Shoot anatomy and secretory structures in Hypericum species

Shoot anatomy and secretory structures in Hypericumspecies (Hypericaceae)boj_1046 70..86

BARBARA ŁOTOCKA1* and EWA OSINSKA2

1Department of Botany, Faculty of Agriculture and Biology, Warsaw University of Life Sciences –SGGW, Nowoursynowska 159, 02-766 Warszawa, Poland2Department of Vegetable and Medicinal Plants, Faculty of Horticulture and Landscape Architecture,Warsaw University of Life Sciences – SGGW, Nowoursynowska 159, 02-766 Warszawa, Poland

Received 14 August 2009; revision 28 February 2010; accepted for publication 10 March 2010

The anatomy and ultrastructure of internodes, leaves and petals were compared in Hypericum elegans, H.inodorum, H. olympicum, H. forrestii and two genotypes of H. perforatum. Internode anatomy was variablebetween species with respect to the structure of the cortical and pith parenchyma, including the presence ofsecretory reservoirs. Also, the secondary growth was more extensive in shrubs, i.e. H. inodorum and H. forrestii.In leaves, phloem secretory reservoirs were formed in all species, mesophyll secretory reservoirs were absent onlyin H. elegans and internal nodules were present only in H. elegans and H. perforatum. The petals differed betweenspecies in the mesophyll structure and the occurrence and location of secretory structures. The phloem secretoryreservoirs lacked sheaths, whereas these were distinct in the mesophyll reservoirs. Other ultrastructural traits ofthe reservoirs were similar in all the species studied, with the exception of the leucoplast ultrastructure. In internalnodules, the inner cells vs. sheath cells differed in the number of vesicles and other membranous structures andplastid ultrastructure. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010,163, 70–86.

ADDITIONAL KEYWORDS: endodermis – hypericin glands – Hypericum elegans – Hypericum forrestii –Hypericum inodorum – Hypericum olympicum – Hypericum perforatum – leaf anatomy – petal anatomy –secretory reservoirs.

INTRODUCTION

Hypericum perforatum L., traditionally used in herbalmedicine as a therapeutic plant, is today widely inves-tigated for its anti-depressant and anti-retroviralactivities (Strzelecka & Kowalski, 2000; Ernst, 2003).Hypericum perforatum produces hyperforins, a familyof antimicrobial acylphloroglucinols, and hypericins, afamily of phototoxic anthraquinones exhibiting anti-microbial, anti-viral and anti-herbivore properties invitro (Sirvent & Gibson, 2002). As therapeutic drugs,hyperforin, hypericin, pseudohypericin and protohy-pericin can act as anti-tumoural, anti-depressant,anti-viral, anti-inflammatory and fungicidal agents

(Onelli et al., 2002 and citations therein). In vivo, thesesecondary metabolites are components of the inducibleplant defence responses of H. perforatum, as inferredfrom their elevated content in response to biotic elici-tors (Sirvent & Gibson, 2002).

As a result of its long history of medicinal use, H.perforatum has been more widely studied than otherHypericum spp., which have usually been surveyedonly as a source of novel chemicals; the structure oftheir organs has been neglected. This work was aimedat gaining a better understanding of the anatomicaland ultrastructural diversity of some of these species.Special attention was paid to the secretory structures(phloem and mesophyll secretory reservoirs and innernodules, appearing as black dots on the shoot)because of their importance for discrimination amongtaxa.*Corresponding author. E-mail: [email protected]

Botanical Journal of the Linnean Society, 2010, 163, 70–86. With 7 figures

© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 70–8670

MATERIAL AND METHODSSPECIES CHOICE AND GROWTH CONDITIONS

For the present study, five Hypericum spp. with dif-ferent life forms were chosen.

1. Two mesophytic hemicryptophytes: H. elegansSteph. ex Willd. and H. perforatum L. (sectionHypericum L.). For the latter species the cultivar‘Topaz’ and a local population from the Sanok townarea in south-eastern Poland were sampled.Hypericum perforatum is common in Poland,whereas H. elegans occurs only at a single site inKaty near Zamosc in eastern Poland (Brzeg, Koc-zewska & Szkudlarz, 1988).

2. A xerophytic hemicryptophyte H. olympicum L.(section Olympia (Spach) Endl.) originating fromthe Balkans and north-western Turkey (FloraEuropaea Database, 2010).

3. Two mesophytic deciduous phanerophytes: H.inodorum Mill. (section Androsaemum (Duhamel)Godr.) and H. forrestii (Chitt.) N.Robson (sectionAscyreia Choisy) originating from south and south-western Europe (Flora Europaea Database, 2010)and temperate China (GRIN, 2008), respectively.

Voucher specimens were deposited in a herbariumat the Department of Botany, WULS.

Seeds of taxa native to Poland were collected fromnatural stands. Seeds of H. inodorum were obtainedfrom the Adam Mickiewicz University BotanicGarden in Poznan, Poland, and seeds of H. olympi-cum and H. forrestii were obtained from the BotanicalGarden of the University Vienna. The plants weregrown in the experimental field of the Department ofVegetable and Medicinal Plants in Wilanów-Zawady(Warsaw). Before planting, fertilizers [50 kg ha-1

nitrogen (N), 50 kg ha-1 phosphorus (P2O5) and100 kg ha-1 potassium (K2O)] were applied andsupplemented with 40 kg ha-1 N in May. In the yearswhich followed, the doses were repeated. During thegrowing period, weeds were removed and the soilsurface was aerated.

MICROSCOPY

In the present study, the aerial organs that are usedas the raw materials in herbal medicine were col-lected. Fragments of lateral and middle leaf bladesfrom the upper part of the shoot, internodes andapical and basal parts of petals from freshly openedflowers were sampled from plants in full bloom(the hemicryptophytes were re-grown after the firstraw material was cut in June) and processed formicroscopy.

The material to be processed for transmission elec-tron microscopy (TEM) was cold-fixed for 24 h at

-0.06 MPa in a mixture of 5% glutaraldehyde and 4%paraformaldehyde in 0.1 M sodium cacodylate buffer(pH 7.2). Samples were then rinsed repeatedly withthe same buffer, post-fixed for 2 h with 2.5% osmiumtetroxide (OsO4) in 0.1 M cacodylate buffer and rinsedagain. They were gradually dehydrated in ethanoland acetone and embedded in glycidether 100 epoxyresin (formerly known as Epon 812; SERVA), gradehard. Approximately half of the samples wereintended to be used only for light microscopy andthese were not treated with OsO4 but were stainedwith a stock solution of safranin (Broda, 1971; afterJensen, 1962) in 50–90% ethanol.

