Journal of Experimental Botany, Vol. 66, No. 16 pp. 4897–4912, 2015
doi:10.1093/jxb/erv189 Advance Access publication 28 April 2015
REVIEW PAPER
RNA processing in auxin and cytokinin pathways
Mónika Hrtyan*, Eva Šliková*, Jan Hejátko and Kamil Růžička†
Department of Functional Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, Brno,
CZ-62500, Czech Republic
* These authors contributed equally to this work.
† To whom correspondence should be addressed. E-mail: [email protected]
Received 9 February 2015; Revised 16 March 2015; Accepted 19 March 2015
Abstract
Auxin and cytokinin belong to the ‘magnificent seven’ plant hormones, having tightly interconnected pathways leading to common as well as opposing effects on plant morphogenesis. Tremendous progress in the past years has
yielded a broad understanding of their signalling, metabolism, regulatory pathways, transcriptional networks, and
signalling cross-talk. One of the rapidly expanding areas of auxin and cytokinin research concerns their RNA regulatory networks. This review summarizes current knowledge about post-transcriptional gene silencing, the role of noncoding RNAs, the regulation of translation, and alternative splicing of auxin- and cytokinin-related genes. In addition,
the role of tRNA-bound cytokinins is also discussed. We highlight the most recent publications dealing with this topic
and underline the role of RNA processing in auxin- and cytokinin-mediated growth and development.
Key words: Auxin, cytokinin, gene silencing, miRNA, non-coding RNA, RNA processing, splicing, tRNA.
Introduction
Auxin is an essential plant hormone responsible for almost
all aspects of plant growth and development, which includes
cell division and expansion, apical–basal axis formation,
embryogenesis, meristem formation, or tropisms (Zazimalova
et al., 2014). Auxin can be produced by multiple biosynthetic
pathways (Brumos et al., 2014) and, according to classical
concepts, it is polarly transported from the source tissues
towards the root (Went, 1927). Auxin enters the cell by passive diffusion or via AUXIN RESISTANT 1/LIKE AUXIN
(AUX1/LAX) transporters (Yang et al., 2006). The direction
of the flow is controlled by polarly localized efflux carriers
of the PIN-FORMED (PIN) family which, together with
PIN-LIKES (PILS) and ATP binding casette B (ABCB)
transporters, maintain auxin gradients that are essential for
pattern formation (Petrasek et al., 2006; Barbez et al., 2012).
Intracellular auxins are perceived by six partially redundant
receptors of the TRANSPORT INHIBITOR RESPONSE 1/
AUXIN SIGNALLING F-BOX (TIR1/AFB) family, consisting of two phylogenetic clades in Arabidopsis thaliana
(TIR1/AFB2 and AFB4; Dharmasiri et al., 2005; Kepinski
and Leyser, 2005; Parry et al., 2009). They form a co-receptor
complex with transcriptional repressors AUXIN/INDOLE3-ACETIC ACID (AUX/IAA) which are ubiquitinated and
degraded after auxin binding. This leads to the release of primary auxin-responsive genes regulated by developmentally
important AUXIN RESPONSE FACTORS (ARFs) that
control primary auxin response genes (Ulmasov et al., 1997;
Gray et al., 2001). Auxin responses in various tissues depend
on a co-ordinated regulation of gene expression, transcript
abundance, and stability of key components of the auxin signalling machinery (Zazimalova et al., 2014).
Cytokinin signalling in plants is mediated by a multistep
phosphorelay pathway. Cytokinins bind to the membranelocalized histidine kinase receptors of the ARABIDOPSIS
Abbreviations: AS, alternative splicing; cZ, cis-zeatin; cZRMP, cis-zeatin riboside monophosphate; dsRNA, double-stranded RNA; DMAPP, dimethylallyl pyrophosphate; eTM, endogenous target mimic; IAA, indole-3-acetic acid; lncRNA, long non-coding RNA; m7G, 7-methylguanosine; pre-miRNA, precursor miRNA;
pri-miRNA, primary miRNA; PTGS, post-transcriptional gene silencing; RMP, riboside monophosphate; siRNA, small interfering RNA; sRNA, small non-coding RNA;
tasiRNA, trans-acting siRNA; tZ, trans-zetin; uORF, upstream open reading frame; UTR, untranslated region.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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4898 | Hrtyan et al.
HISTIDINE KINASE (AHK) family (Inoue et al., 2001;
Higuchi et al., 2004; Nishimura et al., 2005). This, in turn,
leads to phosphorylation of histidine on ARABIDOPSIS
HISTIDINE PHOSPHOTRANSFER PROTEINS (AHPs).
There are five canonical AHPs (AHP1–AHP5) in the A. thaliana genome, and AHP6, a pseudo histidine phosphotransfer
protein which lacks the key histidine residue and inhibits cytokinin signalling (Hutchison et al., 2006; Mähönen et al., 2006).
AHPs mediate the phosphotransfer to a conserved aspartate
residue on nuclear-localized ARABIDOPSIS RESPONSE
REGULATORS (ARRs). There are three types of ARRs:
type-A, -B, and -C. The type-B ARRs act as positive regulators of cytokinin signalling, whereas the type-A ARRs are
cytokinin primary response genes and together with probably
some type-C ARRs are mainly negative regulators of cytokinin signalling (Hwang and Sheen, 2001; Sakai et al., 2001;
To et al., 2007). Other proteins involved in cytokinin signalling
are CYTOKININ RESPONSE FACTORS (CRFs) of the
AP2/ERF-type transcription factor family, which are induced
by cytokinin and share their targets with type-B ARRs
(Rashotte et al., 2006; Kieber and Schaller, 2014).
In eukaryotic cells, the main checkpoints in the regulation
of gene expression occur at the transcriptional, post-transcriptional, translational, and post-translational level. At the co- or
post-transcriptional level, primary transcripts are converted into
functionally competent, mature forms of mRNA by mechanisms
collectively known as RNA processing. This involves the following steps: capping, polyadenylation, and splicing (Proudfoot
et al., 2002). Capping includes the addition of 7-methylguanosine (m7G) at the 5′ end of pre-mRNA during transcription,
which protects the 5′ end of the primary RNA transcript against
degradation and has a positive role in the recognition of mRNA
by the ribosome during the initiation of translation (Lewis and
Izaurralde, 1997; Gu and Lima, 2005). Polyadenylation involves
the endonucleolytic cleavage of a specific site located at the 3’
end of pre-mRNA, followed by the addition of 150–200 adenosine residues. It is essential for mRNA stability, transport to
the cytoplasm, and translation (Colgan and Manley, 1997; Lutz
and Moreira, 2011). During splicing of pre-mRNA, introns are
removed from the primary transcript and exons are assembled
to form mRNA (Stamm et al., 2005; Kelemen et al., 2013).
Subsequently, mRNA is exported from the nucleus and translated, which is coupled with its quality control and the regulation of its degradation (Bassett, 2007; Braun and Young, 2014).
Eukaryotic genes can be functionally separated into two
main groups: protein-coding genes and RNA genes, transcribed into non-protein-coding (or non-coding) RNAs.
