the importance of localized auxin production for morphogenesis of

REVIEW PAPER

The importance of localized auxin production for morphogenesis of reproductive organs and embryos in Arabidopsis

Hélène S. Robert1,*, Lucie Crhak Khaitova1, Souad Mroue1 and Eva Benková2

1 Mendel Centre for Genomics and Proteomics of Plants Systems, CEITEC MU - Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic2 Institute of Science and Technology Austria (IST Austria), 3400 Klosterneuburg, Austria

* To whom correspondence should be addressed. E-mail: [email protected]

Received 17 February 2015; Revised 24 April 2015; Accepted 5 May 2015

Editor: Angus Murphy

Abstract

Plant sexual reproduction involves highly structured and specialized organs: stamens (male) and gynoecia (female, containing ovules). These organs synchronously develop within protective flower buds, until anthesis, via tightly coor-dinated mechanisms that are essential for effective fertilization and production of viable seeds. The phytohormone auxin is one of the key endogenous signalling molecules controlling initiation and development of these, and other, plant organs. In particular, its uneven distribution, resulting from tightly controlled production, metabolism and direc-tional transport, is an important morphogenic factor. In this review we discuss how developmentally controlled and localized auxin biosynthesis and transport contribute to the coordinated development of plants’ reproductive organs, and their fertilized derivatives (embryos) via the regulation of auxin levels and distribution within and around them. Current understanding of the links between de novo local auxin biosynthesis, auxin transport and/or signalling is pre-sented to highlight the importance of the non-cell autonomous action of auxin production on development and mor-phogenesis of reproductive organs and embryos. An overview of transcription factor families, which spatiotemporally define local auxin production by controlling key auxin biosynthetic enzymes, is also presented.

Key words: Arabidopsis, auxin, auxin biosynthesis, auxin signalling, auxin transport, carpel, embryo, gynoecium, ovules, reproductive organs, stamen, transcription factors.

Introduction

Sexual reproduction is a major process in the lifecycle of flowering plants, as it is essential for seed production and the maintenance of floral diversity. It depends on highly specialized reproductive organs protected, until anthesis, by flower buds. Flowers are composed of sepals, petals, stamens (male reproductive organs) and gynoecia (female reproduc-tive organs). These organs are specified during early phases of flower development, and the growth and development of the male and female reproductive organs must be tightly

controlled and coordinated for optimal fertilization success. At anthesis the flowers open, ready for fertilization, and their development ends with fruit growth, maturation and ripening (Smyth et al., 1990).

Stamens, the male reproductive organs, are each composed of an anther (where pollen grains develop) and a filament, which transmits water, nutrients and hormones to the anther, and positions it for optimal pollen dispersion (Scott et al., 2004). Pollen grains are tri-celled male gametophytes that

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Journal of Experimental Botany, Vol. 66, No. 16 pp. 5029–5042, 2015doi:10.1093/jxb/erv256 Advance Access publication 27 May 2015

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originate from meiotic tetrads (via microspore formation and maturation) in a sporangial tissue called the tapetum within the anthers. Stamen primordia and pollen grains are formed during early stages of stamen development. Late stamen development stages involve three coordinated processes: pol-len maturation; elongation of the stamen filament (thereby allowing pollen grains to contact female stigma for pollina-tion); and anther dehiscence, which releases pollen grains (Fig. 1).

The gynoecium, the female reproductive organ, is essen-tial for plants’ sexual reproduction, as it harbours the ovules. Composed of two fused carpels separated by a septum in Arabidopsis, the gynoecium forms a fruit after fertilization, which carries and protects the developing embryos and ulti-mately plays a major role in seed dispersal (Larsson et al., 2013). The gynoecium primordium initially forms as a hollow tube, which elongates until formation of the stigma, the most apical structure of the gynoecium, is initiated. This results in closure of the tube, specified as an ovary. The ovary and stigma are linked by a stalk called the style. Remarkably, during its specification the style typically acquires radial symmetry, in a pronounced shift from the characteristic bilateral symmetry of the ovary and other lower parts of the gynoecium (Moubayidin and Østergaard, 2014). The mature gynoecium is highly specialized, consisting of four main com-ponents formed along an apical-basal axis: (i) a stigma, which receives pollen grains; (ii) a style, which provides a conduit for pollen tubes, (iii) a tube-like ovary, containing ovules and seeds; and (iv) a gynophore, connecting the gynoecium to the stem via a pedicel (Fig. 2) (Larsson et al., 2013). During gynoecium development, in addition to the apical-basal and radial/bilateral polarity axes, another axis of symmetry, the medial-lateral axis, is differentiated during ovary specifica-tion, with the carpel valves forming the lateral domain and

the carpel margins the medial domain (Larsson et al., 2013, 2014). The margins of the carpels, containing the carpel mar-gin meristem (CMM), give rise to the placenta, the septum and the transmitting track. From the placenta ovule pri-mordia emerge. Subsequent ovule development is divided into three main phases: ovule identity establishment, ovule primordia initiation and formation (Cucinotta et al., 2014). A mature ovule primordium is a finger-like structure com-posed of three distinct structures along a proximal (basal)-distal (apical) axis: (i) the funiculus, which connects the ovule to the carpel and conducts nutrients and hormones to and from the ovule, (ii) the chalaza, and (iii) the nucellus (Skinner et al., 2004; Cucinotta et al., 2014). Within the nucellus, a cell differentiates into a female gametophyte, or embryo sac. The mature female gametophyte is composed of an egg cell, two synergids, a central cell with two polar nuclei and three antipodal cells along a micropylar-chalazal axis, aligned with the apical-basal axis (Christensen et al., 1997; Drews and Yadegari, 2002). The embryo sac formation is coordinated with the development of two integuments in Arabidopsis (Fig. 3). The integuments are initiated from the chalazal region and grow to fully envelop and protect the nucellus and embryo sac, apart from the micropyle, which provides an opening for the pollen tube to enter and access the egg cell (Bencivenga et al., 2011).

