Figure 2.

VIGS of OHP2 causes a pale-green phenotype in Arabidopsis. A, Phenotypes of 5-week-old VIGS plants (i.e. 3 weeks after infiltration). Pale-green leaves in the VIGS-OHP2 plants are indicated by white arrows. B, RT-qPCR analysis of transcripts of the LHC gene family. OHP transcript levels are reduced specifically in the respective silencing lines. Total RNA was DNase digested and reverse transcribed. RT-qPCR was performed using ACT and SAND genes as references. Data were normalized to the expression in VIGS-GFP lines using the ΔΔCt method (Pfaffl, 2001). Data represent averages of three biological replicates, and error bars represent the sd. C, Western-blot analysis of OHPs showing reduced accumulation of OHPs in the VIGS lines. Total protein extracts were fractionated by SDS-PAGE or Tricine-SDS-PAGE, transferred onto nitrocellulose membranes, and probed with specific antibodies. The large subunit of Rubisco (RbcL) was used as the loading control. D, Chlorophyll (Chl) quantification by HPLC. Pigments were extracted from freeze-dried leaf material in cold alkaline acetone. Extracts were separated by HPLC, and pigments were quantified using pure standards. The inset shows the ratio of chlorophyll a to b species. Data represent averages of four biological replicates, and error bars represent the sd. DW, Dry weight. E, Status of the xanthophyll cycle, expressed as the ratio of violaxanthin (Vio) to antheraxanthin (Anth) and zeaxanthin (Zea). Data represent averages of four biological replicates, and error bars represent the sd.