Non-coding RNAs represent a significant part of the transcriptome (Zhu and Wang, 2012). They include highly abundant and functionally important RNAs such as tRNAs or
rRNAs, and also RNA species with regulatory functions:
small non-coding RNAs (sRNAs) and long non-coding
RNAs (lncRNAs) (Erdmann and Barciszewski, 2011). Noncoding RNAs are abundant and functionally important, and
are involved in various processes such as the regulation of
translation, RNA splicing, transcriptional regulation, and
antiviral defence (Erdmann and Barciszewski, 2011; Martínez
de Alba et al., 2013).
Modification of nucleotides in tRNA as a
source of bioactive cytokinins
Various RNA species can be covalently modified, which
has diverse effects on their stability and functional properties, including the coding information in mRNA and tRNA
(Machnicka et al., 2013). Natural cytokinins are derivatives of
adenine with an isoprene (isoprenoid cytokinins) or aromatic
(aromatic cytokinins) side chain attached at the N6-terminus.
Such adenine derivatives as isoprene cytokinins were found
to be present in tRNA (Biemann et al., 1966; Chen and Hall,
1969; Kieber and Schaller, 2014).
Isoprenoid cytokinins are among the most abundant
classes of cytokinins in plants. Besides N6-(isopentyl) adenine (iP) and dihydrozeatin (DHZ), the most common representative is zeatin. It exists in two isomers—trans (tZ) and
cis (cZ)—depending on the position of the terminal hydroxyl
group on the isoprenoid side chain. Several studies tend to the
conclusion that, in most plants, cZ shows practically no biological activity compared with tZ (Kieber and Schaller, 2014).
However, it was revealed that cZ is perceived by cytokinin
receptors in rice (Yonekura-Sakakibara et al., 2004) and it
was also found that cZ also has some physiological effects in
eudicots (Gajdošová et al., 2011; Köllmer et al., 2014).
Biosynthesis of free cytokinin occurs principally through
two different pathways: by de novo biosynthesis or by the degradation of tRNA containing cytokinins. The combination
of both mechanisms is also possible. De novo biosynthesis is
a direct and most common pathway. It is based on the conversion of AMP/ADP/ATP and dimethylallyl pyrophosphate
(DMAPP) catalysed by adenylate isopentenyltransferases
(adenylate-IPTs), which leads through several additional
steps to active cytokinins. The last step in this pathway is
catalysed by the LONELY GUY (LOG) enzyme, which was
first identified in rice (Oryza sativa), based on a mutant with
a severely reduced number of floral organs (Kurakawa et al.,
2007; Kamada-Nobusada and Sakakibara, 2009). The second pathway is indirect; it involves the addition of DMAPP
to adenine A37 on tRNA (prenylation of tRNA), followed
by the release of cytokinin nucleotides by tRNA degradation,
and by the removal of the phosphoribosyl moiety by LOG
(Fig. 1; Kurakawa et al., 2007; Kieber and Schaller, 2014).
tRNA prenylation is catalysed by tRNA isopentenyltransferases (tRNA-IPTs), and the IPT2 and IPT9 proteins are
required for this process in A. thaliana (Golovko et al., 2002;
Miyawaki et al., 2006). Importantly, ipt2 ipt9 double mutants
show undetectable levels of cZ-type cytokinins (Miyawaki
et al., 2006).
Molecules identical to cytokinins in their tRNA-bound
form were also found in many other eukaryotes, including
metazoans, and in bacteria (Biemann et al., 1966; Persson
et al., 1994). In plants and other organisms, the modified
adenine base is situated at the conserved position 37 within
tRNA, adjoining the anticodon sequence and showing preference for the UNN codon (NNA anticodon; Fig. 1) consensus (Taller, 1994; Yevdakova et al., 2008). This nucleotide
modification in tRNA may play a role in the regulation of
gene expression. For example, it is well documented that the
Auxin and cytokinin RNA processing | 4899
efficiency in fission yeast (Lamichhane et al., 2013), and yeast
mod5 mutants, affected in a gene homologous to IPT2, were
isolated as extragenic suppressors of a missense gene mutation (Laten, 1984).
Although ipt2 ipt9 double mutants show perturbed cytokinin levels and severe cytokinin-related defects (Miyawaki
et al., 2006; Köllmer et al., 2014), the function of tRNA-IPTs
is deeply conserved, as yeast strains with mutated tRNA-IPT
MOD5 can be functionally complemented by A. thaliana
IPT2 (Golovko et al., 2002). Therefore, it would be interesting to speculate whether tRNA-bound cytokinins may also
affect translation in higher plants, in addition to their role in
maintaining overall cytokinin homeostasis.
Post-transcriptional gene silencing of
auxin- and cytokinin-related genes
Fig. 1. Basic model of cis-zeatin (cZ) synthesis from tRNA in A. thaliana.
tRNA-IPTs (IPT2 and IPT9) catalyse the addition of the isopentenyl moiety
from dimethylallyl pyrophosphate (DMAPP) to position A37 of tRNA.
This results in the formation of prenyl-tRNA. In the next step, cis-zeatin
riboside monophosphate (cZRMP) is released by degradation of prenyltRNA. cZRMP can be converted directly by LONELY GUY (LOG) proteins,
or indirectly through other metabolites, to cZ.
deficiency of methylthiolation in tRNA-Lys (UUU) at the
A37 position leads to a mistranslation of the lysine codon
from the proinsulin gene and causes a decrease in its synthesis (Wei and Tomizawa, 2012). Importantly, it was also
found that the isopentenylation of A37 mediates translation
In most eukaryotes, including plants, the expression of many
genes is regulated by RNA-mediated processes known as
post-transcriptional gene silencing (PTGS). PTGS is frequently accompanied by the presence of ~21–25 nucleotide,
sRNAs, typically represented by microRNAs (miRNA) or
small interfering RNAs (siRNA). A variety of mechanisms
of post-transcriptional control of gene expression by various
sRNAs have been discovered in plants and other organisms,
underlying the complexity and importance of this process
in plant development, including the hormonal pathways
(Martínez de Alba et al., 2013; Bologna and Voinnet, 2014).
Plants exhibit a high diversity of sRNA species and factors which are instrumental in sRNA production. sRNAs are
transcribed as single-stranded transcripts mainly by RNA polymerase II, similar to protein-coding genes (Fig. 2). The primary transcript either contains hairpin structures of variable
lengths, which applies for miRNAs, or they are converted into
double-stranded RNA (dsRNA) by RNA-dependent RNA
polymerases (RDRs) in the case of siRNAs. sRNAs are divided
into several subgroups. Among them, in A. thaliana, miRNAs,
trans-acting (tasi-), and secondary siRNAs have been particularly shown to participate in the auxin response. miRNAs are
20–25 nucleotide sRNAs, which mostly negatively regulate
mRNA stability in plants. tasiRNAs are 21 nucleotide long
and their maturation requires the participation of miRNAs in
the cleavage of non-coding RNA produced from the TAS loci.
In a similar fashion, secondary siRNAs derived as cleavage
products of miRNA-directed PTGS mediate the degradation
of transcripts similar to the original target mRNA and thereby
amplify the miRNA silencing effect (Martínez de Alba et al.,
2013; Bologna and Voinnet, 2014).
