ORIGINAL ARTICLE

Flower development in garlic: the ups and downs of gaLFYexpression

Rotem Neta • Rakefet David-Schwartz •

Yuval Peretz • Ilan Sela • Haim D. Rabinowitch •

Moshe Flaishman • Rina Kamenetsky

Received: 28 October 2010 / Accepted: 12 January 2011 / Published online: 1 February 2011

� Springer-Verlag 2011

Abstract The lack of sexual processes prohibits genetic

studies and conventional breeding in commercial cultivars

of garlic. Recent restoration of garlic flowering ability by

environmental manipulations has opened new avenues for

physiological and genetic studies. The LEAFY homologue

gaLFY has been shown to be involved in the floral devel-

opment, while two alternatively spliced gaLFY transcripts

are expressed in flowering genotypes. In the present work,

quantitative real-time PCR and two techniques of RNA in

situ hybridization were employed to analyze spatiotempo-

ral expression patterns of the gaLFY during consequent

stages of the garlic reproductive process. Temporal accu-

mulation of gaLFY is strongly associated with reproductive

organs, significantly increased during florogenesis and

gametogenesis, and is down-regulated in the vegetative

meristems and topsets in the inflorescence. The two alter-

native transcripts of the gene show different expression

patterns: a high level of the long gaLFY transcript

coincided only with floral transition, while further

up-regulation of this gene in the reproductive organs is

associated mainly with the short gaLFY transcript. It is

concluded that gaLFY is involved at different stages of the

sexual reproduction of garlic. These new insights broaden

our basic understanding of flower biology of garlic and

help to establish conventional and molecular breeding

systems for this important crop.

Keywords Florogenesis � Allium sativum � qPCR � FISH �DIG-labeled � In situ hybridization � LEAFY homologue

Introduction

Modern cultivars of garlic (Allium sativum L.), one of the

most economically important plants, are completely sterile

and thus propagated only vegetatively. The lack of sexual

propagation prohibits genetic studies and conventional

breeding, and fertility restoration should significantly

broaden variability and make the improvement of eco-

nomically important traits possible (Kamenetsky 2007).

Recent introductions of fertile A. sativum accessions from

Central Asia (Etoh 1985; Etoh and Simon 2002), restora-

tion of flowering ability and fertility by environmental

manipulations (Kamenetsky et al. 2004b) and increased

variation by seed propagation (Jenderek and Hannan 2004;

Jenderek and Zewdie 2005; Kamenetsky et al. 2005;

Shemesh et al. 2008) open the way for in-depth physio-

logical and genetic studies, as well as conventional and

molecular breeding in this plant. However, the regulation

of floral development and fertility in geophytes remains

obscure, and fundamental physiological and genetic

knowledge is required for both basic and applied purposes.

In this context, investigation of genes involved in the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00425-011-1361-8) contains supplementarymaterial, which is available to authorized users.

R. Neta � Y. Peretz � I. Sela � H. D. Rabinowitch

The Robert H. Smith Faculty of Agriculture,

Food and Environment, The Hebrew University of Jerusalem,

The Robert H. Smith Institute of Plant Science and Genetics

in Agriculture, 76100 Rehovot, Israel

R. David-Schwartz � M. Flaishman � R. Kamenetsky (&)

Institute of Plant Sciences, ARO, The Volcani Center,

50250 Bet Dagan, Israel

e-mail: vhrkamen@volcani.agri.gov.il

R. David-Schwartz

The Mina and Everard Goodman Faculty of Life Sciences,

Bar-Ilan University, Ramat Gan, Israel

123

Planta (2011) 233:1063–1072

DOI 10.1007/s00425-011-1361-8

control of flowering is of great importance (Benschop et al.

2010; Noy-Porat et al. 2010; Rotem et al. 2007).

In general, the formation of inflorescence and flowers in

plants consists of a series of sequential steps. These pro-

cesses were studied in detail in Arabidopsis and other

model plants. The effect of environmental factors on

flowering involves several signaling pathways that con-

verge towards the regulation of floral meristem identity

(Mouradov et al. 2002). ‘Integrators’ genes, such as

FLOWERING LOCUS T (FT) and SUPPRESSOR OF

OVEREXPRESSION OF CO1 (SOC1) co-operate in the up-

regulation of floral meristem identity genes like LEAFY

(LFY) and APETALA1 (AP1) (Benlloch et al. 2007;

Cockram et al. 2007; Krizek and Fletcher 2005). These

genes up-regulate each other and are essentially expressed

in the shoot apical meristem (SAM), where the transition

from vegetative to reproductive development is followed

by the differentiation of inflorescence and individual

flowers. The activity of LFY/AP1 is antagonized by TFL1

(TERMINAL FLOWER 1) whose expression maintains the

indeterminacy of the SAM (Ratcliffe 1999; Bernier and

Perilleux 2005).