Following fertilization, the egg cell becomes a zygote, which elongates and asymmetrically divides into a basal cell and an apical cell, which have different fates. The basal elongated cell forms a cell file, the suspensor, connecting the integuments to the progeny of the apical cell, the embryo. The small api-cal cell undergoes a series of divisions resulting in formation of a round pre-globular stage embryo of eight cells. Further divisions and morphogenetic changes lead to formation of a heart-shaped embryo and specification of a shoot pole at the

Fig. 1. Auxin production and signalling during stamen development. Drawings showing three stages of stamen development: floral stage 8, when pollen gametophytes are specified (left); floral stage 10, when late stamen development starts with anther dehiscence (middle); and floral stage 13 when stamen development finishes with pollen maturation and release, and filament elongation (right). Upper and lower rows show transversal and longitudinal views of the stamen, respectively. Tissues showing auxin biosynthesis are coloured in blue (left) and response in green (right).

5030 | Robert et al.


apical extremity (marked by the formation of two cotyledon primordia in Arabidopsis) and a root pole at the basal extrem-ity, facing the suspensor (Fig. 3) (Lau et al., 2012). Ultimately, the embryo matures and the seed prepares for desiccation and dormancy.

Clearly, complex developmental programmes are involved in the formation of all the organs and tissues involved in sex-ual plant reproduction, which require precise coordination between cell division and cell differentiation. Phytohormones, particularly auxin, play key roles in the development of these plant organs (and all others), as described in the following sections.

The importance of auxin for organ patterning

The plant hormone auxin is crucial for all plant morphogen-esis processes. Its local accumulation triggers organ initiation and its dynamic and uneven distribution, so-called auxin gra-dients, within plant organs provide spatial information that is a major determinant of tissue patterning (Benková et al., 2003). These gradients are established as a result of the intercon-nected activities of four processes: auxin metabolism (biosyn-thesis, conjugation, degradation), intercellular transport, and signalling (Chapman and Estelle, 2009; Petrásek and Friml, 2009; Vanneste and Friml, 2009; Rosquete et al., 2012; Ljung, 2013; Zhao, 2014). A number of reviews have recently summa-rized current knowledge of auxin biosynthetic pathways and their molecular components (Novak et al., 2012; Korasick et al., 2013; Ljung, 2013; Zhao, 2014). Under normal growth conditions, plants mainly produce indole 3-acetic acid (IAA, the natural auxin) from L-tryptophan (Trp), in a two-step pathway, the IPyA pathway. From Trp, the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1) and

related (TAR1 and TAR2) enzymes produce indole-3-pyru-vic acid (IPyA), which is converted into IAA by a reaction catalysed by YUCCA (YUC) flavin-dependent monooxyge-nases (Novak et al., 2012; Korasick et al., 2013; Ljung, 2013; Zhao, 2014). This Trp-dependent auxin production pathway is essential for many plant developmental processes, including embryogenesis, and development of both flowers and fruits (Table 1).

TAA1 was isolated from four independent forward genetic screens for mutants exhibiting: (i) root growth insensitivity to ethylene (weak ethylene insensitive8/wei8; Stepanova et al., 2008), (ii) resistance to the shade avoidance response (shade avoidance3/sav3; Tao et al., 2008), (iii) root growth resist-ance to the auxin transport inhibitor NPA (transport inhibi-tor response2/tir2; Yamada et al., 2009) and (iv) root growth resistance to cytokinin (cytokinin-induced root curling1/ckrc1; Zhou et al., 2011) (Supplementary Table S1, available at JXB online). The YUC genes were identified from activation tag-ging screens for auxin-related phenotypes (Zhao et al., 2001; Woodward et al., 2005; Kim et al., 2007; Lee et al., 2012) (Supplementary Table S1). Analysis of three clades com-prising YUC1, YUC2, YUC4, YUC6, YUC10 and YUC11 (Cheng et al., 2006, 2007) further confirmed the involvement of these YUC genes in the control of local auxin biosynthesis and regulation of plant development.

During the past few years it has become increasingly clear, from genetic and both in vitro and in vivo biochemistry stud-ies, that these two gene families participate synergistically in the same auxin production pathway, with YUCs acting down-stream of the TAA1/TAR enzymes (Mashiguchi et al., 2011; Phillips et al., 2011; Stepanova et al., 2011; Won et al., 2011; Novak et al., 2012). Various combinations of taa1/tar1/tar2 and yucca multiple mutants show similar phenotypes, nota-bly during flower and embryo development (Table 1). There are also many examples of local auxin biosynthesis affecting

Fig. 2. Auxin production during gynoecium development. Schematic illustrations of gynoecium development from floral stages 6 to 13. Current knowledge of auxin production (via expression of YUC2/4 and TAA1/TAR2) and auxin response (DR5) patterns is colour-coded. Auxin transport flows in early development stages (6 to 9), as recently described (Larsson et al., 2014; Moubayidin and Østergaard, 2014), are illustrated by red arrows.

Auxin production for morphogenesis of reproductive organs and embryos | 5031


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plant patterning during vegetative development (Lin et al., 2005; Ikeda et al., 2009; Vandenbussche et al., 2010; Cheng et al., 2012; Baylis et al., 2013; Cui et al., 2013; Pinon et al., 2013; Ursache et al., 2014). Although the Trp-dependent IPyA auxin biosynthesis pathway is considered as the main source of auxin produced from Trp, required for morphogen-esis of plant reproductive organs and its derivatives (described below), recent work indicated that Trp-independent auxin biosynthesis may participate significantly to patterning of the early embryos (Wang et al., 2015).