Currently, there are 325 precursors and 427 mature
Arabidopsis miRNA sequences available in the specialized
miRNA database (release 21, www.mirbase.org, retrieved
in December 2014). Many miRNAs do not accumulate uniformly, but are expressed in specific cell types or developmental stages and often make gradients across tissues. This is in
accordance with their common regulatory role in morphogenesis and pattern formation as well as in response to environmental stimuli (Hisanaga et al., 2014).
4900 | Hrtyan et al.
Fig. 2. A simplified scheme of miRNA biogenesis. The MIRNA gene is
transcribed by RNA polymerase II (PolII) from its cognate locus. A hairpinforming RNA is capped and polyadenylated, giving rise to pri-miRNA.
The pri-miRNA associates with SERRATE (SE), HYPONASTIC LEAVES
1 (HYL1), and the endonuclease DICER LIKE1 (DCL1). DCL1 in the
first slicing step removes the m7G cap and the poly(A) tail, producing
pre-miRNA. In the second slicing step, the miRNA/miRNA* duplex is
generated. Next, the miRNA/miRNA* duplex is methylated (black dots)
by HUA ENCHANCER1 (HEN1) and transported to the cytoplasm by a
mechanism involving HASTY (HST), a nuclear pore-associated protein.
Subsequently, the target-complementary miRNA strand associates with
ARGONAUTE (AGO), while its opposing miRNA* strand is degraded. AGO
endonuclease and miRNA are part of the RNA-induced silencing complex
(RISC), where the target mRNA is prompted for degradation.
The biogenesis of miRNA starts from a transcript several
hundreds of nucleotides long, called primary miRNA (primiRNA; Fig. 2). When transcribed, it contains extensively
base-paired RNA hairpins of variable length and is capped
and polyadenylated (Xie et al., 2005; Rogers and Chen,
2013). In the next step, leading to precursor miRNA (premiRNA), the cap and the poly(A) tail are removed. PremRNA is processed by DICER-LIKE 1 (DCL1), which
dices pre-miRNA to an miRNA/miRNA* duplex (Kurihara
and Watanabe, 2004; Rogers and Chen, 2013). The process
occurs in the nucleus and it is assisted by dsRNA-binding
protein HYPONASTIC LEAVES 1 (HYL1) and zinc-finger
protein SERRATE (SE). HYL1 and SE improve the effectivity and accuracy of DCL1-mediated cleavage and are
responsible for enhancing the stability of several miRNAs
(Vazquez et al., 2004; Laubinger et al., 2008). The miRNA/
miRNA* duplex is 2’-O-methylated by HUA ENHANCER
1 (HEN1) methyltransferase on the ribose moiety of their
3’ ends, which improves its protection against degradation
(Park et al., 2002; Rogers and Chen, 2013). miRNAs are
then exported to the cytoplasm by machinery which presumably includes HASTY (HST), a homologue of mammalian Exportin 5 (Bollman et al., 2003; Rogers and Chen,
2013). In the cytoplasm, the miRNA/miRNA* duplex associates with proteins of the ARGONAUTE (AGO) family,
endonucleases which are the key part of the RNA-induced
silencing complex (RISC). Here, the miRNA/miRNA*
duplex dissociates and, while the opposing miRNA* strand
gets degraded, the miRNA, together with the AGO protein, recognize its mRNA target (Rogers and Chen, 2013;
Martínez de Alba et al., 2013; Bologna and Voinnet, 2014).
Although most reports show that plant miRNAs induce
cleavage of their cognate mRNA, several studies reveal that
the RISC complex is also able to repress their translation
(Iwakawa and Tomari, 2013).
The ago1, dcl1, hen1, hyl1, and hst mutants in A. thaliana
underline the crucial roles of miRNAs in plant development,
as they display dramatic and pleiotropic developmental defects (Bologna and Voinnet, 2014). Numerous studies demonstrated that miRNAs also directly target several
genes involved in auxin signalling and homeostasis (outlined in Fig. 3). Accordingly, mutants defective in core
miRNA processing genes also show auxin-related phenotypes. For example, loss-of-function hyl1 mutants exhibit
reduced sensitivity to auxin and also additional defects
ascribed to perturbed auxin homeostasis, such as reduced
root gravitropism and apical dominance (Lu and Fedoroff,
2000). The ago1 mutant shows altered sensitivity to exogenously applied auxin and auxin herbicides, and reduced levels of free IAA and its metabolites (Sorin et al., 2005), and
both ago1 and dcl1 show altered expression of several genes
involved in auxin response (Vazquez et al., 2004; Williams
et al., 2005; Sorin et al., 2006).
miRNA circuits controlling auxin signalling
Since the discovery of the first plant miRNAs, several genes
involved in auxin-dependent processes (mainly the components of auxin signalling) have been identified as their targets.
Auxin and cytokinin RNA processing | 4901
Fig. 3. The role of post-transcriptional gene silencing in auxin signalling
pathways. (A) A complex regulatory network among ARFs and their
targeting miRNAs controls adventitious root formation in response to light
(Gutierrez et al., 2009). (B) Osmotic stress up-regulates IAR3 hydrolase by
inhibiting levels of miR167. This leads to the release of free IAA from IAA-Ala
conjugates. By a not entirely clear mechanism, miR167 induced by osmotic
stress targets IAR3, but not ARF6 and ARF8 (Kinoshita et al., 2012). (C)
Several pathways during miR393-mediated silencing of the TIR/AFB2 clade
of auxin receptors. Various inputs, such as salt stress or drought stress as
well as ABA treatment, promote miR393-guided cleavage of TIR1 and AFB2
(Chen et al., 2012; Iglesias et al., 2014); bacterial flagellin treatment leads to
miR393-mediated silencing of TIR1, AFB2, and AFB3 (Navarro et al., 2006);
miR393 does not seem to down-regulate AFB1 directly. In A. thaliana roots,
nitrate induces AFB3 transcription, while nitrate metabolites simultaneously
induce miRNA393, which degrades targets AFB3 (Vidal et al., 2010; green).
During leaf development, miR393 preferentially targets TIR1 and AFB2,
which leads to production of secondary siRNAs which silence all four TIR1/
AFB2 receptors (Si-Ammour et al., 2011; purple). (D) The silencing network
of miR160 and mR167 in A. thaliana. miR160 and miR167 target their ARFs
for cleavage. This leads to the production of secondary siRNAs (Manavella
et al., 2012). Endogenous target mimics (eTMs) can bind miRNAs and
inhibit their silencing effect (Wu et al., 2013). (E) During lateral root formation,
MIR390 expression leads to the production of tasiRNAs (tasiARFs) from the
TAS3 non-coding RNA. tasiARFs promote the cleavage of ARF2, ARF3,
and ARF4. ARF3 and ARF4 also regulate the transcription of MIR390 by
a positive or negative feedback mechanism, respectively (Marin et al.,
2010). MIR390 (Yoon et al., 2010) or ARF4 (Marin et al., 2010) can also be
transcriptionally induced by auxin.
