Wednesday, January 15, 2014

Silica in leaves - low temperature oven

Seven species were chosen to be analysed for silica content in their leaves. Some of the chosen species are well known for their silica content as Curatella americana (silica plant) (Teixeira-da-Silva, 1985), Davilla indica (Dr. José Rubens Pirani, personal communication), Equisetum sp (Kaufman et al. 1971) and Magnolia grandiflora (Magnolia) (Postek, 1981), others showed some silica features in their spectra but no studies were found as Fagus grandifolia (Beech) and Acer rubrum (Red Maple) and one that seemed to lack silica features as Cornus florida (Dogwood).

Leaves were collected in Reston and Lovettsville, VA (USA), in Brotas and São Paulo, SP (Brazil), on October 2005, dried in a regular oven for three days at 50°C, enveloped in aluminum foil. They were then turned into powder in a blender, and sieved first in a 28 (600 µm) sieve and then in a 65 (212 µm). Dry leaves of C. Americana and D. indica were cut into small pieces to explore the silica anatomy without organic matter, having two kinds of samples: one of leaves with main veins, and another without the main veins, to explore the contribution of silica coming from different tissues.

Low temperature ashing of leaf pieces and leaf powder was conducted using a radio-frequency (RF) generated oxygen plasma with the goal to oxidize organic matter (OM) and preserve minerals in their original structure and composition (LTA 504; LFE Corporation) (current < 150 W; O2 between 20 and 100 cm3/min; Torricelli 10-7).  Leaves or powder were put in a Petri dish, and weighted. Samples were put in the low temperature oven and weighted and stirred twice or more per day until the weight stabilized around 1/100 of one gram. 3 sets of samples were ashed through  3 different periods of 2, 7 and 15 days beginning in: October 13, November 17, 2004, and Februrary 1st, 2005 respectively.

Scanning electronMicroscope (SEM). Small pieces of ashed C. Americana and D. indica leaves were mounted on aluminum stubs and covered with vaporized carbon in a vacuum chamber, and analyzed using a JEOL 840 (JEOL USA Inc., Peabody, MA, USA) scanning electron microscope (SEM) at a beam intensity of 15 kV; all the images are secondary electron images (SEI) to be compared with leaves of the same plants previously analyzed.

Attenuated Total reflectance (ATR) spectra were measured using two different crystals: a diamond ( 4000 – 400 cm-1), and Zn-Se (4000 – 650 cm-1). Fresh leaves and minerals were measured with Zn-Se crystal, and dry, powdered and ashed leaves with diamond.

1)      Plasma oven
The stabilization of organic matter loss was variable between species taking between 5 and 15 days (Tables 1 and 2).

Table 1. Weight in grams of powdered leaves of 4 species during ash process during 7 days between October 13 and October 19. Magnolia grandiflora, Equisetum sp, Fagus grandifolia and Acer rubrum
Table 2. Weight in grams of powdered leaves of 3 species during ash process for 15 days (Nov 17- Dec 01). The percent loss of OM during ashing was variable between species. The species that are known for being silica rich as Equisetum sp, C. americana, D. indica had about 20% of minerals left in their ashes. C. florida and M. grandiflora had about 14 - 15% and F. grandifolia and A.rubrum had less than 9% of minerals (Table 3). Samples of powdered leaves of C. americana with and without veins were similar in mineral content, 19.81% and 19.58% respectively. 
Table 3. Table showing the initial and final weight, and percentage of approximate lost organic matter (OM) and left minerals for each species.

ATR measurements of dry leaf surface, powder leaf and ashes of each species are  compared (Figures 1-7) . Dry leaf surfaces show bands from long chain aliphatic compounds typical from cuticular waxes, as methyl and methylene (CH2 and CH3) vibrations and relatively shallow hydroxyl (OH) stretching, differently from spectra of ground leaves where long chain CH features are not as prominent with a wider and strong OH stretching. Spectra from leaf surfaces and ground leaves are always different, showing that spectral features of leaf surface in the TIR are mostly from outer shallow tissues. Spectral features of compounds present inside the leaf are mostly from organic matter because they are not seen in ashed leaves.

Most ash measurements show a predominance of hydrated amorphous silica like opal (SiO2.nH2O) and small amounts to traces of Calcite (CaCO3) and Gypsum (CaSO4.2H20) (Figures 1, 3-7), except C. florida (Figure 2) which spectrum does not show opal features. In C. americana (Figure 3) and D.indica (Figure 4), two species from the Dilleniaceae family known to be a silica rich family, silica features are evident on the leaf surface, powder and ashes showing that the inner leaf is impregnated of silica. The same can be said of F. grandifolia (Figure 6) and M. grandiflora (Figure 7), but not as much of A.rubrum (Figure 1), Equisetum sp (Figure 5) that show silica features mixed with many other unidentified features from inner leaf. Equisetum and C. florida ashes show a predominance of Calcium carbonate as Calcite and C.florida also shows bands probably from Calcium sulfate similar to Gypsum spectrum.

The presence of OH stretching and HOH bending in the mineral phase of all species shows that the minerals are hydrated, as for example amorphous Silica, as opal (Si-O2.nH2O), or it may be water adsorbed on the minerals.

