Quantification of Soluble Sugars and Starch

The soluble sugars were quantified as described in Chow & Landhäusser (2004) (link). Briefly, the sampling was performed at bolting stage and 8 h after illumination of light and shade dried leaves of WT and transgenic plants were utilized for the analysis. The extraction was performed with hot ethanol from 50 mg of powdered leaves. The ethanol mixed with powdered leaves were placed in water bath for 10 min at 95 °C. The extraction was performed three times and supernatant was used for sugars analysis. The absorbance for soluble sugars was recorded at 490 nm by using UV-visible spectrophotometer (HACH DR1900, USA). The pellet was utilized for quantification of starch as described by Chang (1979) (link) and Lafont-Mandoza, Severiche-Sierra & Jaimes-Morales (2018) (link). For this analysis, the crude starch leftover was dried at 50 °C to remove the ethanol. The pellet was dissolved in cold water followed by addition of iodine solution. This mixture was incubated at room temperature for 10 min and absorbance was recorded at 615 nm.