Long branch-chains of amylopectin with B-type crystallinity in rice seed with inhibition of starch branching enzyme I and IIb resist in situ degradation and inhibit plant growth during seedling development : Degradation of rice starch with inhibition of SBEI/IIb during seedling development

Abstract

Background: Endosperm starch provides prime energy for cereal seedling growth. Cereal endosperm with repression of starch branching enzyme (SBE) has been widely studied for its high resistant starch content and health benefit. However, in barley and maize, the repression of SBE changes starch component and amylopectin structure which affects grain germination and seedling establishment. A high resistant starch rice line (TRS) has been developed through inhibiting SBEI/IIb, and its starch has very high resistance to in vitro hydrolysis and digestion. However, it is unclear whether the starch resists in situ degradation in seed and influences seedling growth after grain germination.

Results: In this study, TRS and its wild-type rice cultivar Te-qing (TQ) were used to investigate the seedling growth, starch property changes, and in situ starch degradation during seedling growth. The slow degradation of starch in TRS seed restrained the seedling growth. The starch components including amylose and amylopectin were simultaneously degraded in TQ seeds during seedling growth, but in TRS seeds, the amylose was degraded faster than amylopectin and the amylopectin long branch-chains with B-type crystallinity had high resistance to in situ degradation. TQ starch was gradually degraded from the proximal to distal region of embryo and from the outer to inner in endosperm. However, TRS endosperm contained polygonal, aggregate, elongated and hollow starch from inner to outer. The polygonal starch similar to TQ starch was completely degraded, and the other starches with long branch-chains of amylopectin and B-type crystallinity were degraded faster at the early stage of seedling growth but had high resistance to in situ degradation during TRS seedling growth.

Conclusions: The B-type crystallinity and long branch-chains of amylopectin in TRS seed had high resistance to in situ degradation, which inhibited TRS seedling growth.

Keywords: In situ degradation; Resistant starch; Rice; Seedling growth; Starch properties.

Fig. 1

Photographs of rice seedlings

Fig. 2

Shoot height (a) and the dry weight of shoot (b) and root (c) on 30 seeds basis during seedling growth. Values are means ± SD from 90 seedlings for (a) and three replicates for (b and c). Asterisks (*) highlight significant differences between TQ and TRS by Student’s t test (**P < 0.01; ***P < 0.001)

Fig. 3

In vitro culture of mature embryo. (a), the excised embryos from TQ and TRS mature seeds were separately placed in normal MS medium; (b), seedlings at 6 days after in vitro culture, the scale bar = 1 cm; (c), the shoot height at 6 days after culture, values are means ± SD from 30 seedlings

Fig. 4

Seed and starch weights and their relationships with seedling weight on 30 seeds basis during seedling growth. (a), dry weight of seed without embryo; (b, c), the relationships between the decreased seed weight and the root (a), shoot (b), and seedling (root + shoot) weight (c) in TQ (b) and TRS (c); (d), dry weight of starch in endosperm; (e, f), the relationships between the decreased starch weight and the root (a), shoot (b), and seedling (soot + shoot) weight (c) in TQ (e) and TRS (f). For (a and d), values are means ± SD from three replicates, and asterisks (*) highlight significant differences between TQ and TRS by Student’s t test (**P < 0.01; ***P < 0.001). For (b, c, e, and f), values are the means of three replicates, and R indicates the regression coefficient

Fig. 5

The soluble sugar content of endosperm on 30 seeds basis (a) and on the weight basis of pre-germinated seeds (b) during seedling growth. Values are means ± SD from three replicates, and asterisks (*) highlight significant differences between TQ and TRS by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 6

GPC chromatogram of isoamylase-debranched starch isolated from endosperm at different days after imbibition. The arrows indicate the main differences in GPC chromatogram between TQ and TRS

Fig. 7

XRD pattern of starch isolated from endosperm at different days after imbibition. The arrows indicate the main differences in XRD pattern between TQ and TRS

Fig. 8

FTIR pattern of starch isolated from endosperm at different days after imbibition. The arrows indicate the main differences in FTIR spectrum between TQ and TRS

Fig. 9

In situ degradation of starch granule in endosperm at different days after imbibition. (A-E): TQ seed; (B-E): the magnification of germinated seeds at 8 DAI; (a-e): TRS seed; (b-e): the magnification of germinated seeds at 16 DAI. The whole seed longitudinal section was counterstained with periodic acid-Schiff’s and toluidine blue O. Scale bar = 1 mm (A, a), 500 μm (B, b), 100 μm (C, c), and 20 μm (D, E, d, e)