ORIGINAL PAPER

The effect of arabinogalactan proteins on regeneration potentialof juvenile citrus explants used for genetic transformationby Agrobacterium tumefaciens

Vladimir Orbovic • Esther Marie Gollner •

Patricia Soria

Received: 9 August 2012 / Revised: 28 November 2012 / Accepted: 30 November 2012 / Published online: 12 December 2012

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2012

Abstract A possible role of arabinogalactan proteins in

control of shoot regeneration from stem explants of two

citrus cultivars, Carrizo citrange and ‘Duncan’ grapefruit,

was investigated. Treatment of explants with (b-D-Glc)3

Yariv phenylglycoside, able to bind specifically to AGPs,

led to a decrease of cumulative regeneration potential of

both Carrizo citrange and ‘Duncan’ grapefruit. For Carrizo,

lower cumulative regeneration potential on (b-D-Glc)3

Yariv phenylglycoside-treated explants was the result of

both lower number of shoots on the explants that had

shoots (explant regeneration potential) and decreased per-

centage of explants with shoots. In the case of ‘Duncan’,

treatment with (b-D-Glc)3 Yariv phenylglycoside reduced

cumulative regeneration potential only by lowering the

percentage of explants with shoots, but it did not affect the

number of shoots on the explants with shoots. Citrus

explants treated with (a-D-Man)3 Yariv phenylglycoside,

which does not bind AGPs, responded similarly to

untreated explants. Transformability of cells on the cut

ends of explants was also lower for both cultivars follow-

ing the treatment of explants with (b-D-Glc)3 Yariv phe-

nylglycoside. Our data suggest that arabinogalactan

proteins play important role in processes controlling dif-

ferentiation and genetic transformation of citrus cells by

Agrobacterium.

Keywords Arabinogalactan proteins �Agrobacterium tumefaciens � Citrus �Genetic transformation

Abbreviations

AGP Arabinogalactan protein

aManY (a-D-Man)3 Yariv phenylglycoside

bGlcY (b-D-Glc)3 Yariv phenylglycoside

CCM Co-cultivation medium

CRP Cumulative regeneration potential

ERP Explant regeneration potential

FITC Fluorescein isothiocyanate

GFP Green fluorescent protein

PBS Phosphate buffered saline

MS Murashige–Skoog

BA 6-Benzylaminopurine

NAA Naphthalene acetic acid

Introduction

Arabinogalactan proteins (AGPs) are glycoproteins found

in the cell wall area of plant cells. AGPs are composed

of core protein with one or more of its amino acids

Communicated by J. Van Huylenbroeck.

V. Orbovic (&) � P. Soria

Horticultural Sciences Department,

Citrus Research and Education Center,

University of Florida/IFAS,

700 Experiment Station Road,

Lake Alfred, FL 33850, USA

e-mail: orbovic@ufl.edu

E. M. Gollner

Department of Pharmaceutical Biology,

Pharmaceutical Institute,

Christian-Albrechts-University of Kiel,

Gutenbergstrasse 76, 24118 Kiel, Germany

Present Address:P. Soria

Department of Biological Sciences,

Vanderbilt University,

Nashville, TN 37325, USA

123

Acta Physiol Plant (2013) 35:1409–1419

DOI 10.1007/s11738-012-1179-4

O-glycosylated by complex carbohydrates that consist

mainly of galactan and arabinose (Seifert and Roberts

2007). As the structure of AGPs was being elucidated

(Gaspar et al. 2001; Bossy et al. 2009; Gollner et al. 2010,

2011), the details of their involvement in different pro-

cesses throughout the life cycle of plants have emerged.

Based on the growing body of data, AGPs are implicated in

cell division and apoptosis (Serpe and Nothnagel 1994;

Gao and Showalter 1999), pattern formation (van Hengel

and Roberts 2002; Hu et al. 2006), growth (Seifert and

Roberts 2007; Park et al. 2003), and plant–microbe inter-

actions (van Buuren et al. 1999; Fruhling et al. 2000;

Gaspar et al. 2004). In the study done on the developing

embryos of Arabidopsis, it was shown that AGPs were

closely associated with cells undergoing developmental

changes according to immunolabeling with JIM13 antibody

which reacts with AGP epitope (Hu et al. 2006). Experi-

ments with the AtAGP30 mutant of Arabidopsis strongly

suggested the role of AGPs in postembryonic pattern for-

mation/organ differentiation (van Hengel and Roberts

2002). Disruption in function of AtAGP30 resulted in the

inability of callus cultures to form roots. However, when

AtAGP30 was ectopically expressed in mutant plants, their

ability to regenerate roots was restored; while, at the same

time, shoot regeneration was disrupted (van Hengel and

Roberts 2002).

The T-DNA insertion mutant of Arabidopsis rat1 has

decreased expression of AtAGP17/RAT1 gene (Gaspar

et al. 2004), and as a result, its roots exhibit resistance to

Agrobacterium tumefaciens-mediated transformation.

Although the exact function of AtAGP17 has not yet been

revealed, it appears that it may be necessary for the

attachment of Agrobacterium to root cells. However, there

is also a possibility that this gene product may play a role

in signal transduction (Gaspar et al. 2004).