The meristem identity-genes activate the floral-organ

identity-genes in discrete meristematic regions of the

inflorescence, with the consequent differentiation of flower

primordia. In turn, the floral organ identity genes regulate

the various cell types and tissues’ specialization, including

sepals, petals, stamens and carpels (Benlloch et al. 2007;

Bernier and Perilleux 2005; Moon et al. 2005). Regulatory

genes, involved in this process, act either as activators or as

repressors of floral initiation, and the concerted action of

the regulatory network ensures the correct temporal and

spatial control of floral meristem specification, its mainte-

nance and development (Vijayraghavan et al. 2005).

The gene LFY of Arabidopsis encodes for a unique

plant-specific transcription factor that plays a key role in

plant development (Moyroud et al. 2010). It was proposed

that LFY combines properties of flowering-time and flower-

meristem-identity genes, and provides a direct link between

the global process of floral induction (Benlloch et al. 2007;

Blazquez and Weigel 2000; Nilsson et al. 1998), the

regional events associated with the initiation of individual

flowers (Vijayraghavan et al. 2005), and activation of the

ABC genes such as AGAMOUS (AG), APETALA1 (AP1)

and APETALA3 (AP3) that control floral organ identity

(Lamb et al. 2002; Zik and Irish 2003; Benlloch et al.

2007).

LFY and its homologue FLORICAULA (FLO) were first

discovered in Arabidopsis and in Antirrhinum, respectively

(Coen et al. 1990; Weigel et al. 1992), followed by a

number of homologous genes in rice (Oryza sativa: RFL,

Kyozuka et al. 1998); Darnel ryegrass (Lolium temulentum:

LtLFY, Gocal et al. 2001); Narcissus (NLF, Noy-Porat et al.

2010), and more. Recently, we have demonstrated that the

LFY homologue gaLFY is involved in the control of

florogenesis in garlic. Further, we showed that two alter-

natively spliced gaLFY transcripts are expressed in flow-

ering garlic plants (Rotem et al. 2007).

In the present work, we studied the temporal and spatial

expression patterns of gaLFY during several sequential

stages of garlic flower development. Three methodologies

were employed, to analyze the expression patterns of

gaLFY during the differentiation of garlic inflorescence,

individual flowers and floral organs.

Materials and methods

Plant material and sampling procedures

Bulbs of garlic clone #3028, originated from Central Asia,

were obtained from the Field Gene Bank for Vegetatively

Propagated Short-Day Allium spp., The Robert H. Smith

Faculty of Agriculture, Food and Environment, Rehovot,

Israel (FAHU). In Israel, these plants produce bulbs, fertile

flowers and seeds. In June 2008, mature bulbs were har-

vested, cured and stored in a roofed shed under ambient

conditions until September. Intact bulbs were then placed

in a temperature-controlled room at 4�C for 8 weeks, in

the dark. In November 2008, individual clean, healthy

cloves were sorted for size and planted in the FAHU

experimental farm in Rehovot, and standard agricultural

practices were applied throughout. Destructive morpho-

logical analyses on three randomly selected plants were

performed weekly, and apical meristems were sampled for

scanning electron microscopy (SEM), RNA analysis and

in situ studies.

Scanning electron microscopy

Freshly harvested plants were carefully stripped of their

leaves and the spathes removed from the developing

inflorescences. Samples were fixed in a 5:5:90 mixture of

glacial acetic-acid:formalin (40%):ethanol (70%), dehy-

drated in a graded ethanol series (25, 50, 75, 95, 100%),

dried using liquid CO2 in a Bio-Rad 750 critical-point

dryer, mounted on SEM stubs and gold coated. Images

were obtained directly from the SEM (JSM-35C, JEOL

Japan) at 15 kV.

RNA isolation and cDNA preparation

Entire shoot apices, including reproductive meristems and

inflorescences were collected weekly from three garlic

plants, under binocular (November 2008–June 2009).

