Role of long noncoding RNAs in regulation of

flowering in Arabidopsis
A REVIEW ASSINGMENT FOR PAPER CODE: 2.4.5 submitted to
Ravenshaw University, Cuttack for partial fulfillment of
Degree in Master of Science in Botany

Submitted By
Udit Sahu
Roll No: 13MBO-024

Under the guidance of

Dr. Narendra Nath Mohanty

P.G.DEPARTMENT OF BOTANY AND BIOTECHNOLOGY

RAVENSHAW UNIVERSITY
CUTTACK, ODISHA
 P.G. DEPARTMENT OF BOTANY

Certificate

This to certify that the review entitled “Role of long noncoding RNAs in
regulation of flowering in Arabidopsis is an authentic compilation of review work
carried out by Udit Sahu under my supervision in partial fulfillment of the requirement for
the Master degree in Botany, Ravenshaw University and further state that no part thereof
has been presented earlier for any other degree

Place: Cuttack Dr. N. N. Mohanty Dr. P. K. Jena

Dt :15.05.2015 (Review Guide) HOD
 DECLARATION

I, Udit Sahu, hereby declare that the Review entitled “Role of long
noncoding RNAs in regulation of flowering in Arabidopsis”
Submitted to the Department of Botany and Biotechnology,
Ravenshaw University, Cuttack is a bonafide assignment work
carried out by me under the guidance of Dr. Narendra Nath Mohanty,
PG Department of Botany and Biotechnology, Ravenshaw University,
Cuttack during the period of 2015. To the best of my knowledge and
belief no part of this work has been submitted elsewhere for any
degree or equivalent qualification.

Place and date Signature

Abbreviations

AG-AGAMOUS; AP1-APETALA1; ASI-ANTISENSE I; CO- CONSTANS; COLDAIR- COLD

ASSISTED INTRONIC NONCODING RNA; CLF- CURLY LEAF; COOLAIR- COLD INDUCED
LONG ANTISENSE INTRAGENIC RNA; CUL3-CULLIN3; FD- FLOWERING LOCUS D; FLC-
FLOWERING LOCUS C; FRI- FRIGIDA; FT- FLOWERING LOCUS T; LD
LUMINIDEPENDENS; LFY- LEAFY; lncRNAs- LONG NONCODING RNAs; PHD-PLANT
HOMEODOMAIN; PI-PISTILLATA; PRC2-POLYCOMB REPRESSIVE COMPLEX 2; SOC1-
SUPPRESSOR OF OVEREXPRESSION OF CO1; SEP3- SEPALLATA; SVP-
SHORTVEGETATIVE PHASE; SWN- SWINGER; VIN3 –VERNALIZATION; INSENSITIVE 3;
VRN1-VERNALIZATION 1,

Glossary
Autonomous pathway: genetic pathway that controls flowering time in Arabidopsis. It is
thought to contain several parallel activities all repressing FLC.
COLDAIR: lncRNA expressed from within FLC intron 1 in the sense direction.
COOLAIR: lncRNA fully encompassing FLC in the antisense direction. It is alternatively
polyadenylated and alternatively spliced.
FCA: RNA-binding protein in Arabidopsis, stimulating alternative polyadenyla-tion genome-
wide, and in particular of COOLAIR.
FLOWERING LOCUS C (FLC): a well-studied Arabidopsis gene (~7 kb) that encodes a
MADS-box transcription factor. This is a key repressor of the floral transition and is targeted
by several regulatory pathways.
FRIGIDA (FRI): an Arabidopsis protein determining the need for vernalization through
establishment of high FLC expression.
Plant homeodomain (PHD): a protein domain that is generally associated with the ability to
recognize particular post-translationally modified histones.
PHD–PRC2: PRC2 accompanied by several PHD domain-containing proteins. It is required for
establishing high H3K27me3 at FLC during vernalization.
Polycomb repressive complex 2 (PRC2): PRC2 catalyzes the methylation of histone tails at
H3K27, leading to high levels of the repressive H3K27me3 mark.
Vernalization: a process in plants whereby flowering is accelerated by prolonged exposure to
cold, ensuring that plants align their flowering with spring.

Front page Figure: Arabidopsis thaliana is induced to flower in response to seasonal cues
such as winter chilling. Here a mature plants flowering in spring after exposure to
winter temperatures is shown.
The role of long noncoding RNAs in the regulation of flowering in
Arabidopsis.

