Charles University in Prague, Faculty of Science

Department of Experimental Plant Biology

2012

Roman Skokan

The evolution of auxin transport mechanisms

Bachelor Thesis

Supervisor: RNDr. Jan Petrášek, Ph.D.

Co-supervisor: Mgr. Stanislav Vosolsobě

Acknowledgments

I hereby thank RNDr. Jan Petrášek, Ph.D. for valuable discussions and help with my work and

for his kind effort to introduce me to his colleagues and into the world of science. I also thank the

consultant, Mgr. Stanislav Vosolsobě, for his willingness to help me in any way and to all other

members of the Department of Experimental Plant Biology of Charles University in Prague for their

offers of help.

I also thank the members of the Institute of Experimental Botany AS CR for the intriguing

topic to work on and for the delicious cake.

Abstract

Auxin, the longest studied phytohormone, is distinguished from other phytohormones by its unique directional, so-called polar transport. This feature helps to facilitate the broad range of auxin action at all stages of plant development. The polar auxin transport has been evolving together with plant lineages. By studying the mechanisms of auxin transport, biosynthesis, metabolism and particularly signaling we can perhaps better elucidate many milestones of plant evolution, such as complex multicellularity or transition to land. This bachelor thesis summarizes the available data and gives a basic overview of auxin-related characteristics. As far as we know, the advanced mechanisms of auxin transport and signaling known from land plants are probably not very ancient and are absent in various algae. Auxin biosynthesis, however, is rather common and a lot of green algae contain orthologs of important biosynthetic enzymes from land plants. Based on the available data it seems that a complete auxin signalling pathway coupled with proteasomal degradation and affecting gene expression is not present in algae. The polar auxin transport, so far with the earliest evidence from moss sporophytes, was recently found in the gametophytic thallus of stonewort (Chara) from a green algal clade Streptophyta, which is considered to be directly ancestral land plants. Proteins with similar sequence to important auxin carriers were uncovered not only in Streptophyta, but most recently in some taxa from Chlorophyta clade (the rest of green algae), which apparently lies one more step below in evolution. However, the ability to facilitate a directional flow of auxin between cells was not demonstrated. Future research will require a more complex genomic knowledge of evolutionary interesting taxa, especially the morphologically advanced algae from Streptophyta clade.

Keywords: auxin, auxin transport, plant development

Abstrakt

Auxin, nejdéle studovaný fytohormon, je v porovnání s ostatními fytohormony unikátní svým směrovaným tzv. polárním transportem. Tato vlastnost napomáhá zprostředkovat širokou škálu působnosti auxinu ve všech stadiích života rostliny. Polární transport auxinu se v evoluci vyvíjel společně s rostlinami. Zkoumáním mechanismů transportu auxinu a jeho biosyntézy, metabolismu a především signalizace můžeme snad lépe objasnit mnoho důležitých milníků evoluce rostlin, jako vývoj složitější mnohobuněčnosti či přesun rostlin na souš. Tato bakalářská práce shrnuje dostupná data a podává základní přehled rysů s auxinem spojených. Jak dosud známo, pokročilé mechanismy transportu auxinu a jeho signalizace, známé z vyšších rostlin, nejsou zřejmě fylogeneticky staré a v řadě řas zcela absentují. Biosyntéza auxinu je ovšem vcelku běžná a mnoho zelených řas obsahuje ortology důležitých enzymů spojených s metabolismem auxinu. Dle dostupných informací se zdá, že kompletní auxinová signalizace, spojená s degradací v proteasomu a ovlivňující genovou expresi, v řasách chybí. Polární transport auxinu, dosud znám nejdříve ze sporofytů mechů, byl nedávno zjištěn v gametofytické stélce parožnatky (Chara) z oddělení zelených řas Streptophyta, které jsou považovány za přímé předchůdce vyšších rostlin. Proteiny sekvenčně podobné důležitým auxinovým přenašečům byly odhaleny nejen ve Streptophyta, ale nejnověji i v zástupních Chlorophyta (zbylé zelené řasy), které patrně leží v evoluci ještě o stupeň níže. Jejich schopnost zprostředkovat směrovaný tok auxinu mezi buňkami však prokázána nebyla. Budoucí výzkum bude vyžadovat kompletnější znalost genomů evolučně zajímavých taxonů, především morfologicky pokročilých řas z oddělení Streptophyta.

Klíčová slova: auxin, transport auxinu, rostlinný vývoj

Table of contents

1 Introduction 5

2 Auxin in general 62.1 Chemical structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.2 Auxin biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.3 Auxin signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.4 Methods of auxin research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3 Auxin transport in higher plants 93.1 The chemiosmotic polar diffusion model . . . . . . . . . . . . . . . . . . . . . . . . . . 93.2 Polar auxin transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.3 Cellular auxin carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

3.3.1 AUX1/LAX auxin influx carriers . . . . . . . . . . . . . . . . . . . . . . . . . . 123.3.2 MDR/PGP/ABCB auxin transporters . . . . . . . . . . . . . . . . . . . . . . . 123.3.3 PIN proteins, determinants of auxin transport polarity . . . . . . . . . . 13

3.4 Auxin transport in the development of higher plants . . . . . . . . . . . . . . . . . 15

4 Auxin during the evolution of land plants 184.1 Current opinion on early land plant phylogeny . . . . . . . . . . . . . . . . . . . . . 184.2 Auxin in bryophytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .204.3 Auxin in algae? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

4.3.1 Brown and Red algae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214.3.2 Green algae; Chlorophyta and Streptophyta . . . . . . . . . . . . . . . . . . 22

4.4 PINs again . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234.5 Obstacles and future expectations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5 Discussion 25

Abbreviations 30

References 31

5

1 Introduction

Auxin has been among first discovered plant growth substances with numerous

physiological effects. It affects plants from their very first zygotic division until their last days

alive, and particularly holds control over major developmental processes. Also, the mechanisms

of handling auxin have been evolving together with plant lineages. A common use of auxinic

herbicides in agriculture may represent an example: while dicot weeds die after the application

of auxin-like molecules, monocot grains remain insensitive to them.

Earlier methods of studying auxin in planta consisted of detecting auxin within tissues or

observing the effects of various inhibitors of auxin-related features. Along with the progress in

genetics and molecular biology, the mechanism of auxin effects could finally be studied and its

key players identified. Auxin metabolism, signaling and transport have been well described for

land plants in the last twenty years.

Auxin seems to have been present in some crucial steps of plant evolution. Researching this,

however, is a very difficult matter. We have to step outside the warm, comfortable home of

Arabidopsis thaliana model plant and sail into the unexplored waters of primitive plant and algal

lineages, often with great expectations and little perspectives. The lack of genomic data does not

make our way any easier. The complexity of the regulation of cell-to-cell directional auxin

transport in higher plants suggests that taxa where it is absent had not developed complex auxin

responses. While land plants universally stand on a high level of auxin management, the related

green algae generally do not possess these mechanisms, as far as we know. Likewise do not the

more distantly related taxa. Indeed, it is important to identify mechanisms of auxin management

in both Streptophyta and Chlorophyta algae. Although these mechanisms do not necessarily have

to be identical to land plant actions, there might be some unique characteristics uncovered.

