Thermal Properties, Morphology of Starch Granules and Crystallinity of Endosperm Starch in SSI and BE Isozymes Double Mutant Lines

Natsuko Abe; Yasunori Nakamura; Naoko Fujita

doi:10.5458/jag.jag.JAG-2013_003

Abstract

Starch is an insoluble glucose polymer that forms semi-crystalline granules. Insight into the relationships between the physicochemical properties of starch and its structure is limited. A deeper knowledge of these relationships is necessary for understanding the starch properties of mutant lines. Starch synthase (SS) and starch branching enzyme (BE) play central roles in the biosynthesis of starch. To explore the relationships between the physicochemical properties of starch, morphology of starch granules, starch crystallinity, and the function of SS and BE isozymes expressed in rice endosperm, we analyzed these traits in starches isolated from the double mutant lines [ss1/be1 and ss1^L/be2b (ss1^L means leaky ss1 mutant)]. Deficiency of SSI and BEI lead to an increase and decrease in gelatinization temperature, respectively. A significant increase in gelatinization temperature results from deficiency of BEIIb, as has been shown in previous studies in rice and maize, and this phenomenon is pronounced in the reduction of SSI activity. Deficient BEIIb expression also had a significant impact on the morphology of starch granules in the endosperm and changed the A-type diffraction pattern of the granules to a B-type pattern, while deficiency of SSI and/or BEI did not affect these traits.

Abbreviations

DP, degree of polymerization; DSC, differential scanning calorimeter; SEM, Scanning electron microscopy; SS, starch synthase; BE, branching enzyme.

INTRODUCTION

Starch consists of two homopolymers of α-D-glucosyl units: amylopectin, which is a large highly-branched molecule, and amylose, which is a primarily linear molecule whose content strongly affects the physiochemical properties of starch. At least four enzyme classes comprised of multiple isozymes are related to starch biosynthesis: ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (BE) and starch debranching enzyme (DBE).¹⁾ ²⁾ ³⁾ ⁴⁾ Of these four enzymes, SS and BE play central roles in starch biosynthesis. A good deal of information on the function of each isozyme has been gained from analyses of mutant lines deficient in one or more of these starch biosynthesis-related isozymes. However, insight into the relationships between the physicochemical properties of starch, including thermal and pasting properties of gelatinized starch, and starch structure is limited.⁵⁾ ⁶⁾ ⁷⁾ ⁸⁾ ⁹⁾ A deeper understanding of these relationships is necessary for understanding the starch properties observed in mutant lines.

SSI generates chains with degree of polymerization (DP) 8‒12 from very short DP 6‒7 chains emerging from the branch point in the A and B₁ chains of amylopectin.⁷⁾ A BEI-deficient mutant was equivalent to the wild type with respect to the starch accumulation, whereas the chain-length distribution of amylopectin was different, with a reduction in both long chains with DP ≥ 37 and short chains with DP 12‒21, and an increase in short chains with DP ≤ 10 and in middle chains with DP 24‒34.¹⁰⁾ Relatively mild changes were observed between SSI- and BEI-deficient mutants and wild-type rice, although BEIIb deficiency produced a marked impact on the traits of endosperm starch; BEIIb-deficient rice exhibited a significant decrease in amylopectin short chains with DP ≤ 13. This resulted in a dramatic resistance to gelatinization.¹¹⁾

Double mutant lines [ss1/be1 and ss1^L/be2b (ss1^L means leaky ss1 mutant)] between SSI, which is the largest components of total soluble SS activity, and BEI or BEIIb, BEs that are expressed strongly in developing rice endosperm, were generated by crossing between single mutant lines to examine the function of each isozyme (Abe et al., submitted). The amylopectin structures of these double mutant lines were analyzed and compared to those of the parent lines. Seeds from ss1/be1 mutants were divided into translucent and white core morphologies. F₃ seeds generated by self-pollination of double recessive F₂ plants were segregated translucent (～28%) and white core (～72%) although this ratio varies depending on the different growth years. However, the seeds of both phenotypes were deficient in SSI and BEI, indicating that the seed phenotypes could depend on the environmental condition (Abe et al., submitted). Analysis of the chain-length distribution from ss1/be1 endosperm amylopectin showed an additive effect of loss of SSI activity on the chain-length of synthesized amylopectin in a be1 background. Analysis of ss1^L/be2b amylopectin showed that the short chains of amylopectin were significantly reduced due to BEIIb deficiency. Although the apparent amylose content of the be2b mutant was much higher (29.4%) than that of the wild type, that of ss1/be1 and ss1^L/be2b endosperm starch was nearly the same as that of the wild type (21.3‒21.6%) (Abe et al., submitted).

In the present study, we analyzed starches isolated from these double mutant lines (ss1/be1 and ss1^L/be2b) to explore the relationships between the physicochemical properties, morphology of starch granules, starch crystallinity, and function of SS and BE isozymes expressed in rice endosperm.

