complement among plant cells determinates the type of plastid
substructures for pigments storage [28].
Then, in order to understand the biosynthesis and accumulation of carotenoids in plant cell, and in fruits in particular, it is
essential a reliable visualization of the carotenoids-containing structures in cells and plastids ultrastructure. Moreover, carotenoids
storage has been also demonstrated to be determinant for their
bioaccessibility. In this chapter, we provide a detailed protocol for
transmission electron microscopy (TEM) analysis of plastids accumulating carotenoids, specially focused on fruit tissue, since these
cells have high potential for carotenoid storage, and exhibit large
carotenoid and plastid diversity.
2 Materials
2.1 Reagents
and Solutions
1. PIPES buffer: Mix piperazine-NN
0 -bis-2-ethanesulfonic acid
(PIPES)- 60.48 g with 10 M sodium hydroxide (NaOH) to
bring the pH up to 7.2. Make up to 1 L with distilled water to
produce a 0.2 M solution of PIPES buffer. The buffer can be
divided up into suitable volumes and frozen until required.
2. Fixative solution (4% formaldehyde): Mix 4 g of paraformaldehyde with 20 mL distilled water in a small beaker. Heat to
85
C on a hotplate in the fume cupboard. Add three drops
of 0.1 M NaOH and stir until the liquid clears. Make up to
50 mL with distilled water. This will produce 8% formaldehyde
and it should be then diluted 50/50 in PIPES buffer with
glutaraldehyde (see Note 1).
3. Reynolds lead stain: Weight 1.33 g lead nitrate (Pb(NO 3 ) 2 )
and 1.76 g Tri-sodium citrate and place both into 30 mL
distilled water in a very clean glass container. Shake for1 min
and then intermittently for 30 min to produce a milky suspension. Add 8 mL of freshly prepared 1 M sodium hydroxide and
shake until clear. Make up to 50 mL with fresh distilled water.
The stain can be divided up into suitable volumes and stored in
sealed containers (see Note 2).
4. Uranyl acetate stain: Weight 1–2 g of uranyl acetate (see Note
3) and place it in a centrifuge tube capable of withstanding
repeated centrifugation (i.e. polypropylene). Fill the tube with
50 mL of ethanol 50% and leave to stand in the fridge overnight. Solution reacts with light forming a precipitate, therefore should be stored in the dark. The tube must be centrifuged
before use (see Note 4).
2.2 Equipment
1. Scalpels.
2. Petri dishes.
Structure of Plastid-Containing Carotenoids from Fruits
239
substructures for pigments storage [28].
Then, in order to understand the biosynthesis and accumulation of carotenoids in plant cell, and in fruits in particular, it is
essential a reliable visualization of the carotenoids-containing structures in cells and plastids ultrastructure. Moreover, carotenoids
storage has been also demonstrated to be determinant for their
bioaccessibility. In this chapter, we provide a detailed protocol for
transmission electron microscopy (TEM) analysis of plastids accumulating carotenoids, specially focused on fruit tissue, since these
cells have high potential for carotenoid storage, and exhibit large
carotenoid and plastid diversity.
2 Materials
2.1 Reagents
and Solutions
1. PIPES buffer: Mix piperazine-NN
0 -bis-2-ethanesulfonic acid
(PIPES)- 60.48 g with 10 M sodium hydroxide (NaOH) to
bring the pH up to 7.2. Make up to 1 L with distilled water to
produce a 0.2 M solution of PIPES buffer. The buffer can be
divided up into suitable volumes and frozen until required.
2. Fixative solution (4% formaldehyde): Mix 4 g of paraformaldehyde with 20 mL distilled water in a small beaker. Heat to
85
C on a hotplate in the fume cupboard. Add three drops
of 0.1 M NaOH and stir until the liquid clears. Make up to
50 mL with distilled water. This will produce 8% formaldehyde
and it should be then diluted 50/50 in PIPES buffer with
glutaraldehyde (see Note 1).
3. Reynolds lead stain: Weight 1.33 g lead nitrate (Pb(NO 3 ) 2 )
and 1.76 g Tri-sodium citrate and place both into 30 mL
distilled water in a very clean glass container. Shake for1 min
and then intermittently for 30 min to produce a milky suspension. Add 8 mL of freshly prepared 1 M sodium hydroxide and
shake until clear. Make up to 50 mL with fresh distilled water.
The stain can be divided up into suitable volumes and stored in
sealed containers (see Note 2).
4. Uranyl acetate stain: Weight 1–2 g of uranyl acetate (see Note
3) and place it in a centrifuge tube capable of withstanding
repeated centrifugation (i.e. polypropylene). Fill the tube with
50 mL of ethanol 50% and leave to stand in the fridge overnight. Solution reacts with light forming a precipitate, therefore should be stored in the dark. The tube must be centrifuged
before use (see Note 4).
2.2 Equipment
1. Scalpels.
2. Petri dishes.
Structure of Plastid-Containing Carotenoids from Fruits
239