Additionally, 30–40 flower buds and flowers at the same

1064 Planta (2011) 233:1063–1072

123

developmental stages were carefully detached from the

inflorescence (May and June 2009). All collected samples

were immediately frozen in liquid nitrogen, and RNA

extracted using RNeasy Mini Kit (Qiagen, Hilden,

Germany). Extracts were treated with DNase using

TURBO DNA-free KIT (Ambion Austin, TX, USA) and

tested for DNA contamination. Amount and quality of

RNA were determined by spectrophotometry (NanoDrop

Technologies, Wilmington, DE, USA). Only RNA samples

of sufficient purity with absorbance ratio A260/280 [1.8

were considered for synthesis of cDNA. Total RNA from

individual samples was reverse-transcribed to produce

cDNA in a 20 ll reaction volume containing 1.5 lg of

RNA sample, using EZ-first strand cDNA synthesis kit

(Biological Industries, Israel) according to the manufac-

turer protocol. Each sample of the obtained cDNA was

diluted in a ratio of 1:10 before amplification.

gaLFY expression patterns were analyzed by quantita-

tive real-time PCR (qPCR) and RNA in situ hybridization,

using primers and probes designed from the formerly

described 682-bp fragment of the gene (Rotem et al. 2007;

GenBank AY563104 http://www.ncbi.nlm.nih.gov/nuccore/

48476628).

Quantitative real-time PCR

Quantification of gaLFY (the sum of the two transcripts;

gaLFYl and gaLFYs, Rotem et al. 2007) was determined

using the commercial dual hybridization probe #144

(CCTCTTCC) designed using the Universal Probe Library

Software (http://www.roche-applied-science.com) and

provided as a master PCR mix by Roche. The primers used

for gaLFY are presented in Table S1 and Fig. S1. Several

reference genes were tested with similar results, and actin

(AY821677) transcript was selected for data normalization

and for calculation of the relative mRNA levels. The

commercial dual hybridization probe #64 (CCAGGCTG)

was used to detect qPCR of actin. The qPCR primers used

for forward and reverse actin transcript are depicted in

Table S1. qPCR was carried out in a 20-ll reaction volume

using FastStart Universal Probe Master (92) (Roche,

Indianapolis, IN, USA); the reaction mixture included

0.5 lM primer, and 4 ll cDNA.

Reactions were performed using Light-Cycler 480 Real-

Time PCR (Roche) in multi-well plate. In each PCR plate,

at both primer combinations, two internal controls were

included: a negative control without cDNA template and

a sample from the eighth leaf (February 2, 2009) as a

calibrator (Pfaffl et al. 2002). For each primer set, PCR

efficiency was determined by standard curve method

(Table S1).

The reaction was performed under the following

cycling conditions: an initial denaturing step of 10 min at

95�C, followed by 40 cycles consisting of 10 s at 95�C

and 25 s at 60�C. All qPCR experiments were repeated in

three biological and two technical replications. During

inflorescence development, gaLFY was quantified using

the DDCt method (Pfaffl 2001), and the n-fold change was

calculated with LightCycler 480 software, provided by

the manufacturer, relative to the vegetative sample as

described above.

Quantitative analysis of gaLFYl was performed by

qPCR using the SYBR Green fluorescent dye. Actin was

used as reference gene, and the eighth leaf served as cali-

brator in the QC with the negative control in each qPCR

plate. Primers were designed by Primer3 software (http://

frodo.wi.mit.edu/primer3). Primers for actin were descri-

bed above (Table S1). The reactions were carried out in a

20 ll RT-reaction mixture containing 10 ll of FastStart

SYBR green I Master 910 (Roche), 0.5 lM of each pri-

mer, and 4 ll cDNA template, and performed with the

following cycling conditions: 1 cycle for 10 min at 95�C,

followed by 40 cycles of 10 s at 95�C, 10 s at 58�C and

10 s at 72�C. PCR analyses were performed using 96

samples Light-Cycler 480 and Real-Time PCR software

(Roche) as described above. All PCR experiments were

carried out in three biological and two technical

replications.