The importance of auxin transport for plant development was revealed by studies with inhibitors of auxin transport (Okada et al., 1991; Lomax et al., 1995; Jensen et al., 1998;

Nemhauser et al., 2000) and subsequently the identification and characterization of the components involved in intercel-lular auxin transport (Bennett et al., 1995, 1996; Chen et al., 1998; Gälweiler et al., 1998; Luschnig et al., 1998; Muller et al., 1998; Friml et al., 2002a, b; Péret et al., 2012). At the single-cell level, signalling cascades locally control down-stream responses of auxin involved in developmental adapta-tions to environmental (light, gravity, stress) and endogenous (morphogenesis) stimuli (Baskin et al., 1986; Lincoln et al., 1990; Berleth and Jürgens, 1993; Leyser et al., 1993; Przemeck et al., 1996; Sessions et al., 1997; Hardtke and Berleth, 1998; Hamann et al., 1999; Lee et al., 2012). From its production sites, auxin is redistributed to its action sites by a combination

Fig. 3. Auxin production, transport and signalling during ovule and embryo development. Schematic illustrations of current knowledge regarding auxin production, transport and signalling during indicated stages of female gametophyte (FG, top two rows) and embryo development (bottom row). Expression patterns of auxin biosynthesis (YUC1/2/3/4/8/9 and TAA1) and response (DR5) patterns are colour-coded. Auxin transport directions are indicated by arrows. Please note that the expression pattern of auxin response as reflected by the new and more sensitive DR5v2 reporter (Liao et al., 2015) would be slightly different as the one presented here for the DR5 reporter. The main difference relates to an extended expression pattern of the DR5v2 reporter to surrounding cells as compared with the one of the DR5.



Tab

le 1

. D

evel

opm

enta

l and

non

-cel

l aut

onom

ous

effe

cts

of a

uxin

bio

synt

hesi

s

TAA

1/TA

RY

UC

DR

5

Phe

noty

pe

Mut

ant/

refe

renc

eE

xpre

ssio

nG

enes

Ref

eren

ceE

xpre

ssio

nG

enes

Ref

eren

ceE

xpre

ssio

nR

efer

ence

- C

otyl

edon

vas

cula

ture

at

torp

edo

stag

e an

d se

edlin

gs-

Cot

yled

ons

TAA

1

TAR

2

Tao et al.,

200

8;

Ste

pano

va et al.,

2008

; Zh

ou e

t al.,

201

1

- C

otyl

edon

tips

- C

otyl

edon

vas

cula

ture

YU

C1

YU

C2

Che

ng et al.,

2007

; Li

et al.,

2008

Cot

yled

on ti

psFr

iml et al.,

2003

Cot

yled

on

vasc

ulat

ure

defe

cts

wei

8 ta

r2, w

ei8

tar1

tar2

/

Ste

pano

va et al.,

2008

; tir2

/

Yam

ada et al.,

200

9-

Sho

ot a

pica

l mer

iste

ms

from

gl

obul

ar s

tage

- R

oot a

pica

l mer

iste

m fr

om

hear

t sta

ge-

Vasc

ulat

ure

from

hea

rt s

tage

TAA

1

Ste

pano

va et al.,

2008

; Tao

et al.,

2008

; Ya

mad

a et al.,

200

9

- S

hoot

api

cal m

eris

tem

fro

m g

lobu

lar

stag

e-

Vasc

ulat

ure

from

hea

rt

stag

e-

Sus

pens

or-

End

ospe

rm

YU

C1,

4

8 3, 4

, 910

, 11

Che

ng et al.,

2007

; B

elm

onte

et al.,

2013

; Rob

ert et al.,

2013

Cot

yled

on ti

psR

oot a

pica

l m

eris

tem

, red

uced

in

wei

8 ta

r1 ta

r2,

yuc1

yuc4

Wei

jers

et a

l., 2

006,

S

tepa

nova

et al.,

2008

, Rob

ert et al.,

2013

Em

bryo

pa

tter

ning

wei

8 ta

r2, w

ei8

tar1

tar2

/

Ste

pano

va et al.,

2008

; yu

c1yu

c4yu

c10y

uc11

/

Che

ng et al.,

2007

- O

uter

laye

r of

flow

er

prim

ordi

um, m

edia

l rid

ge o

f gy

noec

ium

- A

ll flo

ral o

rgan

s, g

ynoe

cium

va

lves

TAA

1

TAR

2

Ste

pano

va et al.,

2008

; Zh

ou e

t al.,

201

1-

Inflo

resc

ence

api

cal

mer

iste

m, fl

oral

prim

ordi

a,

stam

ens,

car

pels

- Lo

w e

xpre

ssio

n in

in

flore

scen

ce a

pex

(ste

ms,

yo

ung

flow

er b

uds,

pet

als,

st

amen

s, g

ynoe

cium

)-

Inflo

resc

ence

api

cal

mer

iste

m, fl

oral

prim

ordi

a,

apic

al re

gion

of c

arpe

ls,

stam

ens,

sep

als,

gy

noec

ium

.