In A. thaliana, miR160 and miR167 have been implicated
in the down-regulation of several classes of ARF genes and
miR393 targets auxin receptors of the TIR1/AFB family.
miR390 also controls the expression of several ARFs, but
using a more complicated mechanism, as outlined in the section ‘trans-acting siRNAs in auxin signalling pathways and
development’ (Rhoades et al., 2002; Martínez de Alba et al.,
2013; Bologna and Voinnet, 2014; Fig. 3).
It has been reported that miR160 inhibits the expression
of ARF10, ARF16, and ARF17 auxin transcription factors
(Rhoades et al., 2002; Mallory et al., 2005; Wang et al., 2005)
and thus regulates the transcription of their respective auxin
primary response genes. Plants expressing miR160-resistant
ARF17 show severe aberrations in both vegetative and floral
development, including reduced fertility. They also show several defects ascribed to auxin deficiency, such as altered phyllotaxis, lateral root formation, and shorter hypocotyls, but their
morphological response to exogenously applied auxin IAA is
not dramatically changed (Mallory et al., 2005; Wang et al.,
2005). ARF17 controls the transcription of group II GH3like enzymes, which conjugate IAA to amino acids (Mallory
et al., 2005; Staswick et al., 2005; Gutierrez et al., 2012). This,
together with elevated DR5::GUS auxin reporter in plants
carrying miR160-resistant ARF17, suggests that miR160
regulates endogenous levels of free IAA via ARF expression
(Mallory et al., 2005). ARF10 and ARF16 are involved in
root cap formation and the regulation of columella cell identity, cell division of the root meristem, and root gravitropism,
which is consistent with the phenotype of plants overexpressing MIR160 and arf10 arf16 double mutants (Wang et al.,
2005). Repression of ARF10 by miR160 also appears to have
an important role in the interaction between auxin and abscisic acid (ABA) during germination, as the overaccumulation
of miR160-resistant ARF10 alters ABA sensitivity in seeds
(Liu et al., 2007).
Gutierrez et al. (2009) uncovered a complex regulatory circuit consisting of ARFs, their cognate miRNAs, and the light
signalling pathway, which regulates adventitious root formation (Fig. 3A). ARF6 and ARF8 are positive regulators of
adventitious rooting and are up-regulated by light, whereas
ARF17 is a negative regulator and is down-regulated by
light. The expression of ARF17 is inhibited by miR160, while
ARF6 and ARF8 are targets of another miRNA, miR167.
ARFs mutually regulate their owns expression, directly via
transcription or by changing levels of miR160 and miR167,
in a positive and also in a negative manner (Gutierrez et al.,
2009).
miR167 also down-regulates ARF6 and ARF8 in adult
leaves, inflorescences, and flowers, and its function is also
crucial for ovule and anther formation and other aspects
of flower development, as seen in the phenotypes of transgenic lines with altered miR167-mediated silencing. This
process also shows a high degree of complexity. It has been
shown that during flower development, miR167 expression
is controlled by the transcription factors MYB33 and TCP4,
which are themselves regulated by miR159 and miR319,
respectively (Rubio-Somoza and Weigel, 2013). Because the
reporter signal of the MIR167A promoter fusion seems to
differ from that in miR167 in situ hybridization assays, it has
4902 | Hrtyan et al.
been hypothesized that miR167 stability may vary in different
cell types, or even that miR167 moves between cells (Válóczi
et al., 2006; Wu et al., 2006). It was also found that miR167
levels in the root pericycle (founder tissue of lateral root emergence) are down-regulated during nitrogen treatment. This
leads to the up-regulation of ARF8 expression and thereby
the inhibition of lateral root emergence, thereby contributing to reshape the root system during the nitrogen response
(Gifford et al., 2008).
Besides ARFs, miR167 also targets for degradation IAAALANINE RESISTANT 3 (IAR3), a hydrolase which controls free auxin levels by releasing IAA from its inactive
conjugates, such as IAA-alanine. The miR167–IAR3 pair
shows more mismatches than in the case of silencing of ARF6
and ARF8; therefore, it was not predicted or detected in the
earlier studies (Fig. 3B; Davies et al., 1999; Chorostecki et al.,
2012; Kinoshita et al., 2012). The levels of miR167 decrease
under osmotic stress, which leads to up-regulation of IAR3.
Bioactive IAA produced by IAR3 from inactive IAA-alanine
pools contributes to developmental changes in roots induced
by osmotic stress. This includes a reduction in primary root
growth and the promotion of lateral root development.
Interestingly, ARF6 and ARF8 expression seems to remain
unchanged by miR167 under these conditions and also, in
contrast to iar3, the arf6 arf8 double mutant does not show
altered sensitivity to osmotic stress. This suggests that perhaps the tissue specificity of MIR167 expression, counteracting transcriptional regulation of target genes, and/or an
additional factor specifically regulating the priority of IAR3/
ARF6/8 silencing is required in this process (Kinoshita et al.,
2012).
miR393 has been implicated in down-regulating the expression of various members of the AFB2 clade of the TIR1/AFB
auxin receptor family (TIR1 and AFB1–AFB3). Navarro
et al. (2006) found that miR393 is induced by bacterial flagellin. This leads to inhibition of auxin signalling by silencing
the auxin receptors TIR1, AFB2, and AFB3 at the posttranscriptional level; the remaining gene, AFB1, is probably
repressed by another mechanism (Fig. 3C). Accordingly,
overexpression of miR393 leads to increased resistance
to pathogen infection. miR393 is produced from two loci,
MIR393A and MIR393B, which show highly overlapping
expression patterns (Parry et al., 2009; Si-Ammour et al.,
2011). Although both genes act mainly redundantly, it was
reported that MIR393B expression is rather auxin dependent, while MIR393A plays a role in the response to environmental stress (Chen et al., 2011). miR393-dependent silencing
(mainly from its MIR393B locus) is crucial for auxin-dependent epinasty of cotyledons, root elongation, and lateral root
initiation, and also for leaf development (Chen et al., 2011;
Si-Ammour et al., 2011).
It was found that AFB3 (but not other TIR1/AFB genes) is
induced transcriptionally within <1 h by exogenously applied
nitrate. This acts in concert with specific miR393-guided
cleavage of AFB3, as miR393 transcript peaks 2 h after
nitrate treatment. Interestingly, in contrast to AFB3, miR393
is induced by nitrate metabolites (Fig. 3C). This feed-forward
mechanism is important for auxin-mediated rearrangement
of the root system architecture during the nitrate response,
resulting in the inhibition of primary and the promotion
of lateral root growth (Vidal et al., 2010), similar to effects
described for the action of miR167 (Gifford et al., 2008).
miR393-mediated regulation of TIR1/AFB receptors has also
been shown to play a role in response to oxidative, drought,
and salt stress (Chen et al., 2012; Iglesias et al., 2014).
miR393-dependent targeting of TIR1/AFB also leads to
the production of secondary siRNAs (Fig. 3C). miR393 is
accumulated during leaf development, where it silences TIR1
and AFB2 with higher preference than AFB3 or AFB1. TIR1and AFB2-derived secondary siRNAs, also called siTAAR,
spread the miR393 silencing effect to AFB3 or AFB1, and
probably also to other unrelated genes (Si-Ammour et al.,
2011). It has been shown that miR160 and miR167 produce
secondary siRNAs as well. However, detailed information
about their functions in auxin-dependent processes is still
incomplete (Fig. 3D; Manavella et al., 2012).
