Comparative analysis of electron microscopic structure of seed testa, lignin and biosynthesis-related enzyme activities in hulled and hull-less seeds of Cucurbita moschata

The hull-less seed (thin-coated seeds) Cucurbita moschata is an excellent material for the food and seed oil industry; however, there are very few studies on this important trait. Here, using a scanning electronic microscope, we investigated the anatomical structure of the seed testa in two C. moschata inbred lines with hulled and hull-less seeds. We found that the seed coat of hulled seeds possessed a typical five-layer structure including, from the outside to inside, the epidermis (E), hypodermis (H), sclerenchyma (S), aerenchyma (A) and the chlorenchyma (C). During seed development, significant differences were observed in the seed testa between hulled and hull-less seeds. In the seed coat of hulled seeds, with seed development, there was increasing lignification in the S and H layers which was lacking in that of hull-less seeds. Instead, in the seed coat of hull-less seeds, after 15 days post anthesis(DPA), the five layers exhibited successive degradation, and were devoid of vascular bundle and lipid droplets. The lignin content in the testa of hull-less seeds also showed continued decrease over time, but continued to increase in that of hulled seeds, which was 0.22 mg/g and 64.22 mg/g at 50 DPA for the former and latter, respectively, suggesting lignin plays a critical role in the formation of seed testa. We examined the activities of four enzymes that are important for lignin biosynthesis and found that, consistent with the declined lignin content, there were also decreased activities in the seed coat for the phenylalanine ammonia lyase (PAL), 4-coumaric acid coenzyme A ligase (4CL), cinnamate-4-hydroxylase (C4H), and cinnamyl alcohol dehydrogenase (CAD). We conclude that degeneration or absence of the typical seed testa structure in hull-less seeds of C. moschata is due to reduced activity of key enzymes in the lignin biosynthesis pathway.