Professor Christopher Grof

In plant cells, the molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation and the generation of the vast amount of chemical energy required to drive these processes relies on an adequate supply of the essential macronutrient, phosphorous (P). The requirement of an appropriate level of P in plant cells is evidenced by the intricately linked molecular mechanisms of P sensing, signaling and transport. One such mechanism is the posttranscriptional regulation of the P response pathway by the highly conserved plant microRNA (miRNA), miR399. In addition to miR399, numerous other plant miRNAs are also required to respond to environmental stress, including miR396. Here, we exposed Arabidopsis thaliana (Arabidopsis) transformant lines which harbor molecular modifications to the miR396 and miR399 expression modules to phosphate (PO4) starvation. We show that molecular alteration of either miR396 or miR399 abundance afforded the Arabidopsis transformant lines different degrees of tolerance to PO4 starvation. Furthermore, RT-qPCR assessment of PO4-starved miR396 and miR399 transformants revealed that the tolerance displayed by these plant lines to this form of abiotic stress most likely stemmed from the altered expression of the target genes of these two miRNAs. Therefore, this study forms an early step towards the future development of molecularly modified plant lines which possess a degree of tolerance to growth in a PO4 deficient environment.

We previously demonstrated that microRNA396 (miR396) abundance is altered in 15-day-old Arabidopsis thaliana (Arabidopsis) whole seedlings following their exposure to a 7-day salt stress treatment regime. We, therefore, used a molecular modification approach to generate two new Arabidopsis transformant populations with reduced (MIM396 plants) and elevated (MIR396 plants) miR396 abundance. The exposure of 8-day-old wild-type Arabidopsis whole seedlings and a representative plant line of the MIM396 and MIR396 transformant populations to a 7-day salt stress treatment regime revealed unique phenotypic and physiological responses to the imposed stress by unmodified wild-type Arabidopsis plants and the MIM396 and MIR396 transformat lines. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was, therefore, applied to demonstrate that the plant line specific responses to salt stress likely stemmed from the unique molecular profile of each of the GROWTH REGULATING FACTOR (GRF) transcription factor gene family members which form posttranscriptional targets of miR396-directed expression regulation. RT-qPCR additionally revealed that, in 15-day-old Arabidopsis whole seedlings, the three previously identified putative target genes of miR396 belonging to the NEUTRAL/ALKALINE NONLYSOSOMAL CERAMIDASE-LIKE (NCER) gene family, including NCER1, NCER2, and NCER3, do not form targets of miR396-directed expression regulation at the posttranscriptional level. Taken together, the phenotypic and molecular analyses presented here demonstrate that alteration of the miR396/GRF expression module is central to the molecular response of Arabidopsis to salt stress.

Arabidopsis thaliana (Arabidopsis) has been used extensively as a heterologous system for molecular manipulation to genetically characterize both dicotyledonous and monocotyledonous plant species. Here, we report on Arabidopsis transformant lines molecularly manipulated to overaccumulate the small regulatory RNA microRNA397 (miR397) from the emerging C4 monocotyledonous grass model species Setaria viridis (S. viridis). The generated transformant lines, termed SvMIR397 plants, displayed a range of developmental phenotypes that ranged from a mild, wild-type-like phenotype, to a severe, full dwarfism phenotype. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based profiling of the SvMIR397 transformant population revealed a strong correlation between the degree of miR397 over-accumulation, repressed LACCASE (LAC) target gene expression, reduced lignin content, and the severity of the developmental phenotype displayed by SvMIR397 transformants. Further, exposure of SvMIR397 transformants to a 7-day regime of salt stress revealed the SvMIR397 transformant lines to be more sensitive to the imposed stress than were wild-type Arabidopsis plants. Taken together, the findings reported here via the use of Arabidopsis as a heterologous system show that the S. viridis miR397 small regulatory RNA is able to repress the expression of three Arabidopsis LAC genes which led to reduced lignin content and increased salt stress sensitivity.