Out of different forms of Yariv reagents [generic

structure 1,3,5-tri-(glycosyloxyphenylazo)-2,4,5-trihydrox-

ybenzene], b-D-glucosyl form was shown to bind AGPs

with a high degree of specificity (Serpe and Nothnagel

1994; Gao and Showalter 1999). The cell division in sus-

pension cultures of St. Paul rose cultivar was severely

reduced in the presence of (b-D-Glc)3 Yariv phenylglyco-

side (bGlcY) reagent, while (a-D-Gal)3 Yariv phenylgly-

coside and (b-D-Man)3 Yariv phenylglycoside (aManY)

had no effect (Serpe and Nothnagel 1994). As a result of

adding AGP-binding (b-D-Gal)3 Yariv phenylglycoside to

Arabidopsis suspension cultured cells, a high percentage

underwent programmed cell death (Gao and Showalter

1999). Addition of bGlcY to developing embryos of Ara-

bidopsis halted or negatively impacted their development

(Hu et al. 2006), while the addition to Brassica microsp-

ores induced their death and decreased their rate of division

(Tang et al. 2006).

Juvenile stem explants of Carrizo citrange exhibited

higher shoot regeneration potential and were more easily

transformed than any other citrus cultivar after the treat-

ment with the same Agrobacterium strain (Gutierrez-E

et al. 1997; Yu et al. 2002; Orbovic et al. 2007). These

differences among citrus species are attributed to genetic

differences, without knowledge of biochemical regulation

of this process by particular classes of molecules. As

opposed to Carrizo, in the experiments with other members

of citrus group, the success rates obtained with the stem

explants as starting material are in the range of single digits

which makes introduction of genes into desirable cultivars

still a difficult task. In general, much higher transformation

rates are obtained through the use of protoplasts or cell

suspensions. Among the citrus cultivars used in such pro-

jects were some belonging to mandarins and sweet oranges

(Dutt and Grosser, 2010; Omar et al. 2007; Li et al. 2003).

The major obstacle for a wider use of this technique is the

difficulty in creating an embryogenic cell suspension.

Adequate plant regeneration can be achieved from cell

suspension/protoplasts only if the suspension is fully

embryogenic and relatively young in age (less than 2 years

in culture; Dutt and Grosser 2010). Presently, embryogenic

cell suspensions exist for a small number of citrus cultivars

including commercial sweet oranges, Ruby Red grapefruit,

Meiwa kumquat, and a few mandarin types. Discovery that

AGPs have an effect on regeneration potential of juvenile

explants may suggest similar effect on embryogenic

potential of citrus cell suspension/protoplasts. Ability to

manipulate embryogenic potential of citrus cell suspension/

protoplasts would be a major contribution to efforts

directed towards production of improved citrus cultivars

through genetic modification. In addition, it is known that

AGPs promote proper attachment of Rhizobium, a close

relative of Agrobacterium, to root cells of legume plants

(Xie et al. 2012). Improving attachment of Agrobacterium

cells to ends of citrus explants by supplementing the media

with the appropriate AGPs molecular species would further

boost transformation success rate. To the best of our

knowledge, there were no studies investigating the prop-

erties and function of AGPs and their possible involvement

in developmental processes of citrus cultivars. Proposed

roles of AGPs in cell division/embryo pattern formation

and plant–microbe interactions of other plant species make

them potential candidates for involvement in shoot mor-

phogenesis and mediation of Agrobacterium efficacy dur-

ing the genetic transformation of citrus.

Our goal in this study was to establish the presence of

AGPs in stem explant tissues from two citrus cultivars and

examine their effect on regeneration potential. We also

wanted to evaluate propensity of cells on explants to

undergo Agrobacterium-mediated transformation and to

examine if AGPs play a role in this process.

1410 Acta Physiol Plant (2013) 35:1409–1419

123

Materials and methods

Plant material

Explants used in these experiments were obtained from

stems (mesocotyls) of seedlings of Carrizo citrange [Citrus

sinensis (L.) Osbeck 9 Poncirus trifoliata (L.) Raf.] and

‘Duncan’ grapefruit (Citrus paradisi Macf.). Etiolated

seedlings were grown in darkness for 5 weeks and cut into

15–20-mm segments. For the initial series of experiments

described in Table 1, explants were obtained from etiolated

seedlings (grown in darkness for 29 days) that were

exposed to white light for 5 days.

AGP quantification

Arabinogalactan proteins were isolated and quantified

according to the procedure described by van Holst and

Clarke (1985) with slight modifications. Pieces of

mesocotyls (0.5 g) were grounded in a mortar in liquid

nitrogen and powdered tissue was transferred to a cooled

2-mL plastic tube. 1 mL of extraction buffer (50 mM Tris–

HCl, pH 8.0, 10 mM EDTA, 2 mM Na2S2O5, 1 % v/v

Triton X-100) was added to the tube and vortexed for 60 s.

Samples of AGP-containing extracts were left on ice for at

least 20 min and centrifuged for 15 min at 10,000 rpm.

Supernatants were collected into new tubes and used for

single radial diffusion (‘‘halo’’) assays.

