Masters Degrees (Botany and Zoology)

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Carbon partitioning in sugarcane internodal tissue with special reference to the insoluble fraction
(Stellenbosch : Stellenbosch University, 2000-12) Bindon, Keren (Keren Ann); Botha, F. C.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
ENGLISH ABSTRACT: The changes in carbon allocation to sucrose, hexoses, fibre, starch and respiration were investigated in developing internodes of sugarcane. Radiolabelling studies were conducted on internode 3, 6 and 9 tissue, representing three stages of increasing maturity. It was apparent that a high rate of cycling between triose-phosphate and hexose-phosphate occurred. A maximum of 50% of carbon entering triose-phosphates was returned to hexose-phosphate in internode 3 tissue, and this flux decreased with tissue maturity to 30%. Carbon partitioning into sucrose increased from 34% of total 14C uptake in internode 3, to 61% in internodes 6 and 9. In immature tissue, the protein and fibre components were the dominant competing sinks with sucrose for incoming carbon, to which 14 and 16% of carbon were allocated respectively. Increased carbon allocation to sucrose with tissue maturity, coincided with a decrease in partitioning to fibre. This indicated that previous studies had underestimated total carbon allocation to respiration, since the protein component was not considered. In contrast with earlier work, the respiratory pathway was the strongest competitor with sucrose for incoming carbon, even in mature tissue. Between internodes 3 and 6, carbon allocation to total respiration did not change significantly, but decreased 50% in mature tissue. Starch was a weak competitor with sucrose, for incoming carbon, to which a maximum of 2% of 14Cwas allocated in immature tissue. In cane harvested in early spring, radiolabelled maltose was recovered in internode 3 tissue of ripening cane, indicating that concomitant starch synthesis and degradation occurred. The. redistribution of C-1 and C-6 in starch glucose was analysed following feeding of tissue with [1_14C]_and [6_14C]_glucose. Randomization of label in starch indicated that the pathway for carbon movement into sugarcane plastids for starch synthesis is primarily through the triose-phosphate translocator. Finally, this study indicated that radiolabelling of tissue discs is a suitable experimental system to determine carbon flux in sugarcane. During the 3 h labelling period the rate of 14C02 release became linear, indicating that the system approached isotopic steady state between the external and internal glucose pool; and the respiratory processes involved in CO2 release.
The development of an in situ hybridisation technique to determine the gene expression patterns of UDP-Glucose dehydrogenase, pyrophosphate-dependent phosphofructokinase and UDP-Glucose pyrophosphorylase in sugarcane internodal tissues
(Stellenbosch : Stellenbosch University, 2003-12) Ramoutar, Rakeshnie; Cramer, M. D.; Botha, F. C.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and accumulation in sugarcane is poorly understood. The present study was therefore aimed at the development of an in situ hybridisation (ISH) technique to study differential gene expression among the various cell types of the sugarcane culm. This technique in conjunction with northern and western blotting was then used to determine the sites of cellular and tissue specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose metabolism. This study revealed that the determination of the influencing parameters associated with the development of an ISH protocol was essential for the successful detection of the endogenous RNA sequences in sugarcane internodal tissues. The parameters that were investigated included the type of embedding medium, duration of fixation period, pre-treatment procedures and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the analysis of cytological gene expression at all stages of sugarcane development. The second part of this study revealed very localised transcript expression for UDP-Glc DH, PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all expressed in the parenchyma cells that were associated with the vascular bundles and the stem storage compartment, suggesting that the parenchyma cells distributed throughout the stem in the different tissue types complement each other in function for the purposes of phloem loading, unloading and assimilate transport processes. Complimentary northern and western hybridisations demonstrated that internode 7 represents a shift in the sink from utilisation to storage. This is evident by the observed decline in both the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage of development. The relative mRNA and protein abundances for the three enzymes showed a similar trend. Higher levels of the gene transcripts and translated products were observed in the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was expressed exclusively in parenchyma cells that were associated with the vascular bundles and those serving a storage function in the stem pith and UGPase was found to be localised in the phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such findings have demonstrated an increase in resolution with which gene expression can be examined at a cellular level. Hence, the results from this study have demonstrated that the knowledge of metabolic compartmentation between different tissue and cell types is a requisite to understanding the function(s) of individual enzymes within complex structures such as the sugarcane culm.
