Abstract:
Sucrose-phosphate synthase (SPS, EC 2.4.1.14) plays a key role in sucrose biosynthesis in autotrophic and heterotrophic tissues and is thought to be important in regulation of starch- to-sugar conversion in ripening fruit. The goal of this thesis was to isolate SPS genes from kiwifruit (Actinidia deliciosa var. deliciosa (A. Chev.) C.F, Liang et A.R. Ferguson cv. Hayward], a hexaploid, commercial species, and from A. chinensls Planch. var. chinensis, a diploid and putative ancestor of kiwifruit. The isolated genes would then be used to characterise SPS gene expression, in particular during ripening of A. chinensis fruit. Three partial cDNA clones encoding for SPS (designated KSPSI-KSPS3) with greater than 96% nucleotide identity were isolated from ripening fruit of A. deliciosa. KSPl, a 2584 bp partial cDNA clone was isolated from a cDNA library. KSPS2 and KSPS3, representing the 5'end of SPS, are cloned PCR fragments. These were amplified from the poly-A RNA used to construct the cDNA library. From ripening fruit of A. chinensis, a clone (CKSPS2) was obtained that was closely-related to KSPS2. CKSPS2 comprised a cloned 5'PCR fragment and an 3'PCR fragment that was identified by partial sequencing. The full-length sequence of CKSPS2 was estimated to be 3.6 kb. Phytogenetic analysis of SPS protein sequences from kiwifruit (KSPSI and KSPS2) and other plant species indicated that two distinct groups of SPS genes (A and B) occur in both dicotytedonous and monocotyledonous plant species. This result suggests that the gene amplification giving rise to the two groups predates the divergence of monocots and dicots. All Actinidia clones isolated are members of group A. Southern hybridisations using DNA of the diploid A. chinensis indicated that group A SPS genes were represented by a small gene family, possibly containing 4 -6 genes. Screening of aGenomic library from A. chinensis resulted in isolation of several clones with homology to SPS. One clone, ‘˄SPS2, was closely-related to the cDNA clone CKSPS2. ‘˄SPS2 was 13.7kblong, contained most of the coding region of the SPS gene (including at least 5 introns), and 417 bp upstream of the putative translation initiation codon. Putative promoter regulatory elements (T ATA box and a cold-inducible element) were identified. Four other genomic SPS clones were isolated that contained either 5 'or 3' coding regions. These clones appeared tobe rearranged and may represent library construction artifacts. Northern and Western analysis was performed to estimate the relatives steady-state mRNA and protein levels of SPS in A. chinensis tissues and cell cultures. Western analysis using aspinach SPS polyclonal antibody, detected similar levels of SPS protein in all tissues examined. Northern hybridisation detected mRNA of all isolated cDNA clones (group A SPS genes). SPS steady-state mRNA was detected in all A. chinensis tissues examined and levels increased in leaves during sink-to-source transition, being highest in senescent leaves. In roottissues SPS steady-state mRNA levels were relatively low compared to other tissues whereas they were high in young floral tissues and increased during fruit development. SPS steady-state mRNA levels increased in ripening A chinensis fruit, firstly when fruit reached physiological maturity, secondly in response to ethylene-treatment, and d, thirdly during the main phase of starch degradation before endogenous ethylene was measurable. SPS steady-state mRNA levels increased slightly during cold-storage of fruit (7 days) and increasedfurther after return to 20oC (24 h). A suspension culture was initiated from A. chinensis fruit tissue in order to study turnover rates and stability of SPS mRNA and protein. When transcription was inhibited using actinomycin D, SPS mRNA appeared to be relatively stable with a half-life of 17.5 h, suggesting that mRNAs representing group A SPS genes do not have a high turnover rate. In cell cultures incubated at 0C, the activation state of SPS enzyme (V6/Vn'a\ activity) increased. This increase was prevented in the presence of cycloheximide, suggesting that a protein synthesis step (either of a new SPS protein or a protein that activates pre-existing SPS protein) was required. In conclusion, two divergent groups of SPS genes (A and B) have been postulated to exist in most dicot and monocot species. A. small gene family of closely-related group A SPS genesHas been shown to be expressed during ripening of A. chinensis fruit. It is proposed that SPS group A and B genes are differentially expressed with respect to tissue and environmental conditions in various species. Furthermore, based on Northern hybridisations I suggest that specific group A SPS mRNAs in A. chinensis are expressed differentially in response to various triggers, such as sugar concentrations, ethylene and temperature.