Polyamine profiles and biosynthesis in somatic embryo development

and comparison of germinating somatic and zygotic embryos

of Norway spruce

LENKA GEMPERLOVA,1 LUCIE FISCHEROVA,1 MILENA CVIKROVA,1,2

JANA MALA,3 ZUZANA VONDRAKOVA,1 OLGA MARTINCOVA1

and MARTIN VAGNER1

1 Institute of Experimental Botany v.v.i., Academy of Sciences of the Czech Republic, Rozvojova 236, 16502 Prague 6, Czech Republic

2 Corresponding author (cvikrova@ueb.cas.cz)

3 Forestry and Game Management Research Institute, v.v.i., 156 04 Praha 5, Zbraslav-Strnady, Czech Republic

Received April 27, 2009; accepted July 24, 2009; published online 25 August, 2009

Summary The polyamine (PA) contents and activities of

PA biosynthetic enzymes in Norway spruce somatic

embryos [Picea abies L. (Karst.), genotype AFO 541]

were studied in relation to anatomical changes during

their development, from proliferation to germination, and

changes in these variables associated with the germination

of mature somatic and zygotic embryos were compared.

Activities of PA biosynthetic enzymes steadily increased

during the development of somatic embryos, from

embryogenic suspensor mass until early cotyledonary

stages. In these stages, the spermidine (Spd) level was

significantly higher than the putrescine (Put) level, and the

increases coincided with the sharp increases in S-adeno-

sylmethionine decarboxylase activity in the embryos. The

biosynthetic enzyme activity subsequently declined in

mature cotyledonary embryos, accompanied by sharp

reductions in PA contents, especially in cellular Put

contents in embryos from 6 weeks old through the

desiccation phase (although the spermine level signifi-

cantly increased during the desiccation phase), resulting

in a shift in the Spd/Put ratio from ca. 2 in early

cotyledonary embryos to around 10 after 3 weeks of

desiccation. In mature zygotic embryos, Spd contents

were twofold lower, but Put levels were higher, than in

mature somatic embryos, hence their Spd/Put ratio was

substantially lower (ca. 2, in both embryos and megaga-

metophytes). In addition, the PA synthesis activity

profiles in the embryos differed (ornithine decarboxylase

and arginine decarboxylase activities predominating in

mature somatic and zygotic embryos, respectively). The

start of germination was associated with a rise in PA

biosynthetic activity in the embryos of both origins, which

was accompanied by a marked increase in Put contents in

somatic embryos, resulting in the decline of Spd/Put ratio

to about 2, similar to the ratio in mature and germinating

zygotic embryos. The accumulation of high levels of PAs

in somatic embryos may be causally linked to their lower

germinability than in zygotic embryos.

Keywords: ADC, ODC, Picea abies, SAMDC, somaticembryogenesis.

Introduction

Tissue culture approaches, in particular somatic embryo-

genesis, hold a considerable promise for accelerating breed-

ing programs of woody plants. However, although the

induction, proliferation and maturation of somatic

embryos under suitable conditions can yield sufficient

embryos of some coniferous species, in other species the

germination frequency of somatic embryos is often too

low for practical applications (Igasaki et al. 2003).

Research into somatic embryogenesis of conifers has

increased rapidly ever since the first reliable protocol for

Picea abies L. (Karst.) was published (Chalupa 1985,

Hakman et al. 1985). Coniferous somatic embryogenesis

can be divided into a number of stages, most of which

are comparable to zygotic embryogenesis (Attree and

Fowke 1993). However, since somatic embryos develop

without the protective environment of the surrounding

maternal tissue, there is a need to supply both nutrients

and regulatory compounds exogenously. Induction and

continuous proliferation require auxin and cytokinins,

whereas the further development and maturation of

embryos depends on abscisic acid (Attree et al. 1991). By

the end of the maturation phase, all of the anatomical struc-

tures of the embryo have been established (Find 1997), but

Tree Physiology 29, 1287–1298

doi:10.1093/treephys/tpp063

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the embryo only becomes biochemically mature after a des-

iccation phase (Flinn et al. 1993).

Despite the availability of a successful protocol for gener-

ating Norway spruce plants using a somatic embryogenesis

technique, there is a lack of data concerning the endogenous

composition of biologically active compounds in both

somatic and zygotic embryos. Generally, the development

of embryos, as well as their conversion into complete plant-

lets, is closely associated with the changes in endogenous

phytohormone levels. We have recently reported the

changes in endogenous hormone levels (IAA, ABA and eth-

ylene) during somatic embryo development and maturation

of Norway spruce (Vagner et al. 1998). Another recent study

on hybrid larch embryos has shown pronounced differences

in the concentration of ABA between the mature embryos

of somatic and zygotic origin (Von Aderkas et al. 2001).