RNA fluorescence in situ hybridization

Modified fluorescence in situ hybridization (FISH) tech-

nique was adapted from Gottlieb et al. (2006). Freshly

harvested plants were carefully stripped of leaves and

spathes. Apices’ samples for microscope analysis were

collected from November to June, and fixed overnight in

Carnoy’s fixative (6:3:1 mixture of chloroform:etha-

nol:glacial acetic acid). Thereafter, samples were decolor-

ized twice in 6% H2O2 solution in ethanol for 1 h.

Pre-hybridization in hybridization buffer [20 mM Tris–

HCl, pH 8.0, 0.9 M NaCl, 0. 01% sodium dodecyl sulfate

(SDS), 30% formamide] for 1 h was followed by overnight

hybridization at room temperature using the same buffer

supplemented with 1 pmol fluorescent probe/ml. The

hybridized samples were washed twice in 29 sodium

chlorides/sodium citrate (SSC), 0.1% SDS solution for

10 min, and once with a mixture of 0.59 SSC and 0.1%

SDS for 10 min at room temperature. Meristems or

reproductive tissues similarly hybridized except that the

fluorescent probe was replaced by water, served as con-

trols. Samples were then stored in 29 SSC at 4�C, until

studied by confocal microscope (LSM 510 Axiovert

100 M; ZEISS, USA). The labeled probe 50-CUAUGA

ACGGGUGCUCCCUUUGCCG-30 (Fig. S1) was designed

to detect the two gaLFY transcripts (Bioneer, Munpyeong-

dong, Daedeok-gu, Daejeon 306-220, Korea).

Planta (2011) 233:1063–1072 1065

123

DIG-labeled in situ hybridization

DIG-labeled in situ analysis was performed according to

Szymkowiak and Irish (2006). Meristems were fixed in

FAA (formaldehyde:acetic acid:70% ethanol, 10:5:85,

v/v), dehydrated in a series of ethanol solutions and

embedded in ParaPlast (McCormick Scientific, St. Louis,

MO, USA) according to Johansen (1940), then sliced

(10 lm) by a Leica RM2245 microtome (VectaMount-

Vector Laboratories, Burlingame, CA, USA), and placed

on SuperFrost Plus slides (Menzel-Glaser, Braunschweig,

Germany) for 2 days on a 37�C hot plate.

The 195-bp gaLFY segment (Fig. S1) isolated by RT–

PCR was cloned into a p-Drive cloning vector (Qiagen)

with a T7 promoter attached to the 50-end of the gene and

an SP6 promoter sequence attached to its 30-end. DIG-

labeled RNA sense and antisense probes were generated

using the MEGAscript kit (Ambion) and DIG RNA

labeling mix (Roche Applied Science), purified by

MEGAclear kit (Ambion) and quantified on agarose

gel, 1 ll per sample. Sections were deparaffinized in

Histoclear (Finkelman Chemicals, Yahood, Israel) for

2 9 10 min and rehydrated in a decreasing ethanol series,

followed by 20 min wash with 29 SSC, and by a 30-min

incubation at 37�C with 1 lg ml-1 proteinase K

(Fermentas) in TE buffer. The sections were then fixed

for 10 min in 4% paraformaldehyde in PBS buffer, and

re-dehydrated prior to hybridization. Sections were

hybridized overnight at 50�C with hybridization solution

containing 50% formamide, 10% dextran sulfate,

4 9 SSC, 0.4 mg ml-1 tRNA and 400 ng probe diluted in

100 ll 50% formamide. Following three washing steps

with 0.29 SSC at 50�C for 1 h each, slides were incu-

bated with 10 lg ml-1 RNase A (Sigma–Aldrich, St.

Louis, MO, USA) at 37�C for 30 min, followed by

washing with PBS for 10 min. Sections were blocked for

45 min in Blocking Reagent (Roche) at RT, followed by a

15 min wash. Slides were incubated in BSA solution (1%

BSA, 100 mM Tris pH 7.5, 150 mM NaCl and 0.3%

Triton X-100) for 15 min with mild agitation, followed by

incubation with anti-digoxigenin-AP Fab Fragments

(Roche) (diluted 1:500 in BSA solution) for 2 h and then

washed 3 9 10 min in TBS (100 mM Tris pH 7.5,

150 mM NaCl). Sections were then incubated 2 9 5 min

in detection solution (100 mM Tris pH 9.5, 50 mM

MgCl2, 100 mM NaCl) prior to overnight staining in

detection solution (without MgCl2) containing NBT/BCIP

(Roche). The staining reaction was stopped by washing

the slides with water. Sections were mounted in Vecta-

Mount (Vector Laboratories) and recorded under a

microscope (DMLB, Leica, Germany) using a digital

camera (NikonDs-Fs-fi1).