Interestingly, it was also found that a specific type of
endogenous lncRNA, called endogenous miRNA target
mimic (eTM), is able to mimic the genuine mRNA targets
of miRNA-mediated silencing. For example, the prototypical non-coding RNA of INDUCED BY PHOSPHATE
STARVATION 1 (IPS1) can bind miR399 that degrades
genes regulating phosphate homeostasis (Franco-Zorrilla
et al., 2007). In A. thaliana, similar eTMs were found also
for miR160 and miR167 (eTM160 and eTM167, respectively,
Fig. 3D). Transgenic plants overexpressing eTM160 show an
elevated expression of ARF10, ARF16, and ARF17. They
show small and serrated leaves, which can be reverted by overexpression of MIR160. ETM160 overexpressors also show
decreased levels of miR160, which suggests that eTMs also
promote miRNA degradation. eTMs most probably act in
a dosage-dependent manner, where expression of eTMs has
to reach a threshold to saturate the corresponding miRNAs
effectively. This can fine-tune miRNA action in a more temporal- or cell type-specific manner (Wu et al., 2013).
trans-acting siRNAs regulating auxin signalling
Being initiated by miRNAs, tasiRNAs act on a similar molecular principle as secondary siRNAs. These 21 nucleotide plantspecific sRNAs induce cleavage of their target mRNA, but
their targets are clearly distinct from their transcript of origin
(Montgomery et al., 2008). In A. thaliana, there are four groups
of tasiRNA genes (TAS1–TAS4). TAS1, TAS2, and TAS4 contain one miRNA target site and associate with AGO1. TAS3
(encoded by TAS3a to c) carries two miR390 target sites in its
sequence and is linked with AGO7 action. TAS transcripts are
converted into dsRNA by RDR6 and this process also requires
SUPPRESSOR OF GENE SILENCING 3 (SGS3), which stabilizes and protects the RNA fragments against degradation.
DCL4 is also involved in the biogenesis of tasiRNAs, slicing
the long TAS dsRNA into 21 nucleotide segments (Martínez de
Alba et al., 2013; Bologna and Voinnet, 2014).
The TAS3 locus is dependent specifically on miR390 and
specifies the production of two tasiRNAs (also called tasiARF
genes), that are responsible for repression of ARF3, ARF4,
Auxin and cytokinin RNA processing | 4903
and ARF2 (Allen et al., 2005; Montgomery et al., 2008; Marin
et al., 2010). TAS3-mediated ARF3 and ARF4 silencing has an
important role in leaf development, particularly in the juvenile-to-adult phase shift (Adenot et al., 2006; Fahlgren et al.,
2006; Hunter et al., 2006). Moreover, it was shown that this
process influences leaf adaxial–abaxial polarity, as both ARF3
and ARF4 were shown to be positive regulators of leaf abaxialization. In this manner, TAS3 is expressed in the adaxial side
of early leaf primordia and down-regulates ARF3 and ARF4
there (Garcia et al., 2006). The effect of tasiARF-mediated
ARF3 and ARF4 silencing was also found in flower development, and this developmental pathway seems to be coupled
with responses to several stress stimuli (Fahlgren et al., 2006;
Matsui et al., 2014).
Auxin is essential for lateral root formation, and its local
maxima in their founder pericycle cells act as a signal for their
initiation (Casimiro et al., 2001; Benková et al., 2003). Using
a sensor of miR390 activity, Marin et al. (2010) revealed that
miR390 is expressed in early lateral root primordia (Fig. 3C).
miR390 triggers the biogenesis of tasiRNAs from TAS3,
which is ubiquitously expressed in the stele. tasiARFs promote
lateral root elongation by silencing ARF2, ARF3, and ARF4
(Fig. 3E). Next, in a feedback loop manner, the transcription
of miR390 is repressed by ARF4 and activated by ARF3. In
addition, auxin directly or indirectly induces the expression
of miR390 and ARF4. These data suggest that the mutually
regulated miR390–TAS3–tasiARF–ARF2,3,4 network coordinates lateral root formation and growth. Interestingly, it
has been proposed that both tasiARF genes and miR390 act
non-cell autonomously in these processes, which is in accordance with several lines of evidence that tasiARFs and miR390
act as mobile signals between cells in shoot apical meristem
and leaves (Marin et al., 2010; Yoon et al., 2010; MarínGonzález and Suárez-López, 2012).
Role of sRNAs in cytokinin-mediated processes
In contrast to auxins, the evidence for silencing cytokininrelated genes is rather indirect. hyl1 mutants show reduced
sensitivity to cytokinin (Lu and Fedoroff, 2000), and overexpression of miR160 leads to the hampered induction of several type A ARR genes under cytokinin treatment in soybean
(Turner et al., 2013). However, in A. thaliana, no PTGS of
genes directly involved in the cytokinin response was identified.
In Physcomitrella patens, miR1221 was predicted to target the
AHK4 cytokinin receptor (Talmor-Neiman et al., 2006), and
miR1027 shows possible complementarity to the cytokinin
oxidase gene, CKX1 (Axtell et al., 2007). Also two siRNAs (id4
and id65) which target an orthologue of A. thaliana cytokinin
synthase, IPT3, were identified from a RNA library of Vitis
vinifera (Carra et al., 2009). However, no further experimental
data are available to assess the functionality of these sRNAs in
cytokinin pathways, including an evolutionary impact.
Cytokinin- and auxin-inducible miRNAs
The expression of some miRNAs was seen to be altered by
cytokinin treatment. In P. patens, cytokinins are known to
promote the juvenile-to-adult transition. The moss-specific
miR534a targets BLADE-ON-PETIOLE (BOP) genes,
which are positive regulators of gametophyte morphogenesis.
Consistently, miR534a loss-of-function mutants show accelerated gametophore development, and miR534a is down-regulated by cytokinin treatment (Saleh et al., 2011). In Medicago
truncatula, cytokinin is required for symbiotic nodulation.
miR171h targets the cytokinin-responsive GRAS family
transcription factor, Nodulation Signaling Pathway (NSP2),
which is involved in early nodulation. miR171h expression is
also cytokinin inducible and directly restricts NSP2 expression. miR171h thus mediates feedback control of cytokinininduced nodulation (Ariel et al., 2012). Although miR171
targets genes homologous to NSP2 in A. thaliana (Llave
et al., 2002), no functional relationship of A. thaliana miR171
to the cytokinin response has been reported to date. In addition, several cytokinin-induced miRNAs were identified and
also partially characterized in an RNA sequencing (RNAseq) study dedicated to cytokinin-induced gene responses in
A. thaliana (Bhargava et al., 2013).
It has been also shown that auxin-inducible mir164 mediates expression of NAM/ATAF/CUC (NAC) transcription
factors, among them NAC1 (Mallory et al., 2004), a regulator
of lateral root formation, positively regulated by the TIR1
auxin signalling pathway (Xie et al., 2000). The plants carrying miR164-resistant NAC1 transgenes and also mir164
mutants show enhanced lateral root formation. The expression of MIR164 (and cleavage of NAC1) is also mediated via
the TIR1 pathway (Guo et al., 2005).