Several developmental processes controlled by cytokinins

also appear to involve polyamines (PAs). Polyamines, low

molecular mass polycations, are ubiquitous cell compo-

nents that are essential for normal growth of both eukary-

otic and prokaryotic cells (Tabor and Tabor 1984). Most of

the biological functions of PAs can be explained by their

polycationic nature, which facilitates interactions with anio-

nic macromolecules (such as DNA and RNA) and nega-

tively charged groups of membranes. Biosynthesis of the

three commonly occurring PAs – putrescine (Put), spermi-

dine (Spd) and spermine (Spm) – is initiated in plants either

by direct decarboxylation of ornithine by the enzyme orni-

thine decarboxylase (ODC; EC 4.1.1.17) or by decarboxyl-

ation of arginine by the enzyme arginine decarboxylase

(ADC; EC 4.1.1.19) via agmatine and N-carbomoylputres-

cine intermediates. The exact role of these two pathways in

Put biosynthesis is not clear, but ADC and ODC activities

vary both between species and between plants of the same

species at different stages (Santanen and Simola 1999).

Another essential enzyme for PA synthesis is S-adenosyl-

methionine decarboxylase (SAMDC; EC 4.1.1.50), which

is required for the production of the aminopropyl group

used in the biosynthesis of Spd and Spm (for a review,

see Wallace et al. 2003).

There have been a number of reports, some regarding

conifers, indicating that PAs play a crucial role in somatic

embryo development. The formation of somatic embryos

of wild carrot in tissue culture appears to be associated with

high Spd levels, and much more Spd than Put has been

found in these embryos in the torpedo stage (Mengoli

et al. 1989). The Spd, in particular, has been implicated in

the somatic embryogenesis in tissue cultures ofHevea brasil-

iensisMull. Arg. (El Hadrami andD’Auzac 1992), in the ini-

tiation phase of somatic embryogenesis in Panax ginseng

CA Meyer (Monteiro et al. 2002) and in the development

of globular pro-embryos in alfalfa (Cvikrova et al. 1999).

In addition, Kevers et al. (2000) found that exogenous appli-

cation of Spd in the initiation phase significantly increased

the production of embryos in P. ginseng cultures. The role

of PAs during in vivo and in vitro development, including

somatic embryogenesis, has been recently reviewed (Baron

and Stasolla 2008).

In the studies of conifers, Put has been found to be the

most abundant PA in embryogenic suspension cultures of

Pinus taeda L. (Silveira et al. 2004); levels of Spd are report-

edly high (and increase with time) during the development

of both somatic and zygotic embryos of Pinus radiata

D. Don (Minocha et al. 1999); and high levels of Put have

been found in the pro-embryogenic tissue of Picea rubens

Sarg., but Spd predominates during embryo development

in this species (Minocha et al. 2004).

This study was undertaken to establish changes in the

metabolism of PAs during the development of somatic

embryos of P. abies, (i) to relate PA concentrations to the

development of the embryos; (ii) to compare PA levels in

mature somatic embryos with those in mature zygotic

embryos and megagametophytes; and (iii) to characterize

changes in the PA levels during the germination of both

types of embryos. The acquired data provide a detailed

anatomical characterization of P. abies somatic embryo

development, from proliferation to germination, and com-

parative information on the anatomy and germination of

mature zygotic embryos, related to the changes in free PA

contents and the activities of enzymes involved in their

biosynthesis (ADC, ODC and SAMDC).

Materials and methods

Plant material

An embryogenic culture of P. abies, genotype AFO 541,

was obtained from AFOCEL, France, and dry mature

seeds of P. abies from cones were collected during the sum-

mer of 2007, before the end of July, in the grounds of

the Forestry and Game Management Research Institute,

Strnady, Czech Republic.

Proliferation of embryogenic culture

The embryogenic culture was grown on GD medium

(Gupta and Durzan 1986) solidified by 0.75% agar

(Sigma-Aldrich, Czech Republic), with pH adjusted to 5.8

before autoclaving, supplemented with 5 lM 2,4-D, 2 lMkinetin, 2 lM BAP (all Sigma-Aldrich, Czech Republic)

and 30 g l�1 sucrose (Lachema, Czech Republic). All

organic components, except sucrose, were separately pre-

pared and diluted, filter-sterilized and added to the cooled,

autoclaved media. The embryogenic cultures were main-

tained by subculturing, weekly, in Magenta vessels

(Sigma-Aldrich, Czech Republic) containing 40 ml of fresh

medium and incubated in darkness at 24±1 �C.

Maturation of somatic embryos

In the GD medium used for maturation, the cytokinins and

auxin were substituted by 20 lM ABA (Sigma-Aldrich,

1288 GEMPERLOVA ET AL.

TREE PHYSIOLOGY VOLUME 29, 2009

Czech Republic), which was used in combination with

3.75% polyethylene glycol 4000 (PEG). The PEG and

ABA solutions were separately autoclaved and filter-

sterilized, respectively, then added to the medium after

autoclaving. During maturation, cultures were subcultured,

weekly, in a fresh medium in Magenta vessels and incu-

bated in the dark at 24±1 �C for 5–6 weeks.

Desiccation of somatic embryos

Fully developed embryos after maturation were selected and

desiccated, by transferring them onto dry paper in small

Petri dishes (3 cm diameter), which were placed in large

Petri dishes (18 cm diameter) with several layers of paper

wetted by sterile water (to maintain 100% humidity). The

large Petri dishes were covered by lids and sealed by para-

film, then incubated in a cultivation room with a 12-h

photoperiod (PPFD about 15–25 lmol m�2 s�1, daylight

fluorescent tubes; Osram AG, Winterthur, Switzerland) at

18±1 �C for 3 weeks.