Results

Developmental morphology of garlic inflorescence

Developmental studies of growing garlic plants in Israel

reveal that florogenesis progresses in several sequential

stages, as follows:

1. The vegetative meristem produces leaf primordia

during the first 7–8 weeks after planting (Fig. 1a).

2. After differentiation of the first 10 leaves, the transi-

tion from vegetative to reproductive stage and initia-

tion of the inflorescence meristem occur (Fig. 1b).

3. Flower primordia are initiated in the inflorescence

meristem (Fig. 1b, c).

4. Differentiation of individual flowers, leafy bracts and

vegetative buds (topsets). Flowers and flower primor-

dia at various stages of differentiation are present

inside the bulb (Fig. 1d).

5. Scape elongates, flowers are differentiated,

26–28 weeks after planting (Fig. 1e).

6. Gametogenesis and anthesis occur ca. 30 weeks after

planting (Fig. 1f, g).

Quantification of gaLFY expression by qPCR

Microscope studies of the developing inflorescence were

accompanied by quantitative estimates of the relative

expression of total gaLFY and the long transcript gaLFYl,

using qPCR (Fig. 2). In general, the relative expression of

the gaLFYl was rather low throughout floral development,

when compared with the total gaLFY expression levels.

However, a sharp increase in gaLFYl expression was

recorded during the transition of the apical meristem from

the vegetative to the reproductive stage and at the begin-

ning of inflorescence differentiation. This peak declined

thereafter (Fig. 2a).

Between initiation and anthesis, total gaLFY is up-reg-

ulated at least three times (Figs. 2b, 3). The first peak

coincided with meristem transition to the reproductive

stage and initial differentiation of the inflorescence; the

second with differentiation of the individual flowers; and

the third with differentiation of anthers and gynoecia. It

should be noted that early in January (7–9 weeks after

planting)—during initial stages of the inflorescence

development—a sharp increase in gaLFYl expression

overlap the first peak of total gaLFY.

In individual flowers, gradual increase in gaLFY

expression was recorded from differentiation to maturation

(Fig. 3). At spathe break, flower buds are still closed,

anthers and ovaries are immature and gametophytes’ dif-

ferentiation continues until anthesis (Fig. 1e–g). During

1066 Planta (2011) 233:1063–1072

123

this period, flowers elongate and both perianth lobes and

anthers change color from green to purple. A 600-fold

increase in gaLFY expression was recorded during flower

maturation, compared with the values measured in vege-

tative tissues and sevenfold higher than in previous

developmental stage of flower buds. A few days later, at

anthesis, floral gaLFY expression dropped considerably

(Fig. 3).

Spatial expression of gaLFY in differentiating

inflorescence

In situ hybridization analysis of the vegetative apical

meristems at the fourth leaf stage of development, showed

a very weak fluorescence signal of gaLFY expression

(Fig. 4a). gaLFY expression was first recorded immediately

after the transition and at the early stages of inflorescence

differentiation, in the receptacle, inflorescence meristem

and flower primordia prior to flower differentiation

(Fig. 4b, c, d, f). At this stage, flower primordia consist of

small and dense meristematic cells with rather big nuclei

(Fig. 4g). DIG-labeled in situ hybridization revealed a

gaLFY expression in the cytoplasm of the outer cell layers

of flower primordia (Fig. 4g) concomitantly with the dif-

ferentiation of the first flower organs—perianth lobes and

anthers (Fig. 4d, e, h, i). Later, gaLFY expression became

visible in the pedicel (Fig. 4e, j).

Expression of gaLFY during the differentiation

of individual flowers and topsets

Following the differentiation of the perianth lobes and

anthers, the expression of gaLFY weakens (Fig. 5a, b),

and increases again with the differentiation of the anthers

and gynoecium (Fig. 5c). After spathe’s break, anthers

grow in size and change color from green to purple

(Fig. 1f, g). Strong expression of gaLFY was recorded in

the stamens’ filaments of the purple anthers, just before

pollen shedding (Fig. 5f, g), but not in pollen grains

(Fig. 5e–g).

