Changes in fine structure of amylopectin and internal structures of starch granules in developing endosperms and culms caused by starch branching enzyme mutations of japonica rice.

KEY MESSAGE: BEIIb plays a specific role in determining the structure of amylopectin in rice endosperm, whereas BEIIa plays the similar role in the culm where BEIIb is absent. Cereals have three types of starch branching enzymes (BEs), BEI, BEIIa, and BEIIb. It is widely known that BEIIb is specifically expressed in the endosperm and plays a distinct role in the structure of amylopectin because in its absence the amylopectin type changes to the amylose-extender-type (ae-type) or B-type from the wild-type or A-type and this causes the starch crystalline allomorph to the B-type from the wild-type A-type. This study aimed to clarify the role of BEIIa in the culm where BEIIb is not expressed, by using a be2a mutant in comparison with results with be2b and be1 mutants. The results showed that the amylopectin structure exhibited the B-type in the be2a culm compared with the A-type in the wild-type culm. The starch granules from the be2a culm also showed the B-type like allomorph when examined by X-ray diffraction analysis and optical sum frequency generation spectroscopy. Both amylopectin chain-length profile and starch crystalline properties were found to be the A-type at the very early stage of endosperm development at 4-6 days after pollination (DAP) even in the be2b mutant. All these results support a view that in the culm as well as in the endosperm at 4-6 DAP, BEIIa can play the role of BEIIb which has been well documented in maturing endosperm. The possible mechanism as to how BEIIa can play its role is discussed.

Study of the molecular orientation of steroidal side chains at polyimide surfaces using sum frequency generation vibrational spectroscopy.

The molecular orientation of steroidal side chains at rubbed polyimide (PI) surfaces is studied by sum frequency generation (SFG) vibrational spectroscopy. The main objective is to find a correlation between the molecular structure of the PI film and the liquid crystal alignment on the polymer. Analysis of the SFG spectra shows that rubbing of the polymer film appears to cause conformational changes in the methyl group of the polymer side chain near the steroidal structure. However, rubbing does not significantly influence the orientation of the isopropyl group at the end of the polymer side chain. This shows that the liquid crystal alignment is not correlated with the orientation of the isopropyl group.

Sum frequency generation spectroscopy of the attachment disc of a spider.

The pyriform silk of the attachment disc of a spider was studied using infrared-visible vibrational sum frequency generation (SFG) spectroscopy. The spider can attach dragline and radial lines to many kinds of substrates in nature (concrete, alloy, metal, glass, plant branches, leaves, etc.) with the attachment disc. The adhesion can bear the spider's own weight, and resist the wind on its orb web. From our SFG spectroscopy study, the NH group of arginine side chain and/or NH2 group of arginine and glutamine side chain in the amino acid sequence of the attachment silk proteins are suggested to be oriented in the disc. It was inferred from the observed doublet SFG peaks at around 3300 cm-1 that the oriented peptide contains two kinds of structures.

Effects of BEIIb-Deficiency on the Cluster Structure of Amylopectin and the Internal Structure of Starch Granules in Endosperm and Culm of Japonica-Type Rice.

It is known that one of starch branching enzyme (BE) isoforms, BEIIb, plays a specific role not only in the synthesis of distinct amylopectin cluster structure, but also in the formation of the internal structure of starch granules in rice endosperm because in its absence the starch crystalline polymorph changes to the B-type from the typical A-type found in the wild-type (WT) cereal endosperm starch granules. In the present study, to examine the contribution of BEIIb to the amylopectin cluster structure, the chain-length distributions of amylopectin and its phosphorylase-limit dextrins (Φ-LD) from endosperm and culm of a null be2b mutant called amylose-extender (ae) mutant line, EM10, were compared with those of its WT cultivar, Kinmaze, of japonica rice. The results strongly suggest that BEIIb specifically formed new short chains whose branch points were localized in the basal part of the crystalline lamellae and presumably in the intermediate between the crystalline and amorphous lamellae of amylopectin clusters in the WT endosperm, whereas in its absence branch points which were mainly formed by BEI were only located in the amorphous lamellae of amylopectin. These differences in the cluster structure of amylopectin between Kinmaze and EM10 endosperm were considered to be responsible for the differences in the A-type and B-type crystalline structures of starch granules between Kinmaze and EM10, respectively. The changes in internal structure of starch granules caused by BEIIb were analyzed by wide angle X-ray diffraction, small-angle X-ray scattering, solid state 13C NMR, and optical sum frequency generation spectroscopy. It was noted that the size the amylopectin cluster in ae endosperm (approximately 8.24 nm) was significantly smaller than that in WT endosperm (approximately 8.81 nm). Based on the present results, we proposed a model for the cluster structure of amylopectin in WT and ae mutant of rice endosperm. We also hypothesized the role of BEIIa in amylopectin biosynthesis in culm where BEIIb was not expressed and instead BEIIa was the major BE component in WT of rice.

[Optical Second-harmonic Observation of Stimulated Sacran Aggregates].

Second-order nonlinear optical images of aggregates of the ampholytic megamolecular polysaccharide sacran under various stimuli were observed by optical second-harmonic generation (SHG) microscopy. SHG intensity microspots of several tens of micrometers in size are seen for sacran cotton-like lumps and fibers and they have very clear incident polarization dependence. In these microspots, the sacran molecules are oriented in concentric multilayers. We also observed SHG images of sacran prepared in a needle-ring electrode with applied voltage, where SHG was enhanced near the negatively biased needle electrode. We also observed SHG signals near the cast film edges of sacran. The appearance of the SHG image suggested a phase-separation structure in sacran aggregates. These results show that sacran molecules aggregate in several different ways.