MATERIALS AND METHODS

Plant materials. Two double mutant lines, ss1/be1 (#4011) and ss1^L/be2b (#4017), were generated from crossing (Abe et al., submitted) SSI-deficient (ss1, e7)⁷⁾ and BEI-deficient (be1, EM557)¹⁰⁾ lines and SSI-leaky (ss1^L, i2-1)⁷⁾ and BEIIb-deficient (be2b, EM10)¹¹⁾ lines, respectively. The two phenotypes, translucent and white core seeds of ss1/be1, were combined and used for the analyses in this study, except for the observation of morphology of amyloplasts and starch granules by scanning electron microscope. The parental cultivar Nipponbare (Nip) (the parent of e7 and i2-1), Kinmaze (Kin) (the parent of EM10) and Taichung 65 (T65) (the parent of EM557) were used as controls. Rice plants were grown during the summer months in an experimental paddy field at Akita Prefectural University under natural environmental conditions.

Thermal properties of endosperm starch. The thermal properties of endosperm starch were determined by differential scanning calorimetry (DSC6100, Seiko Instruments Inc., Japan) according to the methods described in a previous report.⁷⁾

SEM observation. Scanning electron microscopy (SEM) observation of purified starch granules and cross-sectioned seeds was performed using the methods described in a previous report.⁸⁾ ¹²⁾

X-ray diffraction. X-ray diffraction was performed as described previously.¹³⁾ The crystallinity of each endosperm starch from the mutant lines was determined by the method described in a previous report¹⁴⁾ using an internal standard.

RESULTS AND DISCUSSION

Thermal properties of endosperm starches from double-recessive mutant lines.

The gelatinization temperature of starches is affected primarily by the chain-length distribution of amylopectin.⁷⁾ ¹⁵⁾ ¹⁶⁾ ¹⁷⁾ The crystalline domains of starch granules are composed of the A chains and the exterior parts of the B chains of amylopectin, which have an average length in the range of DP 12‒16.¹⁸⁾ The abundance or shortage of DP ≥ 12‒16 exterior chains (long chains) within one cluster (DP ≤ 24) results in an increase or decrease, respectively, in the starch gelatinization temperature.⁷⁾ The typical DSC curve and thermal properties [onset temperature (T_o), peak temperature (T_p), conclusion temperature (T_c) and gelatinization enthalpy (ΔH)] of purified starches from various mutant lines and the wild type are shown in Fig. 1 and Table 1, respectively. In the ss1 mutant, amylopectin chains with DP 6‒7 and DP 16‒19 were increased and those with DP 8‒12 were decreased compared to the wild type.⁷⁾ It is possible that the abundance of DP 6‒7 contributes to the reduction in gelatinization temperature, while the shortage of DP 8‒12 and the abundance of DP 16‒19 contribute to the increase in gelatinization temperature, implying that overall, the gelatinization temperature of ss1 starch would increase compared with the wild type. The actual value of gelatinization temperatures (T_p) of ss1 and ss1^L starches determined by DSC were 3.7 and 4.7°C higher, respectively, than that of the wild type (Table 1). Likewise, in the be1 mutant, amylopectin chains with DP 6‒9 and DP 11‒20 were increased and decreased, respectively, compared to wild-type starch,¹⁰⁾ and these changes in amylopectin structure contributed to a reduction in gelatinization temperature. The actual T_p of be1 was 4.1°C lower than that of the wild type (Table 1), and these data agree with previous reports.⁷⁾ ¹⁰⁾ By contrast, the T_p value of ss1/be1 starch was not consistent with the above theory; chains with DP 6‒9 and DP 11‒20 in ss1/be1 starch were increased and decreased compared with those of ss1 starch (Abe et al., submitted), respectively, which, based on the previous observations, would indicate that the gelatinization temperature of ss1/be1 starch would be lower than that of the ss1 starch. However, the actual T_p of ss1/be1 was higher than that of ss1 (Table 1). These results suggested that factors other than chain-length distribution of amylopectin with DP ≤ 24 affect gelatinization temperature. The degree to which double helices including long chains with DP > 24 affect gelatinization temperature remains unknown. Gelatinization is initiated by the absorption of water. Thereafter, the double helices of amylopectin chains must be unwound. The possibility that these steps, including absorption and swelling, affect gelatinization temperature could not be excluded.

Fig. 1.

Typical differential scanning calorimetric (DSC) curve of various mutant lines and the wild types.

Table. 1.

Thermal properties of endosperm starch in various mutant lines of rice and the wild types as determined by DSC.

^a Onset temperature. ^b Peak temperature. ^c Conclusion temperature. ^d Geletinization enthalpy of starch. ^e The values are the average of at least three replications (mean±SE). *Significant differences between single mutant lines and the WT by t-test at p < 0.05. **Significant differences between the parent mutant lines and the double mutant by t-test at p < 0.05. ***Significant differences between the double mutant line and the WT by t-test at p < 0.05. Nip, Nipponbare; T65, Taichung 65; Kin, Kinmaze.

The T_o, T_p, T_c and ΔH of be2b and ss1^L/be2b starch, whose short chains of amylopectin with DP ≤ 14 were significantly decreased, (Abe et al., submitted)¹¹⁾ ¹⁹⁾ were much higher than those of wild-type starch (Fig. 1 and Table 1) ,¹⁷⁾ ¹⁹⁾ and the T_o, T_p and T_c of ss1^L/be2b starch were higher than that of be2b starch (Table 1). This is due to the slight decrease in chains with DP 8‒13 and increase in chains with DP 16‒24 in ss1^L/be2b compared with be2b.

