In order to assess the most effective protocol, we performed a quick spec- troscopic analysis on the samples after each preparation. The principle of the technique relies on a physical process which takes place when a molecule of chlorophyll is excited: the fluorescence. In the next section, the principle of the process is explained.
Chlorophyll Fluorescence
Chlorophyll molecule can absorb light energy and re-emit it as radiation energy. When a photon is absorbed, chlorophyll molecule is excited from the ground state (S0) to the first electronic excited singlet state (S1)(figure
2.3). This process takes about 10−15 seconds. It is important to note that this molecule can be excited to a higher energy levels (S2, Sn) and can relax
to S1 via vibrational relaxation in a time comprised between 10−12 seconds.
Finally, the molecule will relax to the ground state through the emission of a photon. This process is called fluorescence emission and the energy of the emitted photon is equal to the changes in the energy level.
77◦K fluorescence emission
Measure of the fluorescence emission from thylakoid fractions has to be carried out at a liquid-nitrogen temperature (77◦K). In fact, at room- temperature PSI does not emit fluorescence. This is because PSI (and not PSII) is characterised by a great efficiency (∼1) and almost all of the light col- lected is used to perform photochemistry [148]. Moreover, some chlorophyll species are present in the proximity of the PSI-RC characterised by a energy level slightly lower than that of P700 [149]. Room temperature is sufficient to allow free-transfer of photons from these chlorophyll species to the RC and perform photochemistry. At cryogenic temperatures, this energy trans- fer is blocked. High fluorescence emission from PSI finally appears because these special fluorescing Chl species, absorbing at 710-720 nm, compete with P700 in trapping the energy [149]. 77K spectroscopy relies on the fact that when a mixture of thylakoid is excited with blue light, the relative fluores- cence emission spectra collected in the near infrared region is characterised by three main peaks (fig.2.4A). The two peaks between approximately 680 and 695 nm correspond to fluorescence emitted from chlorophyll molecules in PSII, while the peak at 735 nm corresponds to the fluorescence from the chlorophyll molecules in PSI (fig 2.4A). When signals are normalised with respect to a chosen peak (e.g. PSII) amplitudes are proportional to the size of the antenna associated to a given photosystem [150].
We performed the same experiment on our purified sub-thylakoid frac- tions and resulting spectra are reported in figures 2.4 B, C and D. Stroma- lamellae obtained from the purification protocol with the yeda-press display an almost identical amplitude for both peaks of PSI and PSII (fig 2.4B). Moreover, the fact that the PSI-fluorescence peak is detected at a wave- length lower than 735 nm clearly indicates a damage of the antenna appara- tus probably due to the method of purification. By contrast, when stroma lamellae are purified with digitonin, the peak associated to PSII is dramati- cally diminished and, by consequence, that of PSI gains largely in amplitude (fig 2.4B). Different levels of enrichment PSI/PSII were also found in the case of the purification of grana and grana inner discs (BBY). When α-DM was used as detergent, little difference is seen in the amplitude between the peaks PSI/PSII (fig 2.4B). Furthermore, neither the detergent concentration nor the time of centrifugation seem to have a beneficial effect on the quality of the purification (fig 2.4C). Spectrum performed with the grana-BBY is- sued of the TRITON X-100 treatment display the most important difference between the amplitudes of the peaks PSI/PSII. In fact, even if not totally abolished, the peak relative to PSI is largely diminished when compared to that of PSII (fig 2.4D). It is important to note that the peak associated to PSI
Figure 2.4 – 77K chlorophyll fluorescence emission spectra of thylakoid membrane sub- fractions isolated from Arabidopsis with different methods. Thylakoid proteins (or thy- lakoid subfractions) samples were loaded on to a metal cuvette, which was directly bathed into a liquid nitrogen solution. Fluorescence spectra were recorded upon excitation at 470 nm. Peaks were normalized for comparison purposes. A, isolated thylakoids. B, Lamel- lae, margins, grana and thylakoids purified by different protocols. C, grana purified with varying concentration of detergent and time of centrifugation. D, grana inner-discs (BBY) purified with TRITON X-100 10 mg.mgChl−1.
may be in someway over-estimated, since part of the emission of the PSII is re-absorbed by PSI and re-emitted at a longer wavelength. Margins fraction purified with digitonin (fig 2.4B) displays a relative PSI/PSII peak amplitude mostly comparable indicating that both photosystems are probably present.
In conclusion, in order to obtain the purest sample to be analysed by mass-spectrometry, we chose to limit our purification protocols to the use of 5% digitonin in the case of stroma-lamellae and grana margins, and to 10 mg TRITON X-100/ mg Chl for the purification of the inner discs of the grana (grana-BBY).