ABSTRACT:

The deciding factors of flowering in plants are generally environmental factors like
temperature and day length, and endogenous factors such as developmental stage and
phytohormones. Genome-wide transcriptome analysis has revealed that a vast diversity of
non-coding transcripts are involved in flowering in plants. Among these non-coding
transcripts, recent works have focused on the roles of long non coding RNAs (lncRNAs).
There are various types of lncRNAs involved in chromatin regulation under vernalization
condition and induce flowering. Vernalization is an environmentally-induced epigenetic
switch in which prolonged exposure to winter cold triggers epigenetic silencing of floral
repressors and provides competence to flower in spring. Flowering Locus C (FLC) acts as a
floral repressor that require vernalization to promote flowering by repressing FLC expression.
In this process, two lncRNAs play their role, one is COLD INDUCED LONG ANTISENSE
INTRAGENIC RNA (COOLAIR), an antisense transcript and other one is COLD ASSISTED
INTRONIC NONCODING RNA (COLDAIR). In Arabidopsis thaliana, winter temperature
triggers enrichment of H3K27me3 in the chromatin of the floral repressor FLC and results in
epigenetically stable repression of FLC through PRC2 (Polycomb Repressive complex2)
complex. COOLAIR expression promotes 3′ processing at the proximal polyadenylation site
of COOLAIR transcript resulting in H3K4 demethylation of FLC (Down-regulation of FLC
transcription) and promotes flowering. COLDAIR is transcribed in the sense direction from
an intron FLC. COLDAIR promotes vernalization-mediated epigenetic repression of FLC via
the establishment of stable repressive chromatins at FLC through interaction with PRC2. This
review focuses on role of lncRNAs in chromatin modification, and regulation of FLC
expression, one of the key steps in Arabidopsis flowering.

Keywords: Long noncoding RNAs, FLC locus, COOLAIR, COLDAIR, PRC2 complex,
Autonomous pathway, Vernalization pathway, miRNAs,
CONTENTS

1. Introduction

2. Background of lncRNAs

3. Structure of lncRNAs

4. Characteristics of lncRNAs

5. Molecular functions of lncRNAs in plants

5.1. lncRNAs as natural miRNA target mimics

5.2. lncRNAs as molecular cargo for protein re-localization

5.3. lncRNAs and regulation of flowering

5.3.1. Genetic regulation of flowering

5. 3.2. Role of lncRNAs in flowering

5.3.2.1. Action of COOLAIR in autonomous pathway

5.3.2.2. Action of COOLAIR in vernalization pathway

5.3.2.3. Action of COLDAIR in vernalization pathway

5.3.2.4. CUL3 mediated FRI degradation modulates flowering by COLDAIR

6. Identification of new lncRNAs involved in flowering

7. Concluding remarks

8. References
 1. Long noncoding RNAs (lncRNAs): An introduction

Eukaryotic genomes produce transcripts in a wide range of sizes, from long protein-
coding mRNAs to short noncoding transcripts (Ponting et al., 2009). When the whole human
genome was sequenced, it was a surprise that there are only about 20,000-25,000 protein-
coding genes, representing less than 2% of whole human genome. With the rapid
development in high throughput sequencing technologies such as deep sequencing and whole
genome high density tiling array, it is now known that about 98% of the “junk” DNAs are
transcribed as noncoding RNAs (ncRNAs) including short and long ncRNAs (Nie et al.,
2012). Noncoding RNAs include housekeeping RNAs such as ribosomal, transfer and small
nuclear and nucleolar RNAs and regulatory noncoding RNAs. Regulatory ncRNAs are two
types, short noncoding RNAs and long noncoding RNAs (lncRNAs). The short regulatory
noncoding RNAs include microRNAs (miRNAs), small interfering RNAs (siRNAs) and
piwi-associated RNAs (piRNAs) (Moseley et al., 2006). Short noncoding RNAs are less than
200 nucleotide in length, and play crucial role in transcriptional and post transcriptional
regulation of gene expression. Although most of the earlier work on regulatory noncoding
RNAs has focused on short ncRNAs recent work has revealed the importance of lncRNAs.
lncRNAs are defined as RNA transcripts more than 200 nucleotides in length that lack
protein coding capability (Yamaguchi and Abe, 2012).

