Author’s personal copy

Phyton (Horn, Austria) Vol. 54 Fasc. 1 123–133 16. 5. 2014

DOI: 10.12905/0380.phython54(1)2014-0123

Crystal Macropatterns in Vegetative and Reproductive Organs of Trifolium Species

By

Lana Zoric, Ljiljana Merkulov and Jadranka lukovic*)

With 3 Figures

Received March 23, 2011

Accepted April 11, 2013

Key words : Anatomy, calcium oxalate, Fabaceae, Leguminosae, prismatic crystals, Trifolium div. spec.

Summary

Zoric l., Merkulov l. & lukovic J. 2014. Crystal macropatterns in vegetative and reproductive organs of Trifolium species. – Phyton (Horn, Austria) 54 (1): 123–133, with 3 figures.

Crystals are widely distributed in many plant species, some of them being spe-cific for certain taxa. An investigation of type, composition and distribution of crys-tals in vegetative and reproductive organs of twenty Trifolium L. species was car-ried out using Light and Scanning Electron Microscopy and Energy Dispersive Spectroscopy. Solitary, prismatic calcium oxalate crystals, variable in size and number, were present in all examined plant parts. They were distributed in paren-chyma sheath cells along the vascular bundles of lamina, main vein, stipules and calyx and above sclerenchyma groups by phloem in petiole, stem and peduncle. In stipules and calyx they were also present in one layer of mesophyll cells. From taxo-nomical point of view, the type and distribution of crystals were not valuable char-acters in distinguishing species of this genus or taxa of infraspecific rank. They might be more useful at the level of genus or even family. Crystals are less numer-ous, or even absent in organs of forage Trifolium species, which probably also con-tributes to their higher digestibility and forage quality.

*) Dr. L. Zoric, Dr. L. Merkulov, Dr. J. lukovic, University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg D. Obradovica 2, Novi Sad, Serbia. Corresponding author: e-mail: lana.zoric@dbe.uns.ac.rs

124

Author’s personal copy

Zusammenfassung

Zoric l., Merkulov l. & lukovic J. 2014. Crystal macropatterns in vegetative and reproductive organs of Trifolium species. [Kristallmuster in vegetativen und reproduktiven Organen von Trifolium]. – Phyton (Horn, Austria) 54 (1): 123–133, mit 3 Abbildungen.

In vielen Pflanzenarten gibt es Kristalle, einige davon sind charakteristisch für bestimmte Taxa. Eine Untersuchung der Art, der Zusammensetzung und der Ver-teilung der Kristalle in vegetativen und reproduktiven Organen von zwanzig Trifo-lium Arten wurden mittels Licht- und Scanning-Elektronenmikroskopie, sowie mittels energiedispersiver Röntgenspektrographie näher untersucht. Solitäre pris-matische Kalziumoxalatkristalle, unterschiedlich in Größe und Anzahl wurden in allen untersuchten Pflanzenteilen gefunden. Sie wurden in Parenchymscheidezellen entlang der der Leitbündel der Blattspreite, der Hauptader, den Nebenblättern und im Blütenkelch gefunden und waren auch in einer Schicht der Mesophyllzellen vorhanden. Vom taxonomischen Standpunkt her waren Typ und Verteilung der Kristalle keine geeigneten Parameter, um die Arten dieser Familie oder Taxa bestim-men zu können. Möglicherweise sind sie aber auf der Gattung- oder sogar Fami-lienebene in Nutzen. Weniger, oder überhaupt keine Kristalle waren in den Organen von Arten, die als Futter verwendet werden, zu finden. Dies spricht möglicherweise für ihre bessere Eignung als Futterpflanzen.

