bp,编码737个氨基酸,与铁皮石斛、文心兰和蝴蝶兰的蛋白质氨基酸序列的相似性分别为97%、92%和95%。(2)生物信息学分析表明,SuSy蛋白质序列具有较高的亲水性,与拟南芥SuSy蛋白质氨基酸三级结构一致性为75.2%;系统进化树分析发现,白及SuSy蛋白与铁皮石斛处于同一个分支上。(3) qRT-PCR结果表明,SuSy基因在叶片中的表达量最高,块茎中的表达量最低;成熟叶片的表达量高于未成熟叶片的表达量;数据差异性分析显示,SuSy基因在根、块茎中表达量具有极显著性差异,但在一年生叶和二年生叶中的表达量无显著性差异,幼苗叶和一、二年生叶中表达量具有极显著性差异。由此推测,SuSy基因可能受生长发育的诱导,是调控白及生长发育关键基因。     

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.     

Spatial and temporal distribution of sucrose synthase in the radish hypocotyl in relation to thickening growth.

Sucrose synthase (SuSy) is a key enzyme in the development of storage root of radish. Clarification of its spatial and temporal expression during the thickening growth of radish hypocotyl, which later develops into storage root, was carried out immunologically using light microscopy. Sequential harvests at 3, 7, 11 and 13 d after sowing (DAS) were performed on two radish cultivars having different sink capacity. A very low level of SuSy was observed 3 DAS for both cultivars. White Cherrish (WC; strong storage root) showed the maximum level of SuSy between 7 and 11 DAS with increased cell development (thickening), while in Kosena (K; low storage root) the level remained high after 13 d of growth. A high level of SuSy was found in companion cells, which was consistent with previous observations, but SuSy was also found in the xylem parenchyma and in some cortical cells. The level of SuSy differed according to the localization and depended highly on cell development. Both cell division and cell enlargement were stimulated in WC compared with K. The role of SuSy during thickening growth of radish hypocotyl is discussed in terms of utilizing photosynthates.     

Sucrose carrier RcSCR1 is involved in sucrose retrieval, but not in sucrose unloading in growing hypocotyls of Ricinus communis L

The transport of assimilates from source to sink tissues is mediated by the phloem. Along the vascular system the phloem changes its physiological function from loading phloem to transport and unloading phloem. Sucrose carrier proteins have been identified in the transport phloem, but it is unclear whether the physiological role of these transporters is phloem unloading of sucrose or retrieval of apoplasmic sucrose back into the sieve element/companion cell complex. Here, we describe the dynamic expression of the Ricinus communis sucrose carrier RcSCR1 in the hypocotyl at different sink strengths. Our results indicate that phloem unloading in castor bean is not catalysed by the phloem loader RcSCR1. However, this sucrose carrier represents the molecular basis of the sucrose retrieval mechanism along the transport phloem, which is dynamically adjusted to the sink strength. As a consequence, we assume that other release carrier(s) exist in sink tissues, such as the hypocotyl, in R. communis.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed

Developing cotton (Gossypium hirsutum L.) seed exhibits complex patterns of carbon allocation in which incoming sucrose (Suc) is partitioned to three major sinks: the fibers, seed coat, and cotyledons, which synthesize cellulose, starch, and storage proteins or oils, respectively. In this study we investigated the role of Suc synthase (SuSy) in the mobilization of Suc into such sinks. Assessments of SuSy gene expression at various levels led to the surprising conclusion that, in contrast to that found for other plants, SuSy does not appear to play a role in starch synthesis in the cotton seed. However, our demonstration of functional symplastic connections between the phloem-unloading area and the fiber cells, as well as the SuSy expression pattern in fibers, indicates a major role of SuSy in partitioning carbon to fiber cellulose synthesis. SuSy expression is also high in transfer cells of the seed coat facing the cotyledons. Such high levels of SuSy could contribute to the synthesis of the thickened cell walls and to the energy generation for Suc efflux to the seed apoplast. The expression of SuSy in cotyledons also suggests a role in protein and lipid synthesis. In summary, the developing cotton seed provides an excellent example of the diverse roles played by SuSy in carbon metabolism.     

Melon phloem-sap proteome: developmental control and response to viral infection

In addition to small molecules such as sugars and amino acids, phloem sap contains macromolecules, including mRNA and proteins. It is generally assumed that all molecules in the phloem sap are on the move from source to sink, but recent evidence suggests that the macromolecules' direction of movement can be controlled by endogenous plant mechanisms. To test the hypothesis that the phloem-sap protein profile is affected by local metabolic activities, we analyzed the phloem-sap proteome in young and mature tissues of melon plants. We also examined the effect of cucumber mosaic virus (CMV) infection and expression of CMV movement protein in transgenic melon plants on the phloem protein profile. Sap collected from cut sections of young stems or petioles contained specific proteins that were absent from sap collected from mature stems or petioles. Most of these proteins were involved in defense response and protection from oxidative stress, suggesting that they play a role in maintaining safe activity of the sieve tubes in young tissues. Phloem sap collected from CMV-infected plants and transgenic plants expressing the CMV movement protein contained only a few additional proteins with molecular masses of 18 to 75 kDa. Here again, most of the additional proteins were associated with stress responses. Our study indicated that the proteome of phloem sap is dynamic and under developmental control. Entry and exit of proteins from the sieve tube can be regulated at the tissue level. Moreover, the plant can maintain regulation of protein trafficking from companion cells to sieve elements under viral infection or other perturbations in plasmodesmal function.     