Now it is observed that many lncRNAs play critical regulatory roles in diverse cellular
processes such as chromatin remodeling, transcription, post-transcriptional process and
intracellular trafficking (Mercer and Mattick, 2013). Most lncRNAs have been found to be
critical regulator of gene expression. lncRNAs have been found to act nearly every level of
gene regulation; epigenetic, transcriptional, post transcriptional and translational (Wapinski
and Chang, 2011). Research in animal system showed that lncRNAs participate in many
significant biological processes such as X- chromosome inactivation and genomic imprinting
(Ponting et al., 2009). In addition research on lncRNA has application in the treatment of the
diseases such as cancer and Alzheimer's diseases (Wapinski and Chang, 2011). In contrast to
researches in animal, a limited number of publications have revealed the significant role of
lncRNAs in plant gene expression and regulation. These lncRNAs are involved in the
transcriptional and posttranscriptional regulation of gene expression and the modulation of
RNA stability and translation under stress conditions. A set of lncRNAs plays a crucial role
in regulating the expression of the floral inhibitor FLC (FLOWERING LOCUS C)
 2. Background of lncRNAs

Many initial lncRNAs such as XIST and H19 were discovered in the 1980s and 1990s by
searching cDNA libraries for clone of interest. XIST was identified as an effort to characterize
the genomic loci responsible for X-chromosome inactivation (Prensner and Chinnaiyan,
2011). In the past decade large scale analysis was done for identifying lncRNAs including
DNA tiling array and next generation RNA sequencing (NGS). RNA sequencing studies now
suggest that several thousand uncharacterized lncRNAs are present in a given cell type. Large
scale analyses of lncRNAs in stem cell suggest that lncRNAs may be an integral component
of stem cell biology. Tiling array analysis is used to identify the lncRNAs transcribe from
intergenic region (Fatica and Bozzoni, 2014). Whole Genome Array method first detected
large number of unannotated novel transcript in Arabidopsis. These novel transcripts may
represent alternatively spliced forms of known genes, products of antisense or bidirectional
transcription, retained introns. These discovered transcripts were later described as lncRNAs
(Moghe et al., 2013). Using Whole-Genome Tiling Array and RNA-seq approaches, a large
number of uniquely transcribed intergenic regions and stress induced novel transcripts were
found in rice and Arabidopsis respectively. In addition, a single nucleotide resolution array
designed for the Arabidopsis FLC locus (Swiezewski et al., 2009).

3. Structure of lncRNAs

lncRNAs are expressed in lower amount generally compared to their protein coding
counterpart. lncRNAs can be distinguished from mRNA on basis of their high folding energy
and stem loop structure. lncRNAs contain 3 types of regulatory domain RNA binding
domain, protein binding domain and DNA binding domain (Mercer and Mattick, 2013).
(Please refer figure 1)

RNA binding domain

The ability of lncRNA to base pair with other RNAs, lncRNA can act as highly specific
sensor of mRNA, microRNA, and other lncRNA expression. Antisense lncRNAs can regulate
the stability and translation of complementary mRNAs. For example, translation of the
UCHL1 mRNA is regulated under stress by action of an lncRNA with antisense sequence that
is complementary to and encompasses, the UCHL1 start codon (Carrieri et al., 2012).
Figure 1. a and b) lncRNAs contain structural domain that can sense or bind other RNA via
complementary base pair interactions, proteins and possibly DNA that can induce allosteric
conformational changes to other structures in the lncRNA c) binding of RNA induces a
conformational change that prevents protein binding, the protein can bind in the absence of
RNA, inducing the formation of a stem loop secondary structure that can be processed and
cleaved to generate an RNA output. d) lncRNAs can act as molecular scaffold by binding
multiple proteins to form complex ribonucleoprotein structure. e) lncRNAs can target the
catalytic function of proteins to specific sites in the genome as guide [source - (Mercer and
Mattick, 2013)].
 The cleavage of lncRNA can also generate small RNAs that serve as an output signal.
For example, a small tRNA-like sequence is cleaved from the 3′ end of the lncRNA
MALAT1 and trafficked from the nucleus to the cytoplasm (Wilusz et al., 2008). The
formation of stem loop provides a ready substrate for Dicer enzyme to generate multiple
regulatory RNAs with cascading ability to mediate downstream epigenetic changes (Djupedal
and Ekwall, 2009).

