Developmental Changes in the Anatomy of the Sugarcane Stem in Relation to Phloem Unloading and Sucrose Storage

Transverse sections of immature and mature sugarcane internodes were investigated anatomically with white and fluorescence light microscopy. The pattern of lignification and suberization was tested histo-chemically. Lignification began in the xylem of vascular bundles and progressed through the sclerenchymatic bundle sheath into the storage parenchyma. Suberization began in parenchyma cells adjacent to vascular bundle sheaths and spread to the storage parenchyma and outer sheath cells. In mature internodes most of the storage parenchyma was lignified and suberized to a significant degree, except in portions of walls of isolated cells. The pattern of increasing lignification and suberization in maturing internodes more or less paralleled an increase of sucrose in stem tissue. In mature internodes having a high sucrose concentration, the vascular tissue was surrounded by thick-walled, lignified and suberized sclerenchyma cells. The apoplastic tracer dyes triso-dium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) and amido black 10 B, fed into cut ends of the stalk, wereconfined to the vascular bundles in all internodes above the one that was cut — with no dye apparently in storage parenchyma tissue. Thus both structural and experimental evidence is consistent with vascular tissue being increasingly isolated from the storage parenchyma as maturation of the tissue proceeds. We conclude that in mature internodes the pathway for sugars from the phloem to the storage parenchyma is symplastic. The data suggest that an increasingly greater role for a symplastic pathway of sugar transfer occurs as the tissue undergoes lignification/suberization.     

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the pre-storage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.     

The development of phloem anastomoses between vascular bundles and their role in xylem regeneration after wounding in Cucurbita and Dahlia

The differentiation of phloem anastomoses linking the longitudinal vascular bundles has been studied in stem internodes of Cucurbita maxima Duchesne, C. pepo L. and Dahlia pinnata Cav. These anastomoses comprise naturally occurring regenerative sieve tubes which redifferentiate from interfascicular parenchyma cells in the young internodes. In all three species, severing a vascular bundle in a young internode resulted in regeneration of xylem to form a curved by-pass immediately around the wound. The numerous phloem anastomoses in these young internodes were not involved in this process, the regenerated vessels originating from interfascicular parenchyma alone. Conversely, in mature internodes of Dahlia, the regenerated vessels originated from initials of the interfascicular cambia, and their phloem anastomoses did not influence the pattern of xylogenesis. On the other hand, in old internodes of Cucurbita, in which an interfascicular cambium was not yet developed, the parenchyma cells between the bundles had lost the ability to redifferentiate into vessel elements, and instead, regenerated vessels were produced in the phloem anastomoses. Thus, the wounded region of the vascular bundle was not bypassed via the shortest, curved pathway, but by more circuitous routes further away from the wound. Some of the regenerated vessels produced in the phloem anastomoses were extremely wide, and presumably efficient conductors of water. It is proposed that the dense network of phloem anastomoses developed during evolution as a mechanism of adaptation to possible damage in mature internodes by providing flexible alternative pathways for efficient xylem regeneration in plants with limited or no interfascicular cambium.This paper is dedicated to the memory of the late Isaac Blachmann (deceased 19 November 1995), father-in-law of the senior author, for encouragement and advice throughout the yearsThis research was supported by an International Scientific Exchange Award to R.A. from the Israel Academy of Sciences and The Royal Society.     

Oligomerization,Membrane Association,and in Vivo Phosphorylation of Sugarcane UDP-glucose Pyrophosphorylase

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H₂O₂ strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.     

Expression patterns of three genes in the stem of lucerne (Medicago sativa)

We have identified three stem abundantly expressed genes in lucerne (alfalfa, Medicago sativa). A cDNA library, constructed from lucerne stem polyadenylated RNA, was screened by differential hybridization. From this screening, cDNA clones that correspond to genes which are preferentially, or specifically, expressed in the stem were isolated. MsaS1 encodes an unidentified protein, MsaS2 encodes an S-adenosyl-homocysteine hydrolase and MsaS3 encodes an extensin-like protein. Northern blot analysis of RNA isolated from individual stem internodes indicated that the three corresponding genes show differing developmental patterns of expression. The expression of MsaS1 was confined to the youngest stem tissue and may be regulated by sucrose. In stem tissue the level of RNA for the three genes decreased in response to wounding. Tissue print hybridization analysis was used to localize the expression of the genes to the xylem side of vascular bundles in lucerne stems.     

