Flora 233 (2017) 186–193

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Flora

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Anatomical specificities of two paleoendemic flowering desiccation

tolerant of the ()

a,∗ b c

Tamara Rakic´ , Steven Jansen , Dragana Ranciˇ c´

a

Department of Ecology and Phytogeography, Faculty of Biology, University of Belgrade, 11000 Belgrade,

b

Institute of Systematic and Ecology, Ulm University, D-89081 Ulm, Germany

c

Department of Agrobotany, Faculty of Agriculture, University of Belgrade, 11080 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: and R. nathaliae are known as resurrection flowering . Both species are long-

Received 29 August 2016

living chasmophytes that are physiologically inactive during warm summer periods. Besides numerous

Received in revised form 25 May 2017

known adaptations at the physiological level, it is reasonable to expect that these plant species possess

Accepted 3 June 2017

a number of distinctive structural adaptations associated with poikilohydry. Therefore, we analyzed in

Edited by Hermann Heilmeier

detail the anatomy of roots, stem and of both species. Plants were collected from their natural

Available online 9 June 2017

habitat or grown from seeds in controlled conditions. Fresh or fixed plant material was sectioned and

stained by various histochemical reagents. In addition, vascular tissue was investigated on macerated

Keywords:

plant material, and the characteristics of epidermal cells were analyzed on epidermal peelings. Samples

Contractile stem

were investigated by reflected or transmitted light microscopy, or by scanning electron microscopy.

Fluorescence microscopy

Lacunar space Epidermal cells of the leaves have specific anticlinal beaded thickenings, which could enable maintenance

Poikilohydric plant of the epidermis during dehydration and rehydration. Stem vascular tissues form a net-like structure.

Resurrection plant The shape and arrangement of parenchyma cells appear to support axial contraction. At the base of

Vascular system

each adventitious root, a special, thick-walled tissue is observed, which is composed of living cells with

pitted walls and probably not involved in long-distance water transport. Overall, various stem and

adaptations allow effective water transport through the plant and provide mechanical stability during

the relatively fast process of dehydration and rehydration.

© 2017 Elsevier GmbH. All rights reserved.

1. Introduction , including plant families such as Cactaceae, Scrophulari-

aceae, Lamiaceae, , , Gesneriaceae and

The ability of plants to survive complete dehydration and then Myrothamnaceae (Porembski, 2011). In Europe, there are only five

accomplish full recovery after a relatively short period of rehy- eudicotyledonous species known as resurrection plants, all belong-

dration without structural and physiological damage is called ing to the Gesneriaceae family: Ramonda serbica Panc.ˇ , R. nathaliae

poikilohydry, which is a rare phenomenon among vascular flow- Panc.ˇ et Petrov., R. micony (L.) Rchb., rhodopenis Friv.,

ering plants. Considering that drought is one of the most serious and Jankea heldreichii (Bioss.) Bioss. These are tertiary relicts and

ecological and economic problems of the modern world, it is impor- endemics of the Balkan Peninsula, with the exception of R. micony,

tant to understand desiccation tolerance mechanisms in plants. which is found in the Iberian Peninsula. Besides SE Serbia, popula-

Although earlier work has been conducted on the mechanisms tions of Ramonda species can also be found in southern Balkan areas,

involved in desiccation tolerance (Bartels and Salamini, 2001; Vicré with R. serbica growing in Albania, , FYR Macedonia, and

et al., 2004; Lüttge et al., 2011; Moore et al., 2013; Farrant et al., Montenegro, and R. nathaliae occurring in FYR Macedonia, Greece,

2015), structure-functional aspects associated with poikilohydry and Kosovo.

remain poorly understood. Ramonda serbica and R. nathaliae inhabit humid habitats on

From the ca. 300.000 species of flowering plants, only 300 are north exposed slopes of canyons and gorges in rocky outcrops,

known to be poikilohydric, of which about 40 species belong to the often sheltered by surrounding shrubs or in the forest under-

story. Usually, they share their microhabitat with several species

of desiccation-tolerant mosses (e.g., Trichostomum crispulum Bruch,

Ctenidium molluscum Mitten) and (Asplenium ceterach L., A. ∗

Corresponding author.

ruta-muraria L. and A. trichomanes L.).

E-mail address: [email protected] (T. Rakic).´

http://dx.doi.org/10.1016/j.flora.2017.06.003

0367-2530/© 2017 Elsevier GmbH. All rights reserved.