Long non-coding RNAs and their
regulatory role in auxin responses
lncRNAs were shown to dampen gene expression and they
often exhibit tissue- or cell type-specific expression patterns
which suggests their regulatory role in developmentally important processes (Ben Amor et al., 2009). Two related nuclear
speckle RNA-binding proteins (NSRa and b) belong to the
family of serine/arginine-rich family proteins, important factors for alternative splicing (AS) in various eukaryotes. In
A. thaliana, NSRa is constitutively expressed, while NSRb is
inducible by auxin in the root. Auxin-regulated NSRb modulates the AS of several mRNAs. In addition to their target
mRNA, NSRs also bind lncRNA, called Alternative Splicing
Competitor RNA (ASCO-RNA; Ben Amor et al., 2009;
Bardou et al., 2014). ASCO lncRNA competes with NSR target mRNAs altering their expression and AS. NSR function
is required for the proper expression of auxin-related genes
linked to lateral root formation such as ARF7 and ARF19.
Consistently, nsra nsrb double mutants form less lateral roots,
and lateral root formation of nsra nsrb and the ASCO cDNA
overexpressor lines is resistant to auxin treatment (Bardou
et al., 2014). It will therefore be very interesting to test further
how directly this intriguing pathway controls auxin-mediated
lateral root formation.
The dynamics of chromatin structure plays a key role in
gene expression by directly influencing DNA accessibility
4904 | Hrtyan et al.
for transcription machinery. Ariel et al. (2014) uncovered
a molecular switch which regulates expression of PINOID
(PID). PID is a kinase altering auxin gradients by changing the polar localization of PIN auxin transporters (Friml
et al., 2004). The locus of intergenic lncRNA called AUXIN
REGULATED PROMOTER LOOP RNA (APOLO) is situated within the promoter of PID, which is methylated. Auxin
treatment leads to demethylation of this region, relaxation of
the chromatin structure, and an increase of PID expression,
mediated by ARFs. Simultaneously, APOLO is transcribed
as well. APOLO lncRNA subsequently induces methylation
of the APOLO locus, being physically embedded in the chromatin structure, and this also turns off PID expression. It has
been shown that this process also probably alters the auxin
hormonal response, as depletion of APOLO expression by
RNA interference (RNAi) leads to several auxin-related
defects, including an aberrant gravitropic response and lateral
root density (Ariel et al., 2014).
The role of upstream open reading frames
(uORFs) in the regulation of auxin signalling
Translational regulation relies on several control signals,
frequently located inside the 5′-untranslated region (UTR),
upstream of the main open reading frame (ORF). One such
cis-element can be an AUG codon located in the 5′-transcript
leader sequence of eukaryotic mRNAs, upstream from the
main initiation codon. Such upstream AUGs can be associated with upstream open reading frames (uORFs) (Morris
and Geballe, 2000), which can regulate the translation of
the downstream ORF encoding the major gene product. If
the ribosome scanning the mRNA recognizes and translates
the uORF, translation will be prematurely terminated on its
stop codon. For restoring translation of downstream ORFs,
a process known as translation reinitiation is needed, which
requires the formation of a new initiation complex (Zhou
et al., 2010). In A. thaliana, translation reinitiation is controlled by the TARGET OF RAPAMYCIN (TOR) signalling
pathway which acts via phosphorylation of the plant reinitiation factor, EIF3H. The interaction of TOR with ribosomes
is dynamic and it has been shown that it can be influenced
by auxin (Schepetilnikov et al., 2013). According to genomewide analyses, about one-third of the A. thaliana mRNAs
appear to contain uORFs (Takahashi et al., 2012).
ARF3 carries several uORFs that negatively regulate the
translation of its coding region, and the activity of this uORF
affects gynoecium development, consistent with the arf3 (ettin)
loss-of-function mutant phenotype (Nishimura et al., 2005).
A similar uORF mode of action was also observed in the case
of ARF2, ARF5, and ARF6. A role for the ribosomal proteins
RPL24, RPL4D, and RPL5A has been demonstrated in these
processes at the biochemical and genetic levels. rpl mutants,
besides other aberrations, exhibit several auxin-related defects
(e.g. reduced auxin sensitivity or altered expression of auxin
reporter genes), and some of these can be reverted by introducing a uORF-eliminated ARF3 construct (Nishimura et al.,
2005; Rosado et al., 2012). The uORFs present in ARF3 and
ARF5 affect the translation of downstream major ORFs,
even though there is no conservation of the number, length,
or amino acid sequence of the predicted peptides encoded by
their uORFs (Nishimura et al., 2005). In contrast to the situation in ARF genes, uORFs were found to be less common
among transcripts of AUX/IAA ARF-interacting partners
(six genes). They were also predicted in YUC2 and YUC6
from YUCCA auxin biosynthesis genes, TIR1, AFB2, and
AFB5 of the TIR1/AFB auxin receptors and the PIN4 auxin
efflux carrier (Zhou et al., 2010; Goodstein et al., 2012).
Regulation of auxin- and cytokinin-related
genes by alternative splicing
AS is an important post-transcriptional regulatory process
which increases proteome diversity, controls mRNA stability, and also, for example, contributes to the evolutionary
adaptation. According to transcriptome sequencing experiments, >60% of all pre-mRNAs undergo AS in A. thaliana
(Marquez et al., 2012; Merkin et al., 2012; Reddy et al., 2013;
Staiger and Brown, 2013). In plants, it was demonstrated that
AS plays a role in various processes, including responses to
stress, photosynthesis, defence against pathogens, metabolic
pathways, and flowering. AS is also an important regulator
in plant developmental and hormonal regulatory networks;
the importance of AS has been particularly highlighted in
ABA pathways (Staiger and Brown, 2013). Nonetheless, the
amount of solid experimental data about the role of AS in
auxin- and cytokinin-dependent processes is still limited and
our knowledge is largely based on gene predictions (www.
arabidopsis.org; Lamesch et al., 2012) and deep sequencing
data (Marquez et al., 2012; Bhargava et al., 2013).
In general, AS can alter mRNA nucleotide sequences in
both the 5′- and 3′-UTRs as well as in coding sequences. AS
of UTRs modifies regulatory elements which influence transport, translation efficiency, the subcellular localization, and
stability of mRNAs (Stamm et al., 2005; Kelemen et al., 2013).