Differential gene expression in the culm of sugarcane during development, with special emphasis on the storage parenchyma cells
(Stellenbosch : Stellenbosch University, 2002-12) Rogbeer, Omeswaree; Botha, F. C.; Groenewald, S.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
ENGLISH ABSTRACT: For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. While to date the maize ubiquitin (Ubi1) promoter has been the most effective transgene promoter for sugarcane, there is a high demand for tissue and stage specific promoters for localised transgene expression in the mature culm. The present study sought to characterise genes preferentially expressed in the core and peripheral tissues of the mature culm, which can further be used as research tools for specific promoter isolation. cDNA expression arrays containing 3840 clones from a late stage cDNA library representative of the core and peripheral tissues of the mature culm were prepared. The cDNA expression arrays were then differentially screened in independent hybridisation experiments with radioactively-labeled cDNA representations of core and peripheral tissues of internode 7, and peripheral tissues of internode 10. Comparison of the expression profiles of the arrayed cDNA targets in the three probes led to the identification of 60 tissue-specific, 17 stage-specific and 50 selectively expressed cDNAs within the mature sugarcane culm. ~ESTs of 33 chosen selectively expressed cDNAs with a relatively stronger pattern of expression in the core than in the peripheral tissues revealed sequence homology to a diverse collection of genes in the mature culm. These included genes associated with general cellular metabolism such as protein synthesis, protein modification and structural protein. Also identified were stress-responsive genes. The putative translational products of some of these clones had homologs that are involved in cell-wall structure in other species. These included the [acalin homolog, a lectin, hydroxyproline rich glycoprotein and structured polyprotein C. Many of the cDNAs thought to be involved in cell wall structure or stress related responses also accumulate in a developmental manner in other plants. These may indicate that specific mature culm mRNAs accumulate in response to stresses such as rapid cell expansion or as part of the late developmental program. An unexpected observation was that only one gene associated with sucrose metabolism was identified, namely sucrose synthase. These results confirmed that culm maturation was not controlled by sucrose metabolism despite its distinct physiological characteristic of storing high levels of sugars. ESTs analysis further revealed that sequence homology was not obtained for all the cDNAs exhibiting stage and tissue specific expression in the core and peripheral tissues of the mature culm. These could represent novel genes not only from sugarcane but all plants. Northern analysis demonstrated that 9 putatively identified selectively expressed genes tested so far accumulated specifically in the core and peripheral tissues of the mature culm. No expression was detected in root, leaf, leafroll and internode 3. However, their selective expression in a single internode as observed on the arrays (i.e hybridisation signal intensity being higher in the core than in the peripheral tissue) was not detected on the northern blots. These showed that cDNA expression arrays were not a highcapacity gene expression assay since they were prone to false expression analysis. The validity of results obtained through array screening should always be verified in an independent manner, preferably by the northern hybridisation analysis. Hence, the present study shows that the combination of differential screening, northern blot and DNA sequence analysis permits the rapid characterisation of differentially expressed genes in the core and peripheral tissues of the mature sugarcane culm. These can further be used as research tools for mature culm - specific promoter isolation in the sugarcane.
