Silencing of δ-aminolevulinic acid dehydratase via virus induced gene silencing promotes callose deposition in plant phloem

Abstract

The δ-aminolevulinic acid dehydratase (ALAD) enzyme is an intermediate in the biosynthetic pathway of tetrapyrroles. It combines two δ-aminolevulinic acid (δ-ALA) molecules to form the pyrrole, porphobilinogen, an important precursor for plant pigments involved in photosynthesis, respiration, light-sensing, and nutrient uptake. Our recent efforts showed that, in citrus, silencing of ALAD gene via Citrus tristeza virus-induced gene silencing, caused yellow spots and necrosis in leaves and in developing new shoots. Silencing of ALAD gene reduced leaf pigments and altered leaf metabolites. Moreover, total phenolic content, H₂O_2, and reactive oxygen species (ROS) increased, indicating that silencing of ALAD induced severe stress. Herein, we hypothesized that conditions including lower sucrose, elevated ROS, alteration of microRNA involved in RNAi regulatory protein Argonaute 1 (AGO1) and ROS lead to higher deposition of callose in phloem tissues. Using aniline blue staining and gene expression analysis of callose synthases, we showed significant deposition of callose in ALAD-silenced citrus.

Keywords: callose; citrus; citrus tristeza virus; phloem; virus-induced gene silencing; Δ‐aminolevulinic acid dehydratase.

Figure 1.

Silencing of δ‐aminolevulinic acid dehydratase via Citrus tristeza virus-induced gene silencing (CTV-IGS) causes callose deposition in citrus phloem tissue. Note the increased amount of callose deposits in the phloem tissue as seen in peeled stem bark is correlated with the increase in phenotype, but the highest amount is found in the moderate phenotype. Callose deposits were visualized by staining with aniline blue. Zero: control plants (CTV-wt). 1–5: degrees of phenotype in ALAD-silenced plants (CTV-tALAD-as).

Figure 2.

Visualization and quantification of callose deposits in the moderate phenotype of ALAD-silenced plants (CTV-tALAD-as) phloem tissue compared to control plants (CTV-wt). A-H: Visualization of callose deposits by staining with aniline blue. A-D: Control plants. E-H: ALAD-silenced plants. I: Total callose deposits quantified as fluorescence intensity. J: Fold change in callose synthase gene expressions performed with RT-PCR. Arrows indicate callose deposits.

Figure 3.

Proposed model on how silencing of δ‐aminolevulinic acid dehydratase leads to deposition of callose. See main text for details.