2.2.3.1 Measure of the oxygen evolution rate, Chl a/b and P/C ratio
Stroma-lamellae and grana-BBY fractions issued of the selected proto- cols were furtherly challenged by direct measurement of the chlorophyll a/b ratio and oxygen evolution. In plant, chlorophyll molecule is present in two different forms which essentially differ in the composition of a single side chain (figure 2.5). Chlorophyll a is a large molecule that has a porphyrin ring with a magnesium atom at its center. Attached to the porphyrin is a long, insoluble carbon-hydrogen chain which interacts with the proteins of the thylakoids and serves to anchor the molecule in the internal membrane of the chloroplast. Chlorophyll b differs from chlorophyll a only in one of the functional groups bonded to the porphyrin (a -CHO group in place of a -CH3 group). It is an accessory pigment and acts indirectly in photosynthesis
by transferring the light to chlorophyll a [152]. In particular chlorophyll b is mainly associated to the sole PSII-LHCII complex thus more enriched in the grana-BBY fraction. The ratio of chlorophyll a to chlorophyll b in the chloroplast (and thylakoid) is around 1:3 but varies significantly within the thylakoid subfractions [153].
Data summarised in table 2.2 (lane Chl a/b ratio) confirm the successful separation of the two fractions. In fact, stroma-lamellae fraction displays the higher chlorophyll a/b ratio indicating a low content in PSII antenna. By contrast, thylakoid and grana-BBY fraction which contain a higher con- centration of LHCII (this is especially true for grana-BBY) have a lower chlorophyll a/b ratio. Later, we wanted to determine if the water-splitting activity of the PSII was retained after purification. At this purpose, we performed a test in which the oxygen evolution rate was measured by a clark-type oxygraph. Thylakoid and thylakoid subfractions were placed in the oxygraph chamber and artificial electron acceptors were added in order to sustain the primary photosynthetic reaction (ferricyanide and 2,5-dichloro- p-benzoquinone). Oxygen evolution rates were measured in dark conditions and during illumination at 750 µE. Data reported in table 2.2 (column activ- ity) indicate that our grana-BBY fraction was able to retain up to 80% of the activity of the intact thylakoids (57 and 70 nmol O2 h−1. µgChl −1 respec-
Figure 2.5 – Chlorophyll a and b structure
fraction Chl a/b ratio Prot/Chl ratio activity (nmol O2.h−1.µg chl)
thylakoids 2.90 ± 0.01 4.44 ± 0.04 70.0 ± 10.5
grana-BBY 1.73 ± 0.08 2.25 ± 0.18 56.9 ± 6.6
stroma-lamellae 4.28 ± 0.01 6.45 ± 0.17 non detectable Table 2.2 – Measure of the oxygen evolving activity of thylakoids, grana-BBY and stroma-lamellae. Fraction were essayed with ferricyanide (3.5 mM) plus 2,5-dichloro-p- benzoquinone (250 µM).
tively). As expected, stroma-lamellae were not able to evolve any detectable amount of oxygen. This is explained by the fact that only photosystem II can successfully accomplished light-induced water-splitting. Moreover, this finding furtherly demonstrates the good level of purity in our stroma-lamellae preparations since they are particularly deprived of PSII.
2.3
Proteomic Analysis: Introduction
Proteomics is the study of global protein composition within an organ- ism, tissue, cell or organelle. It has delivered important enhancements and unique insights into the characterisation of the biological systems since its in- troduction in the last decades [154]. The characterisation of the proteome is
of prime importance for a complete understanding of plant functions, biosyn- thetic and signaling pathways.
Chloroplast is not only an important center of metabolic reactions but also it is an important signaling hubs that determines the expression of nu- merous nuclear encoded genes in retrograde signaling cascades [155]. Con- sequently, the characterisation and study of its proteome is of crucial im- portance and constitutes an important economic issue in plant biotechnol- ogy. Chloroplast proteome composition and relative expression levels of pro- teins vary in function of developmental stage, as well as environmental con- ditions. Understanding these modifications is essential to improve knowl- edge of the organelle functions and regulation. Proteomics focused on the chloroplast does not only aim at identifying or determining level of protein expression but gives access to a whole wide range of information such as protein-protein interactions (see [156]) or protein sub-localisation within the organelle (see for example [140]). Indeed, Arabidopsis has been the organ- ism of choice for the proteomic analysis of the chloroplasts and is currently the only plant for which a truly comprehensive set of established chloroplast proteins is available. Moreover, Arabidopsis has a wide range of compre- hensive resources already available such as the entire genome sequenced and a large collection of mutants. Several fundamental information platforms and databases exist such as The Arabidopsis Information Resource TAIR (http://www.arabidopsis.org/).
Today, a significant percentage of the chloroplast proteome is charac- terised and much of this progress is certainly due to the improvements of the mass-spectrometry technologies [157]. When compared to those of 10 years ago, latest proteomic instruments are characterised by: (i) much improved sensitivity (routinely at 1-50 fmol) [158][159], (ii) accelerated duty cycle (now tandem mass spectrometry [MS/MS] scans within a few hundred ms), (iii) improved mass accuracy (down to a few ppm for peptides), and (iv) in- creased resolution of the latest generation mass spectrometers. Nevertheless, even if such important technological improvements have been delivered, some weak-points still remain. In particular identification of highly hydrophobic proteins, proteins that are expressed only under particular conditions (e.g. adverse growth conditions, developmental stage, etc.), and proteins with very low expression levels (e.g. more than 10,000-fold lower than RuBisCo) still remain challenging [157]. The following section will give an overview of the scheme that is supposed to be followed during a subcellular-focused proteomic investigation (and of course during chloroplast proteome investigation).