T. Raki´c et al. / Flora 233 (2017) 186–193 187

Fig 1. Well hydrated (A) and dehydrated (B) plants of the desiccation tolerant species Ramonda serbica (Gesneriaceae) in its natural habitat in SE Serbia. The uprooted plant

(C) shows strongly branched and superficial roots.

Although angiosperm resurrection plants share various mech- cally integrated via vascular bundles to tolerate dehydration, while

anisms that enable poikilohydry, they also show distinctive enabling at the same time fast and efficient water uptake during

structural and physiological differences that have evolved in rehydration.

diverse and highly complex environments. While most angiosperm

resurrection plants grow in tropical/subtropical climates of the

2. Material and methods

southern hemisphere, Ramonda species from the Balkan area are

subject to a high seasonality, with a long, warm and dry summer,

One-year-old plants and adult plants were collected from their

and a cold winter with long periods of sub-zero temperatures and

natural habitat in the Jelasnica gorge in SE Serbia. In addition,

a thin snow cover on the steep sides of canyons. Within these cli-

young plantlets were grown from seeds in a sterile culture medium

matic conditions, Ramonda plants go several times per year through

(Murashige and Skoog, 1962) for three months.

dehydration–rehydration cycles. Usually, during the humid and rel-

The whole plant morphology and sections of larger areas were

atively cold periods of the year (i.e., spring, late autumn, and often

observed using a stereomicroscope (Nikon SMZ 18).

winter) both species remain well-hydrated, whereas during the dry

Transverse sections were taken from one-month- and three-

and hot summer they survive in a state of physiological inactivity

months-old plantlets, one-year-old plants, and adult plants. A

or anabiosis.

series of transverse and longitudinal sections, 8–10 ␮m thick, were

Earlier investigations were focused mainly on the physiolog-

obtained from the entire body of the stem, leaves and roots. Plants

ical, biochemical and molecular aspects of desiccation tolerance

were sectioned fresh or fixed in FAA and 50% ethanol prior to prepa-

(Markovska et al., 1994; Bartels and Salamini, 2001; Bartels, 2005;

ration for a standard paraffin method (Ruzin, 1999).

Moore et al., 2007; Jovanovic´ et al., 2011; Morse et al., 2011; Dinakar

Fresh plant material was hand sectioned with a razor blade or

and Bartels, 2013; Rakic´ et al., 2014). So far, only few studies have

40 ␮m thick sections were cut with a cryo-microtome (Leica CM

paid attention to structural features of R. serbica and R. nathaliae, ◦

1850) at −21 C, and observed by epi-fluorescence microscopy to

although it is reasonable to expect that resurrection plants show

detect autofluorescence, or stained with toluidine blue (O’Brien

a number of distinctive structural adaptations at the cell, tissue

et al., 1964). Additional stains included phloroglucinol-HCl to

and organ level that sustain poikilohydry. The morphology and

detect lignified cell walls (Jensen, 1962), Sudan IV for lipids (Pearse,

anatomy of some plant organs have been described in various

1980), Lugol to visualise starch (Johansen, 1940), and ferric trichlo-

resurrection species: leaves in Jankaea heldreichii (Gesneriaceae)

ride to identify phenolic compounds (Johansen, 1940). Paraffin

(Stevanovic´ and Glisic,´ 1997) and Craterostigma plantagineum

sections were double stained in safranin (1%, w/v in 50% ethanol)

(Linderniaceae), Xerophyta humilis (Velloziaceae) (Farrant, 2000),

and alcian blue (1% w/v, aqueous). In addition, root, stem and leaf

leaves, floral structures and stem anatomy in Myrothamnus fla-

tissue was macerated in a mixture of concentrated glacial acetic

bellifolius (Myrothamnaceae) (Sherwin et al., 1998; Farrant, 2000; ◦

acid and 30% hydrogen peroxide (1:1 v/v) at 60 C, until the samples

Moore et al., 2006, 2007), leaves and stems of Reaumuria soongarica

turned white (1–5 days). The macerated cells were stained with

(Tamaricaceae) (Liu et al., 2007) and leaves and roots in the aquatic

safranin. Vessel diameters were measured on transverse sections

resurrection plant Chamaegigas intrepidus (Linderniaceae) (Schiller

at the widest part of the vessel, excluding the cell wall. The length

et al., 1999; Heilmeier et al., 2002). For R. serbica and R. nathaliae

of vessel elements was measured including their tails. Microslides

only leaves have been studied so far (Stevanovic´ and Glisic,´ 1997;

were examined and photographed using a Leica DMLS light micro-

Rascio and La Rocca, 2005).

scope equipped with a Leica DFC295 digital camera.