Solution containing 1 % w/v agarose, 0.15 M NaCl,

0.02 % NaN3, and 50 lg/mL bGlcY (b-D-glucosyl-Yariv

reagent; Biosupplies Australia Ltd., Victoria, Australia)

was heated to boiling and poured into 100 mm

(d) 9 10 mm (h) Petri dishes (8 ml/plate). Aliquots (1 ll)

of AGP extracts were deposited into holes (1.2 mm in

diameter) in the solidified agarose solution, and those

plates were left for 16–18 h at room temperature

(24 ± 2 �C). Surface area of the halos that formed around

the holes where samples were deposited was measured

from the image of the plate captured and analyzed using

Image-Pro Plus image analysis software (Media Cyber-

netics, Silver Spring, MD, USA). Each group of samples

was analyzed and compared with the standard curve with

known concentrations of AGPs from gum arabic (Biosup-

plies Australia Ltd., Victoria, Australia).

AGP isolation for structural analyses

Pieces of mesocotyls were freeze-dried and ground in a

mortar. The powdered tissue was extracted for 12 h with

demineralized water (1:10) at room temperature on a

magnetic stirrer. The solid was filtered off using a washed

paper filter. The freeze-dried extract was dissolved in

demineralized water again and used for Yariv precipitation

with bGlcY according to Kreuger and van Holst (1993).

Neutral sugar analysis

To produce alditol acetates, the bGlcY-precipitated AGPs

were hydrolyzed with trifluoroacetic acid (TFA, 2 mol/L)

at 121 �C. After evaporation of TFA, monosaccharides

were converted to alditol acetates by reduction and acety-

lation. The analysis of the acetylated alditoles was done

according to Gollner et al. (2010) by gas liquid chroma-

tography (GLC) on a fused silica capillary column

(Optima-OV 225–0.25 lm, L 25 m, i.d. 0.25 mm,

Macherey-Nagel, Duren, Germany) using a gas chro-

matograph (HP 5890 Plus Series; Hewlett Packard, Nurn-

berg, Germany) with flame ionization detector. The

nitrogen flow rate was 1.2 ml/min and the oven tempera-

ture isothermal (230 �C); temperature of injector and

detector was 240 �C. For quantitative analysis, a defined

amount of myo-inositol was added to the samples as

internal standard.

Linkage analysis

Methylation was performed with potassium methylsulfinyl

carbanion and methyl iodide in dimethyl sulfoxide (Gollner

et al. 2011). Gas liquid chromatography–mass spectrome-

try of partially methylated alditol acetates was done on a

fused silica capillary column (0.25 i.d. 9 25 m, OV-1701,

Macherey-Nagel, Duren, Germany) using a gas chro-

matograph (HP 5890 Series II, Hewlett Packard, Palo Alto,

CA, USA) with the following temperature program: 2 min

170 �C, increase of 1 �C per min up to 210 �C, 10 min

210 �C. Helium flow was 0.7 ml/min. Mass spectra were

recorded on a HP MS Engine 5898 A (Hewlett Packard,

Palo Alto, CA, USA) instrument.

Identifications were based on peak retention times and

on comparison of mass spectra with the spectra from a

library of undermethylated reference compounds. The

Table 1 Cumulative regeneration potential of pooled Carrizo

citrange and ‘Duncan’ grapefruit explants that were incubated in dif-

ferent formulations of CCM: no supplement (CCM), with a-D-man-

nosyl Yariv (aManY), with b-D-glucosyl-Yariv (bGlcY), and with

gum arabic (gum arabic)

Treatment Carrizo ? ‘Duncan’

CCM 3.75a (365)

aManY 3.45ab (358)

bGlcY 3.30b (357)

Gum arabic 3.75a (342)

Means followed by different letters differ significantly as tested with

DMRT at P \ 0.05. Numbers in parentheses designate the number of

explants used for statistical analyses

Acta Physiol Plant (2013) 35:1409–1419 1411

123

quantification of the partially methylated alditol acetates

was done by integration of the corresponding flame ioni-

zation detection signal areas. Mass percentage was con-

verted into molar percentage using molar response factors

for flame ionization detection (Sweet et al. 1975).

Molecular mass determination

The molecular mass of citrus AGPs was determined by size

exclusion chromatography (Gollner et al. 2011) on two PL

aquagel-OH 40, 8 lm columns and one PL aquagel-OH

MIXED 8 lm column in series (temperature 35 �C, Polymer

Laboratories, Darmstadt, Germany). The samples were

eluted with NaNO3 (0.1 M) with a flow rate of 0.7 mL/min.

The detection system consisted of a multi-angle laser light

scattering instrument (mini DAWN, Wyatt Technology,

Santa Barbara, CA, USA), directly followed by a refractive

index detector (Polymer Laboratories, Darmstadt, Germany).

A value of 0.141 mL/g was used for the refractive index

increment (dn/dc) (Mahendran et al. 2008).

Treatment with Yariv reagents and transformation

experiments

Production and treatment of citrus explants were done as

previously described (Orbovic and Grosser 2006). In brief,

explants were cut and dipped into liquid co-cultivation

medium [CCM: Murashige-Skoog (MS) salts and vitamins

plus 3 mg/L of 6-benzylaminopurine (BA), 0.1 mg/L of

naphthalene acetic acid (NAA), 0.5 mg/L of 2,4 dichloro-

phenoxyacetic acid, and 19.6 mg/L of acetosyringone, pH

6]. Explants incubated only in CCM represented control

treatments in the experiments. For treatment of explants

with different forms of Yariv reagent (bGlcY or aManY;

Biosupplies Australia Ltd., Victoria, Australia), the

chemicals were added into liquid CCM (50 lg/mL).

