4. Discussion
4.1.3. On the development of a method to enrich leaf peroxisomes from
Arabidopsis thaliana is the first higher plant to have its fully sequenced genome publicly accessible. Characterization of the proteome from A. thaliana therefore offers several advantages such as cloning of the genes that encode novel proteins, generation and analysis of mutants for the functional characterization of the genes etc. In the context
of the stress proteome, the situation is even more advantageous since the isoforms specifically involved in a stress response may be identified unambiguously. For example, a
‘digital’ northern analysis suggested significant differences in isoform-specific gene-expression between normal and stressed tissues of A. thaliana (data not shown). About 70% of the ESTs (30 out of 42) of a particular isoform of alanine-glyoxylate aminotransferase (AGT1) gene (acc. no. At2g13360) was represented in libraries constructed from stressed tissues while the distribution of other AGT isoforms (At3g08860, At4g39660 and At4g38400) did not show such a bias in either type of tissues (data not shown) suggesting a possible stress-related function for AGT1 and house keeping roles for the other AGTs. Thus, because of its sequenced genome, a large number of proteomic investigations has been carried out using this organism. Mitochondria (Kruft et al., 2001;
Millar and Heazlewood, 2003; Heazlewood et al., 2003), the plasma membrane (Santoni et al., 1998), Golgi membranes (Prime et al., 2000) and different sub-compartments of chloroplasts (Peltier et al., 2002; Schubert et al., 2002; Froehlich et al., 2003) have been analyzed from Arabidopsis tissues, and heterotrophic cell-suspension cultures served as organelle sources for some of them. When the present work was initiated, no protocol was available for the isolation of pure peroxisomes from leaf tissues of A. thaliana that allowed for proteomic investigations to be carried out; only recently has such a protocol been published (Fukao et al., 2002). This procedure, however, was not as effective in our hands (data not shown). Therefore a great deal of effort was invested during the course of this study into developing an effective method to enrich leaf peroxisomes from Arabidopsis.
Six buffers were shortlisted after an extensive survey of literature for their efficiency in plant peroxisome preparation. The selected buffers allowed for sufficient variations with respect to the osmotica that were employed (different concentrations of sucrose for pea, castor bean and cucumber, mannitol for spinach and sorbitol for yeast) or the pH (pH 7.5 for most of them except for the ‘yeast’ and the Arabidopsis buffer that had a slightly acidic pH of 6). The efficacy of these buffers was tested based on their ability to sustain the stability of leaf peroxisomes over a time course.
Stability was optimal in a hypertonic medium (1 M sucrose), yet, inferior to that sometimes achieved using 0.5 M sucrose buffered with Tris. The inconsistency of results was thought to be related to the greater sensitivity of Tris to fluctuations in temperature.
Therefore the pea- grinding buffer (with tricine) was used to isolate leaf peroxisomes from Arabidopsis. Interestingly, the ‘yeast’-buffer with sorbitol as the osmoticum and a pH at 6 also gave good results with respect to maintenance of stability. The positive influence of KCl and MgCl2 on the stability of leaf peroxisomes may be due to the stabilizing effect of these additives on the peroxisomal membranes. On the other hand, the negative effect of PMSF may be due to its preparation in isopropanol. Glycerol is thought to have resulted in a negative effect due to its profound effect on osmolarity of the buffer. The optimized buffer was also found to be suitable for the isolation of peroxisomes from a number of plant sources including tomato and rape (data not shown).
Further, differential centrifugation, to enrich peroxisomes, while keeping plastid contamination to a minimum, was also optimized.
The sedimentation of peroxisomes by differential centrifugation presented two independent problems. These were:
1. the artificial aggregation of organelles (as already mentioned for the spinach leaf peroxisome isolation procedure) and
2. the necessity to mechanically resuspend the sediment enriched for peroxisomes prior to its homogenization.
In order to overcome these problems simultaneously, attempts were made to enrich peroxisomes over a cushion of sucrose during differential centrifugation. However, a different kind of problem was encountered. This was related to the lack of stability of the organelles when diluting the peroxisomes-containing fraction which was of a high sucrose concentration.
The method that was suitable to isolate highly pure leaf peroxisomes from spinach by the ‘post-plastidic’ supernatant loading analytical method was not efficient for Arabidopsis.
When such a fraction was applied on a shortened sucrose density gradient, the yield of peroxisomes was low, probably, because of very high concentrations of active proteases.
Yields could not be increased even when the grinding buffer was supplemented with protease inhibitors.
Therefore, it was decided to sediment the peroxisomes from the post-plasidic supernatant and then carefully resuspend it prior to gentle homogenization. A relatively short centrifugation time was highly preferred to isolate leaf peroxisomes. Therefore,
several density gradient media such as Percoll (colloidal silica), Ficoll (high molecular weight sucrose-polymers formed by copolymerization of sucrose with epichlorohydrin) and Nycodenz (5-(N-2,3-dihydroxypropylacetamido)2,4,6-triiodo-N, N’-bis (2,3-dihydroxypropyl) isophthalamide) were tested. Hayashi et al. (1975) purified peroxisomes from rat liver using Ficoll as the density gradient medium. Nycodenz has been previously shown to be useful for the preparation of peroxisomes from mammalian tissues (Ghosh and Hajra, 1986; Yoshihara et al., 2001; Kovacs et al., 2001) and from yeast (Erdmann and Bloebel, 1996). While Percoll based density gradients gave good result when used with raffinose, reproducibility was still a major concern. The other media gave poor results. However Ficoll- and Nycodenz–based methods were not pursued intensively due to time constraints.
A sucrose-based density gradient was optimized and used for routine isolation. The peroxisome-enriched fraction obtained from the sucrose density gradient was subjected further to a different kind of density gradient separation. This was because of the difficulty with washing the peroxisomes obtained from the first purification in order to concentrate them. For the second centrifugation, the concentrated 52% (w/w) sucrose fraction containing the peroxisomes was diluted gradually to 48% (w/w) and the contaminants were floated away. When such a method comprising two successive density gradient centrifugations was applied, the extent of purity was quite high and allowed allowed for proteomic analysis.