Germination of somatic embryos

The desiccated embryos were transferred into Magenta

dishes filled with 40 ml of half strength GD medium, sup-

plemented with sucrose (1%) and active charcoal (0.4%),

but no phytohormones, with pH adjusted to 5.8 before

autoclaving. The dishes containing embryos were placed

in a cultivation room with a 12-h photoperiod (PPFD

about 60–80 lmol m�2 s�1, daylight fluorescent tubes) at

24±1 �C for 2 weeks.

Imbibition and germination of seeds

Cold-stored (4 �C) seeds of P. abies were imbibed in dis-

tilled water for 24 h at room temperature. During this time,

hydration of the cotyledons, hypocotyl and finally the rad-

icle was completed, and the zygotic embryos were ready to

germinate (Tillmansutela and Kauppi 1995). This period of

imbibition was necessary to compensate for the extra barri-

ers surrounding zygotic embryos (the seed coat and mainly

the lipophilic layers surrounding the endosperm) compared

to their somatic counterparts. Subsequent germination took

place in unsealed Petri dishes with wet filter paper under a

16-h photoperiod (PPFD about 60–80 lmol m�2 s�1, day-

light fluorescent tubes) at 24 �C. For further analyses, sep-arated zygotic embryos and megagametophytes were

collected after removal of the seed coat.

Material for biochemical analyses

The concentrations of PAs and the activity of biosynthetic

enzymes (ADC, ODC and SAMDC) were measured at

1-week intervals, over the course of the development of

somatic embryos and their desiccation. For the first 3 weeks

of maturation, samples contained the whole embryogenic

suspensor mass (ESM); thereafter (from the fourth week),

the embryos were separated from the remaining mass.

Samples of germinating somatic embryos were collected

on days 1, 2 and 6. Biochemical analyses of zygotic

embryos were performed on separated embryos and on the

megagametophyte tissue isolated from dry mature seeds,

imbibed seeds and seeds germinated for 1, 2 and 6 days.

The samples (25–50 somatic or 50–100 zygotic embryos)

were frozen in liquid nitrogen and stored at �80 �C until

analysis.

Anatomical study

Paraffin sections (12-lm thick) of ESM and embryos of

both origins were prepared according to Johansen (1940)

and stained by the two-step procedure, using alcian blue

and nuclear fast red, described by Svobodova et al.

(1999). Alcian blue binds specifically to polysaccharides

in the cell wall, and nuclear fast red counterstains chroma-

tin structures. At least 10 samples (ESM or embryos)

representing each of the developmental stages, described

above, were used in the biochemical analyses (see below).

ODC, ADC and SAMDC assays

Ornithine decarboxylase (ODC; EC 4.1.1.17), arginine

decarboxylase (ADC; EC 4.1.1.19) and S-adenosylmethio-

nine decarboxylase (SAMDC; EC 4.1.1.50) activities were

determined using a radiochemical method as described by

Tassoni et al. (2000). Samples were extracted in three vol-

umes of ice-cold 0.1 M Tris–HCl buffer, pH 8.5, containing

2 mM b-mercaptoethanol, 1 mM EDTA and 0.1 mM pyr-

idoxal phosphate, and centrifuged at 20,000g for 30 min at

4 �C. Portions (0.1 ml) of both supernatant (soluble frac-

tion) and resuspended pellet (particulate fraction) were used

to determine ODC and ADC activities, by measuring

the 14CO2 evolution from 7.4 kBq L-[1-14C]ornithine

(1.92 GBq mmol�1, Amersham Pharmacia Biotech UK)

or 7.4 kBq L-[U-14C]arginine (11.5 GBq mmol�1

Amersham Pharmacia Biotech UK), for ODC and ADC,

respectively, in the presence of 2-mM unlabeled substrate

during a 1.5-h incubation at 37 �C. The CO2 was trapped

in hyamine hydroxide, and the radioactivity was counted

using a Tri-Carb 2900TR liquid scintillation analyzer

(Packard Instrument Co., Meriden, CT).

To determine SAMDC activities, samples were homoge-

nized in three volumes of 0.1 M phosphate buffer, pH 7.6,

containing 2 mM b-mercaptoethanol and 1 mM EDTA,

and centrifuged at 20,000g for 30 min at 4 �C. The super-

natant and the resuspended pellet (0.1 ml portions) were

incubated separately with 3.7 kBq [1-14C]S-adenosylmethi-

onine (2.15 GBq mmol�1, Amersham Pharmacia Biotech

UK) in the presence of 2.8-mM unlabeled substrate and

3 mM Put. The amount of 14CO2 released during a 1-h

incubation at 37 �C was then measured using a Tri-Carb

2900TR liquid scintillation analyzer.

The protein contents of the samples were measured

according to Bradford’s method, using bovine serum

albumin as a standard (Bradford 1976).