Introduct ion

Crystals are nonprotoplasmic inclusions of plant cells, which are widely distributed in plant species (Fahn 1967, Dickison 2000). The most common crystals are deposits of calcium oxalate (MetcalFe & chalk 1957). They are formed of calcium that is absorbed from the environment, and from endog-enously synthesized oxalic acid (Franceschi & nakata 2005, lersten & horner 2008). Crystals are classified into five categories based on their mor-phology (esau 1977, Dickison 2000). Solitary prisms and crystal druses are relatively widespread, whilst raphides, styloids and crystal sand are more restricted in distribution and therefore of greater taxonomic importance. Constancy of crystal morphology within species, cell specialization, and co-ordination of crystal growth, and cell expansion indicate that crystal forma-tion is genetically regulated. Genetic, as well as environmental factors, de-fine calcium oxalate crystal amount, shape, size and function. Crystal size can be very variable and mostly depends of the type of the cell in which crystal is formed. Ca availability and environmental factors, such as tem-perature, pressure and soil properties (Franceschi & horner 1980). They are found in vegetative and reproductive organs, in photosynthetic and non-photosynthetic tissues. Many plant crystals assumed to be calcium oxalate on the basis of their shape, but without any further positively identification (Franceschi & horner 1980).

Physiological functions of crystals are still not fully explained (korth & al. 2006). A general hypothesis about the function of calcium oxalate crys-

125

Author’s personal copy

tals in plants is that they protect cells from excess calcium, regulate ion balance, are involved in plant defense, tissue support, detoxification and light gathering and reflection (Franceschi & horner 1980, nakata 2002, Franceschi & nakata 2005). Crystals, particularly raphides and styloids, may cause dermal irritation or wounds in mouth and soft tissues of grazing animals, so they have role in preventing plants from herbivory and insects (Park & al. 2009). A negative correlation has been proved between the amount of crystals and grazing (Franceschi & nakata 2005). korth & al. 2006 proved the role of calcium oxalate crystals in plant protection against feeding insects, where they act as antinutritive defense, as well as feeding deterrent. Presence of calcium oxalate crystals in rumen walls, arteries or kidneys of grazing animals may even cause their death.

In the family Fabaceae solitary crystals mostly occur along the vascular bundles of the veins (MetcalFe & chalk 1957). Legume species differ widely in type and location of crystals. Only solitary crystals and druses are known in this plant family, and they usually originate inside the vacuole. Most com-monly they are located along the leaf vascular strands, but may also be pre-sent in leaf mesophyll and epidermal cells. Location and type of crystals are typical for certain taxa and genetically determined. Their presence is proved in leaves, stems, seeds, as well as in tapetal cells and connective tissue of anthers (Franceschi & horner 1980). However, many observations on legume crystals were made without providing proof that they were really of calcium oxalate (ZinDler-Frank 1987). In alfalfa leaves they were located in paren-chymatous sheath surrounding vascular bundles in the leaf, but they were less frequent in stems (WarD & al. 1979). Most of the crystals were calcium oxalate, but potassium oxalate crystals were also recorded. nakata & Mc-conn 2000 identified seven different classes of calcium oxalate defective Medicago truncatula Gaertn. mutants that showed altered crystal morphol-ogy, distribution and amount. One of the mutants even lacked crystals, which did not affect its growth.

The genus Trifolium L. is one of the largest genera in family Fabaceae (cooMbe 1972). The genus is divided into eight sections, on the basis of mor-phological characteristics of species (Zohary & heller 1984). Some of the species, like T. repens L., T. pratense L., T. incarnatum L., T. hybridum L., T. resupinatum L. and T. subterraneum L., are well-known forages, cultivated worldwide (cooMbe 1972, cincovic 1972, krstic & al. 2008). Previously pub-lished data about crystals in Trifolium species were not numerous, and were mostly included in general anatomic studies. None of the authors investi-gated the location and distribution of crystals in detail. ZinDler-Frank 1987 recorded that in T. subterraneum crystals were present along the leaf and petiole veins, adjacent to the sclerenchymatic sheath of the phloem and ap-peared at the time when the leaf unfolds. They were also recorded in T. lupi-naster palisade parenchyma and in T. repens, accompanying the veins. This author does not give any data about the presence of crystals in plant organs

126

Author’s personal copy

other than leaf. el-Fiki & kaDy 1990 recorded crystals in T. resupinatum leaflets and stem cortex, but not in T. alexandrinum and T. polystachyum. Crystals were not found in floral sepals, xylem and pith parenchyma of the stem of these three species. Variations in crystal morphology are observed in the leaves of T. pratense, but mechanisms controlling their morphology are still unknown (nakata 2002). Therefore, the aim of the present paper was to determine the type, the shape and the composition of crystals of Trifolium species and to investigate and describe their macropatterns in vegetative and reproductive organs, in order to give data useful in taxonomy of the genus and breeding of forage species.