The following text starts with a brief description of what we know about auxin in planta. It

then reviews the important information about the evolution of auxin action and auxin transport

mechanisms in particular, most of which has been described only recently. It is not this thesis

goal to bring new information or revolutionary perspectives, rather than to think about the role of

auxin throughout evolution, about what we know and what one day we may find.

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2. Auxin in general

2.1 Chemical structure

Like other so-called “phytohormones”, auxins are rather simple molecules with a broad

range of effects. Though many compounds, both endogenous and exogenous, display an auxinic

activity, the most frequented endogenous auxin of higher plants is the indole-3-acetic acid, IAA.

It is also most commonly used in research. Lately, the other endogenous auxins have received

some attention [1]. There are also synthetic auxins, which are used in agriculture or as a tool for

research (Figure 1). Despite their differences in structure and effects, they all contain an

aromatic ring with a carboxylic acid residue attached to it.

Virtually any data obtained about auxin refers to the endogenous IAA or to synthetic 2,4-

dichloro-phenoxy-acetic acid (2,4-D) and 1-naphthylacetic acid (1-NAA). It is hard to find a

process of major importance, both embryonic and post-embryonic, where auxin would not play

its part.

IAA, a natural auxin 2,4-D, a synthetic auxin 1-NAA, a synthetic auxin

Figure 1: The structure of major endogenous and synthetic auxins.

2.2 Auxin biosynthesis

As reviewed in [2] five pathways of auxin (IAA) biosynthesis have been proposed so far:

one tryptophan (Trp)-independent and four (Trp)-dependent. They are usually named according

to their characteristic intermediate. Some important enzymes are, however, largely unknown.

Moreover, these pathways appear to be redundant and in the end, the importance of each is

difficult to assess. Tryptamine (TAM) and indole-3-pyruvate (IPA) pathways have been defined

as especially important for developmental processes (rev. in [2]). Here, YUCCA enzymes of the

TAM pathway play an important role in both embryonic and post-embryonic development [3].

Other studies on TAA enzyme (tryptophane aminotransferase of Arabidopsis) of the IPA

pathway showed its transcription to be regulated by ethylen [4] and even by light quality changes

as a part of the shade avoidance phenomenon [5]

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2.3 Auxin signaling

Nowadays, the auxin signal transduction pathway and signaling machinery is relatively well

explored. Auxin acts through the regulation of gene expression as well as through the rapid non-

transcriptional regulation, e.g. the upregulation of activities of proton pumps. Both levels of

regulation seem to be mutually interconnected.

The elements of pathway for auxin-regulated gene expression based on the proteasomal

degradation of transcription repressors (Figure 2) are likely to be present in all land plants from

bryophytes onward. This pathway has been reviewed many times and most of auxin-related

articles or reviews at least mention it (e.g. [1-2, 6]). Auxin-regulated genes are under control of

Auxin Response Factors (ARFs), a family of transcription factors that bind to the auxin

responsive element of DNA (TGTCTC) and can dimerize with each other. Some ARFs activate,

i.e. their action promotes transcription, and other repress transcription. ARFs, however, are

themselves inhibited by Aux/IAA transcriptional repressors, which bind to them. Aux/IAAs, are

strongly redundant, as their multiple mutations do not alter phenotypes. They also recruit a co-

repressor TOPLESS (TPL). Under low auxin concentrations, ARFs are inactive. When auxin

concentration increases (through biosynthesis, deconjugation or transport), it is perceived by its

receptor from TIR1/AFB family of F-box proteins that forms a part of the E3 SCF ubiquitin

ligase complex. Auxin then acts as a kind of molecular glue and without inducing a change in

conformation, it binds this E3 ligase to an Aux/IAA. Therefore, the Aux/IAA is marked for

degradation (by chains of ubiquitin) in 26S proteasome. Its respective ARF is then free and can

promote or repress transcription, depending upon its nature. ARF1 can itself be degraded via

proteasome, independently of this pathway [7]. This may be of importance to repress auxin-

induced expression under specific conditions.

Since the discovery of the SCFTIR1/AFB pathway, the role of other auxin-triggered

mechanisms had been rather underestimated. Recent research, however, highlights an

undisputable importance of an alternative auxin receptor ABP1 (rev. in [8]). The ABP1 has been

shown to take part in the majority of known auxin-mediated processes at all stages of

development. The protein itself resides both on plasma membrane (PM) and in the ER. The latter

seems to be only a recent trait, since the C-terminal ER-retention amino-acid sequence (KDEL)

is only found in the ABP1s of flowering plants. There, however, most of the protein localizes to

the ER. It is probably thanks to the association with another protein. ABP1 induces activation of

PM proton pumps and K+ inward rectifying channels. The acidification of apoplast activates

expansins in the cell wall, which cause it to loosen, and K+ uptake enables the water uptake,

necessary for cell expansion. However, some plants (such as pea) seem to have different ways of

executing an auxin-induced cell expansion, independent of ABP1. ABP1 is also essential for cell

8

division. It is particularly involved in the G1/S transition and regulates expression of core cell

cycle genes, like D-type cyclins (CYCD) or RETINOBLASTOMA RELATED (RBR).

Figure 2: Scheme of the SCFTIR1/AFB-mediated auxin signaling pathway. Without an auxin stimulus, transcription factors from ARF family are silenced by Aux/IAA transcription repressors. Incoming auxin, acting as a kind of molecular glue, binds Aux/IAAs to the TIR1/AFB F-Box protein of the SCF ubiquitin ligase. Aux/IAAs are sent into 26S proteasome for degradation and ARFs are free to promote transcription of auxin responsive genes. Reproduced from [79].

Another auxin-triggered fast effect might involve the activity of dual specificity phosphatase

IBR5. It dephosphorylates and therefore inactivates MAPKs (mitogen-activating protein

kinases). Both are found in the nucleus. MAPK-IBR5 pathway may represent an auxin signaling

mechanism, which considerably affects the expression of auxin responsive genes while acting

independently of TIR1 [9-10].

2.4 Methods of auxin research

There are various methods to study auxin related features. The essential proteins of auxin

action can be inhibited or their respective genes silenced, which manifests into an aberrant

phenotype. If not, the protein is not particularly important or there are ways to complement for

this loss. The auxin response of genes can be observed by using a synthetic auxin responsive

element (AuxRE) called DR5 [11]. It contains multiple tandem repeats of a natural auxin

responsive motif TGTCTC. DR5 is usually fused to reporters like β-glucuronidase (GUS) or

Green Fluorescent Protein (GFP) [12], which visualize the level of expression.

The auxin transport (or flow) itself can simply be determined by measuring concentration of

auxin molecules in the opposite sides of an immobilized polarized tissue by using radioactive-

labeled auxin molecules ([12]). Auxin molecules other than IAA exhibit different transport and

signaling properties, which may be used in research [12]. The inhibitors of auxin transport are a

very useful tool as well (e.g. [13]). They act by an increase (auxin efflux inhibitors) or decrease

9

(auxin influx inhibitors) of intracellular accumulation of auxin, possibly by non-competitive

association with auxin carriers or by other mechanisms. It is worth noting that both synthetic and

endogenous auxins are used in agriculture, either as growth stimulants, or more importantly as

selective herbicides [14]. Plants overall develop resistance to these at strikingly low scale. One

of the resistant mutants (wild mustard) has an altered ABP1 auxin receptor [15], though it is not

clear whether this is actually responsible for herbicide resistance

Studying the evolutionary perspectives of auxin transport is quite different. It often equals a

search for the origins of key proteins in taxa of interest. These proteins have been largely

described in Angiosperms, notably Arabidopsis thaliana. This search, however, requires

sufficient genomic knowledge of investigated organisms, which is not always available. Still,

these have to be described for a more in-depth research. Some solution to this problem may

provide the EST (expressed sequence tag) libraries, which at least allow us to see the

transcriptomes of various organisms where genome information is lacking.