YU

C1

YU

C2

YU

C4

Che

ng et al.,

2006

Ovu

le p

rimor

dia

in W

TM

icro

pyle

pol

e ap

ical

tip

of th

e gy

noec

ium

, st

igm

a,

vasc

ulat

ure

of th

e gy

noec

ium

val

ves

Ben

kova

et al.,

2003

; Cec

cato

et al.,

201

3; L

ituie

v et al.,

201

3

Infe

rtile

flow

ers,

de

fect

s in

gy

noec

ium

m

orph

olog

y

wei

8 ta

r2 /

Ste

pano

va et al.,

2008

; yuc

2yuc

6, y

uc1y

uc4

and

trip

le, q

uadr

uple

co

mbi

natio

ns /

Che

ng et al.,

2006

- S

tam

ens

and

polle

nY

UC

2Y

UC

6C

heng

et al.,

2006

; C

ecch

etti et al.,

20

08

Thec

a, p

olle

n gr

ains

Alo

ni e

t al.,

200

5;

Feng

et al.,

2006

; C

ecch

etti et al.,

20

08;

Def

ectiv

e in

la

te a

nthe

r de

velo

pmen

t

yuc2

yuc6

/ C

heng

et al.,

2006



of long distance transport via the vascular system and short cell-to-cell polar auxin transport mediated by membrane-localized influx carriers of the AUX/LAX family (Bennett et al., 1996; Péret et al., 2012) and efflux carriers of both the PIN (Bennett et al., 1995; Chen et al., 1998; Gälweiler et al., 1998; Luschnig et al., 1998; Muller et al., 1998; Friml et al., 2002a, b, 2003) and ABCB/PGP (Noh et al., 2001; Geisler and Murphy, 2006) families.

As a result of the polar transport, auxin accumulates in precise parts of plants, where it participates in signals that coordinate appropriate developmental responses (Chapman and Estelle, 2009). Auxin signals can be perceived by two types of receptors, AUXIN BINDING PROTEIN1 (ABP1), localized in the endoplasmic reticulum and the cell surface; and TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALLING F-BOX (TIR1/AFB) nuclear F-box pro-teins. ABP1 may sense changes in auxin levels at the cell surface and mediate rapid cellular responses (Napier et al., 2002; Robert et al., 2010; Xu et al., 2010), while TIR1/AFB proteins induce auxin-dependent transcriptional cascades (Ruegger et al., 1998; Dharmasiri et al., 2005a, b). Binding of auxin to the TIR1/AFB receptors enhances its affinity to AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcrip-tional repressors, targeting them for proteolytic degradation (Gray et al., 2001; Tiwari et al., 2001; Tan et al., 2007). This results in the derepression of AUXIN RESPONSE transcrip-tion FACTORS (ARFs), which modulate auxin-responsive gene transcription (Weijers et al., 2006).

Local auxin conjugation and degradation are substantial factors of regulation of intracellular auxin level. Fine-tuning of reversible auxin conjugation and irreversible auxin degra-dation is a powerful strategy for the plant to strictly control its cellular pool of active auxin, and the downstream effects on plant development (Rosquete et al., 2012; Barbez and Kleine-Vehn, 2013; Ljung, 2013). However the function of the enzymes involved in these pathways, and of the modi-fied IAA molecules, for plant development is not yet fully uncovered.

It has long been assumed that auxin is produced exclu-sively in young growing parts, such as leaves, flowers and root tips, and is delivered to action sites by a combination of long distance transport and short cell-to-cell polar auxin trans-port, as outlined above. However, recent discoveries (par-ticularly identification of a new family of auxin biosynthetic enzymes in Arabidopsis) have amended our understanding of auxin biosynthesis pathways, highlighting the importance of local auxin production for tissue patterning (Lin et al., 2005; Ikeda et al., 2009; Vandenbussche et al., 2010; Cheng et al., 2012; Baylis et al., 2013; Cui et al., 2013; Pinon et al., 2013; Ursache et al., 2014). The identification of key molecu-lar components of auxin biosynthesis has also deepened our insights into the role of local auxin production for the devel-opment of stamens, gynoecia, ovules and zygotic embryos. In the following section we discuss the importance of tightly spatiotemporally controlled auxin biosynthesis in regulation of the development of reproductive organs and embryos, emphasizing the non-cell autonomous character of its devel-opmental effects.

Developmental role of local auxin production in reproductive tissues

Stamen development

Detailed expression analysis of genes involved in auxin bio-synthesis indicates that auxin is produced locally by YUC1 and YUC4 during stamen primordia formation and by YUC2 and YUC6 during late stamen development (Fig. 1) (Cheng et al., 2006; Cecchetti et al., 2008). To date, no expres-sion of TAA1 and related genes has been detected in stamens. However, genes involved in auxin transport (mostly of the PIN, ABCB1/PGP1, ABCB19/PGP19 families) are expressed in filaments and stamens during all phases of their develop-ment (Noh et al., 2001; Feng et al., 2006; Ding et al., 2012; Cecchetti et al., 2015). Local auxin responses, monitored using the auxin-sensitive DR5 reporter and transcriptional reporter fusions to TIR1, AFB1, AFB2 and AFB3 promot-ers, have been detected in stamens during late stages of their development before filament elongation (Fig. 1) (Aloni et al., 2005; Feng et al., 2006; Cecchetti et al., 2008).

Mutants with impairments in auxin biosynthesis (yuc2 yuc6), auxin signalling (tir1 afb1 afb2 afb3, arf6 arf8) or reduced auxin contents in stamens, obtained by organ-spe-cific expression of iaaL, a bacterial gene responsible for auxin conjugation (pAtPIP5K1-iaaL), have defects in pollen matu-ration and/or anther dehiscence, and shortened filaments at anthesis (Nagpal et al., 2005; Cheng et al., 2006; Feng et al., 2006; Cecchetti et al., 2008, 2015). Similarly, defects in auxin transport due either to a lack of activity of PIN1, PIN2, ABCB1/PGP1 and ABCB19/PGP19 transporters or inhibi-tion of auxin transport by 1-naphthylphthalamic acid (NPA), lead to stamens with short filaments. In addition, absence of auxin transport during early stamen development in pin3 pin5, pin6, pin7 and pin8 mutants affects pollen and stamen development (Cecchetti et al., 2008; Ding et al., 2012).

Altogether these studies suggest a dual role for auxin dur-ing both early (formation of primordia and pollen) and late (pollen maturation, anther dehiscence and filament growth) stamen development phases. The role of auxin in coordina-tion of these stamen developmental steps with the growth and development of gynoecia is particularly essential for suc-cessful pollination.