Materials and Methods

Twenty species were collected during the flowering period (May-June) from native populations in Serbia and Montenegro, and determined at the Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad. Specimens were labeled, numbered, annotated with the date of collection, the locality, and the name of the collector. Voucher specimens were deposited in the Herbarium of the Depart-ment of Biology and Ecology, University of Novi Sad (BUNS) (Table 1). Infrage-neric classification followed Zohary & heller 1984.

Table 1. List of Trifolium species included in the study, with voucher numbers, col-lecting sites and dates.

Section Species Collecting site and dateVoucher number

Lotoidea Chronosemium ParamesusTrifolium Trichocephalum

T. angulatum WalDst. et kit. 1800T. hybridum L. 1753T. montanum L. 1753T. repens L. 1753T. campestre schreb. in sturM 1804T. micrantum viv. 1824T. velenovskyi vanDas 1889T. strictum L. 1755T. alpestre L. 1763T. angustifolium L. 1753T. arvense L. 1753T. dalmaticum vis. 1829T. incarnatum L. 1753T. medium L. 1759T. pannonicum Jacq. 1767T. pratense L. 1753T. stellatum L. 1753T. striatum L. 1753T. trichopterum Pancic 1856T. subterraneum L. 1753

Ostrov, 05. 2002. Rudina planina, 06. 2003.Vrdnik, 05. 2002.Valdanos, 05. 2002. Valdanos, 05. 2002. Rudina planina, 06. 2003.Rudina planina, 06. 2003.Rudina planina, 06. 2003.Vrdnik, 05. 2002. Valdanos, 05. 2002. Goč, 06. 2004.Dimitrovgrad, 06. 2002.Dimitrovgrad, 06. 2002.Vitoša, 06. 2002. Tara, 06. 2002.Novi Sad, 05. 2002.Valdanos, 05. 2003.Ostrov, 05. 2003. Rudina planina, 06. 2003.Valdanos, 05. 2002.

BUNS 2-1942BUNS 2-1956BUNS 2-1938BUNS 2-1935BUNS 2-1936BUNS 2-1987BUNS 2-1951BUNS 2-1953BUNS 2-1939BUNS 2-1937BUNS 2-1959BUNS 2-1985BUNS 2-1943BUNS 2-1944BUNS 2-1946BUNS 2-1941BUNS 2-1949BUNS 2-1988BUNS 2-1952BUNS 2-1934

127

Author’s personal copy

Twenty plants from each population were used for studies of crystals. Crystals of leaf (terminal leaflet of trifoliate leaf), petiole, stipule, stem, peduncle and calyx were observed under light and scanning electron microscope (SEM). Because of the specific nature of plant material, relevant data about crystals could not be obtained for some plant parts of T. dalmaticum, T. striatum (sesile inflorescences, no pedun-cle) and T. micranthum (very delicate plant parts). The segments of the organs, from the middle part of the plants, were separated and fixed in 50% ethanol. For light microscopy cross sections were made using Leica CM 1850 cryostat, at temperature -18 °C to –20 °C, at cutting intervals of 25 mm. Light microscopy observations were made using Image Analyzing System Motic 2000. For SEM analysis dry plant mate-rial of five plants per species was sputter coated with gold for 180 seconds, 30 mA (BAL-TEC SCD 005) and viewed with JEOL JSM-6460LV electron microscope at an acceleration voltage of 20 kV. Composition of crystals was detected by Energy Dispersive Spectroscopy (EDS), using Oxford instruments, Inca X Sight program. For preparation of plant material the same procedure was followed as in SEM.