3 Auxin transport in higher plants

3.1 The chemiosmotic polar diffusion model

There are generally two ways of auxin transport in higher plant body (e.g. rev. in [2]). First,

there is a rapid, long-distance, passive transport via phloem. It runs from highly biosynthetically

active young shoot tissues to the root or young shoot lateral organs. Second, a slower, cell-to-cell

movement, confined to vascular and other tissues, is facilitated by PM-located carrier proteins

that often determine directionality of this transport. The directionality of auxin movement had

been already recognized some time ago. Based on the experiments from early 1970's (as

reviewed e.g. in [2],[16]), the concept of chemiosmotic polar diffusion model for IAA transport

had been proposed. According to this model, the major driving force for auxin transport across

plasma membrane is a proton gradient between a cytoplasm and the extracellular space,

maintained by the action of proton pumps. Acidification of apoplast establishes a difference of

pH~7.0 inside the cell to pH~5.5 in the apoplast. Both synthetic and naturally occurring auxin

molecules are weak organic acids and as such they undergo reversible dissociation. As with any

dissociation, the equilibrium of this reaction depends on pH. With a relatively acidic pH in the

apoplast, one can expect about 20% of IAA molecules to remain undissociated [17]. These

protonated molecules are less polar and therefore can pass through the PM via passive diffusion.

10

Figure 3: The chemiosmotic polar diffusion model of auxin transport and carrier-assisted polar auxin transport. In the relatively acidic pH of extracellular space, maintained by H+-ATPase proton pumps within the PM, some auxin (IAA) molecules remain protonated. These are more hydrophobic and can pass through the membrane passively. In a higher pH value inside, they completely dissociate and anions cannot get outside on their own. Dissociated form is taken from the outside via AUX1/LAX influx transporters. From the inside, auxin anions may too only exit via active transporters, PIN and MDR/PGP/ABCB. The activity of these efflux transporters can me modulated, such as by inhibitors (NPA, flavonoids). These target the carriers themselves or their auxiliary proteins, like TWD1 of the PGPs. A polar subcellular localization of PINs determines the direction of auxin flux. PGP carriers exhibit mostly non-polar localization. Reproduced from [2].

Once inside, they are exposed to the cytoplasmic pH~7.0 and get dissociated (lose their proton),

becoming polar anions, unable to pass through the PM. In this state they can only be excreted via

transporters. This mechanism is sometimes called an “anion trap” [18]. Chemiosmotic polar

diffusion model suggested that the polarity of the auxin flow through a tissue is maintained

mainly by the asymmetric distribution of auxin efflux carriers (Figure 3).

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3.2 Polar auxin transport

The polar auxin transport (PAT) is a unique process that distinguished auxin from other

phytohormones. It is a directional cell-to-cell flow of auxin molecules through individual strands

of cells or within tissues. PAT contributes to the establishment of auxin concentration gradients

that regulate plant morphogenesis through specific gene expression. It has been proposed

recently by modeling of auxin flow in roots that auxin transport alone coupled with site-specific

biosynthesis is sufficient to create and maintain its maxima and gradients [19]. While a plant cell

excretes auxin predominantly in the direction of PAT, this direction itself further promotes this

type of polarity. This model fits with the role of auxin as the self-polarizing signal for its own

transport as the canalization theory postulated for leaf venation development [20].

Figure 4: Schematic representation of auxin fluxes in planta. (A) In shoot and shoot/derived organs (Leaf primordia P1 and P2) auxin is transported towards the tip through the epidermis and refluxed back through the inner tissues (future vasculature) in a reverse fountain manner. In root and root derived organs (lateral root, LR), auxin goes towards the tip through the interior and refluxed back through the epidermis in the fountain manner. (B) A fountain of auxin flow in the root tip with the depicted localizations of individual auxin carriers. lrc-lateral root cap, ep-epidermis, c-cortex, en-endodermis, s-stele. Reproduced from [16].

Due to the sessile nature of plants, they often need to change their growth polarity in order

to respond to their environment. PAT has been identified as the important morphoregulatory

force for the establishment and maintaining of polarity. This is further corroborated by the facts

that the spreading of auxin through apolar callus is primarily diffusive and that the polarity of

plant tissues is disturbed by introducing them to unnaturally high auxin concentrations, as

12

reviewed in [18]. Predominant directions of auxin fluxes with the plant body can be postulated

for its respective tissues (Figure 4). Rich auxin sources, young leaves and shoot meristems,

generate the majority of auxin. From here auxin flows via vascular and surrounding cells, i.e.

plant interior, downward. In root, auxin accumulates mainly in the columella root cap cells and

around the quiescent centre and it is redirected to epidermis and cortex. Once there, the auxin

flow follows an opposite direction; it is directed up, through the periphery, to young shoot. In

root, auxin flow resembles a fountain [21], while in shoot, the process is reverse resembling

reverse fountain. Auxin maxima (sources and sinks) are usually represented by budding young

organs (lateral roots/shoots, leaves, flowers), with the exception of root quiescent centre and, to

some extent, the nearby basal meristem [22].

3.3 Cellular auxin carriers

3.3.1. AUX1/LAX auxin influx carriers

Auxin does not enter the cell just by plain diffusion. A portion of IAA in the apoplast is not

dissociated and cannot passively enter cells. For this purpose the plant cells contain active auxin

influx carriers of this impermeable auxin form, known as AUX1/LAX (AUXIN

RESISTANT 1/LIKE AUX1). AUX1 gene was first identified after the analysis of loss-of-

function aux1 mutant, which is agravitropic and strongly resistant to IAA, suggesting that the

encoded protein is somehow involved in its uptake. More precise description has been carried

out during experiments on root gravitropic response [23] and AUX1 carriers were identified as

proteins similar to amino-acid permeases. The AUX1/LAX carriers function as H+/IAA-

symporters [24]. The Arabidopsis thaliana genome encodes four types of these proteins in total

(AUX1, LAX1-3). They have been shown to be involved in almost any auxin-dependent process,

e.g. lateral root formation [25], gravitropism [23], embryonic development [26] or phyllotaxis

[27].

3.3.2 MDR/PGP/ABCB auxin transporters

The MDR/PGP/ABCB transporters facilitate the transport of IAA to and from the cell (rev.

in [2]). As their name suggests, they are multidrug resistance/phosphoglycoproteins of the

ATP-binding Casette transporter family, subfamily B. They have been discovered and are mostly

associated with the experiments inhibiting the auxin transport (mostly by NPA). These

inhibitors, both synthetic and natural, interfere with the MDR/PGP/ABCB action by disrupting

their binding to TWD1 auxiliary protein (TWISTED DWARF1). The ABC family is overall very

13

ancient; the sequences coding for their nucleotide binding domains are among the most

conserved in living organisms [28]. ABCB19 was shown to work in coordination with PIN1 in

specific PM domains, thereby at least taking part in a directional auxin transport [29].