Gynoecium development

As summarized above, gynoecia consist of a stigma, style, ovary and gynophore formed along an apical-basal axis (Fig. 2). A corresponding auxin gradient, with relatively high concentrations in the apical region and low concentrations in the basal region, hypothetically plays a critical role in the spec-ification and formation of the tissues along the axis (Sessions et al., 1997; Nemhauser et al., 2000; Larsson et al., 2013). This hypothesis is consistent with phenotypic deviations displayed by plants with perturbations in auxin transport or signalling, following treatment with the auxin transport inhibitor NPA (Okada et al., 1991; Nemhauser et al., 2000; Larsson et al., 2014) and in pin1 auxin efflux mutants (Okada et al., 1991;



Gälweiler et al., 1998; Moubayidin and Østergaard, 2014), PINOID protein kinase auxin transport regulator mutants (Bennett et al., 1995; Christensen et al., 2000; Benjamins et al., 2001), and arf5/monopteros (mp), arf3/ettin (ett) and ett arf4 auxin signalling mutants (Przemeck et al., 1996; Sessions et al., 1997; Nemhauser et al., 2000; Pekker et al., 2005). In all these cases, alteration of the gynoecial auxin gradient results in aberrant gynoecium development with perturbed apical-basal tissue repartition.

The Nemhauser-Sessions model, based on observed gynoe-cial morphogenesis effects of the auxin gradient, is also par-tially supported by measurements of local auxin production, local auxin responses and auxin transport along the apical-basal axis. Two main sites of auxin production, the api-cal gynoecium and carpel valves, have been deduced from expression patterns of the TAA1, TAR2, YUC2 and YUC4 auxin biosynthetic genes. This suggests that auxin might be produced in several regions during gynoecium development: apically as hypothesized in the Nemhauser-Sessions model during the early phases (Sessions et al., 1997; Nemhauser et al., 2000; Larsson et al., 2013) and transiently in the car-pel valves during later phases of development. In early stages, TAA1 is expressed at the apex of the gynoecium, extending to the medial domain, while at anthesis its expression is localized to the style. TAR2, a close homologue of TAA1, is transiently expressed in the carpel valves, before anthesis (Stepanova et al., 2008; Moubayidin and Østergaard, 2014). In mature gynoecia, YUC4 and YUC8 are expressed in the style, whereas YUC2 is expressed in carpel valve tissues (Cheng et al., 2006; Martínez-Fernández et al., 2014). Multiple TAA1/TAR and YUC mutants display distorted gynoecia with abnormal for-mation of apical tissues and absence of carpel valve tissue, supporting the importance of local auxin production for car-pel patterning (Cheng et al., 2006; Stepanova et al., 2008).

Expression patterns of the transport machinery compo-nents during gynoecium development indicate that the spati-otemporal dynamics of auxin transport during establishment of the gynoecium polarity axes are complex (Larsson et al., 2014; Moubayidin and Østergaard, 2014). Before specifica-tion of the style and stigma, the PIN1 efflux carrier hypo-thetically directs auxin flow from the base of the gynoecium to the top of the gynoecial tube, where a more apolar ring of PIN1, PIN3 and PIN7 has been detected (Larsson et al., 2014; Moubayidin and Østergaard, 2014). DR5 auxin signals have also been detected in the apical region of the gynoecium, first as two, then four foci that merge into a ring (Benková et al., 2003; Aloni et al., 2005; Larsson et al., 2013, 2014; Moubayidin and Østergaard, 2014). This loss of polarity in auxin transport, and the appearance of a ring of DR5 expres-sion at the gynoecium apex may be involved in the loss of bilateral gynoecial symmetry and transition towards radial symmetry accompanying specification of the style tissue. In addition to this ring at the apex, two strips of a weaker DR5 auxin signal were observed in the lateral domains/valves, before stigma differentiation, presumptively indicative for replum formation (Girin et al., 2011; Grieneisen et al., 2013; Larsson et al., 2014). However the hypothesized auxin gradi-ent along the apical-basal axis of the gynoecium proposed

by the Nemhauser-Sessions model has not yet been experi-mentally confirmed with the currently available experimental tools and computer simulations of auxin distribution within the gynoecium epidermis (Grieneisen et al., 2013).

Along the apical-basal axis of the gynoecium, the direction of auxin flow differs between the medial and lateral domains. PIN1 polarizes to the apical cell sides in the lateral domains, whereas it is more apolar in the medial domains, where it acts together with ABCB1/PGP1 and ABCB19/PGP19. In accordance with the above observations, three ARF pro-teins—ARF3/ETT, ARF4 and ARF5/MONOPTEROS (MP)—locally relay the auxin signal required for gynoecium patterning (Przemeck et al., 1996; Pekker et al., 2005).

Clearly, together with local auxin production and local auxin signalling, the transport of auxin within the gynoecium is crucial for generation of the auxin gradient required for the apical-basal axis specification (Fig. 2). However, the mecha-nisms involved in the downstream responses to the gradient that control gynoecium tissue differentiation are far from understood.

Ovule development

During ovule development, a dynamic pattern of auxin pro-duction, transport and signalling is observed. All three of these processes are required in ovule initiation (i.e. emergence of the ovule primordia from the placenta). TAA1 and ARF5/MP are expressed in the placenta, and TAA1 and PIN1 in primordia (Nole-Wilson et al., 2010; Bencivenga et al., 2012; Galbiati et al., 2013). Perturbations of these auxin dynamics, e.g. reductions in TAA1 expression levels, consistently result in losses of ovules (Nole-Wilson et al., 2010). Gynoecia of weak pin1 mutants produce reduced numbers of ovules, in most of which development is arrested at female gameto-phytic initiation (Bencivenga et al., 2012). Weak mp mutants are characterized by an absence of placental tissue, and hence absence of ovules (Galbiati et al., 2013).