Results

Crystals were recorded in all analyzed organs and plant parts of Trifo-lium species. They were solitary, of prismatic shape and variable in size. The cells contained only a single crystal (Fig. 1). Energy Dispersive Spectroscopy (EDS) showed that crystals were predominantly composed of calcium, which leaded to conclusion that they were calcium oxalate crystals (Fig. 2).

Fig. 1. SEM micrographs of calcium oxalate crystals in sheath cells along vascular bundles of T. campestre calyx (A), T. strictum calyx (B) and T. strictum leaf (C, D).

128

Author’s personal copy

In leaflet lamina, crystals were present in parenchyma sheath cells of vascular bundles, especially by sclerenchyma groups on abaxial side. They were more prominent and larger in sheath cells of the vascular bundle of the main vein (Fig. 3A). In petiole they occurred in parenchyma sheath cells above the sclerenchyma strains of vascular bundles (Fig. 3B). Parenchyma

Fig. 2. Energy Dispersive Spectroscopy (EDS) - SEM determination of calcium oxa-late crystals in T. strictum calyx.

sheath cells by vascular bundles in stipules also contained crystals (Fig. 3E, 3F). In addition to this, in stipules of most of the species of section Trifolium, crystals which were not in connection with veins had also been recorded. They were present in the cells of the first layer of stipule mesophyll, under adaxial epidermis. These cells were smaller, compared to other mesophyll cells, and each contained a single crystal. In stipules of species from other sections crystals were absent, or extremely rare in this layer.

Stem and peduncle showed very similar anatomical structure and the same pattern of distribution of crystals (Fig. 3C, 3D). They were recorded in parenchymatous sheath, which was visible only above phloem sclerenchyma.

129

Author’s personal copy

Fig. 3. Light micrographs of calcium oxalate crystals in different plant organs and parts. A: T. trichopterum, main vein. – B: T. alpestre, petiole. – C: T. alpestre, stem. – D: T. trichopterum, peduncle. – E: T. pratense, stipules. – F: T. hybridum, stipules. – G: T. incarnatum, calyx. – H: T. pannonicum, calyx. Arrows point to the crystals.

130

Author’s personal copy

Crystals were sometimes also present in cylinder parenchyma cells, under the vascular bundles. The first layer of calyx mesophyll, under the inner epi-dermis, was very compact, with closely packed cells that contained pris-matic crystals (Fig. 3G, 3H). In parenchyma sheath cells surrounding perivas-cular sclerenchyma, prismatic crystals also occurred.

In T. alpestre and T. trichopterum crystals were very numerous, espe-cially in the petiole, stem and peduncle (Table 2). On the other hand, a group of species was noticed with crystals that were rarely distributed, or even absent in analyzed plant parts. These were T. repens, T. hybridum, T. campes-tre, T. velenovskyi, T. incarnatum, T. pratense and T. subterraneum. In these species crystals were recorded only in a few cells of parenchymatous sheath by vascular bundles of leaf, petiole, stem or peduncle (Table 2).

Table 2. Presence and abundance of calcium oxalate crystals in analyzed Trifolium species.

Section Species Leaf Petiole Stipules Stem Peduncle Calyx

Lotoidea T. angulatum T. hybridumT. montanum T. repens L.

+*– or ±

+– or ±

±++

– or ±

+, –**+, –+, –+, –

+±+±

+±+±

++++

Chronosemium T. campestre T. micrantum T. velenovskyi

±+

– or ±

+– –+

+, –– –+, –

++±

±+±

+– –+

Paramesus T. strictum + + +, – + + +

Trifolium T. alpestre T. angustifolium T. arvenseT. dalmaticum T. incarnatum T. medium T. pannonicumT. pratense T. stellatum T. striatum T. trichopterum

+±++±±

– or ±– or ±

+++

+++++±++

– or ±++

+ +

+, ++, ++, –– –+, ++, ++, ++, ++, –+, ++, +

+++±+±+±

– or ±+++

+++±

– –±+±

– or ±+

– –++

+++++++++++

Trichocephalum T. subterraneum – or ± ± +, – ± ± ±

* – symbols indicate the presence and abundance of crystals in parenchyma sheath cells of vascular bundles: + – crystals present; ++ – crystals present and very numer-ous; ± – crystals present but rare; – crystals absent; – – – no data. ** – two symbols for stipules assign the presence of crystals in parenchyma sheath cells of vascular bundles and in the first layer of mesophyll, respectively

