ABSTRACT
Storage roots of cassava (Manihot esculenta Crantz) exhibit a rapid post-harvest physiological deterioration (PPD) response that can occur within 24-72 h of harvest. PPD is an enzymatically mediated oxidative process with parallels to plant wound, senescence and defence responses. To characterise those genes that show significant change in expression during the PPD response we have used cDNA microarray technology to carry out a large-scale analysis of the cassava root transcriptome during the post-harvest period. We identified 72 non-redundant expressed sequence tags which showed altered regulation during the post-harvest period. Of these 63 were induced, whilst 9 were down-regulated. RNA blot analysis of selected genes was used to verify and extend the microarray data. Additional microarray hybridisation experiments allowed the identification of 21 root-specific and 24 root-wounding-specific sequences. Many of the up-regulated and PPD-specific expressed sequence tags were predicted to play a role in cellular processes including reactive oxygen species turnover, cell wall repair, programmed cell death, ion, water or metabolite transport, signal transduction or perception, stress response, metabolism and biosynthesis, and activation of protein synthesis.
Subject(s)
Genes, Plant , Manihot/genetics , Apoptosis/genetics , Biological Transport/genetics , Cell Wall/genetics , Cell Wall/metabolism , Expressed Sequence Tags , Gene Expression Profiling , Gene Expression Regulation, Plant , Manihot/growth & development , Oligonucleotide Array Sequence Analysis , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/growth & development , Reactive Oxygen Species/metabolism , Signal Transduction/genetics , Transcription, GeneticABSTRACT
A major constraint to the development of cassava (Manihot esculenta Crantz) as a crop to both farmers and processors is its starchy storage roots' rapid post-harvest deterioration, which can render it unpalatable and un-marketable within 24-72 h. An oxidative burst occurs within 15 min of the root being injured, that is followed by the altered regulation of genes, notably for catalase and peroxidase, related to the modulation of reactive oxygen species, and the accumulation of secondary metabolites, some of which show antioxidant properties. The interactions between these enzymes and compounds, in particular peroxidase and the coumarin, scopoletin, are largely confined to the vascular tissues where the visible symptoms of deterioration are observed. These, together with other data, are used to develop a tentative model of some of the principal events involved in the deterioration process.
Subject(s)
Crops, Agricultural/physiology , Manihot/physiology , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism , Antioxidants/metabolism , Blotting, Northern , Catalase/genetics , Catalase/metabolism , Chromatography, High Pressure Liquid , Coumarins/metabolism , Crops, Agricultural/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Hydrogen Peroxide/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Manihot/genetics , Manihot/metabolism , Models, Biological , Peroxidase/genetics , Peroxidase/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Scopoletin/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxides/metabolism , Time FactorsABSTRACT
BACKGROUND AND AIMS: Control of diseases in the key tropical staple, cassava, is dependent on resistant genotypes, but the innate mechanisms are unknown. The aim was to study phenylpropanoids and associated enzymes as possible defence components. METHODS: Phenylalanine ammonia-lyase (PAL), phenylpropanoids and peroxidases (POD) were investigated in elicited cassava suspension cells and leaves. Yeast elicitor was the most effective of several microbial and endogenous elicitors. Fungitoxicity was determined against the cassava pathogens Fusarium solani, F. oxysporum and the saprotroph Trichoderma harzianum. KEY RESULTS: A single and rapid (> or =2-3 min) oxidative burst, measured as hydrogen peroxide, occurred in elicited cells. PAL activity was induced maximally at 15 h and was preceded by PAL mRNA accumulation, which peaked at 9 h. Symplasmic POD activity increased four-fold in cells, 48 h post-elicitation. POD isoforms (2-7 isoforms, pI 3.1-8.8) were detected in elicited and unelicited cells, extracellular medium and leaves but two extracellular isoforms were enhanced post-elicitation. Also expression of a cassava peroxidase gene MecPOD1 increased in elicited cells. Only anionic forms oxidized scopoletin, with highest activity by isoform pI 3.6, present in all samples. Unidentified phenolics and possibly scopolin increased post-elicitation, but there was no enhancement of scopoletin, rutin or kaempferol-3-O-rutinoside concentration. Fungal germ tube elongation was inhibited more than germination by esculetin, ferulic acid, quercetin and scopoletin. T. harzianum was generally more sensitive than the pathogens and was inhibited by > or =50 microg mL(-1) of ferulic acid and quercetin and > or =10 microg mL(-1) of scopoletin. CONCLUSIONS: Phenolic levels in cells were not enhanced and were, theoretically, too low to be inhibitory. However, in combination and when oxidized they may contribute to defence, because oxidation of esculetin and scopoletin by peroxidase and of esculetin by tyrosinase enhanced their fungitoxicity up to 20-fold.