In early stages of ovule formation (outgrowth of ovule primordium and integument, and initiation of gametophyte development), auxin is produced at the tips of the growing integuments by the action of TAA1, YUC1, YUC2 and YUC4, and may be transported along the nucellus by PIN1 and PIN3 to the distal tips of the ovule primordia where auxin response maxima have been detected using a DR5 reporter (Fig. 3) (Pagnussat et al., 2009; Nole-Wilson et al., 2010; Bencivenga et al., 2011; Ceccato et al., 2013; Lituiev et al., 2013; Larsson et al., 2014). After degradation of the nucellus, initiation of embryo sac development and growth of integu-ments, both local production of auxin mediated by YUC1 and YUC2 and local DR5-detectable auxin responses occur at the micropyle pole. However, unlike YUCs, the DR5 signal at the micropyle is transient as it vanishes when the female gametophyte reaches its final development phase. A loss of PIN1 expression, in pin1-5 mutants or pDEFH9:amiPIN1 mutants (expressing a micro-RNA targeting PIN1 under an ovule-specific promoter), sporophytically affects ovule polar-ity and blocks progression of ovule development (Ceccato et al., 2013). However, a persistent DR5 auxin response signal



in normally developed inner integuments has been detected in the aborted ovules in a pDEFH9:amiPIN1 plant, indicating that auxin transport from the inner integuments to the female gametophyte is necessary for progression of gametophyte development (Ceccato et al., 2013). Furthermore, an initial claim by Pagnussat et al. (2009), that a DR5 signal gradient within the syncytial female gametophyte is required for cell specification, has been recently challenged by several studies in Arabidopsis, maize and Hieracium pilosella (Tucker et al., 2012; Ceccato et al., 2013; Lituiev et al., 2013).

In addition, during the same phase of development a sec-ond source of auxin is localized at the funiculus, according to TAA1 expression analyses, and auxin might be transported away from the ovule towards the gynoecium by PIN1, PIN3, PGP1, PGP19 and AUX1 auxin transporters (Fig. 3) (Ceccato et al., 2013; Lituiev et al., 2013). In late developmental stages (ovule and gametophyte formation), a PGP1-, PGP19- and AUX1-dependent auxin transport system is present in the integuments close to the micropyle pole (Lituiev et al., 2013). Moreover, fertilization of the mature ovule induces an auxin response in micropylar cells surrounding the zygote (Dorcey et al., 2009), which according to the expression pattern of auxin biosynthetic genes in earlier female gametophyte stages, could well be induced by an increase in auxin synthesis rates. Altogether these studies demonstrate that a well-coordinated spatiotemporal network of auxin production, transport and signalling is essential for coordinated development of the sporophytic integuments and embryo sac.

Embryo development

Embryo development after fertilization is also highly depend-ent on auxin (Hardtke and Berleth, 1998; Hamann et al., 1999; Benková et al., 2003; Friml et al., 2003; Bernardi et al., 2012). Auxin biosynthetic genes exhibit a dynamic pattern of expres-sion during embryogenesis (Cheng et al., 2007; Stepanova et al., 2008; Robert et al., 2013). YUC3, YUC4 and YUC9 genes are expressed in the suspensor before the globular stage and might contribute to production of auxin (Fig. 3) (Robert et al., 2013), which is transported by the PIN7 efflux carrier towards the proembryo. There auxin accumulates, and might activate auxin responses (Friml et al., 2003; Robert et al., 2013; Wabnik et al., 2013; Liao et al., 2015). At the globu-lar stage, a new auxin production site might be established through activation of TAA1, YUC1 and YUC4 expression in the most apical cells (Fig. 3) (Cheng et al., 2007; Stepanova et al., 2008; Robert et al., 2013). Auxin accumulation in these auxin-producing cells (where there is no DR5-detectable auxin signalling) has been detected by a new radiometric auxin sensor, R2D2, combining an auxin-sensitive degrada-tion domain II of an Aux/IAA protein and an auxin-resistant version of the protein, both driven by the RPS5A promoter, and fused to fluorescent tags of different colours (Liao et al., 2015). This new embryonic auxin source triggers polariza-tion of the PIN1 proteins towards the basal membranes of the embryonic provascular cells, and consequently auxin transport toward the base of the embryo, thus creating an apical-basal auxin gradient with auxin accumulation in the

uppermost suspensor cells (Friml et al., 2003; Stepanova et al., 2008; Robert et al., 2013; Wabnik et al., 2013; Liao et al., 2015). At the base of the globular embryo the ARF5/MP-dependent signalling pathway induces a developmental programme of embryonic vascular tissue formation and initi-ation of an embryonic root (Schlereth et al., 2010; Saiga et al., 2012; De Rybel et al., 2014). However, this is far from a lin-ear auxin production-transport-signalling pathway. Indeed, auxin signalling by ARF5/MP transcription factors in the lower embryonic tier has feedback effects on auxin transport by maintaining proper levels of PIN and AUX/LAX expres-sion (Schlereth et al., 2010; Robert et al., 2015).

Similarly to embryonic mutations in auxin transport and signalling, reduction of auxin content due to loss of function of TAA1/TAR and YUC genes greatly perturbs embryo devel-opment and specification of root and shoot poles (Berleth and Jürgens, 1993; Hardtke and Berleth, 1998; Hamann et al., 1999; Friml et al., 2004; Cheng et al., 2007; Stepanova et al., 2008; Robert et al., 2013). Hence (as in developmen-tal processes discussed above), during embryo development spatiotemporally tightly coordinated auxin biosynthesis together with polarized transport and local perception is highly important for proper embryo patterning. In embryos and ovules, a PIN-dependent transport mechanism mediates auxin flow from IPyA-dependent auxin sources, thus creat-ing an auxin concentration gradient that is translated into an organ polarity axis (Ceccato et al., 2013; Robert et al., 2013). Importantly, PIN polarization in the embryonic cells depends on the presence of an auxin source (Robert et al., 2013; Wabnik et al., 2013), indicating that auxin produced de novo in source tissues has feedback effects on cells involved in the polarized transport of auxin away from the source, as suggested by the canalization hypothesis (Sachs, 1991; Sauer et al., 2006). This links cell polarity, e.g. asymmetry of auxin efflux carriers’ subcellular localization, to auxin concentra-tion gradients, tissue planar polarity and body axis determi-nation (Nakamura et al., 2012).