For anatomical studies, 3- to 5-mm-thick cross sec-tions were cut using a Jung-RM2065-Supercut micro-tome (Reichert-Jung/Leica) and stained at 70 °C with1% aqueous azure A and 2% methylene blue in 2%borax. For safranin pre-stained sections, stronglydiluted dyes were used resulting in blue coloration ofnon-lignified cell walls and cytoplasm, green colora-tion of lignified cell walls and red coloration of vacu-oles and secretions containing phenolics. A similarstaining method had been previously applied for thedetection of proanthocyanidins (condensed tannins) inH. perforatum (Baroni Fornasiero, Bianchi & Pinetti,1998). For ultrastructural observations, ultra-thinsections were cut using an Ultracut E microtome(Reichert), collected on Formvar-coated slot-grids andcontrasted with lead citrate and uranyl acetate.Observations and micrographs were taken usingbright-field or Nomarski optics on a light microscope(Axioskop, Opton or AX Provis; Olympus) or a trans-mission electron microscope (TEM; JEM 100C or JEM1220; Jeol) operating at 80 kV.

Negatives were scanned into electronic files [1600or 3200 dpi resolution, TEM or light microscope (LM)negatives, respectively] using a CanoScan 9900Fflatbed scanner. The gamma level and contrast of theresulting images were adjusted with Adobe Photoshop7.0 software.

ESSENTIAL OIL ANALYSIS

Samples of leaves, flowers and stems were dried at atemperature of 30 °C. The content of the essential oilin the air-dried samples was determined by themethod of steam distillation according to FarmakopeaPolska VI (2002). The identification of essential oilconstituents was performed by gas chromatographyusing a Hewlett-Packard 6890 gas chromatographand a HP-5 capillary column 30 m in length and0.32 mm in diameter at the detector temperature250 °C and injector temperature 220 °C. Helium wasused as the carrier gas. The column temperature wasprogrammed as follows: 60 °C for 2 min, a tempera-ture increase of 8 °C min-1 and finally 280 °C for

HYPERICUM SHOOT ANATOMY 71


5 min. Compounds were determined on the basis oftheir retention times compared with the standards.

RESULTSGENERAL ANATOMY

The internodes were round or elliptical in crosssection with two wings present in some species(Table 1; Fig. 1A, B). Among the species studied, theinternodes shared general structure and some histo-logical details. All species had a unilayered epidermiswith a thickened outer cell wall. However, they dif-fered in the frequency of stomata and cuticle thick-ness (Table 1). The primary cortex consisted ofchlorenchyma, ground parenchyma and endodermis.In all species, thickening of the cell walls in theground parenchyma (also lignification in somespecies) occurred. Degeneration and obliteration ofthe cells was observed in older internodes. Theprocess was most evident in the phanerophyte speciesH. inodorum and H. forrestii (Fig. 1B) and alsoincluded inner chlorenchyma cells. In the corticaltissues, both parenchymatous zones differed betweenspecies in volume and cell wall thickness (Table 1),whereas the endodermis was uniform. In youngerinternodes, every endodermal cell differentiated aCasparian band in radial and transversal walls. Sub-sequently, the cells underwent non-synchronizeddevelopment (Fig. 1C). The secondary cell wall wasdeposited uniformly around the whole cell wall andan O-thickening was formed. First, a thick lamellateand lignified cell wall zone was formed and, next, athinner, electron-dense and fibrillar inner zone devel-oped. In H. forrestii, both zones were alternatelydeposited two or three times. In older internodes, theouter tangential and radial secondary cell wall sepa-rated from the primary one and sank into the celllumen, which could be an artefact resulting fromdifferential shrinking during dehydration. In suchendodermal cells, the protoplast showed symptoms ofdegeneration. The species investigated exhibited dif-ferent patterns of cortical secretory structure distri-bution, as shown in Table 1. In the stele, the pericyclewas uni- to biseriate (triseriate in H. forrestii) anduniformly parenchymatous (with no fibres). The peri-cycle cells contained large amyloplasts in their basalpart. In phanerophyte species, initiation of peridermfrom inside the pericycle was evident (Fig. 1B). At thetime of fixation (early August), it was composed ofuniseriate phellem, uniseriate phellogen and uni- tobiseriate phelloderm, with the cells arranged intocharacteristic radial files. All peridermal cells werealive (including already suberized phellem cells) andconnected with plasmodesmata, which united thistissue with the adjoining pericycle. Phloem fibres

were absent from internodes of all species examined.In all species, secretory reservoirs were evenly dis-tributed in the outer phloem and, in the two phan-erophytes, which exhibited considerable secondarygrowth, new secretory reservoirs were observed toform close to the cambial zone (Table 1; Fig. 1B, D). Inthe secondary xylem, long-lived fibres were abundantand xylem parenchyma was absent. The medullaryrays were uniseriate within the secondary xylem andphloem. The pith varied in volume, sclerification orcell wall thickening and the presence of crystal-containing idioblasts (Fig. 1E; Table 1). Species-specific patterns of safranin-positive phenolicaccumulation were observed in internodes.