Background: Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and, (iv) is amendable to Agrobacterium tumefaciens-mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis (S. viridis). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Results: Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE/THERONINE-PROTEIN PHOSPHATASE 2A (PP2A), 5'-ADENYLYLSULFATE REDUCTASE 6 (ASPR6) and DUAL SPECIFICITY PHOSPHATASE (DUSP) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A, ASPR6 and DUSP, were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes across the same tissues. Conclusions: This approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C4 crop species.

The role of ShSUT1 in sucrose mobilisation and storage in sugarcane was investigated by employing RNAi technology to reduce the expression of this gene. Transcript profiling in non-transformed plants showed an alignment between expression and sucrose concentration, with strongest expression in source leaves and increasing expression through the daylight period of a diurnal cycle. Five transgenic plant lines were produced with reduced ShSUT1 expression ranging from 52 to 92% lower than control plants. Differential suppression of ShSUT1 sequence variants in the highly polyploid sugarcane genome were also investigated. Amplicon sequencing of the ShSUT1 variants within the transgenic lines and controls showed no preferential suppression with only minor differences in the proportional expression of the variants. A range of altered sugar, fibre and moisture contents were measured in mature leaf and internode samples, but no phenotype was consistently exhibited by all five transgenic lines. Phenotypes observed indicate that ShSUT1does not play a direct role in phloem loading. ShSUT1 is likely involved with retrieving sucrose from intercellular spaces for transport and storage.

Setaria viridis is a C4 grass used as a model for bioenergy feedstocks. The elongating internodes in developing S. viridis stems grow from an intercalary meristem at the base, and progress acropetally toward fully expanded cells that store sugar. During stem development and maturation, water flow is a driver of cell expansion and sugar delivery. As aquaporin proteins are implicated in regulating water flow, we analyzed elongating and mature internode transcriptomes to identify putative aquaporin encoding genes that had particularly high transcript levels during the distinct stages of internode cell expansion and maturation. We observed that SvPIP2;1 was highly expressed in internode regions undergoing cell expansion, and SvNIP2;2 was highly expressed in mature sugar accumulating regions. Gene co-expression analysis revealed SvNIP2;2 expression was highly correlated with the expression of five putative sugar transporters expressed in the S. viridis internode. To explore the function of the proteins encoded by SvPIP2;1 and SvNIP2;2, we expressed them in Xenopus laevis oocytes and tested their permeability to water. SvPIP2;1 and SvNIP2;2 functioned as water channels in X. laevis oocytes and their permeability was gated by pH. Our results indicate that SvPIP2;1 may function as a water channel in developing stems undergoing cell expansion and SvNIP2;2 is a candidate for retrieving water and possibly a yet to be determined solute from mature internodes. Future research will investigate whether changing the function of these proteins influences stem growth and sugar yield in S. viridis.

Cellular pathways of phloem loading in source leaves and phloem unloading in stems of sweet Sorghum bicolor (L.) Moench were deduced from histochemical determinations of cell wall composition and from the relative radial mobilities of fluorescent tracer dyes exiting vascular pipelines. The cell walls of small vascular bundles in source leaves, the predicted site of phloem loading, contained minimal quantities of lignin and suberin. A phloem-loaded symplasmic tracer, carboxyfluorescein, was retained within the collection phloem, indicating symplasmic isolation. Together, these findings suggested that phloem loading in source leaves occurs apoplasmically. Lignin was restricted to the walls of protoxylem elements located in meristematic, elongating and recently elongated regions of the stem. The apoplasmic tracer, 8-hydroxypyrene-1,3,6-trisulfonic acid, moved radially from the transpiration stream, consistent with phloem and storage parenchyma cells being interconnected by an apoplasmic pathway. The major phase of sucrose accumulation in mature stems coincided with heavy lignification and suberisation of sclerenchyma sheath cell walls restricting apoplasmic tracer movement from the phloem to storage parenchyma apoplasms. Phloem unloading at this stage of stem development followed a symplasmic route linking sieve elements and storage parenchyma cells, as confirmed by the phloem-delivered symplasmic tracer, 8-hydroxypyrene-1,3,6-trisulfonic acid, moving radially from the stem phloem.