Local production via local regulation of gene expression

The importance of spatially and temporally fine-tuning local auxin biosynthesis for gynoecium, ovule and embryo pattern-ing raises important questions regarding the upstream regu-latory pathways that determine the expression of key auxin biosynthetic genes. Several recent studies have identified transcription factor families, which through either positive or negative regulation of TAA1 and YUC expression might contribute to the control of auxin production during repro-ductive organ and embryo development.

The SHORT INTERNODES/ STYLISH transcription factors

The SHORT INTERNODES/STYLISH (SHI/STY) tran-scription factor family is one of the most intensively stud-ied regulators of auxin biosynthesis due to its involvement



in auxin-mediated leaf and flower development (Kuusk et al., 2006; Sohlberg et al., 2006). All family members have a char-acteristic, highly similar zinc-finger-RING-like domain, indi-cating that they probably target the same set of promoters (Kuusk et al., 2002; Eklund et al., 2010). A reverse genetic screen for genes related to SHI identified STY1 and STY2 as transcription factors redundantly involved in auxin-depend-ent apical specification of the gynoecium (Kuusk et al., 2002). STY1 locally enhances auxin biosynthesis in gynoe-cia by directly binding the YUC4 auxin biosynthetic gene’s promoter (Sohlberg et al., 2006; Ståldal et al., 2008, 2012; Eklund et al., 2010).

Local auxin application to gynoecia of the double sty1 sty2 mutant rescues the style formation defects, indicating that the developmental alterations in the double mutant are caused by a reduction in auxin levels. These observations also suggest that the primary function of STY1 and STY2 in gynoecium development is in local activation of auxin bio-synthesis (Ståldal et al., 2008). In addition, STY1 genetically interacts with SPATULA, a basic helix-loop-helix transcrip-tion factor (Heisler et al., 2001) involved in gynoecium for-mation (Alvarez and Smyth, 1999). Members of this family of transcription factors potentially bind (ectopically or not) to a consensus sequence in YUC1, YUC5, YUC8 and YUC9 promoters (Eklund et al., 2010; Ståldal et al., 2012), but the developmental function of this regulation is not yet known. STY1 is also expressed in the nucellus of developing ovules and embryos (Kuusk et al., 2002). Determining whether the YUC promoters are also regulated by SHI/STY transcription factors during ovule and embryo patterning will help eluci-date whether common regulatory mechanisms of auxin bio-synthesis act during the development of female reproductive organs.

NGATHA transcription factors

The NGATHA (NGA1, NGA2, NGA4 and NGA3/TOP1) B3-domain transcription factors promote the development of apical gynoecial tissue in Arabidopsis and other eudicots such as Nicotiana benthamiana and Eschscholzia californica (Alvarez et al., 2009; Trigueros et al., 2009). Multiple nga mutants lack styles and stigmas, probably partially due to the altered expression of YUC2, YUC4, YUC8 and TAA1 auxin biosynthetic genes in these tissues (Trigueros et al., 2009; Martínez-Fernández et al., 2014). NGA transcription factors act at different steps of auxin-mediated morphogen-esis by regulating expression of genes involved in auxin bio-synthesis, transport (the auxin transport regulators PINOID and WAG2) and signalling (ARF1, ARF11 and ARF18) (Martínez-Fernández et al., 2014).

All four NGA genes and STY1 have similar expression patterns in the apical gynoecium domain, the ovule nucellus and embryos (Kuusk et al., 2002, 2006; Alvarez et al., 2009; Trigueros et al., 2009; Eklund et al., 2011; Ståldal et al., 2012). Trigueros et al. (2009) suggest that NGA3 and STY1 do not interact or regulate each other’s expression, but synergistically and cooperatively induce YUC-dependent auxin biosynthesis in the gynoecium. However, Alvarez et al. (2009) and Staldal

et al. (2012) suggest that the relationships between STY1 and NGAs are more complex and hierarchical, with STY1 con-ditionally activating expression of NGA2 and NGA4, which in turn activate other STY genes that promote expression of YUC genes.

AP2/ERF and class III HD-ZIP transcription factor families

Several families of AP2/ERF transcription factors indirectly affect auxin biosynthesis. A putative GCC box, recognized by AP2/ERF transcription factors and present in almost all SHI/STY promoters, is required for SHI/STY gene expres-sion in regions where shoot auxin biosynthesis occurs (Eklund et al., 2011). For example, DORNRÖSCHEN and DORNRÖSCHEN-LIKE regulate expression of SHI/STY genes, which regulate YUC genes, suggesting a complex level of regulation for auxin biosynthesis in gynoecia and embryos (Chandler et al., 2011a, b; Eklund et al., 2011). AINTEGUMENTA, another AP2/ERF transcription fac-tor, is involved in gynoecium patterning, redundantly with the class III Homeodomain-Leucine Zipper (HD-ZIP) transcription factors REVOLUTA, PHABULOSA and PHAVOLUTA. AINTEGUMENTA and REVOLUTA reg-ulate TAA1 expression during the development of gynoecia and, possibly, ovules (Nole-Wilson et al., 2010; Bencivenga et al., 2011).