In the coding sequence, AS may modify motifs required for
the protein subcellular localization, fine-tune protein functional activity, or swap important domains. Events leading to
changes in short amino acid sequences often correlate with the
presence of a residue undergoing covalent modification, such
as phosphorylation (Stamm et al., 2005; Merkin et al., 2012;
Kelemen et al., 2013; Reddy et al., 2013). AS can also generate truncated proteins acting as dominant negative repressors
of biological pathways inside the cell. Several splicing events
can lead to non-translated mRNAs which can act in the similar principle at the RNA level. It has also been proposed that
a considerable part of AS events represents noise without any
discernible outcome. However, the steeply increasing number
of research articles dealing with AS in plants suggests that this
might not be a general feature of all isoforms. In general, a
deep evolutionary conservation of a particular splicing event
strongly advocates for its physiological importance (Marquez
et al., 2012; Kelemen et al., 2013; Staiger and Brown, 2013).
In addition to AS per se, transcripts can also be regulated via
alternative promoter selection, or by alternative cleavage and
Auxin and cytokinin RNA processing | 4905
polyadenylation at their 3’ ends (Mayr and Bartel, 2009; Pal
et al., 2011).
AS in auxin-dependent pathways
The importance of AS for auxin transport was demonstrated with ZINC-INDUCED FACILITATOR-LIKE 1
(ZIFL1; Fig. 4). ZIFL1 is a member of the Major Facilitator
Superfamily (MFS) and is required for the precise tuning of
auxin transport. ZIFL1 has a dual role in modulating both
polar auxin transport and drought stress, which is determined
by the AS of its 17 exon-containing mRNA. The ZIFL1.1
isoform corresponds to the full-length transcript. It is localized in the tonoplast and plays a role in the response to auxin
Fig. 4. Genes involved in auxin-dependent processes undergoing alternative splicing (AS). In addition to ZIFL1 (Remy et al., 2013), representative gene
groups involved in auxin synthesis, signalling, and transport (YUCCA genes; IBR5 and ARF genes; PIN genes) were selected and their AS events outlined.
Data for diagrams of alternative splicing were obtained from TAIR (www.arabidopsis.org; darker colours) and extended with RNA-seq-based gene models
(Marquez et al., 2012; lighter colours). Because UTRs were not analysed in the transcriptome sequencing data (Marquez et al., 2012), these regions are
not highlighted. Closely related genes are connected with a dashed line. The ARF family was sorted by the phylogenetic relationships for clarity.
4906 | Hrtyan et al.
treatment, gravitropism, or lateral root development. It does
not function as an auxin transporter itself, but influences
auxin transport indirectly by stabilizing PIN2 on the plasma
membrane during IAA treatment, which accordingly leads
to more rapid PIN2 degradation in its absence. ZIFL1.3 is
a splice variant with an alternative 3’ splice site in the 14th
intron leading to a stop codon in the 15th exon. The resulting
truncated transporter probably retains the functional properties of the MFS transporters, even if probably lacking two
conserved membrane-spanning segments on its C-terminus.
ZIFL1.3 is plasma membrane localized and it is, in contrast
to ZIFL1.1, required in the response to drought and for efficient stomatal closure (Remy et al., 2013).
INDOLE-3-BUTYRIC ACID RESPONSE 5 (IBR5) is
a phosphatase which regulates several aspects of the auxin
response, probably downstream of the TIR/AFB-mediated
auxin signalling module (Strader et al., 2008) and has two
splicing variants, IBR5.1 and IBR5.3 (Fig. 4). IBR5.3 possesses an alternative 3’ splice site in the last intron, which
leads to an early stop codon. IBR5.1, when expressed under
the 35S promoter, is localized to the nucleus and cytoplasm,
while localization of IBR5.3 is exclusively nuclear. They
also seem to have distinct roles. Overexpression of IBR5.1
complements the IAA-resistant root growth of an ibr5-1
knockout as well as its lateral root defects. IBR5.3 overexpression rescues lateral root phenotypes alone (Jayaweera
et al., 2014).
Auxin is synthesized by multiple biochemical pathways
which are utilized differentially, depending on the stage of
organ development, environmental cues, or the subcellular
compartment of biosynthesis. The family of YUCCA genes
codes for flavin-dependent monooxygenases and is involved
in the final step of the developmentally important tryptophan-dependent IAA biosynthetic pathway (Brumos et al.,
2014). Among the 11 members of the family, YUC4 and
YUC6 are each processed by a similar AS mechanism into
two isoforms (Fig. 4; Kriechbaumer et al., 2012). Compared
with canonical YUC4.1, YUC4.2 shows retention of the last
intron which contains an early stop codon. AS does not affect
their enzymatic activity, as both isoforms are able to produce
IAA from its precursor, but it alters the subcellular localization of the YUC4 protein. Canonical YUC4.1 is localized in
the cytosol, while YUC4.2 is anchored to the cytosolic side
of the endoplasmic reticulum. It was previously shown that
YUC4 is an important gene required for flower development
(Cheng et al., 2006) and, interestingly, the YUC4.2 variant
is specifically expressed in these organs (Kriechbaumer et al.,
2012). The TRYPTOPHAN AMINOTRANSFERASE
OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRAN
SFERASE RELATED (TAA1/TAR) proteins function
upstream of YUCCA (Brumos et al., 2014). This family
contains three genes: TAA1, TAR1, and TAR2. It has been
shown that TAA1 is localized in the cytosol (Tao et al., 2008),
while TAR2 resides in the endoplasmic reticulum (Ma et al.,
2014). AS thus might be an interesting mechanism regulating
intracellular pools of free auxin, along with the regulation of
transcription.
Canonical ARFs consist of four domains which are essential for their proper function. However, several of these are
truncated, lacking their C-terminal part with domains dIII
and dIV that are responsible for dimerization with AUX/IAAs
(De Smet et al., 2011). One such truncated ARF is ARF3
(ETTIN), a factor required for floral development. In the
evolutionarily ancient angiosperm Amborella trichopoda, the
ARF3 homologue codes for a non-truncated product, whose
dIII and dIV domains are removed by AS. From domainswapping experiments, it was suggested that the ARF3 truncation is possibly a prerequisite for its proper function in
floral development (Finet et al., 2010). In addition, the ectopic
expression of another isoform (second intron retained), of
related, but non-truncated ARF4 (ΔARF4) also alters flower
development in A. thaliana (Finet et al., 2013). Besides ARF4,
out of the 23 A. thaliana functionally redundant ARFs, 14
genes were found to undergo AS in deep sequencing experiments (Fig. 4; www.arabidopsis.org; Marquez et al., 2012).
For example, both ARF1 and its nearest orthologue, ARF2,
show a broad variety of splicing events (intron 9 retention of
ARF1 is conserved in its orthologue in Solanum lycopersicon;
Zouine et al., 2014), whereas genes of the so-called ‘Class I’
(ARF12–ARF15 and ARF20–ARF23; Remington et al., 2004)
are processed largely constitutively.
The members of the TIR1/AFB auxin receptor family are functionally highly redundant (Wang and Estelle,
2014), but their splicing variants show no common pattern
(Supplementary Fig. S1 at JXB online; Marquez et al., 2012).
Seven out of 29 A. thaliana AUX/IAAs, repressors of auxin
signalling (De Smet et al., 2011), also show AS (www.arabidopsis.org; Marquez et al., 2012). The closely related IAA8
and IAA9 show similar AS patterns. AS of the 3’-UTR in
IAA12, IAA13, and their closest orthologue, IAA11, due to
its deep evolutionary conservation, suggests the existence of
an ancient regulatory mechanism (Supplementary Fig. S1).