In adult plants of R. serbica and R. nathaliae, the apex of the short

In order to observe water absorption and long-distance

aerial part of the stem is covered by 10–15 perennial mesomor-

water transport, dry plants were treated separately with 5(6)-

phous leaves, organized in a rosette (Fig. 1). Beneath these, the dead

Carboxyfluorescein (Grignon et al., 1989; Viola et al., 2001) and

marcescent leaves are firmly attached to the stem and protrude

Eosin Y (Baum et al., 2000) as fluorescent markers of phloem and

above the carpet of mosses that surrounds the stem and covers the

xylem transport, respectively. The stem sections were obtained

soil (Rakic´ et al., 2009). Although both species grow slowly and are

24 h after the beginning of rehydration and observed by epi-

estimated to be long living, their stems usually do not exceed more

fluorescence microscopy.

than three centimetres. It can be presumed that the rehydration

To test for autofluorescence and dye fluorescence an epi-

efficiency of the stem and, to some extent, the rehydration of the

fluorescence Olympus BX51 microscope equipped with an HBO

leaves are largely affected by the organization of the stem vascular

50 W mercury vapour lamp with wide-band BV excitation filter

tissues, which may play a major role during early rehydration.

(excitation filter BP400-440, dichroic beam splitter DM455 and bar-

The aim of the current work is to describe the anatomy of the

rier filter BA475) and wide band Blue excitation filter (excitation

vegetative plant tissues in R. serbica and R. nathaliae. Some of the

filter BP460-490, beam splitter DM500, barrier filter BA520IF) were

questions that will be addressed include: (1) how are the vascular

used. All anatomical parameters were measured using the software

tissues organized, and (2) how are roots and leaves anatomi-

programme Leica Q Win (Leica Microsystems, Germany).

188 T. Raki´c et al. / Flora 233 (2017) 186–193

Fig. 2. Developmental stages of Ramonda serbica showing root and shoot details. One-month-old plantlet (A), three-months-old plantlet with the first nodal root (B), one-

year-old plant with nodal roots (C), the longitudinal cross section of the stem in an adult plant (D) and detailed view with root trace (rt) and leaf petiole base (lt) arising close

to each other from the axial vascular bundle (E).

Fig. 3. Unstained parenchyma cells in root cortex (A); Transverse section through the primary root stained with toluidine blue (B); The orange colour in outer parenchyma

cells within root cortex indicates presence of lipid droplets, based on staining with Sudan (C); Brown colour indicating the presence of phenolic compounds stained with

FeCl3 (D); Ornamentation of a xylem vessel in a macerated root sample stained with safranin (E) and secondary root structure stained with safranin and alcian blue (F). (For

interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In addition, some details regarding leaf and stem anatomy, violet in presence of toluidine blue, a colour that is typical of pri-

xylem elements and vascular sclerenchyma were analyzed using mary cell walls rich in pectins and cellulose (Fig. 3B). Transverse

a JEOL JSM-6460LV scanning electron microscope. sections stained by lugol indicate the absence of starch storage

in the cortex. On the other hand, histochemical staining shows

the presence of lipid droplets in the outer cortical cells (Fig. 3C)

3. Results

and the presence of phenolic compounds (Fig. 3D). In roots show-

ing primary growth, the vessel diameter including the vessel wall

3.1. Root morphology and anatomy

is 12.7 ± 1.6 ␮m and 24.4 ± 3.7 ␮m in R. serbica and R. nathaliae,

respectively, and the vessel element length is 110.9 ± 61.2 ␮m and

The young, one-month-old seedlings possess only a primary

105.7 ± 36.9 ␮m, respectively. The thickness of the secondary cell

root (Fig. 2A). At an early stage of plant development, lateral roots

wall is 2.7 ± 0.8 ␮m in R. serbica and 3.1 ± 0.6 ␮m in R. nathaliae.

as well as adventitious roots arising from the stem node can be

Tracheary elements with both scalariform and reticulate patterns

observed (Fig. 2B, C). During later developmental stages more nodal

of thickening are present in the xylem (Fig. 3E).

roots occur and, on the longitudinal section of the adult plant stem,

Although most roots that spread from the stem have a typical

numerous, thin adventitious roots arise from each node (Fig. 2D,

primary structure, secondary thickening can also be seen in some

E). From the same node from which a root appears, a leaf petiole

roots (Fig. 3F).

arises, which grows in the opposite direction to the root.