Fig. 1 Stages of reproductive development in garlic accessions

#3028. a Vegetative meristem (vm) produces leaf primordia (lp),

6 weeks after planting. Bar 0.5 mm. b Inflorescence meristem (im),

9 weeks after planting. Differentiation of first flower meristems is

visible. Spathe (sp) removed. Bar 0.5 mm. c Inflorescence meristem

produces flower meristems (fm), 12 weeks after planting. Spathe (sp)

removed. Bar 0.3 mm. d Flower primordia at various stages of

differentiation (from 1 to 5) and vegetative topsets (arrows) can be

found simultaneously in the developing inflorescence within the bulb.

Bar 0.5 mm. e Flowers (f) and leaf bracts (lb) are formed, but spathe

is still not open, 25 weeks after planting. In this picture, spathe is

removed. Bar 0.5 cm. f Individual flower prior to anthesis, with

developing anthers (an) and gynoecium. Ovule (ov) is visible inside

carpels. Bar = 0.1 cm. g Anthesis, 30 weeks after planting. Game-

togenesis occurs prior to anthesis of individual flowers. Bar 1 cm

Planta (2011) 233:1063–1072 1067

123

Prior to anthesis, female reproductive organs—style,

stigma and ovaries—showed a definite gaLFY expression.

The ovary wall and integuments, as well as the funicle,

showed strong expression of gaLFY, but no gaLFY mRNA

was present in the ovular nucellus (Fig. 5h).

Following flower differentiation, a number of inflores-

cence vegetative meristems differentiate into topsets and

leafy bracts. No expression was recorded in these vegeta-

tive tissues (Figs. 4f, 5d).

Discussion

In garlic and other bulbous plants, LFY/FLO homologues

are associated with inflorescence and flower development

(Noy-Porat et al. 2010; Rotem et al. 2007; Zaccai et al.

2008). Fertile and sterile garlic genotypes differ in mor-

phology and physiology (Etoh 1985; Kamenetsky and

Rabinowitch 2001, 2002; Kamenetsky et al. 2004a), and

the differential expression of the two alternatively spliced

gaLFY transcripts in fertile and sterile genotypes was

reported (Rotem et al. 2007). Our current findings on

gaLFY expression throughout florogenesis in fertile garlic

suggest that, similar to the LFY homologue NFL in

Narcissus tazetta (Noy-Porat et al. 2010), gaLFY mRNA is

up-regulated in the three main phases of garlic floral

development (Fig. 6).

The first peak of gaLFY expression was recorded con-

currently with floral transition (Phase I, Figs. 2, 6). A

strong expression of the long transcript gaLFYl (Fig. 2a)

coincided with meristem transition, thus supporting our

previous suggestion (Rotem et al. 2007) that the long

transcript is instrumental mainly during the early stages of

meristem transition in fertile garlic. Both transcripts were

detected at the later stages of florogenesis, yet qPCR

measurements revealed only low levels of gaLFYl

expression (Fig. 2a). Therefore, the later expressions of

gaLFY in reproductive meristems and organs are mainly

associated with the short transcript gaLFYs.

The second increase in gaLFY expression coincided

with the initial differentiation of individual flowers

(Fig. 4c–e). Garlic inflorescence consists of a large number

of successively developing flower buds at different stages

of differentiation and growth. Hence, the second phase of

gaLFY expression (Fig. 6) is quite prolonged, stretching

between the 11th and the 18th week after planting

(Fig. 2b). Although at early stages of florogenesis, it is

technically impossible to separate reproductive and vege-

tative organs, morphological observations suggest that the

proportion between floral and vegetative tissues remains

similar. Since vegetative tissues of the inflorescence

showed no expression of gaLFY (Figs. 4f, 5d), we conclude

that the significant increase in the relative expression of

gaLFY is associated with reproductive tissues. This con-

clusion is strongly supported by FISH and DIG analyses.

In Arabidopsis, LFY is expressed throughout the devel-

opment of floral meristems and activates different floral-

organ identity genes in distinct patterns within the flower.