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PA analysis

The cells were ground in liquid nitrogen and extracted over-

night at 4 �C with 1 ml of 5% perchloric acid (PCA) per

100-mg fresh mass tissue. 1,7-Diaminoheptane was added

as an internal standard, and the extracts were centrifuged

at 21,000g for 15 min. Standards (Sigma-Aldrich, Czech

Republic) and PCA-soluble free PAs were benzoylated

according to the method of Slocum et al. (1989), and the

resulting benzoylamines were analyzed by HPLC using a

Beckman-Video Liquid Chromatograph (Beckman Instru-

ments, Inc., Fullerton, CA) equipped with a UV detector

(monitoring the eluate at 254 nm) and a C18 Spherisorb 5

ODS2 column (particle size of 5 lm, column length of

250 · 4.6 mm) according to the method of Slocum et al.

(1989).

Statistical analyses

Two independent experiments were carried out, in which

similar results were obtained. Mean values ± SE obtained

in one experiment (with three replicates) are shown in

the figures. Data were analyzed using Student’s t distribu-

tion criteria.

Results

Anatomical study

During proliferation, the embryogenic culture AFO 541

was characterized by the presence of highly bipolar struc-

tures, consisting of clusters of small meristematic cells with

dense cytoplasm forming meristematic centers, attached to

elongated, highly vacuolized suspensor cells (Figure 1A).

Transition of the proliferating culture to the maturation

medium containing ABA resulted in a rapid growth of

meristematic centers, followed by disintegration of the

suspensor cells. After 3 weeks of maturation, protoderm

was formed as the first inner structure of embryos

(Figure 1B).

Figure 1. Anatomical characterization of somatic and zygotic embryos of P. abies in various developmental stages. (A) Proliferation ofembryogenic culture. (B) Embryogenic culture after 3 weeks of maturation. (C) Fully developed and desiccated somatic embryo. (D)Somatic embryo after 1 day of germination. (E) Apical pole of somatic embryo after 6 days of germination. (F) Root pole of zygoticembryo after 6 days of germination. (G) Zygotic embryo in seed after 1 day of germination. (H) Apical pole of zygotic embryo after6 days of germination. (I) Root pole of zygotic embryo after 6 days of germination. AM, apical meristem; CO, cotyledons; LB, lateralleaf buds; MC, meristematic center; MG, megagametophyte; PC, procambium; PD, protoderm; RC, root cap with collumela; RM,root meristem; and SC, suspensor cells. Bars represent 500 lm (100 lm in section H).

1290 GEMPERLOVA ET AL.

TREE PHYSIOLOGY VOLUME 29, 2009

The establishment of inner structures further continued

until the sixth week of maturation, when somatic embryos

were anatomically fully developed. The mature somatic

embryos had a distinct apical meristem, procambial

strands, well-developed cotyledons and root meristem with

a root cap. During desiccation, virtually no further visible

developmental changes were observed (Figure 1C). The

morphology of mature zygotic embryos was comparable

to that of somatic embryos, except that their shape differed

slightly due to the surrounding megagametophyte (Figure

1G). In the embryos of both origins, the start of germina-

tion was characterized by frequent cell divisions throughout

the embryos. As germination proceeded, the root of

somatic embryo elongated, and in some cases a new root

cap was formed (Figure 1F). The apical pole of germinating

somatic embryos remained unchanged until the sixth day

(Figure 1E). In contrast, the apical meristem of zygotic

embryos had already formed leaf primordia within 6 days

of germination (Figure 1H). At the same time, the root of

zygotic embryo protruded from the megagametophyte

(Figure 1I).

Activities of ODC, ADC and SAMDC biosynthetic

enzymes during development of somatic embryos

The activities of biosynthetic enzymes were measured in

both the soluble and particulate fractions. In ESM, the

activity of ODC continually increased during the culture

period and peaked 3–4 weeks after transfer to the matura-

tion medium (Figure 2A). The level of ODC activity at this

time was 2.5-fold higher than in ESM growing on the pro-

liferation medium. Thereafter, the ODC activity declined

until the end of the maturation phase and remained low

throughout the whole desiccation phase. A further rise in

OD

C (

pkat

mg–1

pro

t)

0

10

20

30

AD

C (

pkat

mg–1

pro

t)

0

1

2

3

4

5

0 14 21 28 35 42 49 56 63 64 65 69

SA

MD

C (

pkat

mg–1

pro

t)

0

2

4

6

8

cytosolicpellet

A

B

C

ESM SE SED SEG

Time (d)

Figure 2. Activities of enzymes involved in PAbiosynthesis during the development of somaticP. abies embryos from proliferation to germination.Biosynthetic enzymes: ODC, A; ADC, B; andSAMDC, C. Bars represent SE of three replicates.Activity of enzymes in the cytosolic (open symbols)and pellet (filled symbols) fractions. ESM, embryo-genic suspensor mass; SE, somatic embryo; SED,somatic embryo during desiccation; SEG, somaticembryo during germination; and d, days.