Fructokinase activity in the sugarcane culm : expression patterns and kinetic properties
(Stellenbosch : Stellenbosch University, 2001-03) Hoepfner, Suzanne Wilmien; Botha, F. C.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
ENGLISH ABSTRACT: Five hexose kinases, two fructokinases and three hexokinases, were identified in sugarcane culm. Fructokinase, a fructose specific hexose phosphorylating enzyme, was further investigated. Two isoforms, FRK1 and FRK2, were found. The isoforms were purified to homogeneity and antibodies raised against each. Both FRK1 and FRK2 have pH optima of 8.0 and both are homodimers of 69 kDa, consisting of subunits of 33 kDa. FRK2 was subject to substrate inhibition by fructose concentrations exceeding 0.1 mM while FRK1 was not inhibited by 1.0 mM fructose. Sugarcane FRK2 is more sensitive to substrate inhibition than FRK2 from other plants. The reaction catalysed by FRK1 is ATP-specific. The FRK2 reaction can utilise a variety of nucleotide triphosphates and no substrate inhibition is apparent when assayed with UTP instead of ATP. We proposed the existence of two nucleotide triphosphate binding sites on the enzymes. One of the sites is an ATP-specific regulatory site while the other is a catalytic site with wide substrate specificity. Additionally two fructose-binding sites are proposed. One is a catalytic site and the other a allosteric regulatory site. Binding of fructose to the allosteric site is only possible if ATP is present in the regulatory ATP-binding site. Such a configuration could explain the kinetic properties of FRK2. Both fructokinase protein expression and total fructokinase activity decreased during development. Consequently the decrease in activity is the result of decreased expression and not inactivation of existing protein. The ratio of FRK2 to FRK1 activity is dependent on the developmental stage of the tissue. FRK1 appears to be the isoform that is preferentially expressed in mature tissue. Previous measurements of fructokinase activity in crude extracts have been inaccurate as a result of the divergent kinetic properties of the isoforms. Based on the findings in this project a novel method is proposed whereby both the activity of each isoform and total fructokinase activity can be accurately calculated using a mathematical equation.
Isolation and characterisation of a culm-specific promoter element from sugarcane
(Stellenbosch : University of Stellenbosch, 2005-03) Goshu Abraha, Tsion; Botha, F. C.; Groenewald, J-H.; University of Stellenbosch. Faculty of Science. Dept of Botany and Zoology. Institute of Plant Biotechnology.
Sugarcane (Saccharum spp) is an important crop worldwide and is cultivated for the high level of sucrose in its mature internodes. Because of the exhaustion of the genetic potential in the commercial sugarcane germplasm conventional breeding has not lately been able to enhance sucrose content. Currently there is a concerted effort to improve culm sucrose content by genetic engineering which will require appropriate transgenes and promoters. One of the major constraints to genetic engineering of sugarcane is the lack of stable promoters required to drive tissue- or organ-specific expression of transgenes. Tissue and developmental stage specific promoters allow targeting of transgene activity and in doing so reduce the impact on non-target tissues. These promoters could also be advantageous to manipulate certain aspects of sucrose metabolism specifically in mature culm tissue. In addition, no promoters are currently freely available to the South African Sugar Industry for use in their transgenic program. The primary goal of this project was therefore to isolate a mature tissue-specific promoter for use in transgenic sugarcane plants. The approach followed was firstly, to identify an endogenous gene expressed in the desired pattern, and then to isolate the corresponding promoter from the sugarcane genome. cDNA macroarrays were initially used to identify differentially expressed sequences. The tissue specificity of potential clones was confirmed using RNA blot analysis. Two clones (c23-a and c22-a) were isolated and confirmed to be mature culm specific. Clone c22-a (putative dirigent-like protein) was selected for promoter isolation based on its culm tissue specific expression pattern and its proximity to the 5’ end of the gene. Furthermore, to confirm the activity of this promoter in the storage parenchyma cells, the exact cellular localisation of the transcript in the mature tissue was determined through in situ hybridisation. In situ hybridisation results confirmed the presence of the transcript in the parenchyma cells of mature culm tissue only. Moreover, the transcript is present in high concentrations in the parenchyma tissues surrounding the vascular bundles and parenchyma cells of the vascular complex. The selected dirigent-like gene was sequenced to allow the design of primers that could be used for the isolation of the corresponding promoter region using a long-range inverse PCR (LR-iPCR) method. Using these we have successfully isolated two highly homologous promoter regions of the dirigent like gene of respectively 1151 and 985 base pairs. In silico analyses confirmed the presence of various transcription motifs, including a TATA-box. However, experimental verification is needed to fully assess the functionality of these promoter regions. Verifying the activity of the isolated promoters through transient expression analysis proved to be problematic because of their highly mature culm specificity. Both constructs are therefore being used to obtain stable transformants in which promoter activity can be evaluated in mature internodal tissues.