3.3.3 PIN proteins, determinants of auxin transport polarity

PIN (PIN-FORMED) auxin efflux carriers are probably the most extensively studied group

of auxin transporters. The functional knock-out of some PINs (PIN1) leads to phenotypes similar

to the effects of auxin transport inhibitors, hinting the same process. PINs are generally

considered to play a major role in the PAT through their polar localization on the plasma

membrane. Auxin itself has the ability to influence PINs' transcription, turn-over and PM

localization [16].

The first described PIN was AtPIN1 from Arabidopsis, discovered through the loss-of-

function mutant pin1. The most important phenotypic trait of pin1 is the inability to develop

floral organs; instead it generates a naked, pin-like stem, hence the name. Its immunolocalization

to the basal end of vascular tissues and the inhibition of auxin flux upon its knock-out suggested

its role in the process [30]. In the same year, AtPIN2 was detected on the membranes of

epidermal and cortical roots cells, localized in a polar manner, and was proposed to control

gravitropic growth (pin2 mutant roots are agravitropic) [31]. The role of PINs as auxin

transporters has been verified in yeast and mammalian HeLa cells, where they were able to

facilitate auxin transport without the need for other plant specific factors [13].

PINs are secondary plasma membrane transporters. They use an already established gradient

of protons (by proton pumps) between the cell and its apoplast. ATP-binding motifs were not

recognized in PIN sequences. They are also subject to rapid vesicular cycling between

endosomal space and PM [32]. This means that a plant cell can swiftly change the PM

localization of PINs and therefore alter the direction of auxin cellular efflux, as demonstrated for

PIN3 in gravitropic response [33]. Auxin can slow down PINs' endocytosis in vesicular cycling,

which stabilizes the auxin flux [34].

Arabidopsis genome contains eight PIN genes. Their products have been functionally

characterized with the exception of PIN6 and PIN8 [35]. Specific types of PINs are expressed in

specific tissues in plant body, but any cell is able to express any type of PIN. Particular roles and

spatial localization of PINs have been very well reviewed in [16].

The structure of PIN proteins has been deduced from their known amino acid sequences. As

studied in Arabidopsis, all PINs share two hydrophobic transmembrane domains (amino- and

carboxy-terminal) separated by one central hydrophilic domain with presumable cytoplasmic

orientation. The transmembrane helices are highly conserved (and tolerate no insertions or

14

deletions) while the hydrophobic loop is not so conserved. It exhibits great variability both in

size and sequence. The loop is probably not essential for transporter function, but rather serves as

an activity regulation site [35]. PIN proteins exhibit functional redundancy [36] and a single

mutation has little or no effect on phenotype while some quadruple pin mutants abort after the

very first zygotic division [36]. .

A plant cell possesses mechanisms to control the PINs at many levels: regulation of gene

expression, protein degradation, intracellular trafficking (exocytosis and clathrin-dependent

endocytosis) and protein activity. Auxin itself upregulates the expression of many PINs [36], but

also was shown to downregulate the expression of PIN5 [37] and to stimulate proteasomal

degradation of PIN2 in gravitropic response [38]. PIN activity is regulated by reversible

phosphorylation via serine/threonine kinases, of which PINOID (PID) has been most extensively

studied. It is necessary for a correct apical localization of PIN1 in root tip cells [39] and to have

its expression inducible by auxin [40]. PID's action is counterbalanced by protein phosphatase

2A (PP2A) and probably controlled by other kinases as well. PID phosphorylates PINs,

promoting their apical localization, whereas PP2A dephosphorylates them, inducing their basal

targeting [41].

Auxinic compounds other than IAA, synthetic (NPA) or natural (flavonoids), can also affect

PINs and PAT, albeit not necessarily through expression, but their mechanism of function

largely remains to be uncovered. And of course, many other compounds may indirectly affect

PINs by targeting common cellular machinery. For instance, a loss-of-function mutation in

GNOM (encodes an ARF-GEF for small GTPases) disturbs correct polar localization of PIN1

from inside the cell to the PM by the means of vesicle trafficking [42], though it seemingly does

not affect all trafficking proteins equally [43]. Similarly, Brefeldin A (an inhibitor of Golgi-PM

vesicular transport) causes gradual decrease in auxin efflux [44]; it does not affect PINs already

present on the PM, but hinders those from the inside to come onto it. Cytoskeletal drugs have

similar effect, as the vesicles use cytoskeleton for movement.

Based on the length of the central loop we can divide the PINs into two subgroups: long

ones and short ones. “Long” PINs share long hydrophilic loop, which separates two hydrophobic

domains of about 5 transmembrane helices each and is also their most divergent part. They

localize predominantly to the PM, which suggests their general function in the transport of auxin

out of the cell. PIN1, 2, 3, 4 and 7 are typical long PINs with numerous roles in auxin transport

described (rev. in [35]). In “short” PINs, the central loop is very short. There is also notable

deletion at the C-terminal hydrophobic domain [45]. These are PIN5, 6 and 8. Research on PIN5

has revealed its localization to the membrane of the ER, probably transporting auxin inside the

lumen. By this action, PIN5 apparently regulates auxin homeostasis within the cell [37]. PIN8 is

15

structurally similar to PIN5 and though it has not yet been fully characterised, similar properties

can be expected. It is known that PIN8 expression is significantly upregulated in male

gametophyte [45]. This is, however, magnified by the fact that (like PIN5) it is otherwise poorly

expressed in general. PIN6 still awaits proper description.

3.4 Auxin transport in the development of higher plants

Auxin influences plant development during both embryonic (establishment of polarity,

specification of cell fates) and post-embryonic events (root and shoot organogenesis, leaf

development, vascular development, etc.) and responds to both internal and external cues. Plant

development is disturbed when auxin transport is inhibited. In general, all processes of axis

formation, de novo organogenesis and organ bending are influenced. These processes also

somehow share the mechanisms by which they are performed, i.e. promotion of cell elongation,

division and very importantly triggering asymmetric cell division. The last is sometimes called

“a driving force of differentiation”, where auxin-induced differential expression results in the

accumulation of distinct transcription factors in two daughter cells that subsequently adopt

different fates [46]. The very first zygotic division is an illustrative example.

During embryogenesis, where the role of auxin was superbly reviewed by Smet and Jürgens

[47], the first asymmetric division of the zygote marks the apical-basal axis of plant body plan

and specifies the different fates of the apical and basal daughter cells. The basal cell has its PIN7

auxin efflux carriers localized to its upper part, therefore transporting auxin up into the apical

cell. The apical cell and its descendants first have their PIN1 localized in a non-polar manner,

but from early globular stage (16-32 cell) onward, the whole apical embryo directs its PIN1 and

PIN7 downwards and transport auxin into the basal part (Figure 5). While the apical cell gives

rise to almost the entire embryo, the basal cell divides only a few times into a chain of cells

called the suspensor. From the uppermost cell of this suspensor, adjacent to the apical embryo

and called the hypophysis, the entire root meristem emerges. The PINs, together with local auxin

biosynthesis and diffusion, are the major determinants of auxin transport in embryogenesis,

although other transporters play a role as well [16].

16

Figure 5: Auxin transport in early embryonic development. The role of three PIN and two ABCB transporters is depicted. Reproduced from [16].

