Analele ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi
Tomul LVII, fasc. 1, s. II a. Biologie vegetală, 2011
ANATOMICAL AND MICROMORPHOLOGICAL ASPECTS AT ARDISIA
CRENATA SIMS
IRINA NETA GOSTIN*, MARIA MAGDALENA ZAMFIRACHE*, M. ŞTEFAN*
Abstract: The paper discusses the structure of foliar node of Ardisia crenata Sims, family
Myrsinaceae, decorative shrub cultivated for ornamental purposes in protected areas, with thick leaves, glossy,
dark green and slightly lacy edges because of foliar nodules well customize at this level of leaf. Anatomical
investigations (optical and electronic microscopy SEM) were performed on leaves with different ages,
highlighting the structural differences of foliar nodules, as well as micromorphologycal characterization of
colonizing microorganisms, analyzed according to age of these formations.
Keywords: Ardisia crenata, foliar nodules, colonizing microorganisms
Introduction
Bacterial nodules on the leaves of some species of the family Myrsinaceae
(inclusive in Ardisia) were first described by Hohnel (1881). They appear on the leaf and
are part of a symbiosis required for the development life cycle of these species; the
bacteria are present in all infected itself the first leaf buds and flowers and seeds as well.
Studies have shown that treatments that destroy bacteria are followed by plant death [10].
Materials and methods
The plant material was collected from Botanical Garden Anastasie Fătu Iaşi.
For histo-anatomical analysis the plant material (10 whole plants in the anthesis phase)
was fixed and conserved in 70% ethylic alcohol. For anatomical analysis, cross sections
of root middle stem (top, middle and basis) and leaves were used. Free hand sections
were performed using a razor blade. The sections were coloured with Iodine Green and
Ruthenium Red. Photographs were taken with an Olympus E-330 photo camera, using
an Olympus BX51 research microscope.
Scanning electron microscopy (SEM) investigations: the investigated material
consists of small leaf pieces. The plant material was fixed in FEA (formol: 70% ethanol:
acetic acid –5:90:5) for 48 hours, washed with distilled water and stored in 70% ethanol.
Some pieces were sectioned under the stereomicroscope with a razor blade. After
dehydration in a graded ethanol series (80%, 90% and 100%) and acetone, the material
was critical point dried with CO2 (using a EMS 850 Critical Point Dryer), sputter-coated
with a thin layer of gold (30 nm) (using a EMS 550X Sputter Coater) and, finally,
examined by scanning electron microscopy (Tescan Vega II SBH) at an acceleration
voltage of 27.88 kV.
*
Alexandru Ioan Cuza University, Faculty of Biology, Bd. Carol I, no. 11, Iasi - 700506 Romania,
[email protected] , [email protected], [email protected]
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Results and discussions
Leaf cross-sections show complete bacterial nodules formed (Plate I). Leaf
infection occurs in early stages of ontogenesis through a stoma-like hydathode which
closes later. This is larger than normal stomata on the leaf. After infection, the
substomatic chamber is closed [9]. The central part of the bacterial nodules is composed
of elongated cells after the penetration of bacteria.
This is almost colourless on fresh sections; in the side areas were seen in fewer
chloroplasts, much smaller than those in mesophyllic adjacent (Plate II). Gardner et al.
(1984) describe by ultra structural point of view, chloroplasts from this region as being
almost completely degenerate, sometimes disorganized inner membrane, with and
without plastoglobuli, but with broken grains. Lersten and Horner (1976) stipulate that
the bacteria begin to divide after they entered the cell; their presence inhibits the
chloroplast. Bacteria are present both intracellular and extracellular spaces. At this stage
the central part of the bacterial nodules consists by central cells and the vascular
connection with the leaf conducting tissue (visible on the external part of the bacterial
nodule).
In mature leaves the bacterial nodules are much larger, has a more organized
structure, compact central area surrounded by a continuous vascular area (Plate III). This
vascular tissue consists from both xylem and phloem and only from phloem tissue.
Outside of the vascular area we see a layer of cells closely united among them,
arranged orderly (a structure reminiscent of endodermoid with vascular bundle, a role,
perhaps in isolation of the nodule). Some of these cells, located near the vascular bundle,
has thickened and lignified cell walls. This protective layer is broken only on the right of
the stomatal chamber, where the infection was achieved. The phloem tissue which is
predominantly in the vascular structure ensures translocation of plant nutrients from the
host to nodules cells [9]. Bacteria found living both in and outside their cells, in close
liaison with the protective cell wall, this would suggest that the bacteria take over the
parietal part of the host material for proper use of the proper wall synthesis [2]. In some
cells mature bacterial nodules the walls destroyed, a fact observed by Gardner (1984). In
the vicinity of the nodules we observed numerous calcium oxalates, so they were
highlighted both in the direct and in the polarized light and scanning electron
microscopy. Although present in all leaf structure, their relatively high number near the
nodules area suggests their protective role, as well as achieving a required deposit of
calcium ion metabolism nodules (calcium is involved in cell wall biogenesis).
Electron investigations using scanning electron microscopy have revealed the
morphology of bacteria nodules both young and in the mature (Plate III). The number of
bacteria observed in the young nodules is very high, they exhibit a visible
pleiomorphism, the majority of rod-shaped, from 1.5 to 2.5 mm, others being in the form
of V or Y. Some of them (few in number) are in stage of division.
In mature bacterial nodules the observed bacteria are fewer in number but larger
in size. Some of them have degenerated during the nodule development; their remnants
are present in intra and inter-cellular spaces. Degeneration of the bacteria was observed
in adult nodule by Gardner (1984) which stipulates that organic material can be reused
by the remaining bacteria.