The cortex of the primary root, which comprises the largest vol-

ume of the root, consists of three regions. Two or three layers of 3.2. Stem morphology and anatomy

tangentially elongated and suberized parenchyma cells form the

outer protective exodermis. The middle region is multi-layered, The underground part constitutes the largest portion of the

with cells elongated in the longitudinal direction, and a third, inner stem, from which the strongly branched roots extend into the shal-

endodermis is composed of an uneven row of smaller cells of low soil (Fig. 1C). The stem of young plants that are about 1–2 years

more or less spherical shape. The cortex cells have unusually thick old is straight, with no wrinkled surface. Longitudinal and trans-

walls (4.6 ± 0.97 ␮m, mean value ± SD; Fig. 3A), which are stained verse sections of the stem and peduncle show regularly positioned,

T. Raki´c et al. / Flora 233 (2017) 186–193 189

Fig. 4. Hand cross-sections of a stem stained with phloroglucinol-HCl: Transverse section of the stem (A), longitudinal section of the stem with pairs of leaf (lt) and root

traces (rt) along the stem (B), longitudinal section at the level of axial vascular bundles with clusters of vascular sclerenchyma arrowed (C). Longitudinal section, double

stained with safranin and alcian blue (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Vascular sclerenchyma visible on transverse sections of the stem: stained intense green with toluidine blue (A) or stained intense red with a double safranin-alcian blue

staining (B, C). Slightly pressed cluster of vascular sclerenchyma tissue observed in macerated stem tissue (D). Unstained, longitudinal section through vascular sclerenchyma

tissue (E) and SEM micrograph of individual cells (F). Vascular sclerenchyma in unstained transverse section observed with a light microscope (G). Autofluorescence of lignin

in vascular sclerenchyma cell walls under BV filter (H) and Blue excitation filter (I), arrowed. (For interpretation of the references to colour in this figure legend, the reader

is referred to the web version of this article.)

individual collateral vascular bundles and therefore a typical pri- parenchyma cells and single vascular bundles showing leaf and root

mary stem anatomy. Over the years, as the stem grows, it contracts traces. Although the age of the plant specimens was estimated to

axially. In older plants, the stem is mostly up to 3 cm long and has a be more than five to ten years, the vascular bundles run separately

noticeably rugged outer surface (Fig. 2D). The surface of the shoot and extend radially during the secondary growth, without connect-

apex and leaf primordia are covered densely with long hairs com- ing laterally and thus never form a continuous ring. Adventitious

posed of some living cells at their base, whereas the upper part is roots originate from the vascular central ring of the stem and pass

made of dead cells (Fig. 2D). through the cortical region during their development. These lat-

Transverse cross-sections show the presence of a periderm on eral roots are visible in both longitudinal and transverse sections,

the surface, a relatively wide cortex zone and a central vascu- almost over the entire length of the stem, and are most abundant in

lar cylinder (Fig. 4A). The outer cortex consists of thin-walled the lower part of the stem. In the longitudinal paradermal sections,

190 T. Raki´c et al. / Flora 233 (2017) 186–193

Fig. 6. SEM micrographs of a stem transverse section (A, B) and pith parenchyma cells observed with SEM (C) and light microscopy (D).

Fig. 7. Leaf cross-section stained with safranin and alcian blue and observed with a light microscope (A) and with SEM (B). Light microscopy of anticlinal epidermal cell walls

in hydrated (C) and dehydrated leaf (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

only root and leaf petiole traces can be observed, and are always walls of this unusual type of lignified cell stain similarly as xylem,

present in pairs (Fig. 4B). but with a different tone: they are more greenish than blue when

Transverse sections of the stem illustrate the anatomical vari- stained with toluidine blue (Fig. 5A), and crimson red when stained

ation along the short stem axis, which derives from a specific with safranin or phloroglucinol (Fig. 5B). These cells show a strong

organization of the vascular tissue at different stem levels. A series autofluorescence under an epifluorescence microscope (Fig. 5G–I).