Polarized cell-to-cell transport of auxin is further important during post-embryonic development.

During lateral root (LR) formation, where the interplay of phytohormones is critical

(rev. in [48]), polarized transport of auxin helps to generate auxin maxima in the pericycle cells

that precede the formation of lateral root. Several auxin transporters were shown to play a role

during LR initiation and growth. For instance, AUX1 influx carrier helps to accumulate auxin to

initialize LR primordia as it probably determines auxin availability by unloading it from the

phloem [49]. The process of LR development could be divided into four phases: priming,

initiation, growth and emergence. During priming of the pericycle cells, there is a critical role of

basal meristem in the root tip. Here, oscillations in the auxin-driven gene expression correlate

with the formation of the consecutive LR. Therefore, priming of pericycle cells is the process

that takes places already in the basal meristem [22]. The primed pericycle cells, once reaching a

certain distance from the root tip, start dividing again, despite already being far away from the

root's meristematic zone. Here, auxin triggers the reactivation of cell cycle events through the

regulation of E2F levels, which then activates the asymmetric cell division of primed pericycle

cells resulting in lateral root formation [22, 50-51]. Once the primed cells' (usually two, but one

is also possible [52]) cell cycle has been activated, they divide asymmetrically and anticlinally,

each resulting in two different daughter cells (shorter and longer). The shorter cells continue

dividing periclinally and after several rounds of division, a new LR primordium is formed. Once

its apex and quiescent centre are established, a LR becomes an auxin sink. By that time it adopts

the same fate as the main root, following a fountain-like manner of auxin flow.

Apart from roots, auxin is also crucial for shoot development, both embryonic and

post-embryonic (rev. in [53]). Formation of cotyledons, leaves and flowers or phyllotaxy are

17

among the controlled traits. The importance of PIN1 transporter, MP transcription factor and PID

kinase has been well demonstrated [54]. Lateral branching of shoot and the emergence of leaves

start with an active, acropetal auxin transport by AUX1/LAX and PIN1 through outer layers up

to the shoot apical meristem, where it is redirected inwards, to the “zone of competence”. There,

its accumulation marks a new primordium. That becomes an auxin sink, draining auxin from its

vicinity and therefore stopping additional primordia from emerging, thereby partly determining

the phyllotactic pattern. The “zone of competence”, where auxin is accumulated, precedes the

formation of future organ.

PAT defines the vascular patterns of leaves, no matter how complex it gets [55]. Vascular

cells develop from procambium, a meristematic tissue which itself originates from a

subepidermal tissue of its leaf primordium, called the ground meristem. Among four PINs found

in leaves, only PIN1 is expressed in a leaf primordium. In epidermal cells, PIN1 drives auxin

into a specific convergence point, where it externally triggers the patterning of a „zone of

competence“ in the basal meristem below, which will become procambium and subsequently a

vein. Once specified, the zone becomes an auxin sink and a vein is formed. Higher order veins

are later established to transport auxin from more peripheral leaf parts to the mid-vein. Despite

the attractive complexity of venous formations, computer modeling suggests only two types of

vein onthogeny are sufficient to cover all venous patterns of Arabidopsis [55]. Furthermore, a

possible auxin-independent determination of zones of competence, as a way to explain venous

complexity, obviously does not take place. In Arabidopsis, the zones' positioning is a self-

organizing, auxin-dependent process. Simply put, auxin is transported and canalized by PIN1

into narrow lines, which later become veins [56].

Auxin transport has been shown to regulate plant growth responses to their environment by

promoting cell elongation on one side of the growing organ (reviewed in [16]). This mostly

includes responding to gravity (Figure 6) and light and results in organ bending. While higher

intracellular auxin concentrations promote elongation in shoots, they inhibit it in roots. Both of

these processes seem to be regulated by PIN3 auxin efflux carrier [33], as pin3 loss-of-function

mutants are defective in both photo- and gravitropism. Root gravity-detecting cells are found in

the root cap (starch sheath, columella) and their PIN3 exhibits uniform localization under normal

conditions. This changes quickly upon gravistimulation, as PIN3 undergoes rapid vesicular

cycling. Auxin is then transported downwards (in respect to gravity) and once it reaches the root

periphery (epidermis and cortex), it joins the classic basipetal transport mediated by PIN2. This

flow is further enhanced by PIN2 degradation on the opposite site [38]. The resulting increased

auxin concentration in the root's lower part inhibits cell elongation, causing the apical part to

outgrow it; a root bends downwards, aka positive gravitropic response. Other types of

18

transporters also play a role here, e.g. the AUX1 knock-out causes an agravitropic phenotype

(distorted root waving) [23]. In shoots, gravity is detected within endodermis. By a similar

mechanism, auxin accumulates to the lower side of the shoot via PIN3 mediated transport and

promotes cell elongation. Thus, the shoot bends upward, a negative gravitropic response.

Actually, little is known about the background of phototropic response, mostly observed on

etiolated seedlings (rev. in [57]). Similarity to gravitropism is expected, but today, it is only clear

that it requires all types of known auxin transporters – PINs [33], AUX1 [58] and ABCB [59].

The phototropic signaling pathway begins with phototropins (phot1 and phot2), which belong to

the AGC VIII family of plant specific protein kinases. Note that PID, a kinase of PIN auxin

efflux carriers, is a member of this family as well. It is expected that auxin transport and

signaling act downstream of phototropins. Yet so far, the only known substrates of phototropins

are phototropins themselves through their autophospohorylation.

Figure. 6: Root gravitropic response. Green gradient - auxin levels. Reproduced from [2].

4 Auxin during the evolution of land plants

4.1 Current opinion on early land plant phylogeny

Many studies have been dedicated to uncovering the divergence points of major plant

lineages and the corresponding events, using both molecular and fossil data. While the fossils

provide a more straightforward information, molecular estimates may largely vary [60]. Using

both approaches is obviously the right way to go.

It is widely accepted that Embryophyta (land plants) evolved from Streptophyta green algae

(also known as Charophyta). These algae possess only one haploid generation in their life cycle

with zygote being the only diploid cell that immediately undergoes meiosis. The streptophytes

19

themselves likely split from Chlorophyta (all other green algae) in between 725-1200 Mya

(different estimates by molecular clock methods) [61]. They both comprise a clade Viridiplantae,

the “green plants” (after chlorophylls a and b). Many important cellular structures

(phragmoplast, plasmodesmata, hexameric cellulose synthase, sporopollenin) and physiological

characters (photorespiration, phytochrome system) originated within the Streptophyta [61]. Two

groups, Charales and Coleochaetales, possess true multicellularity with plasmodesmata, apical

growth and oogamous sexual reproduction. While the phylogeny of the more advanced groups

remains a matter of debate, the simple unicellulate Mesostigma and Chlorokybus reliably

comprise a sister clade to all other Streptophyta and Chlorophyta, i.e. diverged early and stand at

a transition point [62].

The first proto-land plants are believed to have transferred to terrestrial habitat about 500-

470 Mya, i.e. the Ordovican period. Throughout the following ages, they literally created

terrestrial ecosystems, which could later be colonised by animal organisms, and greatly increased

the atmospherical concentration of oxygen. Since then, Bryophyta, Pteridophyta and

Spermatophyta have emerged, the last ones becoming the most successful to these days (rev. in

[63]). This more „recent“ evolution of land plants appears to mainly utilize expansion of genes

and their differential expression rather than sequence variations. Horizontal gene transfer is

extremely rare, except for mitochondrial genes (rev. in [61]).