Generally, leaves had abaxial stomata, outer epi-dermal cell walls that were thicker on the adaxialside, adaxial uniseriate palisade parenchyma andtransitory parenchyma that were more or less clearlydifferentiated (Fig. 2A–E), with the exception of thexerophytic H. olympicum (Fig. 2F). The xeromorphicleaves of this species were isolateral (Table 2) withthick epidermal outer walls and thin cuticles. The leafvenation consisted of three equally thick veins and anetwork of finer branches in H. elegans and H. per-foratum and a main vein with finer branches in theother species. In all species, the main vein(s) wasaccompanied with ground parenchyma, which trans-formed into a few cells with collenchymatous cellwall thickenings at the epidermis (Fig. 2A). In H.inodorum, which has the largest leaves, the structureof the main vein was strengthened with a discontinu-ous layer of fibres (Table 2; Fig. 2B). The veins werealways collateral, but they differed in meristem pres-ence. In large-leaved H. inodorum and H. forrestii,the main vein contained fascicular cambium(Table 2), which increased the volume of vasculartissues, especially the vein phloem. In the samespecies, an adaxial extension of the vein parenchyma-tous sheath in middle-sized veins was observed.Transfer cells were not found in any of the speciesexamined. Secretory reservoirs differentiated in veinphloem (outer phloem in wider veins) in all taxaexcept H. inodorum and H. forrestii. In the main veinof these species, because of the activity of the fascicu-lar cambium, new phloem was formed and new secre-tory reservoirs appeared in the new layers (notshown). In mesophyll, other secretory reservoirs werepresent which differed in their diameter and locationwithin the chlorenchyma (Table 2; Fig. 2A–E), andinternal nodules were also formed in some species(Fig. 2F). As in the internodes, species-specific pat-terns of safranin-positive phenolic accumulation werealso observed in leaves (Table 2). However, largeamounts of safranin-stained material were observedin the vacuoles of palisade parenchyma and paren-chymatous vein sheaths of all species.

72 B. ŁOTOCKA and E. OSINSKA


Tab

le1.

Gen

eral

stru

ctu

reof

stem

inte

rnod

esin

Hyp

eric

um

spec

ies

–an

atom

ical

trai

tsva

riab

lebe

twee

nta

xast

udi

ed

An

atom

ical

trai

tsH

.el

egan

sH

.in

odor

um

H.

olym

picu

mH

.fo

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tii

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perf

orat

um

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win

gsP

rese

nt,

broa

dP

rese

nt

inyo

un

gst

ems

only

,n

arro

w

Pre

sen

t,br

oad

Abs

ent

Pre

sen

t

Per

ider

mA

bsen

tIn

itia

ted

inpe

ricy

cle

Abs

ent

Init

iate

din

peri

cycl

eA

bsen

t

Epi

derm

isS

tom

ata

Nu

mer

ous

Spa

rse

Nu

mer

ous

Spa

rse

Spa

rse

Cu

ticl

eT

hic

kT

hic

kT

hin

Th

inT

hic

k

Cor

tex

Ch

lore

nch

yma

3la

yers

2–3

laye

rs,

cell

sth

ick-

wal

led

3–7

laye

rs2–

4la

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,ce

lls

thic

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alle

d3

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Gro

un

dpa

ren

chym

aS

ever

alla

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;th

icke

ned

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on-l

ign

ified

cell

wal

ls;

obli

tera

tion

inol

der

stem

s

Sev

eral

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loca

lsc

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fica

tion

and

deat

hof

cell

s;ob

lite

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onin

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rst

ems

3–4

laye

rs;

thic

ken

ed,

non

-lig

nifi

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lite

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onin

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rst

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Nu

mer

ous

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loca

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tion

and

subs

equ

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deat

hof

cell

s;ob

lite

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onin

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rst

ems

Sev

eral

laye

rs;

thic

ken

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non

-lig

nifi

edce

llw

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;ob

lite

rati

onin

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rst

ems

Sec

reto

ryre

serv

oirs

Abs

ent

Abs

ent

Abs

ent

Nu

mer

ous

inch

lore

nch

yma

Spa

rse,

sube

pide

rmal

Inn

ern

odu

les

Wit

hin

win

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lyA

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only

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leS

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dan

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in:

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ex,

rays

cort

ex,

phlo

empa

ren

chym

a,ra

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mis

,co

rtex

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loem

pare

nch

yma,

rays

chlo

ren

chym

a,pe

ricy

cle,

rays

,pe

ride

rmin

olde

rst

ems

epid

erm

is,

cort

ex,

phlo

empa

ren

chym

a,ra

ys;

oute

rpi

th



Figure 1. Internode structure in Hypericum. A, general anatomy of the internode with the secondary structure fullydeveloped in Hypericum perforatum ‘Topaz’. B, older internode with crushed chlorenchyma. The cell wall is thickened incortical ground parenchyma and periderm initiated in Hypericum inodorum. Arrows indicate secretory reservoirs inprimary phloem and in young secondary phloem close to the cambium. C, non-synchronous internode endodermisdifferentiation in H. inodorum. Indicated here are the middle lamella (arrow), the Casparian band in a cell at the firstdevelopmental stage (black double arrowheads), remnants of the Casparian band in a cell at the third developmentalstage (white double arrowheads), the lamellate secondary cell wall (black asterisks), the fibrillar secondary cell wall (whiteasterisks) and starch grains in a plastid (rosettes). D, formation of secretory reservoirs within differentiating secondaryphloem in H. inodorum. Rosettes indicate loosening of a middle lamella common to the four epithelial cells of the reservoir.E, thickening of ground parenchyma cell walls in the internode pith in H. perforatum ‘Topaz’. Arrows indicate cell wallthickening with simple pits. Scale bars, 490 mm (A), 260 mm (B), 0.8 mm (C), 6.2 mm (D) and 180 mm (E).



Petals, like leaves, were dorsiventral, were devoidof stomata and had thickened epidermal outer cellwalls on the abaxial side (Fig. 3A–E). In H. olympi-cum, subcuticular mucilage was observed. The speciesdiffered in the presence of epidermal papillae(Table 3). In H. inodorum, H. olympicum and H. for-restii, petals were somewhat fleshy, resulting frommore mesophyll layers and a rather compact arrange-ment of the cells. Species-specific patterns of distri-

bution and type of mesophyll-located secretorystructures were observed in petals. The petal veinswere collateral, ensheathed with parenchyma only,and were devoid of fascicular cambium. They werelocated close to the abaxial epidermis or centrallywithin the mesophyll (Table 3). In H. inodorum, thesheath extension was formed of parenchyma cellsenriched in safranin-positive phenolics. As in theother organs investigated, no transfer cells werefound in the vascular tissue of petals. Secretory res-ervoirs were observed in phloem of veins, but theywere less frequent than in leaf veins (Table 4).Species-specific patterns of safranin-positive pheno-lics were discernible in petals, as they were in leavesand internodes (Table 3).