Phosphoenolpyruvate (PEP) metabolism in roots and nodules of Lupinus angustifolius under P stress
(Stellenbosch : Stellenbosch University, 2004-03) Le Roux, Marcellous Remarque; Valentine, A. J.; Botha, F. C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).
ENGLISH ABSTRACT: This study investigated the activities of several of the enzymes involved in the alternative route of PEP metabolism via PEPc (EC 4.1.1.31). This reaction circumvents the adenyl ate-controlled PK (EC 2.7.1.40) reaction of the conventional glycolytic network under conditions of P stress. It was hypothesized that the synthesis of pyruvate under Pi stress would induce the PEPc alternative route and that C for pyruvate synthesis would primarily be imported via this route. This was assessed by looking at how total enzyme activities are perturbed under P stress and also by following the route of radioactive labelled 14C02 under sufficient (2 mM) and deficient P (2 JlM) conditions in either roots or nodules. The significance of the pathway under P stress, was further assessed by determining pool sizes of pyruvate that was synthesized from PEPc-derived C. The experiments were conducted under glasshouse conditions, as two separate studies: one to investigate the phenomenon of Pi stress and its consequences for PEPc-derived C metabolism, and the other one to study the enzymes involved. Seeds of Lupinus angustifolius (cv. Wonga) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 JlM P04 (LP) or 2 mM P04 (control) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. LP roots synthesized more pyruvate from malate than LP nodules, indicating the engagement of the PEPc route under Pi stress. In this regard, pyruvate is considered as a key metabolite under Pi stress. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the PK and PEPc routes. The enhanced PK activities supported these high pyruvate levels. Under P stress, PEPc activities increased in roots but not in nodules and these changes were not related to the expression of the enzyme. Root and nodular PEPc were not regulated by expression, but possibly by posttranslational control. The novelty of our results for symbiotic roots demonstrates that using metabolically available Pi is indeed a more sensitive indicator ofP stress. These results show that under Pi stress, nodules are able to maintain their Pi and adenylate levels, possibly at the expense of the root. It is suggested that nodules do not experience P stress to the same extent as roots or alternatively function optimally under conditions of low P availability. The increase in concentration of pyruvate synthesized from malate, indeed suggest that under LP conditions there is an increase requirement for pyruvate. It is clear from this data that the operation of bypass route in nodules should be investigated further. Nevertheless, this study provided incentives for understanding the role of C pathways in Ni-fixation, in particular under conditions ofP limitation.
Remobilization of sucrose from the culm during germination of sugarcane setts
(Stellenbosch : University of Stellenbosch, 2005-12) Boussiengui-Boussiengui, Gino; Botha, F. C.; Groenewald, H.; University of Stellenbosch. Faculty of Science. Dept. of Botany and Zoology.
The main substrate use during shoot establishment from the lateral bud of sugarcane setts and enzymes involved in sucrose metabolism were investigated in the shoots and the internodes acting as source of carbohydrates. Radiolabelling studies were conducted to investigate the metabolism of sucrose and glucose during shoot establishment. The internode’s total dry mass over the 21-day of shoot establishment period was reduced by 25% and 30% in plants incubated in dark/light and total darkness, respectively.