Figure 7: Simplified phylogeny of green plants (Viridiplantae). Some representative species are mentioned. Reproduced from [82].

20

It is not uncommon to find a stonewort (Chara sp.) illustrated in a sister relationship to land

plants, based on the assumption that the ancestors of land plants evolved towards rising

morphological and cellular complexity. One report brought support for this claim [64] and other

authors conclude the same, though not very convincingly [61]. Further analysis, however, cast a

shade of doubt on this hypothesis. Reviewing chloroplast genome of Chara vulgaris (and

comparing it to lower and higher taxa) has uncovered rather more distant relationship to land

plants. Zygnematales, the conjugating algae, were identified as the closest relatives of land plants

[65]. Similar study with nuclear genes has determined Coleochaetales as the closest living

relatives [66]. And the most recent research [63], reviewing a large set of nuclear genes for 40

green plant taxa (incl. 6 major Strepthophyta), once again sets Zygnematales (or less probably a

clade of Zygnematales and Coleochaetales) as a sister group to land plants. Yet, all of these data

still do not provide a firm, absolute identification.

4.2 Auxin in bryophytes

The bryophytes (mosses, hornworts and liverworts) represent the most basal lineage of land

plants. They supposedly separated about 400 Mya, a mere 50 Mya after the actual separation of

land plants themselves [60-61]. They also differ quite a lot from the rest of the group, as seen

from the domination of gametophyte in their life cycle and the absence of vascular tissues,

genuine roots, stalks and leaves.

Since the available fossils suggest that the first land plants were moss-like organisms [67],

they would go for a very convenient model to uncover the aspects of floral transition to land.

One moss genome is already known: Physcomitrella patens. It has lost the algal aquatic-related

genes and has not yet developed a plethora of genes for coping with dry land. The putative

moss-like last common ancestor of all land plants has most probably lost aquatic-related genes

and dynein-transport while gaining better tolerance to abiotic stresses, auxin, cytokinin and ABA

signaling [67]. However, even though the number of genes dedicated to auxin is smaller in

comparison with Arabidopsis [68], the essential proteins for advanced auxin biosynthesis

(YUCCA), transport (PINs and AUX/LAX) and signaling are present, including the SCFTIR1/AFB

pathway [67-69]. These facts suggest a probable importance of auxin during and/or for the

conquest of land. The expansion of gene families in more developed (vascular) plants probably

took place after gene duplications of duplications of the entire genomes, which allowed

additional functions and adaptations. The development of rhizoids (muticellular filaments of

epidermal origin) is auxin-dependent in Physcomitrella [70]. In general, it seems that the auxin-

driven mechanisms are already on a very high level in bryophytes. In Physcomitrella patens,

these mechanisms seem to be restricted to rather sporophyte than gametophyte, as indicated by

21

auxin efflux inhibitor treatments [71]. Here, the reduced sporophyte seemingly exhibits polarized

auxin flow and sensitivity to its inhibitor (NPA), while the dominant gametophyte shows apolar

auxin distribution. Moreover, applied auxin transport inhibitors did not induce any

morphological changes.

Altogether, experiments performed in bryophytes suggest a correlation between PAT and

the transition to sporophytic dominance.

4.3 Auxin in algae?

Since the seaweed extracts can successfully be used as a fertilizer, the question arises about

the presence of plant-specific growth stimulants, like phytohormones. Indeed, these have been

found in various algal taxa, or at least a response to its exogenous application was observed

(rev. in [72]). Yet, that was about everything that was known for quite a long time. Although

detailed research is still largely impossible due to lacking genomic data, some intriguing results

have already been obtained by recent studies.

4.3.1 Brown and red algae

Among brown algae (Phaeophyta), the auxin-related physiology has been mostly studied in

rockweed Fucus distichus. Here, IAA has been detected in concentrations very close to those of

higher plant tissues, both in zygotes and in adults. Furthermore, Fucus embryo accumulates

auxin (IAA) in its early stage. The movement of IAA into a cell is probably of a diffusive nature

rather than through a carrier, since its high extracellular concentration does not inhibit auxin

accumulation in a saturable manner. NPA, however, does promote IAA accumulation,

supposedly by blocking an unknown efflux carrier, the way it does in land plants. Growth on

IAA or NPA causes emergence of multiple rhizoids or a branched rhizoid. Normally, there is one

unbranched rhizoid. This could be a result of misdirected axis of division, which then would

have been established in an auxin-dependent manner [73]. This was further corroborated by

finding out that exogenous IAA and NPA disturb the internal polarization of early Fucus

embryos in response to light and gravity, the latter being depended on under dark conditions

[74]. Possible connection of these events with the actin cytoskeleton was also suggested [74], as

latrunculin B inhibits auxin transport. It is tempting to suggest a resemblance to the actin-

mediated vesicular cycling of PINs in land plants.

Apart from Fucus, two studies proposed the involvement of auxin in the development of a

filamentous Ectocarpus [75-76]. They highlighted its auxin biosynthetic capabilities, based on

genomic evidence, plus possible signaling pathway. Auxin is located mainly in the apices of its

filaments.

22

In red algae (Rhodophyta), there has been very little research performed with respect to the

role of auxin so far. There are relatively recent reports on IAA having positive effect on growth

of Gracilaria tissue cultures [77] and of endogenous auxins, cytokonins and even ABA having

been extracted from multiple Rhodophyta species [78], but a mere presence of auxins is not an

uncommon feature.

4.3.2 Green algae; Chlorophyta and Streptophyta

Reports on auxin presence in green algae are numerous and reach far into history.

Experiments on effects of exogenously applied auxin also exist, such as stimulation of cell

division in unicellular Chlorophyta [79] or elongation in filamentous Streptophyta [Aldorfová et.

Vosolsobě, unpublished data]. Yet, the search for possible auxin transport or signaling

machinery by conventional methods has mostly come fruitless. In Chara, however,

distinguishable auxin influxes, possibly mediated by carriers, were recognized [80] and there is

also a most recent evidence for active directional auxin efflux [81], which is a hallmark of auxin

in land plants. During recent years, possible orthologs of auxin-associated genes known from

sequenced land plants are slowly identified. Two major searches in green algae using sequences

from Arabidopsis thaliana, have been performed [79, 82], the latter even considering two

Streptophyta (filamentous Spirogyra pratensis and primitive multicellular discoid Coleochaete

orbicularis). However, these sequences still wait for their functional characterization.

Chlorophyta generally seem to possess many orthologs of auxin biosynthesis, but not of

auxin metabolism [82]. There is no evidence for complete proteins of the SCFTIR1/AFB signaling

pathway in Streptophyta or Chlorophyta, though potential orthologs of single domains can be

found [79]. The available information is, in fact, so convincing to state that the SCFTIR1/AFB

pathway is absent in green algae, at least in the inspected taxa. Interestingly, the orthologs of

TOPLESS, the transcriptional co-repressors of Aux/IAAs, were identified in both investigated

Streptophyta, making them the first lineage with their appearance during evolution. This is very

strange considering that there are probably no Aux/IAAs in Streptophyta. And lastly, the

orthologs of ABP1 and IBR5 were found in both groups, clearly for IBR5 and less clearly for

ABP1 [79, 82].