of longitudinal sections through the stem of both Ramonda species The stem and leaf cross-sections were analyzed after plant rehy-

reveals the net-like organization of vascular tissues that is formed dration, 24 h after the treatment. In stem sections, these lignified

by connection between neighbouring, sinuous axial vascular bun- cells remained unstained with 5,6-Carboxyfluoresceine (phloem

dles (Fig. 4C, D). As a result, some of the longitudinal sections reveal tracer). Only the outer cells became stained with Eosin Y (xylem

continuous strands of vascular bundles, whereas others show sep- tracer), whereas the inner ones remained unstained, showing only

arate vascular bundles in a vertical sequence (Fig. 4B–D). The autofluorescence (not shown). The staining of the outer layer of

vascular network of the stem vascular ring is connected directly these lignified cells is probably a consequence of passive dye dif-

to the leaf and to the root traces at nodes – one vascular trace fusion from the surrounding parenchyma cells that absorb water

diverges to supply both the leaf and the root trace (Fig. 4C). Ves- with diluted dye. The stem pith consists of thin-walled parenchyma

± ␮

sel elements in R. serbica and R. nathaliae are 100.0 36.6 m and with an average transverse diameter of 104.3 ± 16.3 ␮m, length

± ␮ ± ␮ ± ␮

66.0 15.0 m long and 12.8 2.8 m and 12.0 2.1 m wide in of 136.5 ± 40.2 ␮m, and longitudinal width of 56.3 ± 21.4 ␮m. The

their outer diameter, respectively. The thickness of the secondary parenchyma cells have a variable shape and size, there is no clear

± ␮ ± ␮

cell wall is 2.0 0.3 m in R. serbica and 2.2 0.5 m in R. nathaliae. alignment of these cells in a transverse or longitudinal plane, and

Pitting in xylem is reticulate (Fig. 3E). the intercellular spaces are far less spacious in well hydrated plants

Clusters of specific tissue are associated with the stem vascular than in desiccated ones (Fig. 6A–D). In a longitudinal section the

bundles. Clusters are composed of closely packed thick-walled cells cortical parenchyma cells are larger than the pith cells, especially

with pitted walls, which form distinct compact structures that have those lying in-between two branches of vascular tissue. The layers

a patchy, but very regular distribution within the vascular network of parenchyma cells that are directly associated with the vascular

(Fig. 5A–C). This tissue is present only at nodes, always at the base bundles are strikingly compressed (Fig. 4D). In dried plants, wide,

of the root, but never at the base of the leaf trace (Fig. 4C). It can be regular gaps occur between the vascular bundles and the parenchy-

found even in the zone next to the apical meristem. The cells are mal tissue (Fig. 6A, B).

thick-walled, elongated longitudinally, always shorter at the base

of the compact cylindrical structure than at its upper part, and are

3.3. Leaf anatomy

characterized by a pitted-like structure visible in their outer sur-

face (Fig. 5D–F). In R. serbica, the short basal cells are 48.0 ± 7.6 ␮m

The leaf blade is bifacial and the palisade cells are very short

long and 23.4 ± 2.9 ␮m wide in their outer diameter, whereas the

and arranged in one or two layers; the spongy tissue consists of

long cells are 109.9 ± 31.1 ␮m long and 15.0 ± 2.4 ␮m wide. In R.

few cell layers (Fig. 7A, B). Leaves are hypostomatic in R. serbica

nathaliae the length and width of short cells are 43.9 ± 7.2 ␮m and

and amphistomatic in R. nathaliae. Anisocytic stomata are aligned

17.2 ± 2.1 ␮m, whereas those of the long ones are 173.3 ± 26.5 ␮m

with epidermal cells in both species. The dimensions of stomata and

and 16.8 ± 2.5 ␮m. The average diameter of the cell lumen in both

their densities were previously described and discussed by Rakic´

species is 12.9 ± 5.6 ␮m. Most frequently, the cell lumen is filled

et al. (2015).