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ODC activity was connected to the start of germination of

somatic embryos. The ODC activity was equally distrib-

uted between the soluble and pellet fractions within all

monitored phases. Although the activity of ADC was sev-

eral-fold lower than that of ODC on any given day of anal-

ysis, two increases were observed (Figure 2B), both

correlated with the observed maxima of ODC activity, in

week 4 of the maturation phase and on day 6 of germina-

tion. During the desiccation and maturation phases, ADC

activity was two times higher in the soluble fraction than

in the particulate fraction but, conversely, after the start

of germination the activity was two times higher in the

particulate fraction than in the soluble fraction. SAMDC

activity showed a time course similar to that of ODC (Fig-

ure 2C). However, the maximum observed in the embryos

after 4 weeks on the maturation medium was much more

pronounced. This peak of SAMDC activity corresponded

with a marked increase in Spd content (Figure 4). The

SAMDC activity was predominantly found in the

pellet fraction in all developmental stages of somatic

embryos.

Activities of ODC, ADC and SAMDC biosynthetic

enzymes in zygotic embryos

The activities of the three biosynthetic enzymes ODC, ADC

and SAMDC are shown in Figure 3. In zygotic embryos,

ADC was mainly responsible for Put biosynthesis in both

embryos and megagametophytes. However, in megaga-

metophytes, the ADC activity, detected predominantly in

the cytosolic fraction, was significantly lower and repre-

sented only 17–35% that found in embryos (Figure 3B).

In contrast to ADC, ODC activity was detected at very

low levels and was predominantly detected in the pellet

fraction (Figure 3A). Most of the SAMDC activity was

detected in the pellet of megagametophytes, except on

day 6 of germination (Figure 3C), when marked increases

in the activities of all three biosynthetic enzymes were

observed in zygotic embryos, whereas in megagameto-

phytes their levels decreased. This finding correlates with

the high PA levels observed in the embryos during germina-

tion, but not megagametophytes (Figure 6). The imbibition

of mature zygotic embryos resulted in only moderate

increases in the activities of ODC, ADC and SAMDC.

However, marked increases in their activities were observed

on their first day of germination. In the samples of germi-

nating embryos on the first day, ODC activity was detected

mainly in the supernatant, but on days 2 and 6 higher activ-

ities were found in the pellet, in samples from both embryos

and megagametophytes. The ADC activity was very high in

germinating embryos and was detected mainly in the solu-

ble fraction on days 1 and 2.

Comparison of ODC, ADC and SAMDC activities

in somatic and zygotic embryos

In contrast to the predominance of ODC activity found in

somatic embryos, in zygotic embryos ADC played the most

important role in PA biosynthesis (Figures 2A and 3B). In

mature zygotic embryos, the ADC activity was 18- and 34-

fold higher than that of ODC on days 1, 2 and 6, respec-

tively. In somatic embryos, the difference between the

ODC and ADC activities detected at the start of the desic-

cation phase subsequently declined, and there was very little

difference between them at the end of this phase.

OD

C (

pkat

mg–1

pro

t)

0.0

0.2

0.4

0.6

0.8

AD

C (

pkat

mg–1

pro

t)

0

2

4

6

8

10

12

ZE MG ZE I MG I ZE MG ZE MG ZE MG

SA

MD

C (

pkat

mg–1

pro

t)

0

2

4

6 cytosolicpellet

1 1 22 6 6

A

B

C

GERMINATION

Time (d)

Figure 3. Activities of enzymes involved in PA biosynthesis inisolated P. abies zygotic embryos and megagametophytes andduring germination. Biosynthetic enzymes: ADC, A; ODC, B;and SAMDC, C. Bars represent SE of three replicates. Activitiesof enzymes in the cytosolic (open symbols) and pellet (filledsymbols) fractions. ZE, zygotic embryo; MG, megagameto-phyte; I, imbibition; and d, days.

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PA contents during development of somatic embryos

At a very early stage of development, during proliferation

of the embryogenic culture of Norway spruce, ESM con-

tained approximately equal levels of Put and Spd, thus

the Spd/Put ratio was about 1 (Figures 4 and 5). The con-

tent of Spm at this stage was rather low. After 3 weeks of

culture on maturation medium, when the ESM contained

globular and partly polarized embryos, increases in levels

of all three amines were observed, which correlated well

with the observed increases in ODC activity. However, pro-

nounced changes in the PA levels and the Spd/Put ratio

took place after 4 weeks of maturation in torpedo stage

embryos (Figures 4 and 5). From this stage, the embryos

were separated from the associated subtending tissue

(embryogenic and suspensor cell masses), and a marked

increase in PAs was observed in embryos at this stage of

development. The Spd level was significantly higher than

the Put level, and the increase coincidedwith a sharp increase

in SAMDC activity in separated embryos (Figures 2C and

4). In the residual ESM, after separation of somatic

embryos, similar PA contents were found to those detected

in 3-week-old ESM, with approximately equal Put and

Spd levels (data not shown). After 5 weeks of growth on

maturation medium, the level of Put in embryos declined,

and this stage of embryo development was characterized

by very high Spd contents (Spd/Put ratio > 2; Figures 4

and 5). In mature 6-week-old cotyledonary embryos, the

contents of Put and Spd decreased (Figure 4). During the

desiccation period, the Put and Spd levels declined, whereas

the Spm level significantly increased. After 3 weeks of desic-

cation, the Spm level was nearly as high as that of Spd

(Figure 4). The decline in cellular Put contents in embryos

after 3 weeks of desiccation resulted in an increase in the

Spd/Put ratio to nearly 10 (Figure 5). In germinating somatic

embryos, marked increases in Put and Spd levels, correlating

with the increases in biosynthetic enzyme activities on the

first and second day, were detected and the Spd/Put ratio

decreased, but Spm contents remained high (Figures 4

and 5). A strong subsequent increase in the Put content on

day 6was reflected in a decline in the Spd/Put ratio to a value

of about 2 (Figure 5).