PHLOEM SECRETORY RESERVOIRS

In all species investigated, secretory reservoirsformed in phloem of leaves, petals and stems. In crosssection, the reservoirs consisted of four epithelial cells(occasionally more) and no sheath was formed aroundthem (Figs 1D, 4A). Their common middle lamellaand the middle lamella between radial walls of epi-thelial cells gradually disappeared. Thus, a schizog-enous reservoir was formed. Thin, moderatelyelectron-dense cuticle that was discontinuous in someplaces was left in the reservoir after secretion extrac-tion. The walls adjoining the reservoir were consid-erably loosened and they were thinner than the otherwalls of the epithelial cells. Within the fibrils result-ing from cell wall loosening, droplets of moderatelyelectron-dense secretion could be seen (Fig. 4B), butthe reservoir was devoid of such secretion, as itmust have been extracted during sample dehydrationfor microscopy. Occasionally, a different, stronglyelectron-dense substance was present in the reservoir(Fig. 4A). Small granules of a similar substance wereoften present between the plasma membrane of epi-thelial cells and the cell wall. Within the thin parietalcytoplasm of epithelial cells, weakly electron-densedroplets occurred that resembled lipid bodies.Usually, they were associated with multivesicular

Figure 2. Anatomy of leaf in wild-type Hypericum perfo-ratum (A), Hypericum perforatum cv. Topaz (B), Hyperi-cum inodorum (C), Hypericum forrestii (D), Hypericumelegans (E) and xeromorphic Hypericum olympicum (F).Indicated here are the position of phloem reservoirs inveins (arrows), stomata (thin arrows), transitory chloren-chyma (rosettes), mesophyll secretory reservoirs (blackasterisks), epithelial cells (arrowhead), sheath cells(double arrowhead) and inner nodule (white asterisk).Scale bars, 250 mm (A), 160 mm (B), 380 mm (C), 380 mm(D), 285 mm (E) and 285 mm (F).�



Tab

le2.

Gen

eral

stru

ctu

reof

leav

esin

Hyp

eric

um

spec

ies

–an

atom

ical

trai

tsva

riab

lebe

twee

nta

xast

udi

ed

An

atom

ical

trai

tsH

.el

egan

sH

.in

odor

um

H.

olym

picu

mH

.fo

rres

tii

H.

perf

orat

um

Epi

derm

isS

tom

ata

Aba

xial

Aba

xial

Ab-

and

adax

ial

Aba

xial

Aba

xial

Ou

ter

cell

wal

lsA

daxi

alth

icke

rA

daxi

alth

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rA

b-an

dad

axia

lve

ryth

ick

Ada

xial

thic

ker

Ada

xial

thic

ker

Cu

ticl

eA

daxi

alth

ick

Ab-

and

adax

ial

thic

kT

hin

Ada

xial

thic

kA

daxi

alth

ick

Ch

lore

nch

yma

Pal

isad

epa

ren

chym

aA

daxi

alu

nis

eria

teA

daxi

alu

nis

eria

teA

b-an

dad

axia

l,bo

thbi

seri

ate

Ada

xial

un

iser

iate

Ada

xial

un

iser

iate

Tran

sito

rypa

ren

chym

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rese

nt

Poo

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diff

eren

tiat

edN

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scer

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Pre

sen

t

Sec

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ryre

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Abs

ent

Pre

sen

tin

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pare

nch

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smal

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ical

Pre

sen

t,m

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ann

ing,

sph

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nt

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spon

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chym

a(s

mal

l)or

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eir

bou

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ry(l

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),sp

her

ical

and

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gate

Pre

sen

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hyl

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ing,

sph

eric

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Inn

ern

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les

Pre

sen

tA

bsen

tA

bsen

tA

bsen

tP

rese

nt

Vei

ns

Vei

nty

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tera

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scic

ula

rca

mbi

um

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nt

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fasc

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mpr

esen

tin

the

mai

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in

Col

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fasc

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mpr

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tin

the

mai

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Col

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fasc

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cam

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mpr

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tin

the

mai

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Col

late

ral,

fasc

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lar

cam

biu

mab

sen

t

En

shea

thed

wit

h:

pare

nch

yma

only

disc

onti

nu

ous

fibr

ela

yer

inm

ain

and

mid

dle-

size

dve

ins,

pare

nch

yma

infi

ne

vein

s

pare

nch

yma

only

pare

nch

yma

only

pare

nch

yma

only

Ab-

orad

axia

lve

insh

eath

exte

nsi

onA

bsen

tA

daxi

al,

atm

iddl

e-si

zed

vein

sA

bsen

tA

daxi

al,

atm

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e-si

zed

vein

sA

bsen

t

Sec

reto

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serv

oirs

Inph

loem

Inou

ter

phlo

em(a

nd

seco

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ain

vein

)

Inph

loem

Inou

ter

phlo

em(a

nd

seco

nda

ryph

loem

inth

em

ain

vein

)

Inph

loem

Sap

hra

nin

-pos

itiv

eph

enol

icsu

bsta

nce

sab

un

dan

tly

pres

ent

in:

adax

ial

epid

erm

is,

adax

ial

chlo

ren

chym

a,ve

in-a

ssoc

iate

dgr

oun

dpa

ren

chym

a,ve

insh

eath

s

adax

ial

chlo

ren

chym

a,ve

insh

eath

s,va

scu

lar

pare

nch

yma

adax

ial

epid

erm

is,

adax

ial

chlo

ren

chym

a,ve

insh

eath

s

adax

ial

epid

erm

is,

adax

ial

chlo

ren

chym

a,xy

lem

pare

nch

yma,

vein

shea

ths

adax

ial

epid

erm

is,

adax

ial

chlo

ren

chym

a,ve

insh

eath

s



bodies (Fig. 4C). The content of the droplets wassimilar to the secretion present within cell wallfibrils. The unique trait of the epithelial cells wastheir plastids, which did not resemble any other plas-tids in the surrounding phloem or other tissues. Inepithelial cells, small strongly electron-dense leuco-plasts were differentiated. Within their stroma, adelicate reticulum of electron-transparent tubulescould be seen (Fig. 4D, E). However, the presence ofthe reticulum was organ- and species-dependent(Table 5). Typically, the tonoplast underwent earlyfragmentation in epithelial cells (Fig. 4E). Secretoryreservoirs that occurred in the cortical chlorenchymaof some investigated species (Table 1) resembled thephloem reservoirs rather than mesophyll reservoirs ofleaves and petals in H. perforatum, but not in H.forrestii.