The role and regulation of PEPcarboxylase in dissolved inorganic carbon metabolism under Pi starvation in legume root systems
(Stellenbosch : Stellenbosch University, 2004-03) Ward, Caroline (Caroline Linda); Valentine, A. J.; Botha, F. C.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
ENGLISH ABSTRACT: This study aimed to assess the contribution of anaplerotic C prOVISIOn VIa phosphoenolpyruvate carboxylase (pEPc, EC 4.1.1.31), during Pi stress in the root and nodule components of Lupinus angustifolius. The role of PEPc in DIC metabolism in roots and nodules of phosphate-starved plants was studied. The symbioses involving leguminous plants and species of Rhizobium and Bradyrhizobium bacteria form an integral part of effective management ofN in the environment. In agricultural settings, roughly 80% of this biologically fixed N2 comes from this type of symbiotic relationship. Nitrogen-fixing bacteria in concert with legumes fix atmospheric nitrogen, which is then available to the infected plant. Worldwide, legumes are grown on approximately 250 Mha and they fix about 90 Tg (90 billion tons) of N, per year. The overall stoichiometry for nitrogen assimilation in the nodule requires one molecule of oxaloacetate to be converted to one molecule of asparagine per dinitrogen molecule fixed. One possible source for the required oxaloacetate is the reaction catalysed by PEPc. The reaction catalysed by PEPc is a major source of anaplerotic carbon for the plant and it is expected that this reaction will be even more important to plants under Pi stress, as the reaction is not ATP-dependent. Seeds of Lupinus angustifolius (cv. Wong a) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 11MP04 (LP) or 2 mM P04 (HP) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. Under P stress, PEPc activities increased in roots but not in nodules and these changes were not related to the expression of the enzyme. Root and nodular PEPc were not regulated by expression, but possibly by posttranslational control. LP roots also synthesised more pyruvate from malate than LP nodules. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the pyruvate kinase (PK, Ee 2.7.1.40) and PEPc routes. The enhanced PK activities supported these high pyruvate levels. The results show unequivocally that nodules do not experience P stress to the same extent as roots. Implications of the findings are that nodules require low P to function normally. Maintenance of phosphate levels in nodules may be at the expense of host. It can be suggested that when nodules are P-starved they can become aggressive scavengers for available P and even out-compete roots.
Sucrose transporters and sucrose uptake mechanisms in sugarcane
(Stellenbosch : University of Stellenbosch, 2004-12) Titus, Charlene H. A. (Charlene Helecyn Agatha); Cramer, M. D.; Botha, F. C.; University of Stellenbosch. Faculty of Science. Dept. of Botany and Zoology. Institute for Plant Biotechnology.
ENGLISH ABSTRACT: The process of sugar accumulation and transport in sugarcane is still poorly understood. Understanding the processes involved in sucrose transport are important, since membrane transport might be important control points in this pathway. The goals of this project were to unravel the mechanisms of sugar transport in sugarcane culm tissue by using 14C-sugar analysis as well as molecular techniques to identify possible sucrose transporters. Developing (internode 2 and 4) and maturing (internode 8 and 15) culm tissue of sugarcane (Saccharum hybrid) commercial variety N19 was used for all tissue disc experiments. Tissue discs from internodes of different developmental stages were cut from field grown sugarcane plants (cv. N19) and the uptake of 14C-labelled glucose, fructose and sucrose measured. The uptake rates were measured at varying pH, temperature and concentrations of sugars. Hexoses were found to be the major sugar taken up and sucrose was only important when little hexose was available, as was found in the mature ripe internodes. Sucrose uptake differs between tissues and our study showed that sucrose was taken up rapidly at pH 5, similar to the pH optimum of most sucrose transporters Inhibition studies with TRIS (2-amino-2- (hydroxymethyl)-1,3-propanediol) and PCMBS (p-chloromercuribenzenesulphonic acid) indicated that more than one sucrose transporter activity may be present in the sugarcane system at different sucrose concentrations. To date work on sugarcane sucrose transporter expression on DNA and RNA level has been limited. Only recently a sucrose transporter from Saccharum hybrid sugarcane stem cDNA libray, ShSUT1 (Saccharum hybrid Sucrose Transporter ) was isolated and functionally characterized in the yeast strain SEY 6210 (Rae et al., 2004). In an effort to understand sucrose transport in sugarcane culm tissue, a partial sucrose transporter cDNA, ScSUT1(p) from Saccharum hybrid sugarcane a bud cDNA library was isolated, and cloned from a bud cDNA library. The clone was designated ScSUT(p) as a partial Sugarcane Sucrose Transporter. The ScSUT1(p) sequence showed 94% identity to ShSUT1 on nucleotide level over 1258 nucleotides and had an estimated open reading frame of 419 amino acids. Southern blot analysis indicated that the transporter had a low copy number and the ScSUT1(p) transcript expression was constitutive in sucrose accumulating and sucrose storing stem tissue, but was less abundant in immature tissue such as internodes 2 and 3 and in lateral buds. It was concluded that the primary function of ScSUT1(p), was not phloem unloading but that the transporter may be involved in phloem loading, as it is abundant in mature source leaves. ShSUT1 cDNA was obtained from Dr C Grof and the functionality of ShSUT1 as a sucrose transporter in Xenopus leavis oocytes was confirmed. However, electrophysiological measurements on the oocytes demonstrated no measurable current associated with sucrose challenge to the oocytes indicating that the transporter activity was either very low or possibly non-electrogenic. Further investigation is required to characterise the specific mechanism and kinetic properties of this transporter.