PA contents in zygotic embryos

In mature Norway spruce zygotic embryos, the Spd level

was twofold greater than the level of Put (thus the Spd/

Put ratio was slightly > 2, Figure 7), and PA concentra-

tions were higher in embryos than in megagametophytes

(Figure 6). After imbibition, the level of PAs slightly

increased in the embryos and did not change in the imbibed

megagametophytes. Increases in all three PAs were

observed in embryos after 1 day of germination (but the

Spd level was still higher than the Put level), while the con-

tent of all the three PAs in megagametophytes did not

change or declined on day 1. On day 2, the PA contents

in embryos increased slightly, and marked increases were

observed in them after 6 days of germination (Figure 6).

0 14 21 28 35 42 49 56 63 64 65 69

PA

s (n

mol

g–1

DW

)

0

1000

2000

3000

4000

5000PutSpdSpm

SEGSEDSEESM

Time (d)

Figure 4. Changes in cellular levels of Put, Spd andSpm during P. abies somatic embryo developmentfrom proliferation to germination. Bars representSE of three replicates. ESM, embryogenic suspen-sor mass; SE, somatic embryo; SED, somaticembryo during desiccation; SEG, somatic embryoduring germination; and d, days.

0 14 21 28 35 42 49 56 63 64 65 69

Spd

/ Put

rat

io

0

2

4

6

8

10

12

Time (d)

ESM SE SED SEG

Figure 5. Changes in Spd/Put ratios during P. abies somaticembryo development, from proliferation to germination. ESM,embryogenic suspensor mass; SE, somatic embryo; SED,somatic embryo during desiccation; SEG, somatic embryoduring germination; and d, days.

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The Spd/Put ratio was stable in both tissues (embryo and

megagametophyte) during germination, with values ranging

from 1 to 1.5 (Figure 7).

Comparison of PA content in somatic and zygotic embryos

In Figure 8, the Put, Spd and Spm contents in mature des-

iccated somatic embryos, mature zygotic embryos, and

somatic and zygotic embryos 1, 2 and 6 days after germina-

tion are compared. Of note is the very high level of Spm

found in somatic embryos. Pronounced differences between

somatic and zygotic embryos were found not only in PA

contents but also in Spd/Put ratios (Figures 5 and 7).

Discussion

The involvement of PAs in various plant growth and devel-

opmental processes, including a crucial role in somatic

embryo development, has been reported in several conifer-

ous species (Santanen and Simola 1994, Minocha et al.

1999, 2004, Silveira et al. 2004). The results presented here

show that significant, characteristic changes in the endoge-

nous levels of PAs and their biosynthetic enzymes coincide

with the development of somatic embryos from early stages

through maturation, desiccation and germination. On

transfer from proliferation to maturation medium (time

zero in the figures), Put biosynthesis was mediated largely

by the activity of ODC, which continually increased for

4 weeks (Figure 2A). In addition, from 3 weeks ADC

was found to be active in the ESM. Thus, from 3 weeks

both enzymes contributed to PA biosynthesis, and their

activities were highest in the early cotyledonary stage

embryos separated from the associated subtending tissue

at 4 weeks. Activity of the third biosynthetic enzyme,

SAMDC, followed a time course similar to that of ODC

(Figure 2). A transient decline in PA biosynthetic activity

was observed during the late stages of embryo maturation

(5 and 6 weeks) and during the desiccation phase, corre-

sponding well with the observations of Vuosku et al.

(2006) and Loukanina and Thorpe (2008).

Parallel rises in the contents of all three PAs were

observed in the first 3 weeks in ESM cultured on a mat-

uration medium, keeping the Spd/Put ratio close to 1

(Figures 4 and 5). In separated 4- and 5-week-old

embryos, high Spd levels raised the Spd/Put ratio to

about 2. A rapid subsequent decline in cellular Put in

mature 6-week-old embryos and during the desiccation

phase resulted in a shift in their Spd/Put ratios to as high

as 10 at the end of desiccation. The decline in PA contents

ZE MG ZE I MG I ZE MG ZE MG ZE MG

PA

s (n

mol

g–1

DW

)

0

500

1000

1500

2000

2500

3000

3500 PutSpdSpm

1 1 2 2 6 6

Time (d)

GERMINATION

Figure 6. Changes in cellular levels of Put, Spd andSpm in isolated P. abies zygotic embryos andmegagametophytes before and during germination.ZE, zygotic embryo; MG, megagametophyte;I, imbibition; and d, days.