MESOPHYLL SECRETORY RESERVOIRS

Spherical secretory reservoirs occurred in the leafmesophyll of all species examined, with the exceptionof H. elegans (Table 2). In petals, reservoirs of similar

ultrastructure were differentiated as elongated canalsin some species (Table 3). In H. forrestii, in additionto spherical reservoirs, elongated reservoirs werepresent in leaf mesophyll. In the leaves of H. olym-picum and H. perforatum, the secretory reservoirsoccupied the whole thickness of the mesophyll andthus adjoined both ad- and abaxial epidermis (reser-voirs in petals had smaller diameter). In H. inodorumand H. forrestii, the diameter usually corresponded tothe thickness of the palisade or spongy parenchyma.In the mesophyll reservoirs, two or three cell layerswere discernible. The cells that adjoined (and formed)the reservoir, termed epithelial cells, wereensheathed with a uni- or biseriate (rarely) sheath(Fig. 5). At maturity, epithelial cells appeared emptyunder the light microscope, but not with TEM. Thethin-walled epithelial cells were ultrastructurally dif-ferent from the sheath cells. During the formation ofthe schizogenous reservoir, their middle lamellae dis-appeared and cell walls were loosened to form anetwork of delicate fibrils, as in phloem reservoirs.Within the fibrils, and between them and the innercuticle, the secretion was usually present in the form

Figure 3. Anatomy of petal in Hypericum elegans (A), Hypericum inodorum (B), Hypericum olympicum (C), Hypericumforrestii (D) and wild-type Hypericum perforatum (E). Indicated here are the position of phloem reservoirs in veins(arrows), mesophyll secretory reservoirs (thin arrows), adaxial epidermis (papillate in H. elegans and H. perforatum)(rosettes) and inner nodule (asterisk). Scale bars, 360 mm (A), 270 mm (B), 250 mm (C), 500 mm (D) and 330 mm (E).



Tab

le3.

Gen

eral

stru

ctu

reof

peta

lsin

Hyp

eric

um

spec

ies

–an

atom

ical

trai

tsva

riab

lebe

twee

nta

xast

udi

ed

An

atom

ical

trai

tsH

.el

egan

sH

.in

odor

um

H.

olym

picu

mH

.fo

rres

tii

H.

perf

orat

um

Epi

derm

alpa

pill

aeA

daxi

alA

bsen

tA

daxi

al,

peta

lap

ices

only

Abs

ent

Ada

xial

Mes

oph

yll

Par

ench

yma

Few

laye

rs,

un

ifor

m,

spon

gy-l

ike

Nu

mer

ous

laye

rs,

un

ifor

m,

rela

tive

lyco

mpa

ct

Nu

mer

ous

laye

rs,

un

ifor

m,

rela

tive

lyco

mpa

ct

Nu

mer

ous

laye

rs,

un

ifor

m,

rela

tive

lyco

mpa

ct

Few

laye

rs,

un

ifor

m,

spon

gy-l

ike

Sec

reto

ryre

serv

oirs

Pre

sen

t,ab

axia

lly

loca

ted,

sube

pide

rmal

,m

ostl

yel

onga

te

Pre

sen

t,ab

axia

lly

loca

ted,

sube

pide

rmal

,sp

her

ical

Pre

sen

t,ab

axia

lly

loca

ted,

sube

pide

rmal

orw

ith

inm

esop

hyl

l

Pre

sen

t,ab

axia

lly

loca

ted,

wit

hin

mes

oph

yll

Pre

sen

t,ab

axia

lly

loca

ted,

mos

tly

elon

gate

Inn

ern

odu

les

Pre

sen

t,ab

axia

lly

loca

ted,

sph

eric

alor

elon

gate

Abs

ent

Abs

ent

Abs

ent

Pre

sen

t,ab

axia

lly

loca

ted,

sph

eric

alor

elon

gate

Vei

ns

Loc

atio

nA

baxi

alC

entr

alC

entr

alC

entr

alA

baxi

alA

b-or

adax

ial

vein

shea

thex

ten

sion

Abs

ent

Ab-

and/

orad

axia

l,ab

sen

tat

fin

eve

ins

Abs

ent

Abs

ent

Abs

ent

Sec

reto

ryre

serv

oirs

Inph

loem

,ab

sen

tfr

omso

me

vein

sIn

phlo

em,

abse

nt

from

mos

tve

ins

Inph

loem

,ab

sen

tfr

omso

me

vein

sIn

phlo

em,

abse

nt

from

som

eve

ins

Inph

loem

,ab

sen

tfr

omso

me

vein

s

Sap

hra

nin

-pos

itiv

eph

enol

icsu

bsta

nce

sab

un

dan

tly

pres

ent

in:

abax

ial

epid

erm

is,

sube

pide

rmal

mes

oph

yll,

vein

shea

ths

ad-

and

abax

ial

epid

erm

is,

sube

pide

rmal

mes

oph

yll,

vein

shea

ths

and

shea

thex

ten

sion

s,va

scu

lar

pare

nch

yma

ad-

and

abax

ial

epid

erm

is,

sube

pide

rmal

mes

oph

yll,

vein

shea

ths,

vasc

ula

rpa

ren

chym

a

abax

ial

epid

erm

is,

grad

ual

lyle

ssto

war

dsm

iddl

em

esop

hyl

l

abax

ial

epid

erm

is,

sube

pide

rmal

mes

oph

yll,

vein

shea

ths



of (irregular) droplets of moderate electron density. Asimilar substance was usually present in lipid-body-like structures associated with multivesicular bodiesin the epithelial and sheath cells. In the vacuolesof epithelial cells, and especially in sheath cells,safranin-positive globules of phenolics occurred. Thetonoplast was usually fragmented. The ultrastructureof sheath cells and epithelial cells was similar, withthe exception of cell wall, which was thicker in sheathcells, and type of plastids. In epithelial cells, smallleucoplasts occurred that formed a stroma reticulumin some species (Table 4; Fig. 4D). In sheath cells,chloroplasts with distinct grana and (large) starchgrains were observed in leaves and, in some species,also in petals (Fig. 5). In the sheath cells, chloroplastswere always positioned at the cell wall most distantfrom the reservoir.