Sucrose accumulation in sweet sorghum stem internodes in relation to growth

Sweet sorghum (Sorghum bicolor L. Moench) stems of different cultivars (NK 405. Keller and Tracy) reveal a different pattern of sucrose accumulation with respect to in-ternodal sugar content and distribution. The onset of sucrose storage is not necessarily associated with the reproductive stage of the plant, as was hitherto assumed, but obviously occurs after cessation of internodai elongation as was postulated for the sugarcane stem. For at least two of the three cultivars, ripening is an internode to internode process beginning at the lowermost culm parts. Intensive growth of the internodes, combined with a high hexose content in stern parenchyma, shows a strong positive correlation (r |Mg 0.94) to the activity of sucrose synthase (SuSy; EC 2.4.13), but not to invertase (EC 3.2.1.26) which is not present as soluble (neutral and acid) or cell wall-bound, salt-extractable enzyme in the three culsivars investigated. Sucrose synthase measured in sucrose cleavage and synthesis direction reveals divergent activity rates and sensitivity towards exogenously applied Mg²⁺ ions and pH. SuSy activity is connected to the increase of internodai sucrose content in so far as (1) its decline is a prerequisite for the onset of sucrose accumulation and (2) it remains at a constant low level during sucrose storage. Sucrose phosphate synthase (SPS; EC 2.4.1.14) activity in the sorghum stem is low compared to SuSy and uniformly distributed over all inter-nodes. Only source leaves of sorghum show a considerable SPS activity, but neither stem nor leaf SPS reveal a positive correlation to the increase of internodai sucrose content. Sucrose phosphate phosphatase (SPP; EC 3.1.3.24) amounts lo only 24–30% of the respective SPS activity but follows the same distribution pattern. None of the enzymes under study proves to be responsible for the extent of sucrose storage in the stem, so other phenomena such as transport processes within the stern tissue require further investigation.     

Sucrose accumulation and enzyme activities in callus culture of sugarcane

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.The research was funded by the department of Biotechnology and Bioengineering CINVESTAV Mexico City, and F. G.-M. received grant-aided support from CONACyT, Mexico.     

Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K(0.5) = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K(i)) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.     

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form.     

Microautoradiographic studies of phloem loading and transport in the leaf of Zea mays L.

Microautoradiographs showed that [¹⁴C]sucrose taken up in the xylem of small and intermediate (longitudinal) vascular bundles of Zea mays leaf strips was quickly accumulated by vascular parenchyma cells abutting the vessels. The first sieve tubes to exhibit ¹⁴C-labeling during the [¹⁴C]sucrose experiments were thick-walled sieve tubes contiguous to the more heavily labeled vascular parenchyma cells. (These two cell types typically have numerous plasmodesmatal connections.) With increasing [¹⁴C]sucrose feeding periods, greater proportions of thick- and thin-walled sieve tubes became labeled, but few of the labeled thin-walled sieve tubes were associated with labeled companion cells. (Only the thin-walled sieve tubes are associated with companion cells.) When portions of leaf strips were exposed to ¹⁴CO₂ for 5 min, the vascular parenchyma cells-regardless of their location in relation to the vessels or sieve tubes-were the most consistently labeled cells of small and intermediate bundles, and label (¹⁴C-photosynthate) appeared in a greater proportion of thin-walled sieve tubes than thick-walled sieve tubes. After a 5-min chase with ¹²CO₂, the thin-walled sieve tubes were more heavily labeled than any other cell type of the leaf. After a 10-min chase with ¹²CO₂, the thin-walled sieve tubes were even more heavily labeled. The companion cells generally were less heavily labeled than their associated thin-walled sieve tubes. Although all of the thick-walled sieve tubes were labeled in portions of leaf strips fed ¹⁴CO₂ for 5 min and given a 10-min ¹²CO₂ chase, only five of 72 vascular bundles below the ¹⁴CO₂-exposed portions contained labeled thick-walled sieve tubes. Moreover, the few labeled thick-walledsieve tubes of the transport region always abutted ¹⁴C-labeled vascular parenchyma cells. The results of this study indicate that (1) the vascular parenchyma cells are able to retrieve at least sucrose from the vessels and transfer it to the thick-walled sieve tubes, (2) the thick-walled sieve tubes are not involved in long-distance transport, and (3) the thin-walled sieve tubes are capable themselves of accumulating sucrose and photosynthates from the apoplast, without the companion cells serving as intermediary cells.     