Time (d)

ZE MG ZE I MG I ZE MG ZE MG ZE MG

Spd

/ Put

rat

io

0.0

0.5

1.0

1.5

2.0

2.5GERMINATION

1 2 6

Figure 7. Changes in Spd/Put ratios in isolated P. abies zygoticembryos and megagametophytes before and during germination.ZE, zygotic embryo; MG, megagametophyte; I, imbibition;and d, days.

1294 GEMPERLOVA ET AL.

TREE PHYSIOLOGY VOLUME 29, 2009

in mature embryos was partly due to the decrease in bio-

synthetic enzyme activities (Figure 2) and partly due to the

increased catabolism of Put and Spd during the later

stages of embryo development, as previously described

in P. abies (Santanen and Simola 1994). The pattern of

changes in soluble free Put and Spd contents, and the

Spd/Put ratio, during somatic embryo development are

consistent with the previous studies on somatic embryo-

genesis in conifers (Minocha et al. 1999). Results indicat-

ing a direct role for Spd in somatic embryogenesis have

been reported in H. brasiliensis (El Hadrami and D’Auzac

1992), Medicago sativa L. (Cvikrova et al. 1999) and P.

ginseng (Monteiro et al. 2002). Significant increases in

Spd levels are also reportedly associated with the forma-

tion of somatic embryos in P. abies (Santanen and Simola

1992) and P. radiata (Minocha et al. 1999).

Low levels of Spm were found in developing somatic

embryos, corresponding to the levels that were previously

determined in the embryogenic cultures of P. abies and

P. rubens (Minocha et al. 1993). The Spm content slightly

increased only in 4- and 5-week-old embryos, when Put

and Spd reached their maximum levels. Although desic-

cated somatic embryos were anatomically comparable to

the mature ones, a marked increase in the Spm content

was observed in somatic embryos after two and, more

markedly, 3 weeks of desiccation, when its level was com-

parable with that of Spd (Figure 4). Desiccation of zygotic

embryos is, in most seeds, the terminal event in their devel-

opment. In somatic embryos, desiccation for 3 weeks may

represent a sort of osmotic stress, since accumulation of free

Spd and Spm has been recognized as a biochemical indica-

tor of drought-associated osmotic stress (Sanchez et al.

2005). The significant rise of Spm contents in embryos

observed in the process of desiccation might therefore

represent a response to this abiotic stress.

The morphology and the shape of mature somatic and

zygotic embryos showed remarkable similarities, in spite

of the difference in the environments in which they had

developed (Figure 1C and G). However, the comparison

of PA metabolism of mature somatic and zygotic embryos

revealed some differences. In mature zygotic embryos of

P. abies Put was synthesized more actively from arginine

than from ornithine, and ADC activity (predominately

cytosolic) was higher in the embryos than in the megaga-

metophytes (Figure 3B). During somatic embryo develop-

ment, ODC was mainly responsible for Put production,

but in the mature somatic embryos, at the end of the desic-

cation phase, the biosynthetic activities of ODC and ADC

were approximately equal. In mature somatic embryos,

ODC activity was equally distributed between the cytosolic

and pellet fractions, whereas the ADC activity was higher

in the cytosolic fraction (Figure 2A and B). Previous anal-

yses of the subcellular localization of ODC and ADC activ-

ities have indicated that both proteins are active in the

cytosolic as well as in the particulate fractions (Tassoni

et al. 2003). The difference in compartmentalization of bio-

synthetic enzymes may be related to distinct functions in

different cell types (Bortolotti et al. 2004). In accordance

with this hypothesis, assays performed on the leaves of

Arabidopsis thaliana (L.) Heynh. have shown high particu-

late ODC activity to be associated with the nuclei and chlo-

roplast-enriched fractions (Tassoni et al. 2003). However,

the significance of this difference is not clear. Biosynthesis

via ODC is generally connected with cell division, whereas

the ADC activity is often linked to stress responses and cell

elongation (Perez-Amador and Carbonell 1995, Acosta

et al. 2005). Nevertheless, the exact role of these two path-

ways in Put biosynthesis is not clear, and both ADC and

ODC activities vary in plants at different stages of develop-

ment. It should also be noted that the differences in the rel-

ative magnitude of ADC and ODC activities in somatic and

zygotic embryos of P. abies may not be important in the

context of this study, since they both contribute to the

production of Put, and thus to PA synthesis.

Mature zygotic embryos, as well as mature somatic

embryos, contained higher levels of Spd than Put, and

0

500

1000

1500

2000

0

500

1000

1500

2000

PA

s (n

mol

g–1

DW

)

0

1000

2000

3000

4000

Put

Spd

Spm

ZE SE ZE SE

1

ZE SE ZE

2 6

SE

0

Time of germination (d)

Figure 8. Comparison of cellular levels of Put, Spd and Spm inmature somatic and zygotic P. abies embryos before and duringtheir germination. ZE, zygotic embryo; SE, somatic embryo; andd, days.