ESSENTIAL OIL

Significant differences were found in the content andcomposition of the essential oil in leaves, flowers andstems of the investigated species. The essential oilcontent ranged from trace to 0.35% (Table 6). Fromamong 50 detected constituents of the essential oil, 19mono- and sesquiterpenes or their derivatives wereidentified (Table 7). In most of the samples, the domi-nant constituent of essential oil appeared to be2-methyloctane and for the others it was a-pinene.The content of 2-methyloctane in the essential oilranged from 12.33 to 39.43% and the content ofa-pinene ranged from 1.07 to 16.42%. Other com-pounds present in appreciable concentrations were:a-terpineol (1.45–10.09%), b-pinene (0.61–8.90%),b-caryophyllene (0.92–9.73%) and a-humulene (1.05–3.67%).

INTERNAL NODULES

Internal nodules were found in the leaf and petalmesophyll and in stem wings only in H. elegans andH. perforatum (Tables 1–3). In the nodules, a biseri-ate sheath of flat cells surrounded the inner cells,which were rounded, tightly packed and bigger thanthe sheath cells (Fig. 6). During sectioning, the inner

nodule cells often fell out, especially in osmium-contrasted samples of leaves, indicating a limitedpenetration of the embedding resin. All the nodulecells were strongly safranin-positive and extremelyelectron-dense after osmification. Both reactions werestronger in the leaf than in the petal nodules. Inpetals, the outer sheath cells were sometimes onlyweakly coloured.

All the nodule cells were alive prior to fixation andthey contained thin parietal cytoplasm with cytoplas-mic bridges spanning the vacuole. The cytoplasm andorganelles were electron-dense, but less so than thevacuole content. The content was uniformly dark, incontrast to the vacuolar sap of other Hypericum cells,which contained either large globules or a fine pre-cipitate of an electron-dense and safranin-positive (inthe light microscope) substance. The sheath and innercells differed in the appearance of organelles. Insheath cells, all types of organelles were relativelyeasy to observe, including the nucleus, endoplasmicreticulum, Golgi bodies with numerous vesicles, smallmitochondria, chloroplasts with grana, numerousplastoglobules and occasionally starch grains(Fig. 7A, B). Close to the plasma membrane and tono-plast, lipid bodies, numerous vesicles and membra-nous bodies were often seen. In the inner nodule cells,the cytoplasm layer was thinner and the organelleswere more difficult to find, probably because of themuch larger volume of inner vs. sheath cells. Thevesicles and membranous structures were more abun-dant (Fig. 7C, D), and the chloroplasts were ultra-structurally different (Fig. 7E), as they containedfewer thylakoids and more plastoglobuli than those inthe sheath cells. Within the nodule, the cell wallswere always thin (sometimes folded in inner cells)and, between the wall fibrils, small electron-denseparticles were deposited. Plasmodesmata wereobserved within the nodule, including the inner cells(Fig. 7F).

DISCUSSIONGENERAL ANATOMY

Hypericum consists of c. 370 species (Stevens, 2001)and it is the largest genus in Hypericaceae Jussieu.

Table 4. Number of secretory reservoirs in leaf or petal vein phloem

Vein location and size H. elegans H. inodorum H. olympicum H. forrestii H. perforatum

Leaf Main vein(s) 6–7 18–20 2–3 7–8 2–4Middle-sized veins 5–6 2–4 0–2 0–1 0–4Fine veins 0–4 0 0 0 0–3

Petal 0–1 0–1 0–2 0–1 0–1Usually 1 Usually 0 Usually 1 Usually 0 Usually 1



Species are diverse in their habit, ranging from smallherbs to small trees (Metcalfe et al., 1950). Except forH. perforatum, which has been used for centuries asa medicinal herb and has therefore been widely inves-tigated, little is known about the anatomy, and espe-

cially that of the secretory structures, of otherHypericum spp. To the best of our knowledge, this isthe first report on the stem internode, leaf and petalanatomy of H. elegans, H. inodorum, H. olympicumand H. forrestii.

Figure 4. Ultrastructure of phloem secretory reservoirs in Hypericum. A, accumulation of electron-dense secretion inphloem reservoirs in Hypericum elegans. B, accumulation of secretion (asterisk) within a loosened middle lamella and cellwall (rosettes) anticlinal to the reservoir in Hypericum perforatum. C, multivesicular body and secretion at the cell wall(arrow) facing the reservoir lumen in H. perforatum. Indicated here are the cuticle-like layer (double arrowhead) and theelectron-dense deposits at the outer surface of plasma membrane (arrowheads). Note the loosening of the outer cell wall.D, leucoplasts with a reticulate stroma in epithelial cells of H. perforatum ‘Topaz’. Cell wall together with middle lamellaanticlinal to the reservoir lumen (arrows) is loosened until it reaches the position marked (rosette). E, leucoplasts witha non-reticulate stroma in epithelial cells of Hypericum olympicum. Cell wall together with middle lamella anticlinal tothe reservoir lumen (arrows) is loosened until it reaches the position marked (rosette). Scale bars, 9.7 mm (A), 0.6 mm (B),0.4 mm (C), 1.2 mm (D) and 1.1 mm (E).



The general anatomy of shoot organs in Hypericumstudied in this work was consistent with the descrip-tion by Metcalfe et al. (1950), with the exception ofthe stem pericycle which was devoid of sclerenchyma.The structure of vascular tissues was similar betweenthe investigated species, excluding the traits relatedto the secondary growth in the phanerophytes H.inodorum and H. forrestii. In perennial shoots ofthese species, the secondary growth was extensive

Table 5. Plastid types and occurrence of stroma reticulum in leucoplasts (+, present; -, absent) in secretory reservoirsor inner nodules

Cell location and size H. elegans H. inodorum H. olympicum H. forrestii H. perforatum

Phloem reservoirs Internode - - - - -Leaf - - - - +Petal - - - - +Leaf X ch/- ch/- ch/- ch/+Petal ch*/+ chr†/- chr/- chr/+ ch/+

Inner nodules‡ Internode ch/uch§ X X X ch/-Leaf ch/uch X X X ch/-Petal ch/uch X X X ch/uch

X, the secretory structure does not occur in a particular species.*Chloroplasts (ch).†Chromoplast (chr).‡Sheath/epithelial cells or sheath/nodule central cells.§Ultrastructurally changed chloroplasts (uch).