Incubation of explants in CCM supplemented with Yariv

reagent(s) lasted 60–80 min.

For transformation, explants dipped in different formu-

lations of CCM were incubated in suspension of

EHA101 ? pTLAB21 strain (Orbovic et al. 2007) of

A. tumefaciens (optical density = 0.5) for 1–2 min. This

strain harbors binary vector carrying green fluorescent pro-

tein (GFP) as a reporter gene and nptII gene as a selectable

marker. Explants were blot-dried and left in plates with solid

CCM (liquid CCM plus 8 g/L of agar) for 2 days. Following

this period, explants were transferred to plates with regen-

eration medium (MS salts and vitamins plus 3 mg/L of BA,

0.5 mg/L of NAA, 333 mg/L of cefotaxime, 70 mg/L of

kanamycin, 8 g/L of agar, pH 6) where they stayed for an

additional 5 weeks, during which period shoots appeared. At

the end of the experiments, all explants were inspected for

the presence of GFP fluorescence.

Generation of polyclonal antibodies directed

against bGlcY

For the generation of polyclonal antibodies directed against

bGlcY, the small molecule had to be linked to a protein

first to induce a high immune response according to Bossy

et al. (2009). Therefore, 10 mg of bGlcY were dissolved in

water to 1 ml, and 1-h incubation at room temperature with

sodium periodate (0.95 mol/mol) was followed by the

addition of the protein: purified [dialyzed against sodium

hydrogen carbonate buffer (50 mM, pH 8.5)] keyhole

limpet hemocyanin was added up to a concentration of

10 mg/mL to the bGlcY-periodate reaction mixture and

incubated for 5 h at room temperature. Sodium cyano-

borohydride was added in a tenfold higher molar amount,

and after pH adjustment to pH 4.0 with acetic acid,

the reaction mixture incubated another 12 h at room

temperature.

To remove unbound bGlcY, the mixture was ultra-fil-

trated with a molecular weight cut-off of 10 kDa. The

coupled product was dissolved in phosphate buffered saline

(PBS) (pH 7.4) to 1 mg/mL and filtered (0.2 lm, Squarix,

Marl, Germany); 0.3 mL of the bGlcY coupled protein was

emulsified with 0.3 mL of complete Freund’s adjuvant and

injected into two New Zealand white rabbits subcutane-

ously for four times (Charles River, Kisslegg, Germany).

Before immunization, preimmune serum was collected.

The antiserum was precipitated by addition of ammonium

sulfate and afterwards dissolved in PBS-buffer and dia-

lyzed extensively against PBS-buffer with 0.05 % azide.

The IgG-enriched fraction was subsequently purified by

affinity chromatography.

Immunofluorescence labeling for confocal scanning

microscopy

The plant material was treated according to Bossy et al.

(2009) for the confocal laser scanning microscopy: hand-

cut sections of fresh citrus explants were placed in wells of

a 96-well plate (Nunc, Germany). The sections were

washed with PBS for 30 min and blocked afterwards with

blocking buffer (PBS ? 3 % bovine serum albumin) for

1 h. Then, bGlcY (400 lg/mL in 0.15 M sodium chloride)

was added in drops and washed off after 90 min incubation

time with PBS. The following incubation with bGlcY-

antibody (1:25) took 60 min. The sections were washed

again three times for 5 min with PBS before the secondary

fluorescein isothiocyanate (FITC)-labeled anti-rabbit anti-

body (1:100, Sigma-Aldrich, St. Louis, MO, USA) was

added and again incubated for 1 h. After washing with PBS

(containing 1 % Tween 20) to remove last remnants of not-

bound antibodies, the sections were placed on a microscope

slide and embedded in a drop of Mowiol-1,4-diazabicyclo-

1412 Acta Physiol Plant (2013) 35:1409–1419

123

[2,2,2]-octane beneath the cover slip. This was done to

delay the fading of fluorescence during microscopy (Valnes

and Brandtzaeg 1985). Mowiol-1,4-diazabicyclo-[2,2,2]-

octane was prepared following the instruction of the sup-

plier of Mowiol 4-88 (Roth, Germany).

Microscopy

Fluorescence of shoots was evaluated using a Zeiss SV11

epi-fluorescence stereomicroscope equipped with a light

source consisting of a 100 W mercury bulb and a FITC/

GFP filter set with a 480-nm excitation filter and a 515-nm

long-pass emission filter producing a blue light (Chroma

Technology Corp., Brattleboro, VT, USA). For confocal

scanning microscopy of immunolabeled cross-sections, a

TCS SP (Leica, Germany) microscope was used.

Statistical analyses

All of the indices were calculated from the data collected

5 weeks after incubation of explants in different formula-

tion of CCM whether they were exposed to Agrobacterium.

All shoots were examined for the presence of GFP

fluorescence. Cumulative regeneration potential (CRP) is

expressed as the average number of shoots per explant

in studied population. Explant regeneration potential

(ERP = RP per explant) represents average number of

shoots on explants that did produce shoots. Transform-

ability was calculated as the percentage of ends of explants

where transformation event took place regardless of orga-

nizational level of tissue (including fully transformed or

chimeric seedlings, spotty seedlings, transformed calli, and

individual cells; Fig. 1a–e). In all populations of explants

inspected for the presence of transformed tissue, the

number of ends was always double the number of explants,

as each explant has two ends. Both percentage of explants

producing shoots and transformability were calculated by

assigning the value of 1 to the explants (ends) where

examined event took place and 0 where it did not. Sig-

nificant differences among treatments were analyzed using

Duncan’s multiple range test at P \ 0.05 (SAS Institute,

Cary, NC, USA).