SOMATIC AND ZYGOTIC EMBRYOS OF NORWAY SPRUCE 1295

TREE PHYSIOLOGY ONLINE at http://www.treephys.oxfordjournals.org

PA contents were lower in megagametophytes than in

embryos (Figure 6). Higher levels of Spd than Put have also

been observed in zygotic embryos and megagametophytes

of P. radiata (Minocha et al. 1999). However, the compar-

ison of Put, Spd and Spm contents in somatic and zygotic

embryos indicates that there are differences in PA metabo-

lism between these two types of embryos (Figure 8). The

Spd/Put ratio was as high as 10 in somatic embryos after

desiccation, whereas the ratio was much lower (ca. 2) in

zygotic embryos because they had higher Put and lower

Spd contents (Figures 5 and 7). High concentrations of

PAs are commonly observed in tissues undergoing somatic

embryogenesis. However, a high level of free PAs is not the

only important PA-related factor in somatic embryogene-

sis, and (for instance) it has been proposed that an inade-

quate Spd/Put ratio may be causally linked to abnormal

growth and disorganized cell proliferation of grape somatic

embryos with high free PA contents (Faure et al. 1991).

We may hypothesize that the presence of relatively high

concentrations of ABA in the maturation medium, and

consequently high endogenous levels of ABA in developing

somatic embryos of P. abies (Vagner et al. 1998), induces

the biosynthesis and accumulation of PAs. Accordingly,

Von Aderkas et al. (2001) found that ABA concentrations

were about 100 times higher in hybrid larch somatic

embryos than in embryos of zygotic origin, and in embryo-

genic cultures of Araucaria angustifolia (Bertol.) Kuntze the

increases in endogenous ABA contents have been found to

be positively correlated to the increases in Put and Spd lev-

els, showing ABA and accumulation of PAs to be directly

related (Steiner et al. 2007).

We were interested in the changes in PA composition that

may occur during the germination of embryos, especially

somatic embryos, and could potentially be linked to the

developmental events. Morphological differences were

observed between the two types of embryos as germination

proceeded (compare Figure 1D–F and G–I). The start of

germination was characterized by cell division in root mer-

istems of both types of embryos, and in apical meristems of

zygotic embryos, connected with the rise in PA biosynthetic

activity (Figures 2 and 3). We analyzed zygotic embryos

and megagametophytic tissue separately. Whereas ADC

activity increased in zygotic embryos during seed imbibition

and at the beginning of germination, SAMDC activity

markedly increased only in the megagametophytes. In spite

of a rise in Put levels in germinating zygotic embryos, Spd

remained the major PA both in these embryos and megaga-

metophytes (which serve as a storage tissue supporting the

germinating embryos in coniferous seeds; Kong et al. 1997).

The accumulation of Spd in the germinating zygotic

embryos (without a corresponding increase in SAMDC

activity in the embryos themselves) suggests that Spd is sup-

plied from megagametophytes, where we found high SAM-

DC activity, but low Spd levels. However on day 6, the

megagametophytes probably lost this function as their bio-

synthetic activity became much lower than that of the

embryos. The Spd/Put ratio was stable in both the tissues

(embryo and megagametophyte) during germination, with

values ranging from 1 to 1.5 (Figure 7).

Rises in cellular concentrations of Spd, and stronger

increases in Put contents, in somatic embryos during the first

6 days of germination decreased the amount of Spm as a

percentage of total PA contents (to 41% and 21% inmature

somatic embryos, respectively) and reduced the Spd/Put

ratio to about 2, showing a pattern similar to that found

for zygotic embryos (Figures 5 and 7). The accumulation

of high levels of PAs in somatic embryos might contribute

to their ‘reserves’, consisting predominately of proteins

and triglycerides, which are used during their germination.

Another important aspect that should be considered con-

cerns the involvement of PAs in the synthesis of nitric oxide

(NO), which is known to act as a signaling molecule in plant

cells (Besson-Bard et al. 2008). The application of

NO-releasing compounds has been shown to stimulate cell

division and embryogenic cell formation in alfalfa cells in

the presence of auxin (Otvos et al. 2005), and Tun et al.

(2001) reported that adding Spd and Spm resulted in the

rapid production of NO in the elongation zone of the root

tip and primary leaves of Arabidopsis seedlings. This PA-

dependent NO production may be catalyzed by unknown

enzymes or by PA oxidases (Yamasaki and Cohen 2006).

On the other hand, the significantly higher total content of

PAs in somatic embryos (twofold and threefold higher levels

of Spd and Spm, respectively) than in zygotic embryos

might be responsible for their lower germinability.

Acknowledgments

We thank Sees-editing Ltd. for linguistic editing. This work was

supported by the Ministry of Agriculture of the Czech Republic

(Project Nos. QH82303 and 0002070202) and by the Institutional

Grant AV0Z 50380511.

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Appendix

Abbreviations

ADC arginine decarboxylase

BAP 6-benzylaminopurine

2,4-D 2,4-dichlorophenoxyacetic acid

ESM embryogenic suspensor mass

ODC ornithine decarboxylase

PAs polyamines

PCA perchloric acid

PEG polyethylene glycol

Put putrescine

SAMDC S-adenosylmethionine decarboxylase

Spd spermidine

Spm spermine

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