Understanding types and number of genes differentially expressed during seed maturation would help in discerning molecular mechanisms contributing to nutritional superiority, including high rutin content in the seeds of F. tataricum (rice-tartary buckwheat) over F. esculentum. De-novo sequencing of transcripts from flowers of Fagopyrum species ( F. tataricum and F. esculentum) had shown that genes contributing to different biological processes are contributing to variations in the morphology of flowers in Fagopyrum species . Accumulation of higher amounts of rutin during post-flowering stages of F. tataricum (rice-tartary buckwheat) over F. esculentum has also been observed . Overall nutritional superiority of F. tataricum over other Fagopyrum spp has been found in the mature seeds as well as during seed maturation stages. For example, increase in fagopyritols content was shown from immature to mature seeds of buckwheat . In addition, increase in the amount of sucrose and rutin was also reported during seed maturation of Fagopyrum species [15, 20]. Molecular dissection of the genetic machinery contributing to nutritional differences in the seeds of F. tataricum, in particular the rice-tartary type over F. esculentum (common buckwheat) was undertaken through cDNA-AFLP since it is an open architecture technology for global transcriptional analysis in a non-model plant species [21, 22].
Large numbers of TDFs with differential expression pattern were observed in different seed developmental stages of both the Fagopyrum species. TDFs with differential expression patterns belonged to genes involved in primary and secondary metabolism, transportation, signal transduction, gene regulation, photosynthesis & energy, defense and cellular processes in seed developmental stages of the Fagopyrum spp.; Additional file 1: Table S1. Out of 167 differential TDFs, only 22 showed 50–70% identity with the available floral transcriptome of both the Fagopyrum species , thereby, suggesting that most of the TDFs identified in the current study represented new genes. TDFs involved in transport, transcription, secondary metabolism, amino acid & protein metabolism, carbohydrate metabolism and photosynthesis were relatively higher in number and expression pattern in rice-tartary over common buckwheat. Higher expression of TDFs involved in secondary metabolism and transportation such as chalcone synthase, dihydroflavonol reductase, UDP glucosyl transferases, ABC transporters, MATE efflux carrier proteins, which are known to be involved in biosynthesis, accumulation and transportation of flavonoids, indicate their involvement in significantly higher flavonoid content in rice-tartary buckwheat [14, 16, 23]. Increased transcript abundance of TDF encoding Lys/His transporter in rice-tartary buckwheat was implicated for higher amount of histidine in this species . TDFs involved in amino acid & protein metabolism (like Ub protein ligases, alanine glyoxylate amino transferases, cystein proteases), transcriptional regulation (MYB 118, MYB 112, GAMYB, histone acetyl transferases) and signal transduction (calmodulin binding protein, protein kinases, PEP carboxylase) were also found to be abundant in rice-tartary buckwheat. Most of these TDFs represent genes with their direct or indirect role in controlling the growth and development of seeds and/or their nutritional composition. On the basis of differential expression pattern of transcripts in rice-tartary and common buckwheat, the TDFs representing genes which have been implicated in biosynthesis, modification, regulation and transport of secondary metabolites [16, 18, 23] were chosen to investigate their role, through qRT-PCR analysis, in the biosynthesis of higher rutin content in the seeds of rice-tartary over common buckwheat.
The flavonoid content increase in buckwheat seedlings has been attributed to the increase in the concentration of sucrose . In addition, sugars also act as developmental signals regulating seed maturation and accumulation of flavonoids in plants such as Arabidopsis, V. vinifera[26–28]. Exponential increase in the transcript of a sugar transporter (JN982735) from flowers (S6) to immature seeds (S7) of rice-tartary compared to common buckwheat suggests its contribution in higher content of flavonoids and fagopyritols in the seeds of rice-tartary buckwheat. Relatively increased transcript abundance of auxin efflux carrier protein (JN982731) in different seed maturation stages (S8 & S9) of F. esculentum suggests its negative role in the biosynthesis of flavonoids, which are present in lower amounts in different growth stages of F. esculentum. Flavonoids have been implicated as inhibitors of auxin transport in Arabidopsis[18, 29]. The ABC and MATE classes of transporters are known to be involved in the transport of flavonoids from cytosol into vacuoles [16, 23, 30]. In present study the abundance of TDF ABC transporter (JN982732) was relatively higher in flowering to seed maturation stages, S6 (4.7x), S7 (95.4x), and S8 (2.4x) of rice-tartary buckwheat compared to common buckwheat suggested that this gene might be playing a key role in the transport of flavonoids (rutin, quercetin and quercitrin) in rice-tartary buckwheat. Also, it has been shown that biosynthesis of flavonoids takes place in lower parts of Fagopyrum spp. and then gets transported to upper parts .
Significantly higher expression of 4 TDFs representing genes for ubiquitin protein ligase, ABC transporter, sugar transporter and calmodulin binding protein in S7 of rice-tartary buckwheat compared to F. esculentum suggests their major involvement in nutritional superiority of rice-tartary buckwheat. The calmodulin binding proteins regulate diverse cellular processes by interacting with other proteins and help in secondary metabolism by acting as secondary messengers through signal transduction [31, 32]. In addition, calmodulin proteins are also known to induce anthocyanin biosynthesis in V. vinifera. Extensins, the major structural proteins in plant cell wall play important role in various biological processes such as embryo development, root hair growth, seed coat development, defense, etc. [34, 35]. Increased transcript abundance of extensin protein (JQ003863) in mature seeds of rice-tartary buckwheat in comparison to common buckwheat may contribute to development of seeds of both the Fagopyrum species.
Transcription factors are known to play important role in various seed development processes and regulation of secondary metabolism [36–38]. Zn finger binding proteins (JN982723) have been implicated in regulation of important biological processes such as flower and seed development, seed germination, stress tolerance in Arabidopsis[39, 40]. Expression of a TDF representing a gene for Zn finger binding protein was relatively higher in seed developing stages S8 and S9 of common buckwheat compared to rice-tartary buckwheat. Transcript of a TDF encoding for another transcription factor MYB 118 (JN982734) was high in the flowers (S6) of rice-tartary compared to common buckwheat.
Ub protein ligases (JN982742) are known to regulate various biological processes like photomorphogenesis, hormonal response, senescence, floral, embryo and seedling development through degradation of proteins as well as regulates phenylpropanoid pathway during UV stress and sugar signaling during seedling development [41–43]. Relatively higher expression of Ub protein ligase in immature seeds (S7) of rice-tartary buckwheat has been observed in the present study. Significantly higher expression of most of the selected TDFs during early seed formation stage (S7) of rice-tartary buckwheat (Figure 6E) reflects their biological importance in maintaining higher amounts of rutin, which otherwise drops significantly in the same stage of F. esculentum.