Effects of δ-aminolevulinic acid dehydratase silencing on the primary and secondary metabolisms of citrus.

δ-aminolevulinic acid dehydratase (ALAD) is an important enzyme in tetrapyrrole synthesis. ALAD combines two δ-aminolevulinic acid (δ-ALA) molecules to form the pyrrole molecule, porphobilinogen, an important precursor for plant pigments involved in photosynthesis, respiration, and nutrient uptake. In this study, we investigated the effects of silencing of ALAD gene on citrus leaf pigments and metabolites. The ALAD enzyme was inhibited using virus-induced gene silencing (VIGS) technology using citrus tristeza virus (CTV). δ-ALA accumulated in citrus plants inoculated with the recombinant virus (CTV-tALAD) to silence ALAD and resulted in discrete yellow spots (yellow islands) and necrosis in leaves and stems. The levels of chlorophylls, starch, sucrose, trans- and cis-violaxanthin, and α- and β-cryptoxanthin were reduced in CTV-tALAD plants, whereas zeaxanthin was increased. The increase in zeaxanthin and the decrease in its precursors indicated that the reduction in chlorophylls resulted in light damage. Salicylic acid and jasmonic acid levels, as well as emission of (E)-α-bergamotene and (E)-β-farnesene, increased in CTV-tALAD plants indicating these plants were under stress. Our results showed that silencing of ALAD induces stress in plants and that VIGS using mild CTV strains is a promising technique to study biological function of citrus genes.