Finally, the AP2-domain PLETHORA (PLT) gene expres-sion domain overlaps with that of YUC1 and YUC4 in the shoot meristem and flower primordia (Cheng et al., 2006; Prasad et al., 2011). The plt3 plt5 plt7 triple mutant has reduced YUC1 and YUC4 expression levels, and PLT genes rapidly induce expression of YUC genes in the shoot apical meristem (Pinon et al., 2013). However, the role of PLT genes in control of auxin biosynthesis during reproductive organ, ovule and embryo development needs to be experimentally addressed.

The negative regulators: SPOROCYTELESS/NOZZLE and KANADI transcription factors

Two families of transcription factors negatively affect expres-sion of auxin biosynthesis genes. SPOROCYTELESS/NOZZLE (SPL/NZZ), which is essential for male and female gametophyte development in Arabidopsis (Schiefthaler et al., 1999; Yang et al., 1999), acts as a repressor of auxin biosyn-thesis by indirectly down-regulating YUC2 and YUC6 (Li et al., 2008). The KANADI (KAN1 to KAN4) gene family encodes members of the GARP family of MYB-like tran-scription factors and is responsible for the abaxial identity of lateral organs, acting antagonistically to adaxial identity-pro-moting class III HD-ZIP REVOLUTA, PHABULOSA and PHAVOLUTA transcription factors (Eshed et al., 2001, 2004; Kerstetter et al., 2001). ChIP-seq analysis has shown that KAN1 directly represses TAA1 and YUC5 gene expression, antagonistically to REVOLUTA (Brandt et al., 2012; Huang et al., 2014). In addition to auxin production genes, KAN1 represses expression of genes involved in auxin transport



(LAX1, LAX2, LAX3, PIN4) and auxin signalling (IAA11, IAA18, ARF3) (Huang et al., 2014).

LEAFY COTYLEDON and FUSCA transcription factors: induction of somatic embryogenesis

Somatic embryogenesis is the capacity of differentiated cells to acquire embryogenic competences. FUSCA3 (FUS3), and LEAFY COTYLEDON 1 and 2 (LEC1 and LEC2) B3-domain transcription factors are essential for the matu-ration phase of zygotic embryo development (Braybrook and Harada, 2008). When ectopically expressed, these three transcription factors promote embryonic developmental pro-grams in seedlings, i.e. somatic embryogenesis, in the absence of exogenous application of auxin (Lotan et al., 1998; Stone et al., 2001; Gaj, 2004). ChIP and microarray analyses have revealed that LEC2 activates YUC1, YUC2, YUC4 and YUC10 gene expression, thereby inducing auxin production, associated with induction of somatic embryogenesis (Stone et al., 2008; Wójcikowska et al., 2013). Moreover, LEC1, LEC2 and FUS3 are also expressed during early zygotic embryo development (Lotan et al., 1998; Kroj et al., 2003; To et al., 2006), during which they may also influence auxin biosynthesis.

Alternative splicing in regulation of the subcellular localization of YUC4

Notably, in addition to transcription regulation, alternative splicing of YUC genes might play a role in auxin biosynthetic gene activity. The YUC4 gene encodes, via alternative splic-ing, two protein isoforms. The long isoform, YUC4.1, local-ized in the cytosol, is present in all aerial tissues. The other, YUC4.2, localized at the endoplasmic reticulum membrane facing the cytosol domain, has only been detected in flowers (Kriechbaumer et al., 2012). Both of these isoforms are active in plants and can use IPyA as a substrate (Kriechbaumer et al., 2012).

Concluding remarks and future perspectives

For many years the role of auxin biosynthesis in organ mor-phogenesis was overlooked, but recent insights into auxin biosynthesis pathways, particularly the IPyA pathway, illus-trate the importance of local auxin production for reproduc-tive organ and embryo development processes. Local auxin biosynthesis occurs in very few cells in specific developmen-tal windows in stamens, gynoecia, ovules and embryos. These recently detected sources of auxin influence, mostly non-cell autonomously, the flow of auxin within the reproductive tis-sues and embryos, and thus the formation of auxin gradients, which play key roles in organ morphogenesis. Thus local auxin biosynthesis might be an upstream component and trigger of all auxin-mediated developmental programs. The expression of TAA1/TAR and YUC auxin biosynthetic genes is tightly controlled, providing a means of regulating the spatiotemporal

pattern of auxin production within the tissues. As shown in this review, much has been learnt about the complexity of the tran-scriptional regulation of these major aspects of plant repro-duction, but much remains to be learned in future studies. To understand the importance of fine-tuning auxin production, flow, signalling and degradation for the development and the patterning of these organs, one of the major future challenges in the field would be to quantify auxin (and metabolites) levels in situ either by direct measurements or by the use of newly developed tools such as the radiometric R2D2 auxin reporters.

Supplementary material

Supplementary material is available at JXB online.Supplementary Table S1. Known mutant alleles of genes

involved in auxin biosynthesis.

AcknowledgementsThe authors would like to thank Maytham Karaki for his help in the figure design. The work was supported by grants from: the Employment of Best Young Scientists for International Cooperation Empowerment/OPVKII programme (CZ.1.07/2.3.00/30.0037) to HSR and LCK; the Czech Science Foundation (GA13-39982S) to EB, LCK and SM; and the SoMoPro II pro-gramme (3SGA5602), cofinanced by the South-Moravian Region and the EU (FP7/2007–2013 People Programme), to HSR.

Note added to the ProofAfter this review was accepted for publication, a study by Panoli et al (2015) provides new insight into the role of local auxin biosynthesis by TAA1, TAR2, YUC1, YUC2 and YUC8 and auxin import by the AUX1 and LAX1 proteins for female gametophyte development by detailed analysis of their expression patterns and by a functional genetic analysis of their loss-of-function mutants. This study corroborates their previous work (Pagnussat et al, 2009) showing that auxin production inside the female gametophyte is important for its development.

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