This seems to result from interactions between the globally

expressed LFY and the cofactors expressed in more spatially

restricted domains (Krizek and Fletcher 2005; Moyroud

et al. 2010). In combination with UFO and AP1, LFY acti-

vates the class B gene AP3 in the second and third whorls

and interacts with the meristem gene WUSCHEL (WUS) to

Vegetativemeristem

Differentiationof flower primordia

Differentiation of individual flowers

Inflorescencemeristem

0

20

40

60

80

100

120

140

160

180

200

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Rel

ativ

e ex

pres

sion

of g

aLF

Ydu

ring

inflo

resc

ence

dev

elop

men

t

Weeks after planting

0

2

4

6

8

10

12

14R

elat

ive

expr

essi

on o

f gaL

FY

l du

ring

inflo

resc

ence

dev

elop

men

t

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Weeks after planting

a

b

Fig. 2 Relative expression of gaLFY in garlic inflorescence. The

expression analysis of RNA from the entire sampled inflorescence

were measured by qPCR. Error bars indicate standard deviations

(SD; n = 6). a Relative expression of gaLFYl. b Relative expression

of gaLFY

0100

200300400

500600

700800

Flower budSpathe not

open

Flower bud

Open spathe

Green flower

Purple flower

Flower at

anthesis

Rel

ativ

e ex

pres

sion

of g

aLF

Y

21-23 24-25 26 27-28 29-30

Weeks after planting

Fig. 3 Relative expression of gaLFY in garlic flower buds and

mature flowers, measured by qPCR. RNA samples were collected

from 30 to 40 individual flower buds/flowers in May and June 2009,

22–30 weeks after planting. Error bars indicate standard deviations

(SD; n = 6)

1068 Planta (2011) 233:1063–1072

123

turn on AG expression in the inner two whorls. In petunia,

the LFY homologue ALF actively regulates a wide spectrum

of B-, C-, D- and E-type organ identity genes (Souer et al.

2008). Similarly, in garlic, gaLFY is strongly expressed in

differentiating flower primordia, but is down-regulated

when most flowers are formed (Fig. 5b). These results are in

agreement with previously reported data from many plant

species such as, violet (Jonopsidium acaule), wheat

(Triticum spp.) and Monterey Pine (Pinus radiate), all of

which show a clear expression of LFY/FLO homologues

during flower initiation and differentiation (Mouradov et al.

1998; Shitsukawa et al. 2006; Shu et al. 2000).

The third peak of gaLFY expression was recorded prior

to anthesis, in developing stamen and pistils (Fig. 3). The

expression of gaLFY was visualized in stamen primordia

(Fig. 4j) and filaments, but not in mature anther (Fig. 5e–

g). It is therefore concluded that gaLFY is mainly active

at the early stages of stamen development. Expression

was also detected in most parts of the gynoecium,

including style, ovary, inner and outer integuments and

funicle but not in the ovular nucellus (Fig. 5e–h). The

results indicate a general role for gaLFY in gynoecium

development.

It is suggested that similar to Arabidopsis LFY, gaLFY

plays a key role in the early stages of garlic florogenesis:

floral initiation and the differentiation of individual flowers

and floral organs. However, unlike LFY, gaLFY may also

act as a regulator of gametogenesis. Similarly, in radish a

LFY homologue RsLFY is expressed in primordia of sepals,

petals, stamens and gynoecium, and is also detected in

ovule and developing seeds (Oshima and Nomura 2008).

GmLFY, a soybean LFY homologue, was detected in

developing seeds, suggesting that it plays an essential

regulatory role in seed development (Meng et al. 2007).

Our findings conclusively show that the temporal

expression of gaLFY is strongly associated with the

developmental stages of flowers, but not with the vegeta-

tive meristems, topsets and the inflorescence’s leafy bracts

(Fig. 5d). In other plant species, mutations or down-regu-

lation of LFY and its homologues was reported to cause

reversion in flower determination with the consequent

development of vegetative shoots (Coen et al. 1990;

Molinero-Rosales et al. 1999; Wang et al. 2004; Weigel

et al. 1992).

