Quantification of polyphenolic compounds and relative gene expression studies of phenylpropanoid pathway in apple (Malus domestica Borkh) in response to Venturia inaequalis infection

Abstract

Apple (Malus domestica Borkh) is rich in phenolic compounds, which may enhance resistance to scab disease caused by Venturia inaequalis. In present study, apple cv. Golden Delicious was used for estimation of total phenols, flavonoids and quantification of six individual phenolic compounds. between control vs inoculated samples at different inoculation stages. The relative gene expression of phenylalanine ammonia lyase, chalcone synthase and flavanone 3 hydroxylase increased and polyphenolic compounds were constitutively upregulated at different post-inoculation stages. Data suggest that synthesis and accumulation of polyphenols is closely related with disease resistance against Venturia inaequalis. This study may play a vital role in understanding and finding out the governing mechanisms of scab resistance.

Keywords: Apple; Phenylalanine ammonia lyase; Polyphenols; Resistance; Venturia inaequalis.

Fig. 1

One year-old plants grafted on rootstock M9 grown in a greenhouse and shifted to growth chamber where temperature and humidity was maintained.

Fig. 2

Effect of Venturia inaequalis on total phenolics and flavonoids of apple. Total phenols (mg GAE equivalent (gallic acid)/100 g fw) and flavonoids (mg CE equivalent (catechin) /100 g fw) were measured at different time-points (BI, 1, 7 and 24 Days) of pathogen inoculation and sterile water treatment (as the control). Data are shown as mean ± SD (Standard Deviation) of three replicates from one independent experiment.

Fig. 3

Effect of Venturia inaequalis on (µg/g fw) ferulic acid, coumaric acid, catechin, apigenin, rutin and phloridzin content which were measured at different time-points (BI, 1, 7 and 24 Days) of pathogen inoculation and sterile water treatment (as the control). Data are shown as mean ± SD (Standard Deviation) of three replicates from one independent experiment.

Fig. 4

RNA extracted at different stages after inoculation. (A) (1) Before inoculation (Treated), (2) Before inoculation (Control), (3) One day post inoculation (Treated), (4) One day post inoculation (Control), (5) 7- day post inoculation (Treated), (6) 7- day post inoculation (control), (7) 24-days post inoculation (Treated) and (8) 24- days post inoculation (control) and (B) cDNA confirmation of isolated RNA from leaves of apple cultivars using GAPDH primer (Band at 181 bp). L indicates marker DNA ladder starting from 100 bp.

Fig. 5

Gene Expression and Heatmap for (A) PAL (B) CHS and (C) F3H genes representing relative levels of gene expression at different stages before and post inoculation events when compared to corresponding house-keeping gene ACTIN. Where NI- Non inoculated, I- inoculated, BI-Before inoculation and DPI- Days Post inoculation. Each point represents the mean of three replicates and was expressed as mean ± SD (Standard deviation). Asterisks (*) indicate significant differences (P < 0.05) between Venturia inaequalis treatment (inoculated) and control (Non-inoculated).

Fig. 6

Expression levels of all genes and heatmap indicating differential gene expression levels of PAL, F3H, CHS, color codes represent different gene expressions.