Spatial regulation of alpha-galactosidase activity and its influence on raffinose family oligosaccharides during seed maturation and germination in Cicer arietinum

Abstract

Alpha-galactosides or Raffinose Family Oligosaccharides (RFOs) are enriched in legumes and are considered as anti-nutritional factors responsible for inducing flatulence. Due to a lack of alpha-galactosidases in the stomachs of humans and other monogastric animals, these RFOs are not metabolized and are passed to the intestines to be processed by gut bacteria leading to distressing flatulence. In plants, alpha(α)-galactosides are involved in desiccation tolerance during seed maturation and act as a source of stored energy utilized by germinating seeds. The hydrolytic enzyme alpha-galactosidase (α-GAL) can break down RFOs into sucrose and galactose releasing the monosaccharide α-galactose back into the system. Through characterization of RFOs, sucrose, reducing sugars, and α-GAL activity in maturing and germinating chickpeas, we show that stored RFOs are likely required to maintain a steady-state level of reducing sugars. These reducing sugars can then be readily converted to generate energy required for the high energy-demanding germination process. Our observations indicate that RFO levels are lowest in imbibed seeds and rapidly increase post-imbibition. Both RFOs and the α-GAL activity are possibly required to maintain a steady-state level of the reducing monosaccharide sugars, starting from dry seeds all the way through post-germination, to provide the energy for increased germination vigor.

Keywords: Alpha-galactosidase; RFO; Reducing sugar; Sucrose; chickpea; flatulence; raffinose.

Figure 1.

The seed vigor and the percent change in the vigor during various stages of seed germination is presented. The bars represent the vigor during various stages of germination and line graph represents the percent change in vigor during each germination stage.

Figure 2.

Enzymatic assay of alpha-galactosidase during various stages of chickpea seed maturation and germination expressed as enzyme activity in GalU/milligram of protein. The data is a representation of n = 3, error bars represent standard error of mean.

Figure 3.

Estimation of α-galactosides (a) and sucrose (b) during various stages of chickpea seed maturation and germination. The sugars are expressed as milligram per gram of dry seed powder. The data represents three independent replicates, error bars indicate standard error of mean. Different letters over bars indicate significant differences based on Tukey’s multiple comparison analyses of the amount of sugars in various seed stages or phases of germination. (P < .05). (c) Estimation of raffinose through HPLC followed by mass spectrometry during various stages of chickpea seed maturation and germination. The data represents pooled samples from three biological replicates.

Figure 4.

Localization studies with hand sections of germinating chickpea seeds stained with X-alpha-gal for α-GAL activity. The epidermal layer of the dehusked seed showing α-GAL activity after 16 h of imbibition (a). The growing meristematic tips displaying α-GAL activity after 24HAI (b) and 48HAI (c). Hand section of the green pod (d), cotyledon of yellow pod (e), cotyledon of imbibed seed (f), and root 48HAI (g,h) showing α-GAL activity following staining with the substrate X- α-D-Gal. EP – epidermis, RD – radicle, ED – root endodermis, and VS – vascular tissue.

Figure 4.

Localization studies with hand sections of germinating chickpea seeds stained with X-alpha-gal for α-GAL activity. The epidermal layer of the dehusked seed showing α-GAL activity after 16 h of imbibition (a). The growing meristematic tips displaying α-GAL activity after 24HAI (b) and 48HAI (c). Hand section of the green pod (d), cotyledon of yellow pod (e), cotyledon of imbibed seed (f), and root 48HAI (g,h) showing α-GAL activity following staining with the substrate X- α-D-Gal. EP – epidermis, RD – radicle, ED – root endodermis, and VS – vascular tissue.

Figure 5.

Localization studies with hand sections of chickpea seeds 48HAI stained with X-alpha-gal for α-GAL activity. α-GAL activity in root endodermis enclosing the stele (a), epidermal and endodermal root sections 48HAI (40X) (b & c), endodermis of soil-grown older root (40X) (d), and tissue prints of root cross sections on PVDF membrane and stained for α-GAL activity showing two concentric circles representing epidermis and endodermis of the root (e & f).

Figure 5.

Localization studies with hand sections of chickpea seeds 48HAI stained with X-alpha-gal for α-GAL activity. α-GAL activity in root endodermis enclosing the stele (a), epidermal and endodermal root sections 48HAI (40X) (b & c), endodermis of soil-grown older root (40X) (d), and tissue prints of root cross sections on PVDF membrane and stained for α-GAL activity showing two concentric circles representing epidermis and endodermis of the root (e & f).

Figure 6.

Steady-state level of reducing sugar concentration during various stages of chickpea seed maturation and germination. The sugars are expressed as milligram per gram of seed powder. The data represents three independent replicates, error bars indicate standard error of mean. Different letters over bars indicate significant differences based on Tukey’s multiple comparison analyses of the amount of reducing sugars in various seed stages or phases of germination. (P < .05).