Deep EST profiling of developing fenugreek endosperm to investigate galactomannan biosynthesis and its regulation

Galactomannans are hemicellulosic polysaccharides composed of a (1 → 4)-linked β-D-mannan backbone substituted with single-unit (1 → 6)-α-linked D-galactosyl residues. Developing fenugreek (Trigonella foenum-graecum) seeds are known to accumulate large quantities of galactomannans in the endosperm, and were thus used here as a model system to better understand galactomannan biosynthesis and its regulation. We first verified the specific deposition of galactomannans in developing endosperms and determined that active accumulation occurred from 25 to 38 days post anthesis (DPA) under our growth conditions. We then examined the expression levels during seed development of ManS and GMGT, two genes encoding backbone and side chain synthetic enzymes. Based on transcript accumulation dynamics for ManS and GMGT, cDNA libraries were constructed using RNA isolated from endosperms at four ages corresponding to before, at the beginning of, and during active galactomannan deposition. DNA from these libraries was sequenced using the 454 sequencing technology to yield a total of 1.5 million expressed sequence tags (ESTs). Through analysis of the EST profiling data, we identified genes known to be involved in galactomannan biosynthesis, as well as new genes that may be involved in this process, and proposed a model for the flow of carbon from sucrose to galactomannans. Measurement of in vitro ManS and GMGT activities and analysis of sugar phosphate and nucleotide sugar levels in the endosperms of developing fenugreek seeds provided data consistent with this model. In vitro enzymatic assays also revealed that the ManS enzyme from fenugreek endosperm preferentially used GDP-mannose as the substrate for the backbone synthesis.