Results

In the initial series of experiments, the effects of bGlcY,

aManY, and commercially available AGPs (gum arabic, at

1 mg/ml) on CRP of pooled Carrizo and ‘Duncan’ explants

that were not co-incubated with bacteria were examined.

While aManY and gum arabic had no effect, bGlcY

decreased the CRP of explants (Table 1). All other data

presented in this paper were collected from experiments

with explants co-incubated with bacteria.

For Carrizo explants treated with bGlcY reagent, all

three parameters: CRP, ERP, and the percentage of

explants with shoots were significantly lower than in the

other two populations of explants (Table 2). CRP, ERP,

Fig. 1 Photographs of explant-

derived tissue on a different

organizational level that has

undergone transformation:

a single cell transformed with

GFP, b clump of cells

transformed with GFP,

c ‘‘spotty’’ seedling phenotype-

specific cell groups transformed

with GFP, d seedling that is

chimeric-half transformed with

GFP, and e seedling fully

transformed with GFP. Scalelines represent 1 mm.

Photographs depict tissues

5 weeks after the transformation

experiment

Acta Physiol Plant (2013) 35:1409–1419 1413

123

and the percentage of explants with shoots were similar for

CCM-incubated and aManY-treated Carrizo explants. For

‘Duncan’ explants, recorded results were different. First,

aManY-treated explants had CRP and the percentage of

explants with shoots higher than both CCM-incubated and

bGlcY-treated explants (Table 2). Values for CRP and the

percentage of explants with shoots were intermediate for

CCM-incubated explants and the lowest for bGlcY-treated

explants. However, ERP of ‘Duncan’ explants in these

experiments was not affected by treatment with Yariv

reagents (Table 2). For all three parameters, recorded val-

ues were much higher for Carrizo explants than for

‘Duncan’ explants. This difference ranged from about

60 % (ERP of CCM-incubated explants) to about fifteen-

fold (CRP of bGlcY-treated explants; Table 2).

As opposed to parameters describing regeneration

capacity, transformability of both Carrizo and ‘Duncan’

etiolated explants followed the same trend regardless of

treatment. Transformability was the lowest after bGlcY

treatment, a little higher after incubation with CCM, and the

highest after aManY treatment (Table 3). Carrizo main-

tained supremacy over ‘Duncan’ in the transformability as

well. On average, transformability was about four times

higher for Carrizo than for ‘Duncan’ explants (Table 3).

For both cultivars, concentration of AGPs in etiolated

tissue was similar: 0.22 mg/g FW for Carrizo and 0.23

mg/g FW for ‘Duncan’ (Table 4).

The analyses of composition of neutral monosaccharides

of AGPs precipitated with bGlcY (Table 5) yielded fol-

lowing results: galactose (Gal) and arabinose (Ara) were

the main monosaccharides in both isolated AGPs, whereas

Ara and Gal were present in higher relative amounts in

AGP isolated of ‘Duncan’ (Gal 58.0 vs. 47.2 %, Ara 34.2

vs. 25.9 %) than in isolated AGPs of Carrizo. There was

much more glucose (Glc) in AGP of Carrizo (20.8 %) than

AGP of ‘Duncan’ (3.6 %); fucose (Fuc) was present at a

higher relative amount (3.7 %) in ‘Duncan’ than in Carrizo

(1.9 %); rhamnose (Rha) was present only in Carrizo AGP

preparation (2.3 %); mannose (Man) was present in a low

relative amount in both AGP preparations (Carrizo 1.4 %

vs. ‘Duncan’ 0.5 %); and xylose (Xyl) was found in traces

in both cultivars (Carrizo 0.5 % vs. ‘Duncan’ 0 %).

Table 2 The effect of b-D-glucosyl-Yariv (bGlcY) and a-D-mannosyl Yariv (aManY) on regeneration potential and percentage of explants with

shoots of Carrizo citrange and ‘Duncan’ grapefruit explants co-incubated with Agrobacterium

Treatment CRP avg ERP avg % explants with shoots

Carrizo ‘Duncan’ Carrizo ‘Duncan’ Carrizo ‘Duncan’

CCM 4.38a (78) 0.79b (112) 4.82a (71) 3.07 ns (29) 91.0a (78) 25.9b (112)

aManY 4.35a (101) 1.20a (117) 4.72a (93) 2.75 (51) 92.1a (101) 43.5a (117)

bGlcY 1.95b (86) 0.13c (115) 3.17b (53) 1.88 (8) 61.6b (86) 7.0c (115)

Means followed by different letters differ significantly as tested with DMRT at P \ 0.05. Numbers in parentheses designate the number of

explants used for statistical analyses

CCM CCM with no supplement, aManY CCM with a-D-mannosyl Yariv, bGlcY CCM with b-D-glucosyl-Yariv, ns non-significant

Table 3 The effect of a-D-mannosyl Yariv (aManY) and b-D-glu-

cosyl-Yariv (bGlcY) on transformability (in %) of explant tissue of

Carrizo citrange and ‘Duncan’ grapefruit explants co-incubated with

Agrobacterium

Treatment Carrizo ‘Duncan’

