Microscope analyses

To analyze the chloroplast arrangement on O. streptacantha cells, segments of cladode (5 × 8 × 0.3 mm) subjected to the treatments described below were observed without fixation under a light microscope Leica DM2000 (Leica, Wetzlar, Germany). The photographs were obtained and digitized with LAS Imaging Software (Leica). For the transmission electron microscope (TEM), the segments of cladodes were treated with a fixative solution (10% glutaraldehyde in 0.1 M sodium phosphate, pH 7.4) during overnight at 4 °C. Then, they were washed using Sorensen’s phosphate buffer and dehydrated with ethanol; subsequently, the samples were polymerized in a fresh resin at 60 °C as described by Cocoletzi et al.⁷⁴. Ultrathin sections were contrasted using aqueous uranyl acetate (2% w/v) and aqueous lead citrate (2% w/v). Samples were examined with TEM JEOL 200CX (JEOL, Welwyn Garden City, UK) using a 100 kV acceleration voltage.

The ultrastructural analysis of chloroplasts to determine the thickness of the thylakoid lumen was realized using the methodology reported by Kirchhoff et al.³⁸. The distances of stacking repeat unit (R), which includes the two thylakoid membrane bilayers (M) and the widths of one partition gap (P) were realized with Image J software⁷⁵. Subsequent, the thickness of the thylakoid lumen (L) were estimated using the equations proposed by Kirchhoff et al.³⁸: L = R − M − P. The measurements of lumen thickness were made on grana lamellae (GL) using three separate micrographs and we take three grana lamellae per micrographs for each treatment (n = 9).