Variants of the gene encoding the beta subunit of pyrophosphate dependent phosphofructokinase (PFP) and their transcriptional expression in sugarcane
(Stellenbosch : Stellenbosch University, 2001-12) Reddy, Sanushka; Huckett, B.; Botha, F. C.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
ENGLISH ABSTRACT: Sugarcane, a complex polyploid, may theoretically contain up to 12 alleles for a particular gene at a single locus. The number of alleles and the extent of their variation is of particular importance due to the potential for the exploitation of genetic variation through breeding. Also, allelic variation has implications for the manipulation of gene function via genetic engineering. Pyrophosphate dependent phosphofructokinase (PFP) is considered a key regulatory enzyme involved in sucrose biosynthesis, which may provide a target for genetic manipulation to increase sucrose yields in sugarcane. The enzyme is composed of a regulatory (alpha) and catalytic (beta) protein subunit encoded by the PFP-a and PFP-p genes respectively. The PFP-p gene, which has been shown to be a single locus gene in other plant species, was used in this study as a model for allelic variation in sugarcane. Two main areas of investigation involved genomic and expression analyses to further characterise the gene. Polymerase Chain Reaction (PCR) using specific primers previously designed from conserved regions of the PFP-p gene from different plant sources, were used to amplify across exons 10 to 12 of the sugarcane PFP-p gene. Two PCR products, designated PFP-81/881250bp and PFP-81/881100bp respectively, were obtained from commercial cultivars N19 and N21. Numerous clones of the fragments were obtained and sequenced. International database searches confirmed that both amplicons were identifiable as PFP-p. Comparative sequence analysis indicated that the PFP-81/881250bP and PFP-81/881100bp fragments were poorly homologous to each other, with higher regions of homology residing in the putative exon regions (77-78%) compared to the intron regions (34- 56%). Although minor sequence variation was detected within the amplicon populations, it was evident that two major variants of the PFP-p gene are present in sugarcane. Southern hybridisation analysis revealed a simple banding pattern for PFP-p. Also, there are DNA polymorph isms for the genomic regions corresponding to the PFP-81/881250bP and PFP-81/881100bPfragments. Previous evidence indicates that both variants are also present in the ancestral sugarcane germplasm and maintain the same level of heterozygosity. The presence of both gene forms in the ancestral and commercial germplasm prompts speculation that the two variants may not segregate. This theory, together with the simple Southern hybridisation pattern obtained for PFP-p, leads to the hypothesis that the two gene forms are at separate loci in the sugarcane genome, which may be closely linked on the same chromosome. The expression of the variants was investigated during different stages of sucrose accumulation in the sugarcane culm using a Reverse Transcription (RT)- peR approach. A single, identical transcription product was isolated from these and other selected tissues of the plant. In addition, the same transcript was obtained from the ancestral species representatives of modern sugarcane, Saccharum officinarum and Saccharum spontaneum. Sequence comparison of the transcribed product and the derived exon regions of the two variants implies that the PFP-p gene represented by the PFP-B 1/B81250bpvariant is being expressed in sugarcane while the gene form characterised by the PFP-B1/B81100bp amplicon is silent. Northern hybridisation analysis indicates that PFP-p is differentially expressed at different stages of sucrose accumulation. PFP-p expression is higher in the immature culm tissue of sugarcane and low in the mature culm, which suggests that PFP-p is highly regulated during maturation. It is hypothesised that the PFP-p gene underwent duplication and that one gene form was subject to accumulative mutations evolving into a pseudogene. On the basis of present results, it can be suggested that future genetic manipulation of PFP-p should involve the gene variant characterised by the PFP-B 1/B81250bp fragment.

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Browsing Masters Degrees (Botany and Zoology) by browse.metadata.advisor "Botha, F. C."

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