CCM 76.9b (156) 19.2b (224)

aManY 86.8a (204) 27.7a (234)

bGlcY 47.1c (170) 10.8c (232)

Means followed by different letters differ significantly as tested with

DMRT at P \ 0.05. Numbers in parentheses designate the number of

explant ends used for statistical analyses

Table 4 Concentration of AGPs in explants of Carrizo citrange and

‘Duncan’ grapefruit that have not been exposed to Agrobacterium

Cultivar AGP (mg/g FW)

Carrizo 0.22a (14)

‘Duncan’ 0.23a (14)

Means followed by different letters differ significantly as tested with

DMRT at P \ 0.05. Numbers in parentheses designate the number of

‘halo assays’

Table 5 Neutral monosaccharide composition of Carrizo and

‘Duncan’ AGPs precipitated with bGlcY

Monosaccharide Relative amount (% w/w)

Carrizo ‘Duncan’

Gal 47.2 58.0

Ara 25.9 34.2

Glc 20.8 3.6

Fuc 1.9 3.7

Rha 2.3 0.0

Man 1.4 0.5

Xyl 0.5 0.0

1414 Acta Physiol Plant (2013) 35:1409–1419

123

Furthermore, analysis of glycosyl linkage composition

of Carrizo and ‘Duncan’ AGPs (Table 6) revealed a higher

relative amount of terminal Ara for AGP of ‘Duncan’ (24.4

vs. 18.8 %), whereas only for AGP of Carrizo, a small

relative amount of terminal Glc was detected. The other

linkage types were present in comparable amounts.

Discussion

In our study, bGlcY reduced CRP of both Carrizo and

‘Duncan’ explants whether they were co-incubated with

bacteria or not (Tables 1, 2), which suggests an inhibitory

effect of bGlcY on the proliferation and/or differentiation

of cambial cells due to binding to AGPs on cell surfaces.

Shoot sprouting from citrus explants is preceded by the

formation of callus which originates from cells of the

cambial region that proliferate under specific tissue culture

conditions (Pena et al. 2004). Our results showing a

decrease in percentage of explants with shoots for both

cultivars treated with bGlcY offer support to previous

hypothesis that binding of bGlcY to AGPs on cell surface

negatively affects cell division (Serpe and Nothnagel

1994). However, there is another step in the shoot regen-

eration process where the role of AGPs can be hindered by

bGlcY binding, and that is organogenesis. AGPs isolated

from carrot seeds were shown to have a promotional effect

on somatic embryogenesis from carrot cell suspensions

(Kreuger and van Holst 1993). Similar results were

obtained when AGPs isolated from the medium used to

grow embryogenic cultures obtained from ovules were

supplied to protoplasts obtained from beet guard cells

(Wisniewska and Majewska-Sawka 2007). Embryo differ-

entiation in microspore cultures of maize also increased

upon addition of exogenous AGPs (Borderies et al. 2004).

On the contrary, Brassica microspores-supplied bGlcY

exhibited higher death rate and lower embryogenesis rate

(Tang et al. 2006). Because of AGP presence in the region

rich with cambial cells which give rise to calli and shoots

in citrus seedlings (Figs. 2, 3), it can be hypothesized that

bGlcY could also negatively affect shoot morphogenesis as

well. Sometimes, the same callus can produce multiple

shoots and any inhibitory effect will thereby affect the

value of ERP. That is exactly what we recorded. Decrease

in CRP of Carrizo explants following incubation with

bGlcY was a result of lower percentage of explants pro-

ducing shoots and also smaller number of shoots sprouting

from explants that produced shoots. For ‘Duncan’ explants

treated with bGlcY, lower CRP was a consequence only of

decreased percentage of explants where shoot sprouting

took place. In Carrizo explants, bGlcY probably affected

proliferation of cambial cells leading to formation of calli.

Furthermore, bGlcY might have also inhibited differenti-

ation of callus cells into shoots as exemplified by lower

Table 6 Glycosyl linkage composition of Carrizo and ‘Duncan’

AGPs

Monosaccharide Deduced linkage Relative amount (mol %)

Carrizo ‘Duncan’

Gal tp 3.7 2.4

3p 15.0 15.6

4p 2.9 1.3

6p 11.3 10.2

3,6p 25.5 24.5

Ara tf 18.8 24.4

5f 17.9 18.8

Glc tp 2.1 0.0

Man 6p 2.8 2.8

Fig. 2 Photographs of ends of ‘Duncan’ explants that were incubated

for 75 min in: (a) CCM, (b) aManY, and (c) bGlcY. Photographs

were taken immediately following the incubation. Vertical lines at the

top of panel c are ruler ticks separated by 1 mm

Acta Physiol Plant (2013) 35:1409–1419 1415

123

ERP (Table 2). Lower percentage of ‘Duncan’ explants

producing shoots following the treatment with bGlcY

(Table 2) was probably due to decreased cambial prolif-

eration and calli production. Our method of data collection

did not allow us to distinguish whether bGlcY exerted its

effect on cambial cells present on the cut surface of

explants, on the callus formed from cambial cells, or on

both of them. Although we have not counted the number of

calli on the ends of explants that have undergone different

treatments, our observations are that callus production was

affected to a lesser degree than ERP. That would suggest

bGlcY has a stronger influence on determining future

developmental program of cells/calli produced from cam-

bium than on the ability of cambial cells to proliferate.