Sugarcane is an ideal candidate as a biofactory for the production of alternate higher value products. One way of achieving this is to direct useful proteins into the vacuoles within the sugarcane storage parenchyma tissue. By bioinformatic analysis of gene sequences from putative sugarcane vacuolar proteins a motif has been identified that displays high conservation across plant legumain homologues that are known to function within vacuolar compartments. This five amino acid motif, represented by the sequence IRLPS in sugarcane is shown to direct an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP appeared to be dependent on cell type, suggesting that the vacuolar environment can be hostile to introduced proteins. This targeting motif will be a valuable tool for engineering plants such as sugarcane for production of novel products. © CSIRO 2007.

A novel method using an HPAE-PAD system, which is routinely applied to detect carbohydrates at low levels (ng per sample injection), has been applied to the measurement of key sucrose metabolising enzyme activities in partially purified extracts of sugarcane tissues. Extraction and assay procedures tailored for the HPAE-PAD system enabled the accurate measurement of enzyme activities in more mature internodes than had previously been possible using enzyme coupled assay methodology. A major advantage of the HPAE-PAD method is the capability to monitor a broad range of sugars in each assay and provides an overarching perspective of the mix of competing enzymes that may be operating simultaneously in crude extracts. The technique has been successfully applied to measuring the activity of key sucrose metabolising enzymes in sugarcane stem tissue that is generally low in protein and high in endogenous sugars, primarily sucrose. © 2006 Elsevier B.V. All rights reserved.

Increasing sucrose content is a major objective of sugarcane breeding programs. One approach employed by breeders is to introgress new genes from genotypes of Saccharum officinarum L. not previously used in breeding. The activity of a suite of key sucrose metabolizing enzymes was measured in progeny of an introgression program to find biochemical markers associated with high sucrose content, measured as commercial cane sugar (CCS). The enzymes sucrose-phosphate synthase (SPS), three isoforms of invertase, and sucrose synthase were measured in four high and four low CCS clones from an initial cross between a S. officinarum and the commercial cultivar Q165. Subsequently, SPS and the two soluble isoforms of invertase were measured in clones derived from a backcross of one of the progeny to another commercial cultivar Mida. Enzyme activities were measured in tissue from internodes taken from four different positions down the stem profile. Of particular significance was the finding that the activity of a key enzyme involved in sucrose synthesis, SPS, was significantly higher in the upper internodes (one to three) of high CCS clones as compared with low CCS clones in both populations, suggesting that this enzyme may have a key role in establishing metabolic and developmental processes necessary for high sugar accumulation during stem growth and maturation. © Crop Science Society of America.

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 a-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 ß-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 Sue 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 Sue at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition. © 2006 American Society of Plant Biologists.

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 (Ki) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular. © 2006 The Authors.

Rapid and efficient in vitro regeneration methods that minimise somaclonal variation are critical for the genetic transformation and mass propagation of commercial varieties. Using a transverse thin cell layer culture system, we have identified some of the developmental and physiological constraints that limit high-frequency regeneration in sugarcane leaf tissue. Tissue polarity and consequently the orientation of the explant in culture, size and developmental phase of explant, and auxin concentration play a significant role in determining the organogenic potential of leaf tissue in culture. Both adventitious shoot production and somatic embryogenesis occurred on the proximal cut surface of the explant, and a regeneration gradient, decreasing gradually from the basal to the distal end, exists in the leaf roll. Importantly, auxin, when added to the culture medium, reduced this spatial developmental constraint, as well as the effect of genotype on plant regeneration. Transverse sections (1-2 mm thick) obtained from young leaf spindle rolls and orienting explants with its distal end facing the medium (directly in contact with medium) are critical for maximum regeneration. Shoot regeneration was observed as early as 3 weeks on MS medium supplemented with a-naphthalenencetic acid (NAA) and 6-benzyladenine, while somatic embryogenesis or both adventitious shoot organogenesis and somatic embryogenesis occurred on medium with NAA and chlorophenoxyacetic acid. Twenty shoots or more could be generated from a single transverse section explant. These shoots regenerated roots and successfully established after transplanted to pots. Large numbers of plantlets can be regenerated directly and rapidly using this system. SmartSett®, the registered name for this process and the plants produced, will have significant practical applications for the mass propagation of new cultivars and in genetic modification programs. The SmartSett® system has already been used commercially to produce substantial numbers of plants of orange rust-resistant and new cultivars in Australia. © 2006 Springer-Verlag.

