Christopher P. L. Grof

Sucrose transporters of higher plants.

Recent advances have provided new insights into how sucrose is moved from sites of synthesis to sites of utilisation or storage in sink organs. Sucrose transporters play a central role, as they orchestrate sucrose allocation both intracellularly and at the whole plant level. Sucrose produced in mesophyll cells of leaves may be effluxed into the apoplasm of mesophyll or phloem parenchyma cells by a mechanism that remains elusive, but experimentally consistent with facilitated transport or energy-dependent sucrose/H(+) antiport. From the apoplasm, sucrose/H(+) symporters transport sucrose across the plasma membrane of cells making up the sieve element/companion cell (SE/CC) complex, the long distance conduits of the phloem. Phloem unloading of sucrose in key sinks such as developing seeds involves two sequential transport steps, sucrose efflux followed by sucrose influx. Besides plasma membrane specific sucrose transporters, sucrose transporters on the tonoplast contribute to the capacity for elevated sucrose accumulation in storage organs such as sugar beet roots or sugarcane culms. Except for several sucrose facilitators from seed coats of some leguminous plants all sucrose transporters cloned to date, including recently identified vacuolar sucrose transporters, have been characterised as sucrose/H(+) symporters. Transporters functioning to efflux sucrose into source or sink apoplasms as well as those supporting sucrose/H(+) antiport on tonoplasts, remain to be identified. Sucrose transporter expression and activity is tightly regulated at the transcriptional, post-transcriptional as well as post-translational levels. Light quality and phytohormones play essential regulatory roles and the sucrose molecule itself functions as a signal.

Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis

The ability of sugarcane to accumulate sucrose provides an experimental system for the study of gene expression determining carbohydrate partitioning and metabolism. A sequence survey of 7242 ESTs derived from the sucrose-accumulating, maturing stem revealed that transcripts for carbohydrate metabolism gene sequences (CMGs) are relatively rare in this tissue. However, within the CMG group, putative sugar transporter ESTs form one of the most abundant classes observed. A combination of EST analysis and microarray and northern hybridization revealed that one of the putative sugar transporter types, designated PST type 2a, was the most abundant and most strongly differentially expressed CMG in maturing stem tissue. PST type 2a is homologous to members of the major facilitator super-family of transporters, possessing 12 predicted transmembrane domains and a sugar transport conserved domain, interrupted by a large cytoplasmic loop. Its transcript was localized to phloem companion cells and associated parenchyma in maturing stem, suggesting a role in sugar translocation rather than storage. In addition, other categories of CMGs show evidence of coordinated expression, such as enzymes involved in sucrose synthesis and cleavage, and a majority of enzymes involved in glycolysis and the pentose phosphate pathway. This study demonstrates the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques for studies of the developmental regulation of metabolic enzymes and potential transporters in sugarcane.

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 (K0.5 = 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 α-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 β-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.

Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells

Abstract Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro.

Identification of differentially expressed transcripts from maturing stem of sugarcane by in silico analysis of stem expressed sequence tags and gene expression profiling

Sugarcane accumulates high concentrations of sucrose in the mature stem and a number of physiological processes on-going in maturing stem tissue both directly and indirectly allow this process. To identify transcripts that are associated with stem maturation, we compared patterns of gene expression in maturing and immature stem tissue by expression profiling and bioinformatic analysis of sets of stem ESTs. This study complements a previous study of gene expression associated directly with sugar metabolism in sugarcane. A survey of sequences derived from stem tissue identified an abundance of several classes of sequence that are associated with fibre biosynthesis in the maturing stem. A combination of EST analyses and microarray hybridization revealed that genes encoding homologues of the dirigent protein, a protein that assists in the stereospecificity of lignin assembly, were the most abundant and most strongly differentially expressed transcripts in maturing stem tissue. There was also evidence of coordinated expression of other categories of fibre biosynthesis and putative defence- and stress-related transcripts in the maturing stem. This study has demonstrated the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques to highlight the very significant transcriptional investment the maturing stem of sugarcane has placed in fibre biosynthesis and stress tolerance, in addition to its already well-documented role in sugar accumulation.

