ABSTRACT
Raffinose, stachyose and verbascose form the three major members of the raffinose family oligosaccharides (RFO) accumulated during seed development. Raffinose synthase (RS; EC 2.4.1.82) and stachyose synthase (STS; EC 2.4.1.67) have been associated with raffinose and stachyose synthesis, but the precise mechanism for verbascose synthesis is not well understood. In this study, full-length RS (2.7 kb) and STS (2.6 kb) clones were isolated by screening a cDNA library prepared from developing lentil seeds (18, 20, 22 and 24 days after flowering [DAF]) to understand the roles of RS and STS in RFO accumulation in developing lentil seeds. The nucleotide sequences of RS and STS genes were similar to those reported for Pisum sativum. Patterns of transcript accumulation, enzyme activities and RFO concentrations were also comparable to P. sativum. However, during lentil seed development raffinose, stachyose and verbascose accumulation corresponded to transcript accumulation for RS and STS, with peak transcript abundance occurring at about 22-24 DAF, generally followed by a sequential increase in raffinose, stachyose and verbascose concentrations followed by a steady level thereafter. Enzyme activities for RS, STS and verbascose synthase (VS) also indicated a sudden increase at around 24-26 DAF, but with an abrupt decline again coinciding with the subsequent steady state increase in the RFO. Galactan:galactan galactosyl transferase (GGT), the galactinol-independent pathway enzyme, however, exhibited steady increase in activity from 24 DAF onwards before abruptly decreasing at 34 DAF. Although GGT activity was detected, isolation of a GGT sequence from the cDNA library was not successful.
Subject(s)
Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Lens Plant/enzymology , Lens Plant/genetics , Oligosaccharides/biosynthesis , Raffinose/biosynthesis , Seeds/growth & development , Gene Expression Regulation, Plant , Genes, Plant , Lens Plant/growth & development , Oligosaccharides/genetics , Raffinose/genetics , Seeds/enzymology , Seeds/geneticsABSTRACT
Galactinol synthase (GS, EC 2.4.1.123) catalyzes the transfer of a galactosyl residue from UDP-galactose to myo-inositol to synthesize galactinol, a precursor for raffinose family oligosaccharides (RFO) biosynthesis. Screening, a cDNA library constructed with RNA isolated from developing lentil seeds, with partial GS genes resulted in identification of cDNA clones for two isoforms of GS, LcGolS1 (1336 bp, ORF-1002 bp, 334 amino acids) and LcGolS2 (1324bp, ORF-975bp, 325 amino acids) with predicted molecular weights of 38.7 kDa and 37.6 kDa, respectively. During lentil seed development, LcGolS1 transcripts showed higher accumulation during 26-32 days after flowering (DAF) corresponding to seed desiccation, while LcGolS2 showed maximum accumulation at 24 DAF, prior to increase in LcGolS1 transcripts. GS enzyme activity was maximum at 26 and 28 DAF and corresponded to galactinol accumulation, which also increased rapidly at 22 DAF with maximum accumulation at 26 DAF. Substrates for GS activity, myo-inositol and glucose/galactose were present in high concentrations during early stages of seed development but gradually decreased from 20 DAF to 32 DAF when galactinol concentration increased coinciding with increased GS enzyme activity.
Subject(s)
Galactosyltransferases/metabolism , Lens Plant/enzymology , Plant Proteins/metabolism , Seeds/enzymology , Seeds/growth & development , Cloning, Molecular , DNA, Complementary , Disaccharides/metabolism , Galactosyltransferases/chemistry , Galactosyltransferases/genetics , Gene Expression Regulation, Plant , Gene Library , Isoenzymes/genetics , Isoenzymes/metabolism , Lens Plant/genetics , Lens Plant/growth & development , Phylogeny , Plant Proteins/genetics , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/standards , Protein Conformation , Reference Standards , Reproducibility of Results , Seeds/geneticsABSTRACT
Podostemaceae is an interesting family of angiosperms with unusual development and morphology. Among these, double fertilization, a defining feature of angiosperms is invariably missing in the family. Consequently, embryo development in the seeds takes place without endosperm. In recent years, the role of polycomb genes has garnered much interest because of their crucial role in seed development. Some of these genes have been reported from many unrelated species, underlining their high conservation. Thus, it becomes exciting to know the role of these genes in podostemads, which are devoid of double fertilization and endosperm. Here, we report the isolation, characterization and expression patterns of homologs of Fertilization Independent Endosperm (FIE) in two species of Podostemaceae, Zeylanidium olivaceum and Polypleurum stylosum. FIE like homologs could be identified in Z. olivaceum (ZoFIE) and P. stylosum (PsFIE). The predicted amino acid sequence of FIE homologs showed similarity to other homologs, containing the conserved seven WD40 repeats. Expression studies revealed that ZoFIE and PsFIE transcripts were present in the vegetative tissue (thallus in Podostemaceae) and the seedlings, similar to the model plants. However, the ZoFIE and PsFIE expression disappeared in the flowering stages. This unique pattern of expression suggests that in the absence of double fertilization and endosperm the expression of FIS complex genes perhaps is obliterated in Podostemaceae.