The AUX1/LAX influx carriers were not identified in Chlorophyta, except for one clear

ortholog in Chlorella [82], which is certainly odd. The MDR/PGP/ABCB efflux carrier

orthologs were identified in all inspected green algae [82]. This is not surprising, as the ABC

family is one of the most ancient in all living organisms. It is also so functionally diverse that it

is almost impossible to identify strict orthologs based solely on sequence data. As for PINs, there

is no evidence of their presence in Chlorophyta. However, a putative PIN gene ortholog has been

23

found in the genome of Spirogyra pratensis [82]. We do not know anything about its real

function, but it gives us hope and inspiration at least.

4.4 PINs again

So far, auxin efflux carriers from PIN family seem to be restricted to land plants. Inside this

group, however, they have undergone changes during evolution, both in structure and in number.

A notable example is the difference between the PINs of dicots and monocots. While the dicot

PINs have been broadly conserved, the monocot PIN family shows much more divergent

phylogenetic structure. For instance, wheat (Triticum aestivum) possesses three orthologs of the

dicot PIN1 in its genome (Figure 8). Moreover, there is one specific monocot PIN (TaPIN9 in

wheat, aka OsPIN9 in rice), which does not cluster with any dicot sequences. A role in some

monocot-specific feature is expectable, such as the characteristic root system or phyllotaxy [45].

Figure 8: Phylogenetic tree for PINs of four dicots. The early divergence of short PINs 5, 8 and 6 can be seen, as the similarity of long PINs. Reproduced from [45] At: Arabidopsis thaliana, Mt: Medicago truncatula, Gm: Glycine maxsequence, St: Solanum tuberosum

AtPINs of Arabidopsis thaliana share similarity ranging from 32% between AtPIN5 and

AtPIN8 to 85% between AtPIN3 and AtPIN7. These values are high and suggest an evolutional

divergence of PINs from one ancestral sequence [45]. That one was probably similar to PIN5, a

short PIN. The short PINs, relatively less similar to each other in sequence, appear to be

ancestral (Figure 8). Their role in intracellular auxin homeostatis might very well have been their

original function, before they were „chosen“ to direct PAT at the plasma membrane.

An interesting phylogenetic study has been carried out on the plant specific AGC VIII

24

protein kinases [83], of which the PID kinase of PIN1 is a member. Phototropins, the blue light

sensors involved in phototropic response, belong here as well. Of the known two, PHOT2 is

found in Chlorophyta, while PHOT1 is restricted to Spermatophyta and probably originated from

a duplication of the previous. They are generally of an ancient origin and a phototropin of

Chlamydomonas can partially restore Arabidopsis double mutant phot1/phot2. As the light-

activated phototropins control tropic responses by regulating the levels of Ca2+ and PID is

regulated by calcium-binding proteins [84], it is probable that one type of AGC kinases acts

downstream of others. Other AGC kinases related to PID (called WAG) also localize to the

plasma membrane and are likely to at least take part in PAT, as the wag loss-of-function

phenotypes suggest. Apart from using the AGC VIII to reconstruct plant phylogeny, the study

brings the idea on how phototropins used auxin to expand their functions, further contributing to

the co-evolution of related genes together with those of PAT.

A very recent study credibly demonstrated a new type of carriers similar to the ancestral

short PINs and named them PIN-LIKES (PILS) [85]. Like PIN5, 6 and 8, they also localize to

the membranes of ER and could be important for control of the intracellular availability of auxin

molecules. Unlike PINs, however, they are found even in unicellular Chlorophyta. This will

boost the hope of those who seek to uncover the evolution of auxin transport and corroborates

the hypothesis that storing auxin within the ER is one of the most ancestral features in green

plants.

4.5 Obstacles and future expectations

Although the idea of directional auxin transport and its possible evolutional importance is

old, the key representatives in the related processes have been identified only recently and many

auxiliary proteins are still unknown. On one hand, we have mosses, which already seem to

possess a high level of auxin transport and signaling. On the other hand, there are green algae,

which in comparison seem to only have some components of this machinery. While the protein

sequences from land plants like Physcomitrella, Oryza or Arabidopsis are known, very few

genomes of green algae have been described so far. The available data concerns almost

uniformly unicellular organisms. Except for EST libraries and chloroplast sequences, there is

essentially no genomic mapping made on Streptophyta. Another obstacle is that methods, which

work well in land plants do not necessarily have to work in algae. For instance, classical studies

on polar auxin transport with NPA will produce little results for multicellular thalloid Chara

vulgaris, as it obviously lacks phytotropin receptors [80]. Also, a courageous approach of trying

to express land plant carrier proteins in algae may crash on the inability of their expressing

machinery to cope with it, as there are expectable differences in promoter or regulatory domains.

25

Only once a lot more genomic information is available we can uncover sequence of analogous

proteins and test their function. It is not unlikely that many plant “innovations” will actually turn

out to be those of Streptophyta, including auxin machinery. For now, we stand at the start of our

long trip back in time. The author of this bachelor thesis plans to isolate genes for auxin carrier

homologs from streptophyte algae as a part of his diploma studies.

Figure 9: Phylogenetic scheme of green plants in regard to auxin-related mechanisms and proteins.Notes indicate the earliest evidence. See the text for corresponding references. Keep in mind that most of the information is based on genomic data and has not been verified experimentally.

5 Discussion

Although direct evidence is missing, auxin is sometimes proposed to play a role in some

major evolutionary event in plant lineage. One suggestion would be the transition of plants to

land (e.g. [83]). This can be true for the sophisticated auxin signaling and directional auxin

transport, since these mechanisms are largely absent in green algae (Figure 9). However, with the

recent evidence of PAT in stonewort and discoveries of various possible orthologs in algae and

even the ER-resident PIN-like proteins in chlorophytes and streptophytes, auxin might have been

establishing itself long before that. Auxin was perhaps a key player in the evolution of complex

multicellularity within the green plants. During the events like transition to land and/or the

evolution of more complex sporophytic plant body plans the importance of phytohormones

probably has been extended.

Chlorophyta Streptophyta green algae green algae

Embryophyta

auxin biosynthesis, metabolism, response to exogenous auxin, ABP1 and IBR5 signaling, PILs, ABCB/PGP efflux, AUX1/LAX influx (Chlorella)

polar auxin transport in gametophyte (Chara), PINs (Spirogyra)

polar auxin transport in sporophyte, Aux/IAA-ARFs

Streptophyta

Viridiplantae

26

Figure 10: Diversification of green plants (Viridiplantae) and colonisation of terrestrial habitats by streptophyte algae. (A) Early evolution of viridiplant algae; (B) Colonisation of land by streptophytes; (C) Origin of current freshwater ecosysthems. The smaller freshwater body represents an acidic bog/pool with Zygnema-like algae. Different green shades illustrate the two major groups: dark green (Streptophyta) vs. bright green (Chlorophyta). Reproduced from [61].

It is quite surprising that the “land plants” have evolved solely within the Streptophyta clade.