131

Author’s personal copy

Discussion

Plant crystals are one of the important anatomical parameters that may have application in taxonomy. Their type and distribution are typical for some taxa and therefore of taxonomic value. None of the authors that previ-ously examined crystals of Trifolium species gave data about their presence and distribution in plant organs other than leaf, and only seven species were mentioned in their papers (ZinDler-Frank 1987, el-Fiki & kaDy 1990, na-kata 2002). el-Fiki & kaDy 1990 recorded crystals in T. resupinatum stem cortex, but without giving an explanation in which tissue. Our observations agreed only partially with previous reports, which motivated us to conduct further analyzes of crystal macropatterns. The results showed that prismat-ic, solitary crystals were present in all examined plant parts of Trifolium species – leaf (lamina and the main vein), petiole, stipule, stem, peduncle and calyx. Using EDS method it was proved that crystals had been composed of calcium oxalate.

They were recorded in parenchyma sheath cells around or above vascu-lar bundles in all examined organs. Their position near vascular tissue might be explained by the assumption that calcium, which is transported via xy-lem, meets oxalate producing cells in this zone, and forms crystals of very low solubility (ZinDler-Frank 1987). Precipitation of Ca in these cells pre-vents its accumulation in photosynthetic tissue (Franceschi & nakata 2005).

Besides the crystals along the vascular tissue, the ones which were not in connection with veins had also been recorded. In calyx they were present in the first layer of mesophyll cells, under the inner epidermis. In stipules, the first layer of mesophyll cells under adaxial epidermis also contained small solitary crystals, but they were only recorded in some of the species of section Trifolium. In stipules of the species from other sections, crystals were absent or rare in this layer. As the crystals were recorded in only some of the species of the section Trifolium, but not in all of them, this character could not be discussed as taxonomically important for identification of species of this section. Some other anatomical parameters, like the characteristics of leaf epidermal tissue, were more taxonomically informative for Trifolium species (Zoric & al. 2009). However, crystals proved to be of restricted value in distinguishing species or taxa of infraspecific rank. They might be more useful at the level of genus or even family.

Bearing in mind that some of the Trifolium species are very important forages, it is important to discuss the presence of crystals in tissues of these species from agronomical point of view. WarD & al. 1979 found that 20-33% of calcium in alfalfa is in the form of oxalate, and therefore unavailable to ruminants. Crystals pass from the rumen largely intact and appear in the feces unchanged. Birds more effectively removed the crystals from alfalfa leaves, whilst nonruminal herbivores the least effectively (harbers & al. 1980). Since digestion of oxalate is poor, calcium in that form appears to be

132

Author’s personal copy

unavailable to the animals. It could be predicted that presence of such crys-tals in Trifolium forage species is one of the factors that limits their digest-ibility. Digestibility is one of the most important characteristics that deter-mines the quality of forages (buxton & reDFearn 1997, krstic & al. 2008). Selection of anatomical components that affect forage quality is the base of many forage breeding programs. Data about type and distribution of indi-gestible crystals in plant organs that are frequently grazed by animals, to-gether with data about distribution of other indigestible compounds (like tissues composed of cells with lignified walls-xylem, sclerenchyma and scle-renchymatous parenchyma), must be taken into account in improvement of digestibility and nutritive value of forage legumes. The results of our study showed that most of the species with rare crystals in analysed organs were the high quality forages (T. repens, T. hybridum, T. pratense, T. incarnatum and T. subterraneum). As these species are already known as the ones with high digestibility rates, we suppose that such low amount of crystals also contributed to their high forage value. The results of genetic investigations of nakata & Mcconn 2000 on Medicago species showed that presence of crystals could be genetically manipulated and that mutants that lack crys-tals do not differ in growth and development from control plants. This find-ing opened the possibility of new breeding strategies in improvement of di-gestibility of forage legumes.