Sucrose-phosphate synthase (SPS) is a key enzyme in the pathway of sucrose synthesis. Five different gene families encoding SPS have been reported in the Poaceae [Castleden CK, Aoki N, Gillespie VJ, MacRae EA, Quick WP, Buchner P, Foyer CH, Furbank RT, Lunn JE (2004) Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses. Plant Physiology 135, 1753?1764]. Expression of the five families in leaf and stem tissues of Saccharum spp. at different stages of development was determined by quantitative real-time PCR. The type B and C families of SPS genes were predominantly expressed in both immature and mature leaves, whereas the two subfamilies making up the type D family were expressed at similar levels in all tissues examined. In the type A family, expression was lowest in leaves and increased from the meristem region down to internode 7 of the stem. © CSIRO 2006.

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. © CSIRO 2005.

Color intensity of raw sugar is, in part, a result of the activity of the enzyme polyphenol oxidase (PPO) acting on phenolic compounds to produce dark colored polymers when sugarcane (Saccharum spp.) is crushed to release the juice. Paler colored sugar has a potential market premium over darker sugar. In an attempt to alter the level of PPO activity in transgenic plants, sense and antisense constructs containing the native sugarcane PPO gene were introduced into sugarcane by biolistics. In a series of field experiments, it was demonstrated that PPO activity among clones correlated significantly with juice color. In laboratory crystallizations of raw sugar using juice derived from clones with high and low PPO activity, the juice with the higher PPO activity produced darker colored crystals. PPO activity was elevated and juice color was darker in all types of transgenic plants. However, clones derived from a sense construct had higher PPO activity than the other transgenic clones, tissue culture control clones, or cultivars. Furthermore, northern blot analysis demonstrated that PPO sense transgenics had much higher levels of PPO transcripts in the stem than other clones. This is the first targeted manipulation of an endogenous metabolic enzyme-encoding gene in sugarcane that leads to altered enzyme activity. Although low PPO lines with good agronomic performance were not generated, this research demonstrates the principle that juice and sugar color are correlated with PPO activity, consistent with the hypothesis that lowering PPO activity in sugarcane could reduce the color intensity of juice and raw sugar.

The promoter regions of plant pararetroviruses direct transcription of the full-length viral genome into a pregenomic RNA that is an intermediate in the replication of the virus. It serves as template for reverse transcription and as polycistronic mRNA for translation to viral proteins. We have identified functional promoter elements in the intergenic region of the Cavendish isolate of Banana streak virus (BSV-Cav), a member of the genus Badnavirus. Potential binding sites for plant transcription factors were found both upstream and downstream of the transcription start site by homology search in the PLACE database of plant cis-acting elements. The functionality of these putative cis-acting elements was tested by constructing loss-of-function and "regain"-of-function mutant promoters whose activity was quantified in embryogenic sugarcane suspension cells. Four regions that are important for activity of the BSV-Cav promoter were identified: the region containing an as-1-like element, the region around -141 and down to -77, containing several putative transcription factor binding sites, the region including the CAAT-box, and the leader region. The results could help explain the high BSV-Cav promoter activity that was observed previously in transgenic sugarcane plants and give more insight into the plant cell-mediated replication of the viral genome in banana streak disease. © 2004 Elsevier B.V. All rights reserved.

