Frederik C. Botha

Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data

Sucrose accumulation in developing sugar cane (Saccharum officinarum) is accompanied by a continuous synthesis and cleavage of sucrose in the storage tissues. Despite numerous studies, the factors affecting sucrose accumulation are still poorly understood, and no consistent pattern has emerged which pinpoints certain enzyme activities as important controlling steps. Here, we develop an approach based on pathway analysis and kinetic modelling to assess the biochemical control of sucrose accumulation and futile cycling in sugar cane. By using the concept of elementary flux modes, all possible routes of futile cycling of sucrose were enumerated in the metabolic system. The available kinetic data for the pathway enzymes were then collected and assembled in a kinetic model of sucrose accumulation in sugar cane culm tissue. Although no data were fitted, the model agreed well with independent experimental results: in no case was the difference between calculated and measured fluxes and concentrations greater than 2-fold. The model thus validated was then used to assess different enhancement strategies for increasing sucrose accumulation. First, the control coefficient of each enzyme in the system on futile cycling of sucrose was calculated. Secondly, the activities of those enzymes with the numerically largest control coefficients were varied over a 5-fold range to determine the effect on the degree of futile cycling, the conversion efficiency from hexoses into sucrose, and the net sucrose accumulation rate. In view of the modelling results, overexpression of the fructose or glucose transporter or the vacuolar sucrose import protein, as well as reduction of cytosolic neutral invertase levels, appear to be the most promising targets for genetic manipulation. This offers a more directed improvement strategy than cumbersome gene-by-gene manipulation. The kinetic model can be viewed and interrogated on the World Wide Web at http://jjj.biochem.sun.ac.za.

Sucrose phosphate synthase and sucrose synthase activity during maturation of internodal tissue in sugarcane

Sucrose accumulation rates, sucrose-phosphate synthase (SPS, EC 2.4.1.14) and soluble sucrose synthase (SuSy, EC 2.4.1.13) activities were measured in internodal tissue from a sugarcane (Saccharum species hybrids) variety N19. The sucrose accumulation rate sharply increases between internodes 3 to 11. In the older internodes SPS activity was at least three times higher than the SuSy activity. A highly significant positive correlation was found between SPS activity and sucrose content. In contrast, no significant correlation was observed between SuSy and sucrose content. In agreement, when radiolabelled glucose was fed to internodes with a high sucrose accumulation rate, label was equally distributed in the hexose moieties of sucrose. This clearly indicates that SPS is the major sucrose synthesis activity in the culm of sugarcane. Different kinetic forms of SPS apparently exist in the internodal tissue at different stages of development.

Respiratory metabolism and gene expression during seed germination

Oxygen uptake and carbon dioxide release rapidly increase in seeds during imbibition. The oxygen uptake is associated with oxidative phosphorylation through cytochrome oxidase. During the early stage of germination substrate level phosphorylation may also contribute to ATP production. All indications suggest that this route of ATP production is insignificant during aerobic germination. However, during oxygen stress, substrate level phosphorylation does significantly contribute to ATP production in some species. Carbohydrate oxidation plays a significant role in the germination process. Up to two thirds of the carbon from carbohydrate breakdown enters the tricarboxylic acid cycle through the phosphoenolpyruvate carboxylase reaction. This anapleurotic input into the Krebs cycle most probably reflects the high demand on intermediates from the cycle for biosynthesis. The extent to which other substrates are utilized for respiration is uncertain. Information regarding the levels of key metabolites and enzymes, as well as their cellular distribution is limited. The involvement of gene expression in the regulation of respiratory metabolism is poorly characterised. Several genes which have been cloned are only expressed during germination. With the exception of the early methionine labeled polypeptide, little is known about the function of these genes.

Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase

Fertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T1 and T2 progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.

Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was successfully down-regulated in sugarcane using constitutively expressed antisense and untranslatable forms of the sugarcane PFP-β gene. In young internodal tissue activity was reduced by up to 70% while no residual activity could be detected in mature tissues. The transgenic plants showed no visible phenotype or significant differences in growth and development under greenhouse and field conditions. Sucrose concentrations were significantly increased in the immature internodes of the transgenic plants but not in the mature internodes. This contributed to an increase in the purity of the immature tissues, resembling an early ripening phenotype. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. These findings suggest that PFP influences the ability of young, biosynthetically active sugarcane culm tissue to accumulate sucrose but that the equilibrium of the glycolytic intermediates, including the stored sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP activity is sufficient to sustain the required glycolytic flux as the tissue matures. Moreover, it suggests a role for PFP in glycolytic carbon flow, which could be rate limiting under conditions of high metabolic activity.