Ungerminated Rice Grains Observed by Femtosecond Pulse Laser Second-Harmonic Generation Microscopy.

As a demonstration that second-order nonlinear optical microscopy is a powerful tool for rice grain science, we observed second-harmonic generation (SHG) images of amylose-free glutinous rice and amylose-containing nonglutinous rice grains. The images obtained from SHG microscopy and photographs of the iodine-stained starch granules indicate that the distribution of starch types in the embryo-facing endosperm region (EFR) depends on the type of rice and suggests that glucose, maltose, or both are localized on the testa side of the embryo. In the testa side of the embryo, crystallized glucose or maltose are judged to be detected by SHG. These monosaccharides and disaccharides play an important role, as they trigger energy in the initial stage of germination. These results confirm SHG microscopy is a good method to monitor the distribution of such sugars and amylopectin in the embryo and its neighboring regions of rice grains.

Optical second-harmonic images of sacran megamolecule aggregates.

We have detected a second-order nonlinear optical response from aggregates of the ampholytic megamolecular polysaccharide sacran extracted from cyanobacterial biomaterials by using optical second-harmonic-generation (SHG) microscopy. The SHG images of sacran cotton-like lump, fibers, and cast films showed SHG intensity microspots of several tens of micrometers in size. The dependence of the SHG spot intensity on an excitation light polarization angle was observed to illustrate sacran molecular orientation in these microdomains. We also observed SHG signals around a special region of the cast film edges of sacran. These results show that sacran megamolecules aggregate in several different ways.

Wetting effect on optical sum frequency generation (SFG) spectra of d-glucose, d-fructose, and sucrose.

We report a sum frequency generation (SFG) spectroscopy study of d-glucose, d-fructose and sucrose in the CH stretching vibration regime. Wetting effect on the SFG spectra was investigated. The SFG spectrum of d-glucose changed from that of α-d-glucose into those of α-d-glucose monohydrate by wetting. The SFG spectra showed evidence of a small change of ß-d-fructopyranose into other anomers by wetting. SFG spectra of sucrose did not change by wetting. Assignments of the vibrational peaks in the SFG spectra of the three sugars in the dry and wet states were performed in the CH stretching vibration region near 3000cm(-1).

Sum frequency generation microscopy study of cellulose fibers.

Sum frequency generation (SFG) microscopy images of cotton cellulose fibers were observed at the infrared wavenumber of ~ 2945 cm(-1) and with a spatial resolution of 2 µm. Domains of different cellulose microfibril bunches were observed and they showed different second-order nonlinear responses. The intensity of the peak of the asymmetric CH(2) stretching mode at 2945 cm(-1) depended strongly on the orientation of the electric fields of the incident visible and infrared light with respect to the cellulose fiber axis. The second-order nonlinear susceptibility arising from the chirality in the cellulose structure was found to be dominant. The SFG of the cross section of the cellulose fiber was relatively weak and showed a different spectrum from that measured from the side of the fiber axis.

Discovery of deep and shallow trap states from step structures of rutile TiO2 vicinal surfaces by second harmonic and sum frequency generation spectroscopy.

In this report, local electronic structures of steps and terraces on rutile TiO(2) single crystal faces were studied by second harmonic and sum frequency generation (SHG∕SFG) spectroscopy. We attained selective measurement of the local electronic states of the step bunches formed on the vicinal (17 18 1) and (15 13 0) surfaces using a recently developed step-selective probing technique. The electronic structures of the flat (110)-(1×1) (the terrace face of the vicinal surfaces) and (011)-(2×1) surfaces were also discussed. The SHG∕SFG spectra showed that step structures are mainly responsible for the formation of trap states, since significant resonances from the trap states were observed only from the vicinal surfaces. We detected deep hole trap (DHT) states and shallow electron trap (SET) states selectively from the step bunches on the vicinal surfaces. Detailed analysis of the SHG∕SFG spectra showed that the DHT and SET states are more likely to be induced at the top edges of the step bunches than on their hillsides. Unlike the SET states, the DHT states were observed only at the step bunches parallel to [1 1 1] [equivalent to the step bunches formed on the (17 18 1) surface]. Photocatalytic activity for each TiO(2) sample was also measured through methylene blue photodegradation reactions and was found to follow the sequence: (110) < (17 18 1) < (15 13 0) < (011), indicating that steps along [0 0 1] are more reactive than steps along [1 1 1]. This result implies that the presence of the DHT states observed from the step bunches parallel to [1 1 1] did not effectively contribute to the methylene blue photodegradation reactions.

Effect of the mobility of ligands in polyrotaxanes on order structure of water clusters.

The effect of the mobility of ligands (maltose groups) in the polyrotaxanes (pRXs) on the structure of the surrounding water molecules was investigated. Raman spectra of collective OH stretching vibration of water molecules in aqueous solutions of maltose-pRX conjugates with different alpha-cyclodextrin (alpha-CD) threading on a poly(ethylene glycol) (PEG) chain was measured. The mobility of maltose groups was estimated by measuring the relaxation time T2 of the C1 protons in maltose groups bound on alpha-CD by NMR experiment. A positive correlation between the Raman intensity of the collective band and the relaxation time T2 was obtained. This result indicates that the degree of order of the water clusters is higher as the mobility of maltose groups increases in these conjugate solutions. It is suggested that rapid motion of maltose groups in the pRX conjugate can contribute to preserving ordered structure of the bulk water clusters.