The raw and retrograded starch of be2b was digested by pancreatic α-amylase in vitro at a markedly slower rate than the wild type,¹⁹⁾ and rats fed be2b starch showed slowly increasing levels blood glucose at a lower amounts than rats fed wild-type starch. Thus, it is possible that ss1^L/be2b starch, which has a higher gelatinization temperature than be2b starch, is more resistant to degradation by α-amylase.

Morphology of amyloplasts and starch granules from double mutant lines.

Cross-sections of seeds (Fig. 2) and purified starch granules (Fig. 3) from various mutant lines and wild-type lines were observed by SEM. Several polygonal starch granules were packed in the amyloplasts of ss1, ss1^L and be1 mutant lines. The size and morphology of the starch granules and amyloplasts of these mutant lines were similar (3‒5 μm) to those of the wild type (Figs. 2 and 3). By contrast, amyloplasts in the white core of the ss1/be1 seeds were loosely packed and rounded, while those from translucent ss1/be1 seeds were similar to those of the wild type (Fig. 2). As described above, seeds from ss1/be1 mutants were divided into white core and translucent morphologies; however, the seeds of both phenotypes were deficient in SSI and BEI (Abe et al., submitted). It is possible that the reason for the white core is the loose packing of amyloplasts. This phenomenon might have occur due to the deficiency of important two isozyme activities (SSI and BEI) in endosperm cells.

Fig. 2.

Representative scanning electron microscopy (SEM) images of the center regions in the cross-sections of mature seeds from various mutant lines and the wild types.

Fig. 3.

Representative SEM images of purified starch granules in various mutant lines and the wild types.

The size and shape of starch granules and amyloplasts of be2b and ss1^L/be2b mutants were quite different from those of the wild type; larger and smaller starch granules were observed and amyloplasts were loosely packed in the endosperm cells of these mutant lines (Figs. 2 and 3). These observations suggest that BEIIb deficiency significantly impacts the morphology of starch granules and amyloplasts, while SSI and/or BEI deficiency do not.

Crystallinity of starch granules from double mutant lines.

Figure 4 shows the X-ray diffraction patterns of each of the mutant lines. Wild-type, ss1, ss1^L, be1 and ss1/be1 starches produced four strong reflections at 2θ values of 15, 17, 18 and 23°, which are typical A-type diffraction patterns (Fig. 4). By contrast, be2b and ss1^L/be2b produced seven strong reflections at 2θ values of 5, 15, 17, 20, 23, 24 and 26°, which are typical B-type diffraction patterns (Fig. 4). To calculate the relative starch crystallinity (RSC), calcium fluoride (CaF₂) was added as an internal standard (2θ = 28.3°). The deficiency or reduction of SSI (ss1 or ss1^L) showed almost the same or lower RSC compared to the wild type, respectively (Table 2). The deficiency of BEI (be1 and ss1/be1) leads to lower RSC than that of the wild type (Table 2). The RSC of ss1^L/be2b (2.5±0.1) was similar to that of the be2b mutant (2.4±0.0), although the amylose content of ss1^L/be2b (21.6%) was much lower than that of be2b (29.4%, Abe et al., submitted). Amylopectin is the main component responsible for the crystallinity of starch.²⁰⁾ Therefore RSC of be2b and ss1^L/be2b were converted to show the relative amylopectin crystallinity (RAC). RAC of ss1^L/be2b (3.2) was slightly lower than that of the be2b (3.4), indicating that the additional reduction of SSI activity in a be2b background might lead to the decrease of RAC although the analyses of the relationship between starch structure and crystallinity are necessary.

Fig. 4.

X-ray diffraction patterns of purified starch granules in various mutant lines and the wild types.

Table. 2.

Relative starch crystallinity (RSC) of various mutant lines of rice, and the wild types.

^aMean±SE of three replicates. ^bPercent of wild type (Nipponbare). ^cPercent of wild type (Taichung 65). ^dPercent of wild type (Kinmaze). *Significant differences between single mutant lines and the WT by t-test at p < 0.05. **Significant differences between the parent mutant lines and the double mutant by t-test at p < 0.05. ***Significant differences between the double mutant line and the WT by t-test at p < 0.05. Nip, Nipponbare; T65, Taichung 65; Kin, Kinmaze.

ACKNOWLEDGMENTS

The authors are grateful to Professor Hikaru Satoh (Kyushu University) for providing the BE mutant lines (EM10 and EM557) and Professor Yoshinobu Akiyama (Akita Prefectural University) and Dr. Toru Takahashi (Akita Research Institute of Food & Brewing) for instruction of the method for calculation and measurement of the crystallinity of starches from various mutant lines. The authors are also grateful to Ms. Yuko Nakaizumi and Naoko Crofts (Akita Prefectural University) for technical support and reading manuscript, respectively. This work was partly supported by the Program for the Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry and Research and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry.

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