Among eight A. thaliana PIN auxin efflux carriers, PIN4
and PIN7 (Fig. 4) undergo AS, which results in two isoforms
differing in 12 nucleotides at the end of the first exon (TAIR).
PIN3, belonging to the same phylogenetic clade as PIN4
and PIN7 (De Smet et al., 2011), is processed only constitutively. Deep sequencing data suggest that other isoforms may
also exist, including the relatively highly abundant PIN4.3,
which lacks 57 nucleotides corresponding to part of the large
intracellular loop of the transporter (Marquez et al., 2012;
M. Hrtyan et al., unpublished).
Four out of seven A. thaliana PIN-LIKES (PILS;
Supplementary Fig. S1 at JXB online), endoplasmic reticulum-located auxin transport facilitators (Barbez et al., 2012),
are also processed by AS, and the closely related PILS3 and
PILS4 show similar AS patterns. The family of A. thaliana
AUXIN1/LIKE-AUX1 (AUX/LAX) auxin influx carriers consists of four genes, and three of them are processed
into various transcripts. LAX1 has two alternative initiation
codons which affects the length of its intracellular N-terminal
part, but does not affect the overall structure of the transmembrane regions (Supplementary Fig. S1, Swarup and
Péret, 2012).
Auxin and cytokinin RNA processing | 4907
In summary, despite a few recent reports, the functional
role of AS of auxin-related genes is largely unknown.
Based on the predicted and also verified gene models, these
genes appear to be post-transcriptionally regulated at surprisingly many levels. No direct upstream regulator of AS
has been identified in these processes. Several reports have
shown that impairing several regulators of splicing (such
as SR proteins RSZ33 and MERISTEM DEFECTIVE or
DEAH and DEAX box helicases) leads to alteration in
the expression of the auxin reporter and genes involved
in the auxin response, as well as several defects resembling
perturbed auxin perception and homeostasis (Kalyna
et al., 2003; Casson et al., 2009; He et al., 2012; Tsugeki
et al., 2015). The application of modern transcriptomics
approaches will significantly advance our understanding
of the purpose of the numerous splicing events outlined
in this section.
AS in cytokinin-dependent pathways
The cytokinin receptor AHK4 gene is processed into three
splicing variants (Fig. 5; www.arabidopsis.org), which differ
in the position of the alternative start codon or in the length
of the 5′-UTR. Genes coding for cytokinin AHP phosphotransfer proteins are composed of six exons (Fig. 5). AHP5
has two variants (termed AHP5 and AHP5L), which are
expressed in comparable levels in most organs. The longer
isoform, AHP5L, shows retention of the second intron,
which leads to a premature stop codon. It was predicted that
in this region, AHP5L contains an additional downstream
ORF which encodes crucial residues for phosphotransfer
protein functionality (Hradilová and Brzobohatý, 2007). The
potential functional importance of this splicing event is supported by the conservation of this ORF in a Glycine max
homologue (www.phytozome.org; Goodstein et al., 2012).
Fig. 5. Genes involved in cytokinin-dependent processes undergoing AS. Representative gene groups involved in cytokinin signalling and synthesis
(AHK, AHP, IPT, and LOG genes) were selected and their AS events outlined. Data for the diagrams were obtained from TAIR (darker colours) and
extended with RNA-seq-based gene models (Marquez et al., 2012; Bhargava et al., 2013). Closely related genes are connected with a dashed line.
4908 | Hrtyan et al.
The AS of AHP6, a pseudophosphotransfer protein, results
in canonical AHP6b and the alternative AHP6a. They differ
in the position of the donor splice site in their first intron
(Fig. 5). The shorter AHP6a isoform is weakly expressed in
all organs. The AHP6 protein is required for vascular formation in roots, and ahp6-1 knockout shows strong protoxylem
defects. The ahp6-2 allele exclusively expresses the AHP6a
variant and shows an intermediate phenotype with weak protoxylem defects. This suggests that the additive contribution
of both isoforms is required for proper protoxylem development (Mähönen et al., 2006). Several type-B ARRs also show
AS and some of the isoforms have also been confirmed by
RT-PCR (Supplementary Fig. S2 at JXB online; Marquez
et al., 2012; Bhargava et al., 2013). However, the physiological
relevance of these splicing events has yet to be tested.
Within nine A. thaliana tRNA-IPTs, genes IPT2 and IPT9
are composed of 10 and 11 exons, respectively, and are processed by AS into several transcripts with comparable RNA
abundance (Fig. 5; Bhargava et al., 2013). The remaining IPT
genes coding for adenylate-IPTs consist of only one or two
exons, which is consistent with their putative prokaryotic origin (Frébort et al., 2011). Several of nine LOG cytokinin synthesis genes are also processed into numerous splicing variants
(Fig. 5), which suggests that the regulation of 5′-UTR splicing
might be a common regulatory mechanism. In addition, two
of seven A. thaliana CYTOKININ OXIDASE (CKX) genes
show regulation in their 3’-UTR by AS (Supplementary Fig.
S2 at JXB online; Kieber and Schaller, 2014).
Conclusions
Auxin-dependent processes appear to be better described
than cytokinin pathways (Schaller et al., 2015). This is evident from PubMed searches with the keywords ‘auxin’ and
‘cytokinin’ which return ~20 000 and <5000 citations, respectively. This imbalanced ratio is also reflected in the number
of published papers concerning the role of RNA processing
of auxin and cytokinin pathways. It concerns particularly the
regulation of translation by uORFs, recent findings about
lncRNAs in auxin-dependent processes, and also partially the
functional role of AS in auxin-related processes.
Apart from that, it appears from the (probably unbiased) genomic information that auxin-related genes are
subjected to PTGS more frequently than cytokinin genes.
In A. thaliana, no function of sRNA which would specifically regulate cytokinin response genes sensu stricto has been
demonstrated conclusively to date. Several components of
the PTGS silencing machinery show auxin-related defects,
but also deficiencies in cytokinin-dependent processes,
which perhaps suggests the existence of additional unknown
PTGS pathways or underlines the role of some cytokinincontrolled miRNA. Alternatively, proteins regulating PTGS
may constitute hubs where auxin and cytokinin (and other)
pathways interact.
Supplementary data
Supplementary data are available at JXB online.
Figure S1. Additional genes involved in auxin-dependent
processes, which undergo AS.
Figure S2. Additional genes involved in cytokinin-dependent processes, which undergo AS.
Acknowledgements
Supported by the Czech Science Foundation (P501/12/0934, to MH, EŠ,
and KR) and by the European Social Fund (CZ.1.07/2.3.00/20.0189 and
CEITEC). We thank Phil Jackson for the language correction and his comments on the manuscript.
Note Added in Proof
While this article was in proof, it has been reported that also AUX/IAAs
can be regulated by miRNAs. It was found that auxin inducible miR847 targets IAA28, regulating various auxin dependent processes (Wang and Guo,
2015). Reader is referred to this article for further information.
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