Figure 5. Ultrastructure of mesophyll secretory reservoirin Hypericum forrestii. Indicated here are the cuticle-likelayers within the reservoir (double arrowheads), secretionwithin the reservoir or epithelial cells (asterisks) andchloroplasts in sheath cells positioned most distant toepithelial cells (arrows). Scale bar, 6.4 mm.

Figure 6. Ultrastructure of internal nodule in Hypericumperforatum ‘Topaz’. Scale bar, 16.7 mm.



and it involved formation of new secretory reservoirswithin the secondary phloem. In the annual shoots ofthe other species, such reservoirs were formed in theprimary phloem only. Consistent with the differentlife form of H. inodorum and H. forrestii, the initia-tion of periderm from the inner pericycle was evidentin the stems. The cortical parenchyma underwentconcurrent degeneration prior to being shed. Poly-derm (mentioned by Metcalfe et al., 1950) initiationwas not observed, as the new protective layer had acell wall ultrastructure typical of phellem cells. In allspecies investigated, an endodermis was formed thatunderwent ultrastructural differentiation identical tothat typical for this tissue in roots (Clarkson &Robards, 1975), where it serves as an effective barrierfor radial solute transport in the apoplast. The trans-port of particular substances in a particular directionpossibly necessitates the formation of such a barrierin Hypericum stems, but the nature of these sub-stances and their transport remains to be examined.The function of this apoplastic barrier may not

include gravitropic function in Hypericum endoder-mal cells because the large basally positioned amylo-plasts, which are part of the gravitropic reactionmechanism (Sack, 1997), are observed in the pericycleand not in the endodermis. In the shoot, the latter istypically the site of gravitropic stimulus perception(Tasaka, Kato & Fukaki, 1999).

All organs investigated exhibited a specific patternof safranin-positive phenolic accumulation in vacu-oles. The form of accumulation varied between organs(fine precipitate to large globuli). In leaves and petals,the accumulation pattern was reversed in response toirradiation. In leaves, the adaxial cell layers (usuallythe epidermis and palisade parenchyma) accumulatedlarge amounts of these substances, as did the petalsand the abaxial side cells. Until anthesis, petalsexpose their abaxial side to the environment (espe-cially to ultraviolet radiation) and they wither andgradually die approximately 1 day after anthesis inspecies having delicate flowers (H. elegans and H.perforatum). Therefore, the exposure of the adaxial

Table 6. Essential oil content in dry leaves, flowers and stems (%)

Organs H. elegans H. inodorum H. olympicum H. forrestii H. perforatum

Leaves 0.20 Trace 0.10 Trace 0.33Flowers 0.05 Trace Trace Trace 0.12Stems Trace Trace Trace Trace Trace

Table 7. Content of identified compounds in essential oil (%)

Compound

H. elegans H. olympicum H. perforatum

Leaves Flowers Leaves Flowers Leaves Flowers

2-methyloctane 12.33 15.23 19.46 19.04 39.43 14.17a-pinene 2.92 1.07 14.01 14.14 7.09 16.42Camphene 0.77 0.47 1.26 1.70 2.48 1.05b-pinene 0.61 1.23 5.94 6.34 4.50 8.90Sabinene 1.42 1.17 0.76 1.27 0.39 0.63b-myrcene 0.73 0.41 0.32 0.63 0.33 0.58a-terpinene 0.34 0.34 0.60 0.45 0.17 0.24Limonene 0.21 0.15 0.26 0.33 0.12 0.31Cyneole 0.03 0.29 0.26 0.31 0.09 0.23g-terpinene 1.11 0.63 1.23 1.43 0.21 0.86o-cymene 1.08 0.26 1.23 1.05 – 0.84p-cymene 0.23 0.52 0.20 2.16 0.05 0.16Camphor 0.35 0.24 0.23 – 0.18 0.25Linalool 0.43 0.70 – 0.13 0.60 0.15b-caryophyllene 9.73 0.92 9.67 6.38 7.52 5.73Terpinen-4-ol 0.36 1.09 – 0.37 0.48 0.37a-humulene 1.05 1.69 2.99 2.64 2.64 3.67a-terpineol 1.45 2.08 10.00 6.22 3.67 10.09Caryophyllene oxide 0.60 0.98 0.67 3.09 0.53 0.91



tissue layers to the direct sunlight is irrelevant fortheir functioning in the long term.

SECRETORY RESERVOIRS

The presence of secretory structures producing a non-nectar secretion is generally considered to serve adefence function (Palo & Robbins, 1991). Specifically,the secretions (essential oil) may protect the plantsagainst excessive feeding by herbivores. The essentialoil was produced in measurable amounts only in

several Hypericum spp. investigated in this work andthe oil concentration or content was comparable withprevious reports (Hoelzl & Petersen, 2003; Schwobet al., 2004; Demirci et al., 2005; Radusiene, Judzen-tiene & Bernotiene, 2005; Smelcerovic et al., 2007).The secretory reservoirs associated with phloem arethe only ubiquitous and constant secretory structure inthe Hypericum spp. investigated in this study. Such alocation might provide some protection from phloem-feeding herbivores (Lersten & Curtis, 1989). Signifi-cantly, these reservoirs were located in the outer

Figure 7. Ultrastructure of internal nodule in Hypericum. A–B, chloroplasts in a nodule sheath cell in Hypericum elegansand Hypericum perforatum ‘Topaz’, respectively. C–D, membranous structures (double arrowheads) and endoplasmicreticulum (arrowheads) in the cytoplasm of inner cells of the nodule in H. elegans and H. perforatum ‘Topaz’, respectively.E, modified chloroplasts with sparse grana (arrows) and numerous plastoglobules (asterisks) visible within the inner cellsof the nodule in H. elegans. F, plasmodesmata between the inner cells of the nodule in H. perforatum ‘Topaz’. Doublearrowheads indicate the membranous structures in the cytoplasm. Scale bars, 1.3 mm (A), 0.6 mm (B), 0.6 mm (C), 0.9 mm(D), 0.9 mm (E) and 1.6 mm (F).



phloem in stems and abaxially to the vein phloem inleaves and petals, thus increasing the possibility ofreservoir puncture during feeding attempts. In thespecies with perennial shoots (H. inodorum and H.forrestii), which produce more secondary vasculartissues in the stem than species with annual shoots,new phloem secretory reservoirs were formed, replac-ing those that became non-functional with the sur-rounding phloem. It seems that the anti-herbivorousfunction is achieved by means of different compoundsin different organs (stems vs. leaves and petals) andspecies of Hypericum. Stems yielded only traces ofmono- and sesquiterpene-containing essential oils, incontrast to leaves and petals in H. elegans and H.perforatum. In H. inodorum and H. forrestii, despitethe ubiquitous presence of secretory reservoirs, allorgans analysed here yielded only traces of the oil.

