Abstract
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.
Highlights
Tetrapyrroles are macrocyclic compounds that contain four pyrrole rings with various structural alternatives and numerous biological functions in prokaryotes and eukaryotes
Higher plants possess four classes of tetrapyrroles that play important roles in many biological processes; chlorophyll is essential for photosynthesis, heme is necessary for respiration, siroheme is necessary for sulfur and nitrogen assimilation, and phytochromobilin is required for light sensing (Tanaka & Tanaka, 2007)
To investigate the effect of silencing of the ALAD gene on the other genes implicated in the biosynthesis pathway of leaf pigments, the transcription levels of 46 genes involved in biosynthetic pathways of chlorophylls (17 genes) and carotenoids (29 genes) were investigated in C. macrophylla, citrus tristeza virus (CTV)‐wt, and CTV‐tALAD plants (Figure 4)
Summary
Tetrapyrroles are macrocyclic compounds that contain four pyrrole rings with various structural alternatives and numerous biological functions in prokaryotes and eukaryotes. Functional genomics using mutant plants have been extensively used to investigate the regulatory mechanism of tetrapyrrole synthesis and the role of various enzymes in this pathway Most of these studies were performed on Arabidopsis thaliana (Espineda, Linford, Devine, & Brusslan, 1999; Frick, Su, Apel, & Armstrong, 2003; Hirono & Rédei, 1963; Ishikawa, Okamoto, Iwasaki, & Asahi, 2001; Kumar & Söll, 2000; Molina et al, 1999; Nagata, Tanaka, Satoh, & Tanaka, 2005; Tanaka & Tanaka, 2005) and Nicotiana tabacum (Alawady & Grimm, 2005; Höfgen et al, 1994; Kruse, Mock, & Grimm, 1995; Mock, Keetman, Kruse, Rank, & Grimm, 1998; Mock et al, 1999; Papenbrock, Mock, Tanaka, Kruse, & Grimm, 2000; Papenbrock et al, 2001). We hypothesize that silencing of ALAD will affect chlorophyll biosynthesis, but can affect the synthesis of other pigments and metabolites
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