with dense, red-brown substances. At higher magnification, their

Epidermal cells, viewed from the surface, are more or less

inner cell wall surface is seen to be undulated and, despite much

polygonal or undulated with very conspicuous beaded cell wall

reduced cell volume, these cells are alive, possessing a cytoplasm

thickenings, which extend as short columns and are more promi-

with a nucleus (Fig. 5D). Their cell walls are 9.3 ± 1.8 ␮m thick,

nent in the lower epidermis (Fig. 7C, D). The latter is loosely

much thicker than those of ordinary stem xylem vessels. The cell

attached to the mesophyll of the leaf between major leaf veins and

T. Raki´c et al. / Flora 233 (2017) 186–193 191

Fig. 8. Two types of glandular trichomes present on the leaf surface of a three-month-old plantlet (A, B). Longitudinal section of the shoot apex showing leaf petioles, densely

covered with long, uniseriate hairs and glandular hairs (C).

can therefore be very easily peeled off from the main body of the roots, stem and leaves. A specific criss-cross organization of axial

leaf (Fig. 7A, B). This is characteristic of both fully developed leaves bundles may also offer high mechanical stability of the stem dur-

and young leaves during earlier developmental stages. The epider- ing volume changes provoked by fluctuations in its water content

mal detachment is most evident in R. serbica. Vessels in the midribs due to numerous dehydrations and rehydrations to which plants

and leaf petiole are with helical thickenings and are 23.9 ± 6.4 ␮m are exposed repeatedly during their life span.

wide (including vessel walls). The vessel elements in R. serbica and Clusters of thick-walled cells with circular pits have a patchy but

in R. nathaliae are 280.0 ± 101.8 ␮m and 242.1 ± 106.4 ␮m long, regular position within the stem, on the outer side of the vascular

respectively. network, and occur exclusively at the basal part of adventitious

The indumentum on leaves and leaf petioles is composed of mul- roots. Upon maturation these cells are as long as vessel elements

ticellular, unbranched trichomes (Fig. 8A–C), which are especially (in R. serbica) and even three times as long as vessel elements (in

numerous on the lower epidermis. The stalked glandular trichomes R. nathaliae). The difference in the size of cells within one patch,

are randomly scattered over the leaf surface. Some glandular hairs which varies from two to almost four times, could be explained

have a longer stalk with two apical glandular cells (Fig. 8A), whereas by differences in their ageing, and therefore different effects of

others have a short stalk and a head divided by vertical walls into environmental conditions on their growth and development. Their

four cells (Fig. 8B). The basal part of the leaf petiole is covered by an highly lignified and pitted cell walls, apparently thicker than vessel

extremely dense indumentum of linear, non-glandular trichomes walls, and the patchy occurrence of this specific tissue along the

(Fig. 8C). stem, even next to the apical meristem, indicate that it differenti-

ates rather early during stem development relative to xylem. The

histochemical staining and autofluorescence observations show

4. Discussion that the chemical composition of their cell walls and that of the

xylem differs. This tissue seems to be similar to what was previ-

One of the striking morphological features of the resurrection ously described in the stem of the monocot species Eriophorum

chasmophytic plants Ramonda serbica and R. nathaliae is the very vaginatum L. (Cyperaceae) (Cholewa and Griffth, 2004). Although

short stem, which is mainly located below- ground in adult plants Cholewa and Griffth (2004) described it as “vascular sclerenchyma”

and not longer than 3 cm. From most of its conspicuously rugged and proposed it to have the ability to aid in xylem and phloem long-

surface, various thin and highly branched roots grow. These fine distance transport, we could not confirm a xylem or phloem link

roots strongly permeate the shallow organo-mineral substrate and because fluorescent dyes were not observed within this tissue 24 h

in the presence of vesicular-arbuscular mycorrhizal fungi (Rakic´ after re-watering. However, it is possible that it retains water for

et al., 2009, 2013) are highly effective in absorbing water and nutri- some time even after the vessel elements lost the water column

ents from the soil. In both species, the stem shows nonspecific upon desiccation, and that it offers higher strength to the stem.

secondary growth characterized by radial extension of individual Whereas connectivity exists between all stem tissues in well

vascular bundles, which do not join laterally during plant growth hydrated plants, dried stems show gaps between vascular bundles

and ageing. Axial vascular bundles show a net-like organization and the surrounding parenchyma tissue. Difference in hardness and

within the stem and are sinuously folded, suggesting that there is elasticity between thin-walled parenchyma and thick-walled vas-

a permanent and vertical stem contraction. Moreover, the strik- cular tissues and therefore in the level of volume reduction lead to

ingly pressed layers of parenchyma cells that rest directly against their separation. The several layers of collapsible parenchyma tis-

the vascular bundles and the unequally oriented pith parenchyma sue, detected along the vascular bundles, facilitate a volume change

cells point to stem compression. The extremely similar, net-like in parenchyma tissue provoked by plant desiccation and rewater-

organization, as well as the permanent vertical contraction of the ing. Although gaps might represent spots of potential pathogen

stem, has only been described in detail in the Pleuro- attack to plant tissues, intensive histochemical black staining ver-

phyllum hookeri Buchanan from the Macquarie Island (Briggs et al., ifies the presence of large amounts of phenolic compounds, which