Two types of secretory reservoirs have beendescribed in vegetative organs of H. perforatum:phloem reservoirs and mesophyll reservoirs (Curtis &Lersten, 1990; Ciccarelli, Andreucci & Pagni, 2001b).Both types were also found in the species in this workand their structure was consistent with that previ-ously described. Histochemical tests showed thatphloem reservoirs never contained essential oils, butalkaloids, lipids and resins accumulated in them.Tannins also accumulated in stems (Ciccarelli et al.,2001b). The content of the mesophyll reservoirs wasmore organ-dependent. Alkaloids and lipids werealways present in these reservoirs, whereas essentialoils were produced in petals and leaves but not instems, and resins were not present in leaves. Here, ithas been shown that if the essential oil is produced,its composition is organ-dependent.

Previously, contradictory data were published con-cerning the mode of the formation of reservoirs(Siersch, 1927; Curtis & Lersten, 1990 and the cita-tions therein). As Curtis & Lersten (1990) showed, themesophyll reservoirs are undoubtedly of schizogenousorigin. Because the ultrastructure of the cell wallsfacing the reservoir cavity and anticlinal to it isidentical in the mesophyll and phloem reservoirsobserved in all species included in this work, it isreasonable to assume that they are formed in thesame way. Additionally, this hypothesis is supportedby the ultrastructure of secondary phloem reservoirsin H. inodorum and H. forrestii. During the formationof the secretory cavity, no remnants of lysed cells wereever found within it and the cavity was initiatedbecause of the loosening of the middle lamellabetween the four epithelial cells.

Usually, endoplasmic reticulum and Golgi bodiesare implicated in plant cell secretory activity, with themultivesicular bodies being formed as a by-product(Battey et al., 1999). In molecular studies it has beenshown that mono- and diterpene synthesis occurs in

leucoplasts and chloroplasts, whereas sesquiterpenesare synthesized in cytoplasm and smooth endoplasmicreticulum (McCaskill & Croteau, 1995; Turner et al.,1999; Bouvier et al., 2000; Turner & Croteau, 2004).In the epithelial cells of the secretory reservoirsobserved in this work, leucoplasts were present,which, in some species, formed a delicate reticulumwithin their stroma. The structure cannot beunequivocally associated with the production ofmono- or diterpenes, because, of the three species thatproduce a significant amount of essential oil (H.elegans, H. olympicum and H. perforatum), the seconddoes not form reticulum.

The epithelial cells observed in this work had afragmented tonoplast in contrast to the adjacent cellsof different types. The fragmentation may have arisenas a fixation artefact or it could serve a specificfunction. Specialized cells, which during their differ-entiation undergo tonoplast fragmentation that doesnot lead to their degeneration, form the so-calledboundary layer in the cortex of legume root nodules(Parsons & Day, 1990). Because of the turgor lossafter tonoplast fragmentation (and also cell wallmodifications), the cells form an efficient apoplastbarrier (Brown & Walsh, 1996). In Hypericum secre-tory reservoirs, the loss of turgor in epithelial cellsmay decrease the mechanical stress within the middlelamella that bonds their anticlinal walls and thusprevent the escape of toxic secretions into the sur-rounding tissue.

INTERNAL NODULES

The inner nodules of Hypericum do not conform toany internal secretory structures known from anyother group of plants, as claimed by Curtis & Lersten(1990). However, the same authors described thetransition between mesophyll secretory reservoirsand inner (elongated) nodules in petals of H. perfora-tum. The presence of such hybrid structures suggeststhat reservoirs and nodules share some genetic infor-mation. Additionally, in H. balearicum L. leaves,bulges and pustular cavities are present that areinitiated as large cellular nodules. Later, the centralcells separate unevenly to form an irregular cavityinto which isolated cells and clusters of cells intrude(Curtis & Lersten, 1990). These data suggest thatinner nodules are an evolutionary modification ofcommon secretory reservoirs.

In H. perforatum, the inner nodules lack intercel-lular spaces and they are composed of several largecells surrounded by a mono- or biseriate sheath offlattened cells (Curtis & Lersten, 1990; Baroni For-nasiero et al., 1998; Ciccarelli, Andreucci & Pagni,2001a). This work shows that the (ultra)structure ofinner nodules in H. elegans does not differ from H.



perforatum. Histochemical tests show that thenodules are negative for the presence of lipids, essen-tial oils, sesquiterpene lactones, steroids and pro-teins, and they are positive for pectic-like substances,tannins and alkaloids (Ciccarelli et al., 2001a).However, their most interesting feature is the synthe-sis and accumulation of hypericin and pseudohyperi-cin (Zobayed et al., 2006; Kornfeld et al., 2007).

The structure of Hypericum inner nodules has beenstudied since the 19th century, but, because of thecompact arrangement of the nodule cells and darkcontent, they were recognized relatively late and con-tradictory reports were published concerning thenodule ultrastructure. Specifically, several authorsreported that the inner cells became non-functionaland disassembled towards the end of their differen-tiation and that they were used only as reservoirs forsecretion products (Baroni Fornasiero et al., 1998;Onelli et al., 2002 in H. perforatum; Baroni Fornasi-ero et al., 2000 in H. richeri Vill.). In this work, innernodules were investigated in mature leaves of H.elegans and H. perforatum. At maturity in bothspecies, the inner nodule cells retain the cytoplasmwith organelles that do not exhibit any clear degen-erative symptoms. Also, in contrast to the data byOnelli et al. (2002), the inner nodule cells remainconnected with plasmodesmata at maturity. The pari-etal cytoplasm layer is thin and in some cells difficultto differentiate from the vacuole because of theextreme electron density of the nodule. This mayexplain the contradictory earlier observations.

ACKNOWLEDGEMENTS

The authors thank Ewa Znojek, Małgorzata Gzowskaand Alicja Zuchowska for expert technical assistance.

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Shoot anatomy and secretory structures in Hypericum species