Rice Soluble Starch Synthase I: Allelic Variation, Expression, Function, and Interaction With Waxy

Abstract

Starch, which is composed of amylose and amylopectin, is the key determinant of rice quality. Amylose is regulated by the Waxy (Wx) gene, whereas amylopectin is coordinated by various enzymes including eight soluble starch synthases (SSSs), of which SSSI accounts for ∼70% of the total SSS activity in cereal endosperm. Although great progress has been made in understanding SSSI gene expression and function, allelic variation and its effects on gene expression, rice physicochemical properties and qualities, and interactions with the Wx gene remain unclear. Herein, SSSI nucleotide polymorphisms were analyzed in 165 rice varieties using five distinct molecular markers, three of which reside in an SSSI promoter and might account for a higher expression of the SSSIⁱ allele in indica ssp. than of the SSSI^j allele in japonica ssp. The results of SSSI promoter-Beta-Glucuronidase (β-GUS) analysis were consistent with the expression results. Moreover, analysis of near isogenic lines (NILs) in the Nipponbare (Nip) background showed that Nip (SSSIⁱ ) and Nip (SSSI^j ) differed in their thermal properties, gel consistency (GC), and granule crystal structure. Knockdown of SSSI expression using the SSSI-RNA interference (RNAi) construct in both japonica and indica backgrounds caused consistent changes in most tested physicochemical characteristics except GC. Moreover, taste value analysis (TVA) showed that introduction of the SSSI allele in indica or knockdown of SSSI expression in japonica cultivars significantly reduced the comprehensive taste value, which was consistent with the superior taste of japonica against indica. Furthermore, to test the potential interaction between SSSI and different Wx alleles, three NILs within the Wx locus were generated in the indica cv. Longtefu (LTF) background, which were designated as LTF (Wx^a ), LTF (Wx^b ), and LTF (wx). The SSSI-RNAi construct was also introduced into these three NILs, and physiochemical analysis confirmed that the knockdown of SSSI significantly increased the rice apparent amylose content (AAC) only in the Wx^a and Wx^b background and caused different changes in GC in the NILs. Therefore, the effect of SSSI variation on rice quality also depends on its crosstalk with other factors, especially the Wx gene. These findings provide fundamental knowledge for future breeding of rice with premium eating and cooking qualities.

Keywords: SSSI; allelic variation; eating and cooking quality; physiochemical properties; rice (Oryza sativa L.); soluble starch synthase; waxy.

FIGURE 1

Schematic map of allelic variation of Soluble Starch Synthases I (SSSI) (A) and expression of SSSI in different rice varieties (B). Total RNA was extracted from caryopses at 10 DAF and analyzed. A housekeeping gene Actin1 was used as an internal control.

FIGURE 2

T-DNA structure of the pSSSI::GUS construct (A) and GUS activity in the endosperm of pSSSI::GUS transgenic rice (B). WT indicates the untransformed wild-type plant. The seed samples analyzed here were collected at 10 DAF. Error bars represent the standard error.

FIGURE 3

Generation and physicochemical analysis of SSSI-RNAi transgenic rice in both japonica and indica backgrounds. (A) An SSSI-RNAi construct used for suppression of SSSI expression in rice. (B) Confirmation of transgenic rice by PCR. (C) Expression of SSSI analyzed by northern blotting. Apparent Amylose Content (AAC) (D), Gel Consistency (GC) (E), and Rapid Visco Analyzer (RVA) (F) analysis of SSSI-RNAi transgenic rice in the Nip (japonica) background. AAC (G), GC (H), and RVA (I) analysis of SSSI-RNAi transgenic rice in the LTF (indica) background. Asterisks indicate significant differences according to Student’s t-tests (^∗∗p < 0.01).

FIGURE 4

Rapid Visco Analyzer (RVA) and Differential Scanning Calorimeter (DSC) analysis of rice in following suppression of SSSI expression or allelic substitution. RVA (A) and DSC (C) analysis of SSSI-RNAi transgenic rice and the NIL-SSSIⁱ line in the Nip background. RVA (B) and DSC (D) analysis of SSSI-RNAi transgenic rice in the LTF background. The SSSI-RNAi transgenic lines in Nip-SSSI^j and LTF-SSSIⁱ genetic background were designated as Nip-SSSI^j- and LTF-SSS^i-, respectively.

FIGURE 5

Changes in rice starch chain length distribution following suppression of SSSI expression or allelic substitution. GPC analysis of native (A) and debranched (C) starch from SSSI-RNAi transgenic rice and the NIL-SSSIⁱ line in the Nip background. GPC analysis of native (B) and debranched (D) starch from SSSI-RNAi transgenic rice in the LTF background.

FIGURE 6

Changes in rice starch crystallinity following suppression of SSSI expression or allelic substitution. X-ray Powder Diffraction (XRD) (A) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) (C) analysis of SSSI-RNAi transgenic rice and the NIL-SSSIⁱ line in the Nip background. XRD (B) and ATR-FTIR (D) analysis of SSSI-RNAi transgenic rice in the LTF background.

FIGURE 7

Study the potential genetic interaction of SSSI and different alleles of Wx gene. The changes of AAC (A) and GC (B) of rice in response to decreased SSSI expression under different Wx allele background. Asterisks indicate significant differences according to Student’s t-tests (^∗∗p < 0.01).