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Conclusions
Initiation of nodule bacteria is made early in ontogenesis, by infecting leaves
with bacteria that are on their surface [6]. The mature leaves can not be embedding the
bacteria to make this type of symbiosis (modified stomata through which the infection is
close in the meantime). Nodules of bacterial species were considered modified
hydathode [7]. Their role is still controversial. Initially it was believed that they had a
role in nitrogen fixation, in a manner similar to those of the genus Rhizobium from the
Leguminosae. Later, other researchers have assumed that bacterial nodules as a source of
plant growth substances [1] the required substances they would not be able to produce.
But investigations revealed ultra structural morphological similarities between bacteria
from Ardisia nodules and from the leaf of nitrogen fixing (genus Rhizobioum). Recent
researches [10] seem to deny the obligatory symbiosis between bacteria and leave the
Ardisia.
Acknowledgements
This study was supported by a research grant (IDEI 2100, no. 1040/2009)
funded by the National Council of Scientific Research and University Education
(CNCSIS), Romania.
REFERENCES
1. BECKING J. H., 1977 – Dinitrogen fixing associations. In: Proc. of the 1st International Symposium on
Nitrogen –Fixation, Washington State University Press, Pullman, 2: 556-580.
2. GARDNER G. Y., 1984 – Simulation of natural scenes using textured quadric surfaces. Computer Graphics,
18, 3: 11-20.
3. HORNER H. T., LERSTEN N. R., 1972 – Nomenclature of bacteria in leaf nodules of the families
Myrsinaceae and Rubiaceae. Int. J. Syst. Bacteriol, 22: 117-122.
4. LEBARD S., BELIN-DEPOUX M., 2003 – Structure and ontogeny of foliar bacterial nodules of Ardisia
crenata Sims (Myrsinaceae). Acta Botanica Gallica, 150: 19–33.
5. LERSTEN N. R., HORNER H. T. 1967 – Development and structure of bacterial leaf nodules in Psychotria
bacteriophila Val. (Rubiaceae). J. Bacteriol, 94: 2027-2036.
6. LERSTEN N. R., HORNER H. T., 1976 – Bacterial leaf nodule symbiosis in angiosperms with emphasis on
Rubiaceae and Myrsinaceae. Botanical Review, 42: 145-214.
7. MIEHE H., 1911 – Die sogenannten Eiweissdrusen an den Blittern von Ardisia crispa A. Ber. deut. Bot.
Ges., 29: 156-157.
8. MILLER I. M., SCOTT A., GARDNER I. C., 1978 – Pleomorphism of the endophyte in leaf nodules of
Ardisia crispa. Microbios Letters, 9: 133-137.
9. MILLER I. M., 1990 – Bacterial leaf nodule symbiosis. Advances in Botanical Research Incorporating
Advances in Plant Pathology, 17: 163–234.
10. NAKAHASHI C. D., FROLE K., SACK L. 2005 – Bacterial leaf nodule symbiosis in Ardisia
(Myrsinaceae): does it contribute to seedling growth capacity? Plant Biology, 7: 495-500.
11. SAIKKONEN K., WALI P., HELANDER M., FAETH S. H., 2004 – Evolution of endophyte-plant
symbioses. Trends in Plant Science, 9: 275–280.
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Explanation of plates
PLATE I - A - Ardisia crenata leaf morphology (abaxial part) - Note the bacterial nodules located on the edge,
B - cross section through a very young leaf (x20) the nodule is in an incipient stage, C - cross section through
a young leaf (x20) - tubular cell, elongated to the place of penetration of bacteria, D – details from the bacterial
nodules openings (x40) - modified stomata (where infection has been achieved) is yet open, we can also see the
vascular bundle will ensure the nodules vasculature (original).
PLATE II - A - cross section through mature nodules (ruthenium red and Green iodine coloration) (x20) – the
vascular link could be observed and the protective layer surrounding the nodule, B - cross section through the
edge of the Ardis crenata leaf (x20) (uncoloured) – the central part of the nodules is colourless; cells with a
reduced number of small chloroplasts, C - detail of mature nodule (x40) - small phloem bundles surrounding
the nodule, inside of the protector layer could be observed, D - cross section through mature nodule (x20) connection is apparent with the leaf vascular system, E - detail of the vascular connection (x40) - you can see
wooden vessels sectioned longitudinally, F - transverse section through leaf between veins (x20) (original).
PLATE III - A - cross section through the leaves mature nodules observed in polarised ligth (x20) - Counting
visible calcium oxalate crystals in the vicinity of the nodule, B - detail of leaf between veins (observation in
polarized light) (x40), C, D - cross sections through mature leaf (x40) - secretor hair with unicellular base and
multicellular gland (original).
PLATE IV - Electron-microscopic aspects of the bacterial leaf of Ardisia crenata: A - Cross section through
young leaf (bar = 1 mm), B - cross section through the mature leaf – secretory hair with pluricellular gland is
visible (bar = 20 μm), C - cross section through the mature leaf bacterial nodule (bar = 500 μm), D – external
part of an bacterial nodule on a mature leaf (bar = 20 μm), E - bacteria from a young leaf nodule (bar = 5 m), F
viable and degenerated bacteria in mature leaf (bar = 10 μm) (original).
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Irina Neta Gostin, Maria Magdalena Zamfirache, M. Ştefan
PLATE I
A
IRINA NETA GOSTIN
PLATE I
B
C
B
A
C
D
E
D
9
Irina Neta Gostin, Maria Magdalena Zamfirache, M. Ştefan
A
PLATE II
B
C
D
E
10
F
Irina Neta Gostin, Maria Magdalena Zamfirache, M. Ştefan
PLATE III
B
A
C
D
11
Irina Neta Gostin, Maria Magdalena Zamfirache, M. Ştefan
A
B
C
D
E
F
12
PLATE IV