Molecular analysis of genes involved in pineapple fruit development

Koia, Jonni (2012). Molecular analysis of genes involved in pineapple fruit development PhD Thesis, School of Biological Sciences, The University of Queensland.

       
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Author Koia, Jonni
Thesis Title Molecular analysis of genes involved in pineapple fruit development
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Jose Botella
Richard Moyle
Total pages 122
Total colour pages 20
Total black and white pages 102
Language eng
Subjects 0607 Plant Biology
1001 Agricultural Biotechnology
0703 Crop and Pasture Production
Formatted abstract
Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial value, yet surprisingly little research has been undertaken to understand the molecular basis of pineapple fruit ripening. A microarray and bioinformatic study was performed to identify genes involved in pineapple fruit development. The microarray includes a set of EST clones isolated from pineapple fruit and root cDNA libraries. The genes were annotated and assigned putative functions using online tools from NCBI GenBank (BlastX), The Arabidopsis Information Resource (TAIR) database, The Database for Annotation, Visualisation and Integrated Discovery v6.7b (DAVID) and Gene Ontology (GO) classification systems. Quantitative real-time PCR analysis was used to confirm differential gene expression patterns identified within the microarray data. MapMan (v3.1.1), The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway resource and Heat Maps generated through DAVID were further used to visualise clusters of genes involved in biological pathways and processes during pineapple fruit development.

Microarray analyses identified 271 unique cDNAs differentially expressed during pineapple fruit ripening by 1.5-fold or higher. Among the 271 sequences, 184 shared significant homology with known gene products, 53 shared homology with proteins of unknown function and 34 shared no significant homology with any database accession. From the 237 pineapple ESTs with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. All 237 pineapple ESTs were assigned an Arabidopsis thaliana homolog tag for further bioinformatic analysis. DAVID Functional Annotation Cluster (FAC) analysis of all 237 homolog tags revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Real-time PCR analysis confirmed the microarray expression results for nine out of the ten genes assayed.

This is the first large scale gene expression study and bioinformatic analysis that identify a large number of genes with putative involvement in pineapple fruit ripening. Redox activity and the threshold of oxidation state may be useful to determine the cellular mechanisms that differentiate climacteric and non-climacteric fruit ripening. As such, it is possible that non-climacteric fruits such as pineapple have an oxidation threshold that is significantly different to climacteric fruit species, which may explain the absence of high levels of ethylene and rates of respiration at the onset of non-climacteric fruit ripening.

The demand for new plant-based constitutive promoters without patent protection is of particular interest among the research and biotechnology community. This study isolated and characterised the putative promoter sequences of a 60S ribosomal protein L36 gene (AcL36) and a protein translation factor SUI1 gene (AcSUI1) from non-climacteric monocot pineapple. Ribosomal proteins and protein translation factors are highly conserved proteins involved in the universal mechanism of protein synthesis. The AcL36 and AcSUI1 promoters drove constitutive transgene expression in the heterologous dicot plant system Arabidopsis thaliana, at levels comparable to the CaMV35S promoter. The identification of introns in the 5'UTR region of both promoters suggests that intron mediated enhancement activity may account for the constitutive nature of both promoters. AcSUI1 also drove transient expression in a wide range of climacteric and non-climacteric fruit species. These results demonstrate the potential for using ribosomal protein and translation factor gene promoters to drive constitutive gene expression.

A MADS box transcription factor, AcMADS1, was previously identified to be strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses placed AcMADS1 in the same monoclade as LeMADS-RIN, a master regulator of fruit ripening in climacteric tomato upstream of ethylene. LeMADS-RIN has been proposed to be a global ripening regulator shared among climacteric and non-climacteric species. Besides strawberry FvMADS-9, orthologues to LeMADS-RIN have not yet been identified in non-climacteric species. This study showed that AcMADS1 was not able to complement the tomato rin mutant phenotype, suggesting that is not a LeMADS-RIN ortholog. This study further isolated and characterised the AcMADS1 promoter which was found to be active in fruits of rin mutants as well as wild type tomatoes, indicating that the climacteric transcriptional machinery can recognise cis elements in this promoter. AcMADS1 also showed strong homology to Arabidopsis AGL6, banana MaMADS3, and tomato TDR5, a SEPALLATA3 homolog. AGL6 and SEPALLATA3 are involved in flowering time and floral organ development. In planta GUS staining assays showed that the AcMADS1 promoter directs strong gene expression in flowers of tomato and Arabidopsis thaliana. The strong flower expression was confirmed by fluorometric MUG assays and quantitative real time PCR analyses. The results suggest that AcMADS1 may play a dual role in flower development and fruitlet ripening in pineapple.
Keyword Pineapples
Microarray analysis
Nonclimacteric fruit
Redox
Antioxidant
L-ascorbate
Vitamin C
Bromelain
Metalloenzymes
MADS box transcription factor
SUI translation homolog
L36 ribosomal protein

 
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Created: Thu, 15 Nov 2012, 11:18:19 EST by Jonni Koia on behalf of Scholarly Communication and Digitisation Service