Uridine 5′-diphospho-D-glucose-dependent vectorial sucrose synthesis in tonoplast vesicles of the storage hypocotyl of red beet (Beta vulgaris L. ssp. conditiva)

Tonoplast vesicles were prepared from red-beet (Beta vulgaris L. ssp. conditiva) hypocotyl tubers (beetroot) known to store sucrose. Uptake experiments, employing uridine 5-diphospho-[¹⁴C]glucose (UDP-[¹⁴C]glucose) showed the operation of an UDP-glucose-dependent group translocator for vectorial synthesis and accumulation of sucrose, recently described for sugarcane and red-beet vacuoles and for tonoplast vesicles prepared from sugarcane suspension cells. Characterization of the kinetic properties yielded the following results. Uptake of UDP-glucose was linear for 15 min. The apparent K _m was 0.75 mM for UDP-glucose (at pH 7.2, 1 mM Mg²⁺), V _max was 32 nmol·(mg protein)^-1·min^-1. The incorporation of UDP-glucose exhibited a sigmoidal substrate-saturation curve in the absence of Mg²⁺, the Hill coefficient (n _H) was 1.33; Michaelis-Menten kinetics were obtained, however, in the presence of 1 mM MgCl₂. For the reaction sequence under the control of the group translocator a dual pH optimum was found at pH 7.2 and 7.9, respectively. All reaction intermediates and the end product sucrose could be identified by two-dimensional high-performance thin-layer chromatography and autoradiography. The distribution pattern of radioactivity showed almost uniformly high labeling of all intermediates and sucrose. The physiological relevance of the results is discussed in the light of the fact that the tonoplast of red-beet storage cells accommodates two mechanisms of sucrose uptake (i) vectorial sucrose synthesis and (ii) direct ATP-dependent sucrose assimilation.Abbreviations HPTLC High-performance thin-layer chromatography - UDP uridine 5-diphosphate - SDS sodium dodecyl sulfate     

Expression analysis of genes associated with sucrose accumulation in sugarcane (Saccharum spp. hybrids) varieties differing in content and time of peak sucrose storage

Sucrose synthesis/accumulation in sugarcane is a complex process involving many genes and regulatory sequences that control biochemical events in source–sink tissues. Among these, sucrose synthase (SuSy), sucrose phosphate synthase (SPS), soluble acid (SAI) and cell wall (CWI) invertases are important. Expression of these enzymes was compared in an early (CoJ64) and late (BO91) maturing sugarcane variety using end‐point and qRT‐PCR. Quantitative RT‐PCR at four crop stages revealed high CWI expression in upper internodes of CoJ64, which declined significantly in both top and bottom internodes with maturity. In BO91, CWI expression was high in top and bottom internodes and declined significantly only in top internodes as the crop matured. Overall, CWI expression was higher in CoJ64 than in BO91. During crop growth, there was no significant change in SPS expression in bottom internodes in CoJ64, whereas in BO91 it decreased significantly. Apart from a significant decrease in expression of SuSy in mature bottom internodes of BO91, there was no significant change. Similar SAI expression was observed with both end‐point and RT‐PCR, except for significantly increased expression in top internodes of CoJ64 with maturity. SAI, being a major sucrose hydrolysing enzyme, was also monitored with end‐point PCR expression in internode tissues of CoJ64 and BO91, with higher expression of SAI in BO91 at early crop stages. Enzyme inhibitors, e.g. manganese chloride (Mn⁺⁺), significantly suppressed expression of SAI in both early‐ and late‐maturing varieties. Present findings enhance understanding of critical sucrose metabolic gene expression in sugarcane varieties differing in content and time of peak sucrose storage. Thus, through employing these genes, improvement of sugarcane sucrose content is possible.     

