Regulation of storage protein synthesis by nitrogen and sulfur nutrients in alfalfa ( Medicago sativa L.) somatic embryos

Abstract

The low vigor of seedlings from dry somatic embryos of alfalfa ( Medicago sativa L.) limits the establishment of plants from artificial seeds. slow growth and development may be associated with low storage protein deposition in the somatic embryo. The nutritional regulation of storage protein accumulation by nitrogen and sulfur nutrients was investigated to improve ultimately the vigor of dry somatic embryos. All somatic embryos were induced from petiole explants on 2,4-dichlorophenoxyacetic acid-containing medium, dispersed in a suspension, sieved, and developed to the torpedo stage before application of the treatments. During the period of reserve deposition (phase I maturation), different forms and levels of nitrogen and sulfur nutrients were supplied. Subsequently, all somatic embryos were matured on phase II maturation medium for induction of desiccation tolerance, and then dried at a relative humidity of 43%. Somatic embryo quality was assessed by germination (root elongation) and conversion (root and leaf development) after imbibition on moist filter paper. The addition of 50 mM glutamine to the maturation medium prior to autoclaving enhanced accumulation of the sulfur-poor 7S alfin and 11S medicagin storage proteins. Autoclaving the medium converted glutamine to pyroglutamic acid (5-oxoproline) and ammonia. Addition of these end-products simulated the autoclaved-glutamine effect, whereas filter-sterilized glutamine promoted cell expansion and precocious germination. The addition of inorganic sulfates (10–25 mM) increased the accumulation of the sulfur-rich 2S storage proteins and the B 1 and B 2 subunits of the 11S medicagin. The combination of pyroglutamic acid, ammonium and sulfate in the maturation phase I medium dramatically increased accumulation of the 2S proteins and all subunits of the 11S medicagin. Application of ammonium nitrate as the major inorganic nitrogen source did not replace the requirement for pyroglutamic acid. The results indicate that pyroglutamic acid, ammonia and sulfate had nutritive and regulatory roles in storage protein accumulation. The increased accumulation of storage proteins, especially low molecular weight 2S and 11S medicagin, was correlated positively with quality of somatic embryos. The sulfur nutrients had an additional unexpected effect. Somatic embryos that were matured on high sulfate containing media were unable to germinate unless they were desiccated, indicating some form of metabolically-imposed quiescence. This confirms that sulfate has an important regulatory role in somatic embryo development.