2006). At the same position near the nodes, the vascular network in may have antimicrobial properties. The high amounts of phenolic

Ramonda plants diverges to supply both the leaf and the root trace. acids were found in the Ramonda leaves, with the most abun-

This kind of organization of the stem vascular tissue and highly dant ones being protocatechuic, p-hydroxybenzoic and chlorogenic

integrated root–leaf vascular system could be beneficial in water acids (Sgherri et al., 2004), which are known to have an antibacte-

absorption and a very efficient distribution of water between the

192 T. Raki´c et al. / Flora 233 (2017) 186–193

rial activity (Tuncel and Nergiz, 1993; Taguri et al., 2006; Landete The partial detachment of a lower epidermis from mesophyll could

et al., 2008; Campos et al., 2009; Cueva et al., 2010; Merkl et al., enable their independent movement during changes in leaf rehy-

2010; Xia et al., 2010). dration and avoidance of mechanical disruption of mesophyll and

The anatomy of xylem is known to be correlated closely epidermal cells.

with the plant habit and its habitat (Carlquist, 2008). In the

case of vascular resurrection plants, it is also finely attuned to

5. Conclusions

the efficient water economy within the plant during repeated

dehydration–rehydration cycles. The vessel diameter in root, stem

The sinuously folded axial vascular bundles, the shape of the

and leaves in both Ramonda species are, on average, less than

elongated pith parenchyma cells, and the number of strikingly

25 ␮m, which is similar to the value reported for the stem ves-

pressed layers of parenchyma cells that are positioned next to the

sels in the resurrection plant Myrothamnus flabellifolius Welw.

vascular bundles are characteristic stem features in R. serbica and

(Myrothamnaceae), whose stem xylem vessels are ca. 14 ␮m in

R. nathaliae that may enable stem contraction. The short stem, with

diameter (Sherwin et al., 1998). The narrow vessels in Ramonda

the specific vascular tissue organization, is connected to numerous

can be suggested to avoid embolism resistance, which would be

and thin adventitious roots that arise from each stem node, and

strengthened by the presence of surface active lipids in xylem sap

is in immediate proximity to root and leaf traces. This vascular tis-

(Wagner et al., 2000; Schenk et al., 2017).

sue organization may contribute to efficient water transport, which

In contrast to the shorter, reticulate xylem elements in the stem,

would be important for fast rehydration.

the helical xylem elements in the leaf midrib and in the leaf petiole

Various structural adaptations, such as pitting of the stem and

are relatively long and act like strings, without limiting the leaf

leaf xylem elements, organization of stem vascular tissues, loose

movements. However, despite their spiral support and elasticity,

connection of lower leaf epidermis to mesophyll, and specific thick-

the helical structure may endure contractions to a certain degree

enings in anticlinal cell walls of epidermal cells, allow direct volume

and thus limits and directs the shrinking process of the leaf during

and shape changes and might offer high mechanical stability to the

desiccation, as shown in the aquatic resurrection plant Chamaegigas

stem and leaves, and effective re-establishment of water uptake

intrepidus Dinter ex Heil (Schiller et al., 1999).

during rehydration. However, the function of specific “vascular

The upper and the lower leaf epidermis in both plant species

sclerenchyma” tissue observed in the stem of both Ramonda species

is characterized by distinct cell wall thickenings with an indented

remains unclear.

appearance of their anticlinal cell walls, as previously reported for

R. nathaliae by Rascio and La Rocca (2005). There is remarkable Acknowledgement

similarity between the regularly beaded appearance of the epi-

dermal cell walls in the two Ramonda species and the sward-like

This work was supported by the Ministry of Education, Science

formations (resembling “jigsaw pieces”) described in epidermal cell

and Technological Development of Serbia (Grants No. 173030 and

walls in flower petals of Petunia hybrida (Lysenko and Varduny, TR31005).

2013). The authors proposed a potential relationship between an

anthocyanin-dependent photochemical process and the cell wall

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