Functional Specialization of Vacuoles in Sugarcane Leaf and Stem

Plant vacuoles are frequently targeted as a storage site for novel products. We have used environment-sensitive fluorescent dyes and the expression of vacuolar marker proteins to characterize the vacuoles in different organs and cell types of sugarcane. The results demonstrated that the lumen of the vacuole in the parenchyma cells of the stem is acidic (<pH 5) and contains active proteases, characteristic of lytic vacuoles. Western blots and tissue labelling with antibodies to vacuolar H⁺-ATPase suggest that this proton pump is involved in acidification of the vacuolar lumen. Quantitative real-time PCR was used to show that the expression of vacuolar proteases and a vacuolar sorting receptor is also coordinately regulated. In contrast to the stem parenchyma cells, the cells of sugarcane leaves contain diverse types of vacuoles. The pH of these vacuoles and their capacity to hydrolyze protease substrates varies according to cell type and developmental stage. Sugarcane suspension-cultures contain cells with vacuoles that resemble those of stem parenchyma cells and are thus a useful model system for investigating the properties of the vacuole. Understanding the growth and development of storage capacity will be useful in designing strategies to maximize the production of sucrose or alternative bioproducts.     

Rice sucrose transporter1 (OsSUT1) up‐regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles

The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3‐week‐old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1‐ to 10‐day infestation time course, using a combination of gene expression and β‐glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide‐field fluorescence microscopy was used to confirm the expression of OsSUT1‐promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up‐regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up‐regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid‐related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves.     

Tissue-specific expression of the rolA gene mediates morphological changes in transgenic tobacco

The spatial and temporal activity of the entire and individual promoter domains of the rolA gene of Agrobacterium rhizogenes was investigated and correlated with the distinctive features of the phenotypes of transgenic tobacco plants. The GUS assay was performed in the presence of an oxidative catalyst during the development of transgenic plants expressing chimeric genes containing the -glucuronidase coding sequence under the control of the different promoter domains. In situ hybridization was also used on transgenic plants harbouring rolA under the control of the entire or deleted promoter. This paper demonstrates for the first time that the entire rolA promoter, composed of domains, A, B and C, is silent in seeds, then activated at the onset of germination in the cotyledons and in the elengation zone of the radicle and is finally expressed throughout the vegetative and floral phases. Domains B+C, which were sufficient to induce wrinkled leaves and short internodes, were active in all the stem tissues, but only in the companion cells of the phloem strands of the leaves. Domain C, which specified a dwarf phenotype with normal leaves, was weakly expressed in the stem vascular bundles and in the leaf internal phloem. These results indicate that the vascular bundles are the primary targets for the generation of the short internode phenotype. Furthermore, the local expression of rolA in the stem vascular bundles induced a size reduction of the surrounding parenchyma cells, suggesting the existence of some diffusible factor(s) associated with the expression of the rolA gene.     

Immunocytochemical localisation of lectins in cells of Phaseolus vulgaris L. seeds

Antibodies against pure E₄- and L₄-lectins from the seeds of Phaseolus vulgaris L. raised in rabbits were made monospecific by immunoaffinity chromatography on E₄- or L₄-lectin Sepharose 4B columns. Localisation of lectins in bean seeds was investigated by indirect immunofluorescence and by electron microscopy on sections stained with colloidal gold particles coated with monospecific anti-E₄- and anti-L₄-IgG. In parenchyma cells from the cotyledons both E- and L-type lectins were found inside the protein bodies. Apparently the matrix of all protein bodies contained both types of lectins. On the other hand in vascular and in axis cells the two types of lectins were localised in the cytoplasm, outside the protein bodies. Thus these findings suggest different roles for the lectins: in cotyledons this may be a specific form of N storage, while in vascular and axis cells lectins may have a more direct metabolic part to play.