Sucrose Hydrolysis in Relation to Phloem Translocation in Beta vulgaris

Asymmetrically labeled sucrose, ¹⁴C(fructosyl)sucrose, was used to determine whether sucrose undergoes extracellular hydrolysis during phloem translocation in the sugar beet, Beta vulgaris. In addition, the metabolism of various sugars accumulated and translocated was determined in various regious of the plant. These processes were studied in detached regions as well as in the intact, translocating plant in the source leaf, along the translocation path, and in a rapidly growing sink leaf and storage beet. The data show that, unlike sucrose accumulation into the sink tissue of sugarcane, sucrose is neither hydrolzyed prior to phloem loading or during transit, nor is it extracellularly hydrolyzed during accumulation into sink leaves or the storage beet.     

A DNA element between At4g28630 and At4g28640 confers companion-cell specific expression following the sink-to-source transition in mature minor vein phloem

The collection phloem in minor veins is distinct from other vein classes in that the minor veins mature during the sink to source transition and are the primary sites of phloem loading. After maturation, minor vein phloem maintains its character in part through minor-vein specific regulatory cascades; however despite its physiological significance, little of these developmental programs is understood. From an Arabidopsis enhancer trap screen, we identified MATURE MINOR VEIN ELEMENT1 (MMVE1) in the intergenic region between two oppositely oriented genes, the ABC transporter ATM1 (At4g28630) and IAA11 (At4g28640). MMVE1 promotes reporter gene activity in minor vein phloem in a pattern resembling the sink to source transition. Promoter truncation experiments and phylogenetic footprinting demonstrate sequences proximal to ATM1 promote minor vein expression whereas sequences closer to IAA11 repress it. Both orientations of the promoter were used to drive expression of CONSTANS to generate a phloem mobile signal conferring early flowering under non-inductive conditions. Tandem copies of MMVE1 increase minor vein expression strength and specificity. MMVE1 is the first minor vein enhancer characterized from a species that loads from the apoplast, and supports the presence of unique regulatory cascades operating in minor vein phloem.     

Evidence for symplastic phloem unloading in sink leaves of barley

The pathway of phloem unloading in sink barley (Hordeum vulgare) leaves was studied using a combination of electron microscopy, carboxyfluorescein transport, and systemic movement of barley stripe mosaic virus expressing the green fluorescent protein. Studies of plasmodesmatal frequencies between the phloem and mesophyll indicated a symplastic sieve element- (SE) unloading pathway involving thick-walled and thin-walled SEs. Phloem-translocated carboxyfluorescein was unloaded rapidly from major longitudinal veins and entered the mesophyll cells of sink leaves. Unloading was "patchy" along the length of a vein, indicating that sieve element unloading may be discontinuous along a single vascular bundle. This pattern was mirrored precisely by the unloading of barley stripe mosaic virus expressing the green fluorescent protein. Transverse veins were not utilized in the unloading process. The data collectively indicate a symplastic mechanism of SE unloading in the sink barley leaf.     

Activity of membrane-associated sucrose synthase is regulated by its phosphorylation status in cultured cells of sycamore (Acer pseudoplatanus)

Changes in sucrose synthase (SuSy) activity, protein level and degree of phosphorylation were investigated in plasmalemma and tonoplast of sycamore cells cultured either in the presence of sucrose or after 24 h of starvation. SuSy activity was shown to be higher in the plasmalemma than in the tonoplast of cells cultured in the presence of sucrose. In clear contrast, SuSy was shown to be more active in the tonoplast than in the plasmalemma of starved cells. Western blot analyses on both membrane types did not show noticeable differences in SuSy protein levels under the two different regimes. However, phosphorylation state at the serine moieties of the enzyme was shown to be different in the presence or in the absence of sucrose. Plasmalemma-associated SuSy is not phosphorylated in the presence of sucrose, whereas tonoplast-associated SuSy is phosphorylated under similar conditions. Starvation brought about a reverse in phosphorylation state of membrane-bound SuSy. Whereas plasmalemma-associated SuSy became phosphorylated, tonoplast-associated SuSy was completely de-phosphorylated. Together, the data demonstrate that SuSy is simultaneously present in various cell membranes and also demonstrate a lack of direct relationship between membrane type location, and degree of phosphorylation, but substantiate the relevance of phosphorylation to enzymatic activity.