In the annual Arabidopsis, flowering is irreversible

(Amasino 2010); however, reversion to vegetative growth

Fig. 4 gaLFY expression in the vegetative meristem and during the

early florogenesis stages in garlic. a–e FISH, as observed by confocal

microscopy. f–j DIG-labeled in situ hybridization, as observed by

light microscopy. Bars a–e 200 lm, f, h–j 100 lm, g = 50 lm.

a Vegetative meristem (vm) in plants at the fourth foliage leaf stage of

development, November 2008. b Meristem transition and initiation of

the inflorescence. Note the high level of fluorescence signal in the

receptacle (rc), inflorescence meristem (im) and flower primordia (fp).

c Differentiation of flower primordia (fp). A weaker signal in the

receptacle (rc). d Early developmental stages of flower buds (stages

1–3). gaLFY expression is evident in the center of the reproductive

meristem at stage 1; in the apical and lateral parts of the primordium

at stage 2, and in flower organ primordia at stage 3. e Early

developmental stages of flower buds (stage 4). Perianth lobes (pr) and

anthers (an) are visible. f Initiation of flower primordia in the

inflorescence, comparable to b and c. Note absence of gaLFYexpression in the leafy bracts (lb). g Reproductive meristem prior to

differentiation of flower organs, comparable to stage 1 in d. gaLFYexpression is visible in the cytoplasm (blue staining). Note the small

meristematic cells with large nuclei. h Initial differentiation of

individual flower. The apical and lateral parts of primordium are

stained blue, comparable with stage 2 in d. i Flower with perianth and

anther primordia, comparable with stage 3 in d. j Individual flower:

perianth lobes (pr) and anthers (an) are differentiated, comparable

with stage 4 in e

Planta (2011) 233:1063–1072 1069

123

is common in quite a few plant species when inductive

environmental signals is not maintained (Zeevaart 1985;

Tooke et al. 2005). In garlic, topsets’ formation in the

inflorescence can be considered as a reversion in flower

determination, yet the role gaLFY plays in this process, as

well as the question of environmental regulation deserve

further elucidation.

In this study, gaLFY mRNA levels of expression were

recorded during reproductive developmental stages, using

three methodologies, as well as primers and probes from

Fig. 5 gaLFY expression pattern during development of individual

flower. a–d FISH, as observed by confocal microscopy. e–h DIG-

labeled in situ hybridization, as observed by light microscopy Bars a–

d 200 lm, e–g 100 lm, h 50 lm. a Individual flower—differentiation

of perianth lobes (pl) and anthers (a). A strong gaLFY expression is

obvious in the differentiating organs. b Following differentiation of

perianth lobes (pl), gaLFY expression decreases. c Individual flower

with differentiating anthers. gaLFY is expressed in the filament (fi)

and in the seam between the perianth lobes and the pedicel (pd).

d Differentiation of topsets (t) and leafy bracts (l) in the inflorescence.

Note absence of gaLFY expression. e gaLFY expression in maturing

flowers. f gaLFY expression in the ovary (ov) and the filaments (fi)

during microgametogenesis. No gaLFY expression is visible in pollen

grains (p). g gaLFY expression in the style (s), ovary and ovules. No

expression is detectable in the center of ovule. h Close-up of the

ovarian wall (ow) shows a strong gaLFY expression in both the outer

and inner integuments (oi and ii) and in the funicle (f), but not in the

nucellus (n)

Fig. 6 Scheme of the three

main phases of gaLFYexpression during florogenesis

of fertile garlic. Initially, mRNA

is detected during meristem

transition from the vegetative to

the reproductive phase (phaseI). Following initiation of the

individual flower primordia,

gaLFY is down-regulated in the

inflorescence meristem and

expressed again during organ

differentiation in the individual

flowers (phase II). The third

peak is detected in anthers and

ovules of the fully differentiated

mature flowers (phase III)

1070 Planta (2011) 233:1063–1072

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various fragments of the gene. The results support our

assumption that gaLFY is in fact involved in the regulation

of a number of florogenetic processes in garlic, from floral

transition to flower maturation and gametogenesis. Indeed,

the up- and down-regulation of gaLFY coincided with tis-

sue differentiation and various physiological stages of

garlic inflorescence.

Numerous reports suggest that LFY and its homologues

are multi-functional genes, possibly involved in a variety of

morphogenetic stages, and thus can be considered as a

master regulator of flower development (Albert et al. 2002;

Moyroud et al. 2010). Our results obtained from flowering

garlic support this wisdom for florogenesis. Further studies,

however, are required to elucidate the additional (up- and

downstream) molecular and genetic mechanisms involved

in garlic flowering and seed production, as well as general

aspects of florogenesis in herbaceous perennial plants.

Acknowledgments We thank E. Belausov, E. Shemesh and

H. Zemach, The Institute of Plant Science, Agricultural Research

Organization, The Volcani Center, for their assistance in microscopic

observations.

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