Sugarcane sucrose metabolism: scope for molecular manipulation

Improvement of the sucrose content of commercial sugarcane by conventional breeding has reached a plateau, primarily due to the narrow gene pool, and the potential to introduce novel genes or manipulate native genes to influence metabolism may have significant application. This review reports on progress in developing new, and optimising existing, transformation processes for sugarcane, and confirms that the requisite molecular tools for modifying sugarcane metabolism are as yet poorly developed when compared with those currently being applied to dicotyledonous model and crop species. Drawing from the considerable base of biochemical research into sucrose metabolism in sugarcane, a number of target steps for metabolic manipulation are reviewed. Specifically, we review current research into the physiological and biochemical elucidation of the key processes of sucrose synthesis, transport and cleavage. Given the focus of this review on molecular manipulation, particular emphasis is placed on the status of research into the isolation of genes encoding the key enzymes and transporters in the sucrose accumulation process.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the β-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

Effects of tissue culture, biolistic transformation, and introduction of PPO and SPS gene constructs on performance of sugarcane clones in the field

Stably transformed sugarcane plants were produced by the biolistic introduction of DNA into tissue-cultured cells. Constructs containing genes in sense and antisense orientation of polyphenol oxidase and sense orientation of sucrose phosphate synthase were used in the transformations. Regenerated plants were grown in a series of field experiments that incorporated commercial varieties, including Q117, from which the transgenic clones were derived and plants regenerated from tissue culture but not subjected to biolistic bombardment. In all experiments, the mean yield of transgenic sugarcane was lower than commercial varieties and the transgenic clones often exhibited lower sugar content, although individual transgenic clones in some experiments were not significantly different from Q117. Those plants regenerated from tissue culture but not bombarded were intermediate in their yield, and more clones were equivalent to Q117 in agronomic performance. Transformed plants produced by the bombardment of callus performed poorly but the results from the tissue-cultured controls indicated that not all of this could be due to somaclonal variation. Some aspect(s) of the process of transformation itself was deleterious and in most cases more significant than the effects due to tissue culture. Of the transgenic clones grown at Ayr, Queensland, 1.6% were equivalent to Q117 in sugar content and yield, suggesting that large numbers of transgenic clones would have to be generated using the current method in order to allow for selection of clones with acceptable agronomic performance.

A modified assay method shows leaf sucrose-phosphate synthase activity is correlated with leaf sucrose content across a range of sugarcane varieties

Eight different commercial and breeding varieties of sugarcane (Saccharum spp.) grown in controlled conditions were assayed for leaf sucrose-phosphate synthase (SPS) (EC 2.1.4.14) activity and leaf sucrose content. Leaf SPS activity measured at 25˚C ranged between 0.06 and 0.14 nmol sucrose formed mg protein -1 min-1. The cross-varietal average for leaf SPS activity was 0.10 nmol µg protein-1 min-1 (equivalent to 63.4 µmol h-1 g FW-1 or 17.6 nkat g FW-1) which is consistent with previously published leaf SPS activities for sugarcane; however, previous studies have assayed leaf SPS activity at either 30 or 37˚C. The range of leaf sucrose content across varieties (5.5–18.0 mg sucrose g FW-1, average 11.3 mg g FW-1) was consistent with all but one of four previously published reports. Leaf SPS activity and leaf sucrose content were significantly correlated across the eight varieties examined (r2 =0.877, d.f. =7,P<0.001). Whilst previous reports have indicated a co-relationship between leaf SPS activity and leaf sucrose content in single sugarcane varieties both diurnally and with different nutrient regimes, this study shows, for the first time, that this co-relationship also holds true across a range of sugarcane varieties.

PEA-CLARITY: 3D molecular imaging of whole plant organs.

Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.