Terrestrial microalgae are present in several other groups and marine macroalgae dominate

shallow seawaters to these days. Why have they never followed the early streptophyte

algae/plants to land? Speculations to this question have been superbly brought together by

Becker and Marin (2009) [61]. Ever since their appearance, the streptophyte algae had favoured

freshwater habitat and have not abandoned it to this day (Figure 10). It is interesting to note that

30

27

the animal groups, which dominate land habitats to date are also believed to have transferred to

land from freshwater; even more, they are still completely (amphibians, insects) or largely

(mammals, birds, reptiles) absent from marine environment. In contrast to freshwater/land, the

marine/land differences reach much further; salinity, temperature extremes or exposure to

radiation. It is tempting to suggest the freshwater ancestry as a necessity for the transition to dry

land, as the border between a beach and the ocean might have been an uncrossable line. The fact

that land plants have never been defeated by any other group seems to originate from them

originally colonising a relatively empty niche. If any other algae could really emerge from the

salty sea after them, it would lose the competition with the already well established and adapted

embryophytes.

While the streptophytes have certainly outdone themselves on land, their original freshwater

habitat has been largely taken from them. Today, merely 122 of their algal genera remain. As it

appears from Charales fossil record, they were decimated by two mass extinction events; the

Permian/Triassic (250 Mya) and Cretaceous/Tertiary (65 Mya), by the way marking the rise and

fall of a dinosaur age. After the first extinction, a tragedy struck; the primarily marine

Chlorophyta invaded freshwater on a large scale. Chlorophyceae and Trebouxiophyceae almost

entirely switched their environment. Soon they dominated freshwater phytoplankton and

apparently ended the supreme rule of Streptophyta. Their timely invention of phycoplast, which

can enable more complex multicellular body plan based on cellular division, also might have

coincided. Unfortunately, only Charales display a satisfactory fossil record for any conclusion. It

is also unknown whether the recent Quarternary ice ages played any significant role. Yet,

compared to what they once might have been, recent Streptophyta probably represent a ghost of

a past age.

Zygnematales are the most species-rich group of streptophyte algae to date. They also have

been recently proposed to be a sister group to embryophytes. Being both unicellular and

multicellular, they discarded flagellate cells and use conjugation as means of sexual

reproduction. This and their overall morphological simplicity in fact might have been a

secondary adaptation to enable sexual reproduction outside of free water. Alternatively, the

complexity of Coleochaetales and Charales might have evolved independently, as evolution of

complex structures seems to rely on modification of genes in a not necessarily complex ancestor

(as shown for Volvox [86]). It is also noteworthy that Zygnematales (similarly to embryophytes)

have been proposed to evolve faster than the rest of Streptophyta; for instance twice as fast as

Coleochaete [63]. If auxin signaling and regulated auxin transport is really connected to the

evolution of complex multicellularity, it would have appeared firstly in these or similar taxa.

While auxin is not necessary for establishing simple filamentous or mat-like body plans (like

28

Spyrogyra or Coleochaete), it would lead them towards the stem like structure with rhizoids and

phylloids, as in the first bryophyte-like land plants.

The recent discoveries of a putative PIN ortholog in Spirogyra and PIN-like sequences in a

wide range of embryopytes, streptophytes and chlorophytes (even unicellular) opens a new door

to study the origins of auxin transport. These PIN-like genes, based on both predictions and

experimental results, display a characteristic ER-localization. Just like the short PINs from

Arabidopsis. Therefore, it seems that auxin homeostasis and compartmelization into cellular

organelles, where it is converted into intermediates, is the ancient function, to which the green

plant cells have developed specific mechanisms. In fact, it is not even surprising. Being a simple

organic acid, auxin initially may have been just a metabolic intermediate, possibly of the

tryptophane metabolism. Beside exocytic pathway and evolution of regulated efflux, the means

of intracellular compartmelization were introduced. Later, within the primitive multicellular

colonies, the cells were still getting rid of auxin. Yet, it could become a feasible marker of

environmental conditions. When some of the cells grew and divided, their metabolism would

also increase, as would the excretion of auxin. This would give a clue to all other cells, in the

inner layers of a colony for instance, that the time is right to divide and grow. In this moment,

the first signaling pathway would have been established. The fact that auxin goes in via passive

diffusion makes it even more feasible. The first of many roles of auxin, i.e. promoting cell

elongation and division, would be underway. Auxin would become more than just a

“metabolite”. During subsequent evolution, auxin would be recruited for a plethora of other

functions that would arise. Using auxin signaling pathways would be convenient, because the

appropriate pathways would already be present. Let us not forget that there are also other

phytohormones that differentiate plants through their diversification, like ABA or ethylen, and

display a broad variety of functions. Regarding the evolutionary diversification of auxin carrier

proteins, the agents involved are numerous and often functionally redundant, at least in

Angiosperms. In Arabidopsis auxin is mobilized by ATP-driven transport (ABC), H+-symporters

(AUX1/LAX) and gradient driven-carriers (PIN). It was suggested by Zažímalová et. al. [87]

that this had been a way to adapt to evolutionary changes. When one type of carrier would be

insufficient or inhibited by a newly acquired function, another would have to substitute for it.

One very interesting matter, which unfortunately does not raise much attention, would be

Phaeophyta, the brown algae. Some experiments have shown the effects of auxin on their

development, particularly in Fucus, which also somehow appears to be the only brown alga used

in research so far, though Ectocarpus was recently introduced. Despite the great evolutionary

distance from green plants (they even developed multicellularity independently), auxin does not

seem to be unfamiliar to them. It even looks like there might be elements of both auxin signaling

29

and transport present, but completely different from those of land plants. Only the studies on

Ectocarpus suggested similarity of auxin biosynthesis genes to land plants. However, there has

been little or no research so far and it is even not quite possible yet, due to the lack of genetic

information. The conventional methods of exogenous auxins and PAT inhibitors applications

known in higher plants cannot produce many reliable conclusions in such a distant lineage. If the

brown algae do have their own auxin signaling and transport, it would be a remarkable example

of a convergent evolution, which would perhaps allow for the development of complex

multicellularity like it might have in land plants, resulting in the breath taking kelp forests of

California. If they do not possess these auxinic tools, they would then develop all of these traits

without the need for auxin, which would also be a valuable find, alhough it might have cost them

their place on the dry slopes of Sequoia National Park.

In the end, there is much to uncover and more to be known yet before we can do it. Auxin

certainly offers a life-long engagement to any interested researcher. Whatever we may find can

contribute to our better understanding of plant lineage, its evolution or otherwise.

30

Abbreviations

PAT = Polar Auxin Transport

PM = plasma membrane

ER = endoplasmic reticulum

ABA = abscisic acid; a phytohormone of inhibitive nature

NPA = naphtylphtalamic acid; widely used inhibitor of PAT

2,4-D = 2,4-dichlorophenoxyacetic acid; synthetic auxin used in research and as herbicide

NAA = naphtalene acetic acid; another synthetic auxin

BFA = brefeldin A; inhibitor of trans-Golgi vesicular transport

TIR1/AFB = Transport inhibitor response 1/Auxin-binding F-Box protein

Aux/IAA = Auxin/indole-3-acetic acid

ARF = Auxin response factor

SCF = (S-phase kinase-associated protein 1)-cullin-F-box protein

ABP1 = Auxin binding protein 1

IBR5 = Indole-3-butyric acid response 5

MDR/PGP/ABCB = Multidrug resistant/phosphoglycoprotein/ATP-binding cassette subfamily B

TPL = TOPLESS; knocked-out embryos develop a second root instead of shoot

31

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