Discussion

Fig. 4.22 The analysis of size dependence.

To interpret the granum size-dependence of the Soret band of the spectrum, first we should consider what important factors determine the Soret band. Size dependence of electronic spectra could be discussed based upon optical property. Here we list three possibilities that might be the effective factors of size dependence; chemical composition, optical effect and molecular arrangement (Fig. 4.22). The further detail of each element

will be discussed in the following sections, which includes the computational calculation and experimental evidences.

size dependence of optical properties

optical effect

Reabsorption

by itself

by other particles calculatoin

chemical composition

molecular arrangement

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When mentioning molecular electronic spectra, chemical composition of the species usually comes up. It is known that various pigments in the grana and their compositions differ for each granum. The fluctuation of chlorophyll a usually varies with different extracting procedures or solvents, but the main pigment composition is still chlorophyll a [33, 34]. This finding is not only limited to E. densa but also presented in other plants [35]. In other words, although the spectra vary slightly from granum to granum due to different composition, the primary shape should retain the same. Since the composition of granum only has minor influences on the spectrum, the factor of chemical composition can be excluded from the possibilities.

Optical effect can be divided into two parts, reabsorption and calculation. Reabsorption denotes that the scattered light from a sample is reabsorbed by surrounding or the sample itself. In this study, reabsorption means the scattered light from granum is reabsorbed by other grana or itself. Reabsorption by granum itself can be neglected because the grana are small enough (average size is 273 nm). In section 4.2-2, we have already proven the position dependence. Actually, the position dependence showed the indirect verification that reabsorption by other grana was very small. The spectra of grana in arbitrary site discriminating non-dissimilarities implied that the reabsorption effect was so unobvious that could be ignored.

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Next, we used Mie theory to calculate the scattering efficiencies and optical resonance wavelengths of grana for various dimensions, so as to clarify the size dependence of grana. To simplify the calculation, we assumed that granum is a spherical NP full of chlorophyll a and the refractive index of surrounding media is homogeneous, equals to 1.33 for water. Fig. 4.23 shows the scattering spectra of the chlorophyll a NPs. Size dependences of experimental result and calculation are plotted in the Soret band of spectra against size in Fig. 4.24 and the inconsistency is clearly depicted. Experimental result revealed positive correlation between size and spectrum, while calculation presented size independence, i.e. the spectral shift of the Soret band does not occur due to the diverse size of granum.

The gap between experimental result and calculation shown in Fig. 4.24 can be explained by the presumed refractive index. We supposed the chlorophyll a NPs are dispersed in solution. As a matter of fact, the surrounding media is more similar to solid since molecular pigments are embedded in the LHC bound to thylakoid membrane. Based on Mie theory, a red-shift would come in the optical spectra with larger refractive index of environment. If we had learned the real refractive index of granum, the gap would be much smaller than the result.

Hence, optical effect can be excluded from the possible explanations.

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The aggregation and dimerization of porphyrins and metalloporphyrins in aqueous solution have been widely described. As the natural chlorophyll aggregates in the light-harvesting proteins or chlorosomes have a strong transition dipole moment aligned in the

“head-to-tail” direction, porphyrin J-aggregates are important for the study of the excited-state

model of the organisms [36]. J-aggregate is a one-dimensional molecular arrangement in which the transition moments of individual monomers are aligned in parallel.The strong coupling of several self-similar monomers results in a coherent excitation at red-shifted wavelengths relative to the monomer [37]. The following inference is reasonable that chlorophyll pigments form aggregation like a J-aggregate. That is to say, we can consider that there are more molecules in larger granum and align much uniformly. As a consequence, redshift of optical spectra is clearly observed (Fig. 4.14).

Analyses of polarization dependence (Fig. 4.20) also support the above assumption. If the molecules align one-dimensionally perfectly, the interaction between irradiation and molecules should be strongly clear, i.e. the polarization plot (Fig. 4.19) would show a narrow peak at certain angle while others stay level. On the other hand, the narrower the FWHM is, the better molecules align. This is what parameter R stands for. Fig. 4.20 (A) indicates R decreases with respect to size, reflecting size dependence. Same meaning as Fig. 4.20 (B) represented. FWHM decreases against the increase of size.

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All of the experimental results illustrate size dependence, and consequently we could confidently suggest that pigment arrangement changes depending on the size of granum.

Because the aggregation of chlorophyll leading to exciton generation are able to capture the incident light and transfer the energy to “reaction center” with higher efficiency compared to isolated condition, it can be considered that our finding suggests the inhomogeneity of chloroplast in view of light-harvesting.

400 500 600 700

Figure 4.23 The calculation of chlorophyll a NP. Calculating scattering spectrum of granum

which contains lots of chlorophyll pigments is complicated, so we calculated chlorophyll a, the main component of granum instead. Based on Mie theory, calculated scattering spectra of chlorophyll a NPs, from 200 nm to 500 nm, were obtained.

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Figure 4.24 The plot of peak wavelength of the Soret band against size. The plot showed size

dependence of experimental result and calculation. Calculation represented size independence and the Soret band located at around 450 nm differing from experimental result. The difference between the Soret band of experimental result and calculation is contributed to the refractive index of media. Chlorophyll pigments embedded in protein bound to membrane, so the environmental area can be considered as solid, while we assumed water as media.

Redshift presents when the environmental refractive index increases.

440 460 480 500 520 540

200 300 400 500

w avele n gth / n m

size / nm Size dependence

single peak (exp) split peak (exp) calculation

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