Coordinated changes in storage proteins during development and germination of elite seeds of Pongamia pinnata, a versatile biodiesel legume

Abstract

The oleaginous legume Pongamia pinnata is a rapidly growing and economically important tree. The seeds are used increasingly as feedstock for biodiesel production, with the protein-rich residue providing valuable supplement to farm animal diets. However, little is known about seed development and the characteristics of germination. We therefore studied morphological, protein and ultrastructural changes during seed maturation and germination using seeds from a tree selected for superior morphological and reproductive characters (candidate plus tree). Phenology, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and scanning and transmission electron microscopy were used to investigate seed development from 90 to 350 days after flowering (DAF), and germination and seedling development from 0 to 45 days after the start of imbibition (DAI) (Stages 0-VII). Seven distinct developmental stages were identified during seed development. Fresh weight, length, breadth and thickness increased from Stage I (90 DAF) to V (270 DAF) and decreased at Stages VI (315 DAF) and VII (350 DAF), when the seeds were fully ripe. Marked changes in total soluble protein content and SDS-PAGE profile were observed in vegetative and reproductive tissues and in the cotyledons of germinating seedlings. Polypeptide fragments of 150-14 kDa were observed during seed maturation and germination. In SDS-PAGE the expression of three main polypeptide bands (50, 18 and 14 kDa) increased from Stage I to Stage V and then almost became the same until Stage VII during seed maturation. During germination the expression of 50 kDa polypeptide decreased and that of 18 and 14 kDa increased from Stage 0 (ungerminated seed) to Stage VI (30 DAI), respectively; however, all three polypeptides (50, 18 and 14 kDa) completely disappeared at Stage VII (45 DAI). Ultrastructural changes during four stages of seed maturation (early immature, 90-135 DAF; late immature, 180-225 DAF; early mature, 225-270 DAF; and late mature, 315-350 DAF) and three stages of germination and seedling development (early 10 DAI to late 45 DAI) localized marked gradients in protein storage reserves. Increasing the knowledge base for P. pinnata, especially for its seeds, is an essential prerequisite for rapid and successful exploitation of this promising energy and animal feed crop. Our findings contribute to this by establishing key developmental features of the seeds as they form and germinate.