Figure 1. Acer rubrum. Dry adaxial surface (top) OH 3363 cm-1 (orange). CH 2917, 2849, 1462, 730 and 719 cm-1(pink). Si-O at 1053 and 453 cm-1 (green). Ground leaf (middle) OH 3296 cm-1 (orange). CH 2918, 2849, and 719 cm-1 (pink). C-O/Si-O 1033 cm-1 (black). Ashes (bottom). OH 3345 cm-1(orange). HOH bending at 1631 cm-1 (orange). SiO2.nH2O 1082, 784 and 457 cm-1 (green). CaCO3 1408 and 868 cm-1 (yellow). Unidentified double band at 626 and 598 cm-1 (purple).

Figure 2. Cornus florida. Dry adaxial surface. OH 3332 cm-1 (orange).CH 2919, 2850, 1462 and 719 cm-1(pink). Ground leaf OH 3291 cm-1 (orange). CH 2920 and 2851 cm-1 (pink).  Unidentified 1604 and 1028 cm-1 (purple). Ashes (bottom). OH 3379 cm-1 (orange). HOH 1634 cm-1 (orange), CaCO3  1405 and 865 (yellow). CaSO4 1087, 865. Unidentified 626 and 598 cm-1 (purple).

Figure 3. Curatella.americana. Dry adaxial  surface (top) OH 3323 cm-1 (orange). CH at 2917 and 2849, 1463 and 719 cm-1 (pink). Si-O + C-O at 1046 cm-1(black), Si-O at 788 and 449 cm-1(green). Ground leaf (middle). OH centered at 3298 cm-1 (orange).CH 2919 anf 2851 cm-1 (pink). C-O/Si-O at 1031 cm-1 (black). Ashes (w/veins) (bottom). OH 3432 cm-1 (orange). HOH 1620 cm-1 (orange). Si-O 1063, 783 and 441 cm-1 (green). Unidentified  1431, 1320 and 626 cm-1 (black).

Figure 4. Davila indica. Dry adaxial surface (top) OH 3281 cm-1 (orange). CH at 2918 and 2850, 1463 and 717 cm-1 (pink). C-O/Si-O at 1045 cm-1(black). Si-O at 783 and 456 cm-1(green). Ground leaf (middle). OH 3296 cm-1 (orange). CH 2919 and 2851 cm-1 (pink). C-O/Si-O at 1044 cm-1 (black). Si-O at 766 and 444 cm-1 (green). Ashes (bottom). OH 3411 cm-1 (orange). HOH 1620 cm-1 (orange). Si-O 1086, 782 and 450 cm-1 (green). CaSO4 shoulder around 1149 cm-1 and  661 cm-1 (blue). Unidentified 1424, 1323and 598 cm-1 (purple).

Figure 5. Equisetum sp. Dry stem surface (top) OH 3292 cm-1 (orange). CH at 2918 and 2850 cm-1, (pink), C-O/Si-O at 1023 cm-1 (black). Si-O 453 cm-1 (green). Ground stem (middle). Wide and strong OH centered at 3291 cm-1 (orange). C-O/Si-O at 1017 cm-1 (black). Ashes (bottom). OH 3259 cm-1 (orange). HOH 1620 cm-1 (orange). Si-O 1082, 785, 453 cm-1 (green).
Calcite 1391 and 867 cm-1 (yellow). Unidentified 1106 and 617 cm-1 (black).

Figure 6. Fagus grandifolia. Dry adaxial surface (top) OH 3319 cm-1 (orange). CH 2917 and 2849, 1462 and 719 cm-1(pink), C-O/Si-O at 1046 cm-1 (black). Si-O 454 (green). Ground leaf (middle). OH 3324 cm-1 (orange). CH 2920 and 2851 cm-1(pink). C-O/Si-O at 1040 cm-1 (black). Si-O 451 (green).  Ashes (bottom). OH 3344 cm-1 (orange). HOH 1620 cm-1 (orange). Si-O 1069, 781, 454 cm-1 (green).

Figure 7. Magnolia grandiflora. Ground leaf (top). OH 3293 cm-1 (orange). CH 2918 and 2850 cm-1(pink). HOH 1620 cm-1 (orange). C-O/Si-O at 1033 cm-1 (black). Si-O 454 (green).  Ashes (bottom). OH 3356 cm-1 (orange). HOH 1620 cm-1 (orange). Si-O 1082, 786, 465 cm-1 (green).
Calcite 1413 and 865 cm-1 (yellow). Unidentified 600 cm-1 (black).

3) SEM

SEM images of adaxial (Figure 8) and abaxial (Figure 9) of  C. americana, show that cells and trichomes morphology is preserved in the dry  leaf (left) as well as in the ashed leaf (right) surfaces.


Figure 8. C. americana adaxial surface. Left - dry leaf showing epidermal cells contour and short and long trichomes.Right. Piece of ashed leaf showing a very similar structure to the dry leaf.

Figure 9. C. americana abaxial surface. Left - dry leaf showing epidermal cells contour, stomata and trichomes with short and long tentacles.Right. Pieces of ashed leaf showing a very similar strucuture to the dry leaf.

Figure 10. C. americana ashed leaf. Upper left. Inner side of adaxial surface with palissade cells (white arrow) and vases (red arrow). Below left. Adaxial epidermis. Upper right. Inner side of abaxial surface with stomata (white arrow) and vases (red arrow). Below right. Three stomata.

Figure 11. D. indica ashed leaf. Left. External adaxial surface with epidermal cells (red arrow). Right. Inner surface of abaxial epidermis with a smooth layer with grooves (red arrow) and two stoma cavities (black arrows).