The accumulation of high concentrations of sucrose in the stem of sugarcane has been the subject of many studies. Although models have been constructed from the available information, many steps in the transport and accumulation pathway remain unknown. Recent advances in molecular approaches may elucidate some of these processes. Genes encoding proteins associated with sugar synthesis and storage will provide valuable tools. In particular, the use of techniques to localize the sites of expression of sugar transporters and metabolic enzymes will assist in defining possible routes of sugar movement. When combined with an analysis of metabolite concentrations and enzyme activities in cellular and subcellular compartments, these novel approaches will contribute to an integrated picture of stem function. Control points identified will provide useful tools for selection of efficient genotypes and targets for molecular manipulations. © 2005 Elsevier B.V. All rights reserved.

Sugarcane is a genetically complex polyploid grass, which makes the identification of associations between genes and traits difficult. Genomics science facilitates characterization of entire eukaryote genomes at the DNA sequence level, but for crop plants with complex genomes such as sugarcane, gene characterization is currently best achieved via expressed sequence tag (EST) analysis where sequence information is restricted to genes that are actually functioning in a particular tissue or situation. DNA microarrays allow simultaneous expression analysis of thousands of genes. Current work on EST and array analysis of gene expression in sugarcane is reviewed and insights on stem functions associated with maturation and sucrose accumulation are discussed. A strategy for associating gene expression with a trait is described in which individuals exhibiting particular traits are selected from segregating populations of sugarcane and their gene expression profiles compared. A preliminary experiment to test the feasibility and experimental design for this 'genetical genomics' strategy on a population segregating for sugar content is described. Given the complex genetics of sugarcane, this strategy and refinements of it, represent an attractive pathway to the identification of candidate genes that may control sugar accumulation and other traits in sugarcane. © 2005 Elsevier B.V. All rights reserved.

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the "Goldfinger" isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter function. The "selective-shotgun" approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional "linker-scanning" deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the prospect of identifying interacting promoter regions.

Commercial sugarcane, belonging to the genus Saccharum (Poaceae), is an important industrial crop accounting for nearly 70% of sugar produced worldwide. Compared to other major crops, efforts to improve sugarcane are limited and relatively recent, with the first introduction of interspecific hybrids about 80 yr ago. Progress in traditional breeding of sugarcane, a highly polyploid and frequently aneuploid plant, is impeded by its narrow gene pool, complex genome, poor fertility, and the long breeding/selection cycle. These constraints, however, make sugarcane a good candidate for molecular breeding. In the past decade considerable progress has been made in understanding and manipulating the sugarcane genome using various biotechnological and cell biological approaches. Notable among them are the creation of transgenic plants with improved agronomic or other important traits, advances in genomics and molecular markers, and progress in understanding the molecular aspects of sucrose transport and accumulation. More recently, substantial effort has been directed towards developing sugarcane as a biofactory for high-value products. While these achievements are commendable, a greater understanding of the sugarcane genome, and cell and whole plant physiology, will accelerate the implementation of commercially significant biotechnology outcomes. We anticipate that the rapid advancements in molecular biology and emerging biotechnology innovations would play a significant role in the future sugarcane crop improvement programs and offer many new opportunities to develop it as a new-generation industrial crop. © 2005 Society for In Vitro Biology.

A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K m for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes. © Springer-Verlag 2004.

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.