For both cultivars used in our experiments, incubation

with Yariv reagents resulted in differential binding to cells

on cut ends of explants. While binding of aManY was weak

and spread equally on the cut surface of explants, bGlcY

was more localized in the region with vascular tissue that

included cambial cells (Fig. 2). Immunolabeling of cross-

sections of stems of both Carrizo and ‘Duncan’ with poly-

clonal antibodies raised against bGlcY confirmed the

increased presence of AGPs in the area where phloem and

xylem are divided by layers of cambial cells (Fig. 3b, d).

Fig. 3 Photographs of cross-

sections of Carrizo (a medium

and b higher magnification) and

‘Duncan’ (c medium and

d higher magnification) stems.

Sections were exposed to

bGlcY which was allowed to

bind to AGPs. Afterwards,

polyclonal antibodies against

bGlcY were applied to the

surface of explants followed by

the secondary FITC labeled

antibody. Higher presence of

AGPs was detected in the area

of vascular tissue. Although

AGPs were detected in all types

of vascular tissue, they seem to

be more prevalent in phloem.

Scale lines represent 200 lm

1416 Acta Physiol Plant (2013) 35:1409–1419

123

Intensive fluorescence is also present in the outermost layer

of epidermal cells (Fig. 3a, c), and this can be traced back to

autofluorescence. Using both of these methods, bGlcY

staining and immunolabeling, we have clearly established a

presence of AGPs in Carrizo and ‘Duncan’ stem explants

and especially in the region of vascular tissue where cam-

bial cells responsible for the shoot morphogenesis are

situated.

Our data revealed that transformability of cells on cut

surfaces of explants decreased following the incubation of

explants in bGlcY supplemented medium (50 lg/ml,

Table 3). This inhibition was about 40 % for Carrizo and

45 % for ‘Duncan’ when compared to explants incubated

in CCM (Table 3). In the small series of experiments, we

used bGlcY at the concentration of 100 lg/ml and recor-

ded inhibition of transformability of 91.5 % for Carrizo

and 94.4 % for ‘Duncan’ (data not shown). All these results

are in accord with data obtained in experiments with rat1

mutant of Arabidopsis. Roots of rat1 mutant, that is unable

to synthesize Lys-rich AGP, exhibited resistance to Agro-

bacterium-mediated transformation due to decreased

attachment of Agrobacterium to root cells (Gaspar et al.

2004). Effects of rat1 mutation were mimicked in wild

type plants by the treatment of roots with bGlcY resulting

in significant decrease in percentage of cells transformed

with Agrobacterium. For a favorable outcome of transfor-

mation, Agrobacterium has to be able to attach itself to

plant cells. Binding of bGlcY to cambial cells on explants

we used probably limited the ability of Agrobacterium to

attach to them, and that in turn resulted in lower trans-

formability (Table 3). Most recently, a research group in

England has shown that AGPs are responsible for proper

polar attachment of Rhizobium leguminosarum bacteria to

roots of legume and non-legume plants (Xie et al. 2012).

These results offer the strongest support to the idea that

AGPs play essential role in host–pathogen interaction and

degree of attachment of Rhizobia-like bacteria to plant

cells. A. tumefaciens belongs to the Rhizobiaceae family of

bacteria and also exhibits polar attachment to the cells of

its plant host (Tomlinson and Fuqua 2009).

The aManY that we used does not bind specifically to

AGPs (Nothnagel 1997). Therefore, aManY served as a

negative control in our experiments with bGlcY, and

because of its inactivity against AGPs, we did not expect

any significant effect on RP or transformability. For

explants not co-incubated with bacteria, aManY had no

effect on RP (Table 1), but a slight positive effect on CRP

and the percentage of explants with shoots were recorded

for ‘Duncan’ explants that were co-incubated with bacteria

(Table 2). aManY also improved transformability of

explants of both cultivars when compared to CCM treat-

ment (Table 2). The positive effects of aManY might be

the consequence of the presence of mannosyl residues in

the medium as breakdown products of aManY. We have

shown that the substitution of sucrose with mannose in

co-cultivation medium used in the process of genetic

transformation increased slightly the number of shoot

sprouting from ‘Duncan’ and Carrizo explants and trans-

formation rate was enhanced to the levels of statistical

significance (Orbovic et al. 2008).

Different responses of these two citrus cultivars to

treatments with bGlcY (Tables 1, 2, 3) are raising a

question about the processes mediating shoot morphogen-

esis and cell transformation and the role AGPs play in

them. A comparison of the amounts of AGPs between

cultivars used in our experiments showed they were almost

identical (Table 4), while big differences were recorded in

CRPs and transformability of cells (Tables 1, 2, 3) which

seems contradictory. If AGPs are indeed important for cell

division/shoot regeneration and transformation event as our

data suggest, then significantly higher amounts of AGPs

should be present in Carrizo than in ‘Duncan’ seedlings

and that was not the case. However, involvement of AGPs

cannot be considered outside of the context of the physi-

ological state of tissue where they are synthesized. While

in our experiments, we controlled exogenous factors that

could influence organogenesis; no efforts were made to

interfere with internal factors such as hormone production/

presence. Shoot regeneration is influenced by hormonal

balance within the tissue of plants and differs between

representatives from different species or, in some cases,

even within the same species. During the procedure for

production of transgenic sweet orange seedlings, hormonal

composition of the media had to be modified significantly

to induce comparable responses in the tissue of two sweet

orange cultivars (Rodrıguez et al. 2008). Despite the fact

that these two cultivars of sweet orange belong to the same

species and are related, the balance of endogenous hor-

mones in their tissue was significantly different and

required a correspondingly different supply of exogenously

supplied hormones to induce a similar regeneration rate. It

is likely that hormonal balance in Carrizo was such to

allow for higher regeneration potential than in ‘Duncan’

with the same available amount of AGPs present.