Acknowledgements

The authors would like to thank Mr. M. bokorov from University Center for Electron Microscopy, Novi Sad, for his technical assistance, SEM microscopy and EDS analysis. This work was financially supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, Grants No. 31024 and 173002.

References

buxton D. r. & reDFearn D. D. 1997. Plant limitations to fiber digestion and utiliza-tion. – J. Nutr. 127: 814–818.

cincovic T. 1972. Genus Trifolium. – In: JosiFovic M. (ed.), Flora SR Srbije IV, p. 424–471. – SANU, Beograd.

cooMbe D. E. 1972. Trifolium L. – In: tutin T. G. & al. (eds.), Flora europaea, Vol 2, p. 157–172. – University Press, Cambridge.

Dickison W. 2000. Integrative plant anatomy. – Academic Press, San Diego.el-Fiki M. a. & kaDy k. A. 1990. Comparative morphological and anatomical stud-

ies on some Phaseolae and Trifolieae. – Zagazig J. Agric. Res. 17 (2): 313–324.esau K. 1977. Anatomy of seed plants, 2nd ed. – John Wiley and Sons, New York –

Santa Barbara – London – Sydney – Toronto.Fahn A. 1967. Plant anatomy. – Pergamon Press, Oxford – New York – Toronto – Syd-

ney – Braunschweig.Franceschi v. r. & horner h. t. 1980. Calcium oxalate crystals in plants. – Bot. Rev.

46 (4): 361–427.

133

Author’s personal copy

Franceschi v. r. & nakata P. 2005. Calcium oxalate in plants: formation and func-tion. – Annu. Rev. Plant Biol. 56: 41–71.

harbers l. h., callahan s. l. & WarD G. M. 1980. Release of calcium oxalate crys-tals from alfalfa in the digestive tracts of domestic and zoo animals. – J. Zoo Anim. Med. 11: 52–56.

korth k. l., DoeGe s. J., Park s. h., GoGGin F. l., WanG q., GoMeZ s. k., liu G., Jia l. & nakata P. a. 2006. Medicago truncatula mutants demonstrate the role of plant calcium oxalate crystals as an effective defense against chewing in-sects. – Plant Physiol. 141: 188–195.

krstic l., Merkulov l., lukovic J. & boZa P. 2008. Histological components of Trifo-lium L. species related to digestive quality of forage. – Euphytica 160: 277–286.

lersten n. r. & horner h. T. 2008. Crystal macropatterns in leaves of Fagaceae and Nothofagaceae: a comparative study. – Plant Syst. Evol. 271: 239–253.

MetcalFe c. r. & chalk l. 1957. Anatomy of the dicotyledons, Vol I. – Clarendon Press, Oxford.

nakata P. a. & Mcconn M. M. 2000. Isolation of Medicago truncatula mutants defec-tive in calcium oxalate crystal formation. – Plant Physiol. 124: 1097–1104.

nakata P. A. 2002. Calcium oxalate crystal morphology. – Trends Plant Sci. 7(7): 324.Park s. h., DoeGe s. J., nakata P. a. & korth k. l. 2009. Medicago truncatula – de-

rived calcium oxalate crystals have a negative impact on chewing insect per-formance via their physical properties. – Entomologia Exp. Appl. 131: 208–215.

WarD G., harbers l. h. & blaha J. J. 1979. Calcium-containing crystals in alfalfa: their fate in cattle. – J. Dairy Sci. 62 (5): 715–722.

ZinDler-Frank e. 1987. Calcium oxalate crystals in legumes. – In: stirton C. H. (ed.), Advances in legume systematics, part 3, p. 279–311. – Royal Botanic Gardens, Kew.

Zohary M. & heller D. 1984. The genus Trifolium. – The Israel Academy of Sciences and Humanities, Jerusalem.

Zoric l., Merkulov lJ., lukovic J., boZa P. & Polic D. 2009. Leaf epidermal charac-teristics of Trifolium L. species from Serbia and Montenegro. – Flora 204: 198–209.

Author’s personal copy