Genes expressed in sugarcane maturing internodal tissue

Abstract. To explore gene expression during sugarcane culm maturation, we performed a partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases, while 111 matched uncharacterised plant expressed sequence tags (ESTs) only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. This research has provided a framework for functional gene analysis in sugarcane sucrose-accumulating tissues.

Distribution patterns of neutral invertase and sugar contentin sugarcane internodal tissues

Neutral invertase (NI, EC 3.2.1.26) and sugars were extracted from the developing culm tissues of sugarcane. Tissues were divided according to developmental stage (internodes 3, 6 and 9) and anatomical differentiation (enriching for elongating, vascular or storage tissues). The lowest sucrose content was found in the core of the bottom of each of the internodes. The ratio between the two hexoses, glucose and fructose, and sucrose was highest in the young internodes and was also significantly different between the top and bottom parts of the young internodes. There was a significant correlation between sucrose content and NI and this was largely due to a tighter association between the two components in the bottom of the internodes.

Sugarcane ESTs differentially expressed in immature and maturing internodal tissue

Abstract Two subtracted cDNA libraries were constructed by reciprocal subtractive hybridisation between sugarcane immature (low sucrose-accumulating) and maturing (high sucrose-accumulating) internodal tissue. The subtracted libraries contained high, moderate and low abundance transcripts. To isolate cDNAs differentially expressed during culm maturation, 400 random clones (200 from each library) were systematically arrayed onto nylon filters and screened with total cDNA probes prepared from immature and maturing culm poly (A) + RNA. Results indicated that 36 and 30% of the total number of cDNAs analysed were preferentially expressed in the immature and maturing culm, respectively. Northern analysis of selected clones confirmed culm developmental stage-specific and -preferential expression for most of the clones tested. Expressed Sequence Tags (ESTs) generated by partial sequence analysis for all 132 differentially expressed clones indicated 95 unique transcripts. Partial sequence information could assign putative identities to 66% of the differentially expressed ESTs. The majority of ESTs with a putative identity were homologous to genes associated with cell wall metabolism, carbohydrate metabolism, stress responses and regulation, where the specific ESTs identified in the immature and maturing culm were distinct from each other. No developmentally regulated ESTs directly associated with sucrose metabolism were detected. This suggests that growth and maturation of the sugarcane culm is associated with the expression of genes for a variety of processes. This study demonstrates that a combination of cDNA subtraction with macroarray screening is an effective strategy to identify and analyse candidate developmentally regulated genes in sugarcane.

Sugarcane Internodal Invertases and Tissue Maturity

Summary The relationship between extractable invertase activities and sucrose accumulation in the sugarcane ( Saccharum officinarum ) stalk, and in vivo invertase mediated sucrose hydrolysis was investigated to determine the significance of invertases in sucrose utilisation and turnover. In vitro activities were determined by assaying the soluble acid, cell wall bound acid and neutral invertases from internodes three to ten in mature sugarcane plants of cultivar NCo376. Extractable activities were verified by immunoblotting. In vivo invertase mediated sucrose hydrolysis was investigated in tissue discs prepared from mature stalk tissue of the same cultivar. Sugarcane neutral invertase had a higher specific activity than soluble acid invertase (apoplastic and vacuolar) in the sucrose accumulating region of the sugarcane stem. Cell wall bound acid invertase was also present in significant quantities in both immature and mature tissue. Recombination experiments indicated the absence of activators and inhibitors, and immunodetection of neutral and soluble acid invertase followed comparable trends to that of activity expressed on a fresh mass or protein basis. When radiolabelled fructose was fed to both young and mature internodal tissue, label appeared in glucose which demonstrates that invertase mediated hydrolysis of sucrose occurs. In vitro invertase activities were used to calculate the theoretical turnover-times for the endogenous sucrose pool.

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.