The effects of different factors, including those that

modify the media composition, on transformations success

rate were examined in multiple studies (Gutierrez-E et al.

1997; Yu et al. 2002; Rodrıguez et al. 2008). As expected,

the most effective media supplements were antibiotics

because of their detrimental effect on growth, followed by

the plant growth regulators. Increase from 0 to 10 mg/L

kanamycin in the media resulted in a 70 % decrease in

percentage of rooted Carrizo shoots. However, it took

much higher increase in concentration of synthetic auxin

2,4-D from 0 to 18.1 lM to significantly decrease shoot

regeneration frequency (Yu et al. 2002). Changes in

Acta Physiol Plant (2013) 35:1409–1419 1417

123

concentration of one growth regulators coupled with steady

presence of the other(s) can have somewhat non-specific

effect on plant tissue. In the experiments where bGlcY is

used, such outcome is not probable. Specificity of binding

of bGlcY to AGPs is extremely high and it can be safely

assumed that the results we recorded are consequence of

inability of AGPs to perform their function.

Addition of gum arabic (commercially available source

of AGPs from acacia tree) to the batch of citrus explants

did not affect their CRP (Table 1). This result is not

unexpected since acacias belong to the family of Fabaceae

which is not close to the Rutaceae family of citrus. The

structure of AGPs is very complex (Gaspar et al. 2001) and

varies between species. Significant differences that exist in

the structure of AGPs prevented any biological activity of

acacia tree-derived molecules in processes we tested in

citrus explants. bGlcY, which specifically binds to AGPs

and can inhibit their role, obstructed shoot regeneration and

cell transformation to a different degree in citrus explants

of two cultivars (Tables 2, 3) despite high similarity in

levels of AGPs found in their tissue (Table 4). Therefore,

the effects AGPs exert on the tissue may be determined

more by their structure than their concentration. Most

recently, detailed structural analyses of AGPs were repor-

ted from medicinal plants like Echinacea or Cassia (Bossy

et al. 2009; Shina et al. 2011), from cereal species (Gollner

et al. 2011; Tryfona et al. 2010), and from plant-derived

products (Steinhorn et al. 2011). Description of detailed

structure of AGPs and especially of their sugar moiety will

facilitate elucidation of AGPs function in the cell life cycle

(Ellis et al. 2010). Presently, two models are suggested for

the spatial structure of AGPs: wattle blossom and a hairy

rope (Ellis et al. 2010). Determination of absolute molec-

ular mass of Carrizo and ‘Duncan’ AGPs showed that they

weighed 168 and 162 kDa, respectively. Hydrodynamic

volume of Carrizo AGPs was 80 kDa and for ‘Duncan’

AGPs it was 82 kDa. The discrepancy between the abso-

lute molecular mass and the hydrodynamic volume indi-

cates that citrus AGPs are rather globular molecules with

branched carbohydrate units. For gum arabic, a rodlike

structure with a similar molecular mass was depicted by Qi

et al. (1991). Analyses of neutral monosaccharide compo-

sition of crude citrus AGPs revealed the differences

between two cultivars. Sugar moiety of Carrizo AGPs had

lower amount of Gal and Ara than ‘Duncan’ (Table 5). At

the same time, amount of Glc was much higher in Carrizo

than in ‘Duncan’ (Table 5).

Through our analyses, we also discovered variances in

fine structure of AGPs, even between the members of the

citrus group (Tables 5, 6), with the striking difference of a

higher relative amount of terminal Ara for AGP of ‘Dun-

can’. Terminal Ara is probably bound in the periphery of

the molecule, the perfect position for interaction with other

molecules. These variations may be sufficient enough to

affect the ability of Agrobacterium to attach to cells of

different citrus cultivars and as a result affect transforma-

tion success rate.

In conclusion, we believe that our data offer strong

support to the hypothesis that AGPs play an important role

in processes that control the cells’ ability to undergo

division and/or differentiation. In concert with other

physiological factors, AGPs seem to be involved in control

of Agrobacterium-mediated transformation. Our data also

reveal a necessity for more detailed analyses of AGPs

synthesis and their presence in tissues of different citrus

cultivars, and for further studies on the effect AGPs have in

the interaction of pathogens with host cell.

Author contributions Vladimir Orbovic: Design and

performance of experiments concerning AGP quantifica-

tion, transformation and microscopy of explant-derived

tissues, and end of explants; data analysis; creation of main

part of the manuscript. Esther Marie Gollner: Design and

performance of experiments concerning isolation and

structural analysis of isolated AGPs; microscopy of cross-

sections of stems, contribution to written manuscript.

Patricia Soria: Performance of experiments concerning

AGP quantification, transformation and microscopy of

explant-derived tissues, and end of explants.

Acknowledgments We would like to thank Jill Dunlop for her

excellent help with statistical analyses.

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