Conflicting reports of the distribution of sugarcane neutral invertase (SNI) activity in sugarcane, and the complete lack of genetic information have been the impetus for this investigation. Using a hetrologous sequence from Lolium temulentum a clone, which contained a 1716bp open reading frame, was isolated from a mature culm sugarcane cDNA library. The SNI amino acid sequence had at least 53% homologous with sequences from Daucus carota and L. temulentum neutral invertases. The predicted molecular weight of the polypeptide is 64300Da. Southern blot analysis showed that SNI represents a low copy number gene in sugarcane. Both the SNI transcript and protein were present in all analysed tissues of the sugarcane plants and the data are consistent with transcriptional control of SNI activity. In culm tissues where sucrose content was low and hexose contents were high, SNI transcript and protein levels were higher than in tissues dedicated to sucrose storage. © 2003 Published by Elsevier Ireland Ltd.

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.

Extraction and assay methods were developed for the determination of both soluble and cell wall invertase activity in sugarcane (Saccharum sp.) from minimal (0.5 g) tissue. Cell wall invertase (CWI) was measured using a pellet mix procedure and the pH optima ranged between pH 3.2 and 3.6. The pH optima for the soluble invertases were 4.5 and 7.3 for soluble acid (SAI) and neutral (NI) invertase, respectively. At low pH, acid hydrolysis of sucrose was observed and its spurious effect on measured enzyme activity was removed by the inclusion of additional controls run in parallel, which lacked crude plant extract. Invertase activity was examined in sugarcane tissues of varying ages. In leaves and stem, the SAI activity was greatly reduced in mature tissue extracts. Similarly, the CWI activity was reduced in older leaves. In contrast, a less marked drop in NI activity was observed in extracts from old leaves and activity from stem extracts remained constant irrespective of tissue age. The role of SAI has been linked to growth and differentiation and these observations suggest that CWI may also be intrinsically involved in these processes. NI appears to have a housekeeping role in maintaining hexose concentrations within the cytosol.

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.

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.

The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor¿ Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 ± 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 µg/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 µg and 2.11 µg sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 µg mGFP5-ER per mg extractable protein.

The use of the green fluorescent protein (GFP) and in vivo screening for transgenic cells has enabled the first visual selection of transgenic sugarcane (Saccharum L. hybrid) transformed by Agrobacterium-mediated techniques. Selection of GFP-positive embryogenic callus transformed using Agrobacterium tumefaciens strain AGL0 was performed by fluorescence microscopy for 6 weeks after transformation. The use of GFP as a screenable marker aided the rapid segregation of individual transformation events and facilitated the initial visual selection of transgenic sugarcane callus without antibiotics, herbicides or an assay. The binary vector used in transformation was pTO134, which contains a synthetic g/p gene (sgfpS65T) and the bialaphos resistance gene (bar) both controlled by CaMV 35S promoters. The appearance of GFP-expressing cells observed on embryogenic callus was 5.3%. Subsequently, GFP-positive calli grew in the presence of a level of the herbicide bialaphos, that was toxic to untransformed calli. GFP-positive shoots were regenerated and one sugarcane plant expressed GFP in the initial stages. Molecular analysis confirmed the presence of the sgfpS65T coding region in the genome of regenerated plants. Visual screening for Agrobacterium-mediated transformation events using GFP was an efficient technique, which may be applied for the transformation of other plants.

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 µg protein-1 min-1. The cross-varietal average for leaf SPS activity was 0.10 nmol µm 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.

Plants of the Australian commercial sugarcane varieties Q117 and Q138 were grown to 6 months age in a controlled environment at temperatures of 14, 18, 22 and 26°C. The rate of node appearance, which equates to the rate of leaf appearance, was significantly correlated with temperature across the temperature range examined. Analysis of the varietal rates of node deposition as a function of time allowed determination of both base temperature for node (hence leaf) appearance and phyllochron. The base temperatures for node appearance were 7.8°C for Q 117 and 7.6°C for Q138, significantly lower than previously published base temperatures for leaf appearance in sugarcane. During the developmental stages covered by this study, phyllochron differed between the two varieties with Q117 requiring 108.7 °Cd per node, whilst Q138 required 126.6°Cd per node. This work reinforces the value of controlled environment research as a way of elucidating basic functions of plant growth and development.