Protein binding domain

Proteins are the major part of lncRNA with complex ribonucleoprotein (RNP)
particles acting as chaperones, transport aids or effectors (Riordan et al., 2008). Proteins tend
to interact with RNA where it forms complex secondary structures, positioning protein
structure into the groove of an RNA stem loop helix or providing a binding pocket in β-sheets
for unpaired RNA nucleotides. Such interactions provide conformational changes to the
protein, the RNA or both (Reiter et al., 2011). Photoactive ribonucleoside enhanced
crosslinking and immunoprecipitation (PAR-CLIP) studies provide the interaction between
lncRNA and protein (Scheibe et al., 2012).

DNA binding domain

There is a little evidence for direct interaction between lncRNA and DNA. RNA-DNA
hybrids or triplex structure can allow single strand of RNA to interact with DNA duplexes by
base pair interactions. These RNA-DNA interactions could efficiently and selectively target
RNA signals togenomic loci. Such interactions may expose the genome to deamination and
damage (Buske et al. 2011). Similar to protein transcription factors, an enriched DNA
sequence motif has been identified in the binding sites of an lncRNAs, HOTAIR (Chu et al.,
2011).

4. Characteristics of lncRNAs

lncRNAs can be divided into four rough categories according to their relationship to nearby
protein coding gene. Sense lncRNAs overlap with one or more exon of a transcript on the
same strand. Antisense lncRNAs overlap with one or more exons of a transcript on the
opposite strand, intronic lncRNAs derived from an intron within another transcript, and
intergenic lncRNAs occur in the interval between genes on the same strand (Ma et al., 2013).
Most of the lncRNAs are transcribed by RNA polymerase II in eukaryotes. The biogenesis
pathway of lncRNA is similar as mRNA and other noncoding RNA. These lncRNAs may be
located within the nucleus or cytosol and may or may not have poly (A) tail. lncRNAs may
originate from intronic, exonic, intergenic, intragenic, promoter region, 3'and 5'UTR and
enhancer sequence and are sometimes bidirectional transcript. These can be transcribed in
sense or antisense direction. Both lncRNAs and short ncRNAs generally transcribe away
from the 5' or 3' ends of genes, but lncRNA transcripts originate near the promoters and the
first exons or introns of genes (Do Toit, 2013).

5. Molecular functions of lncRNAs in plants

The lncRNAs have been shown to act in various cellular and biological functions such as
miRNA target mimic, substrate for chromatin modification, molecular cargo for protein re-
localization, biotic and abiotic stress etc.

5.1. lncRNAs as natural miRNA target mimics

Phosphate is an essential macronutrient for plant growth and development. Plants not
only absorb phosphate from the soil but must also have a regulatory mechanism to maintain
phosphate homeostasis throughout the plant for the growth and metabolic requirements of
each tissue. In phosphate starvation miRNA399 is expressed in companion cells and phloem
(Aung et al., 2006). In pho2 mutant Arabidopsis, the target of miR399 and encoding an E2
ubiquitin conjugase related enzyme (UBC24) with a single point mutation is repressed due to
miR399 mediated mRNA cleavage (Aung at al., 2006). Consequently, the gene pho2 is now
found to be UCB24. Low UCB24 activity leads to enhancer expression levels of two root
specific phosphate transporter gene, pht8 and pht9, resulting in increased phosphate uptake.
Besides miR399, lncRNA Induced by phosphate starvation 1 (IPS1) is also induced by low
UCB24 activity. IPS1 is a member of the TPS1/Mt4 gene family which was first lncRNA
found in tomato and Medicago truncatula and then in rice and Arabidopsis. IPS1 does not
encode a protein, and 23 nucleotide long sequence motif is conserved among the member of
different plant species. This 23-nt motif is partially complementary to miR399 with a 3
nucleotide central mismatch corresponding to position 11-13 of miR399. As miRNA
mediated RNA cleavage usually occurs between nucleotide 10 and 11 relative to the 5'end of
the miRNA, this central mismatch disrupt crucial base-pairing between miR399 and IPS1 and
hence inhibits miR399 mediated cleavage of IPS1.
 From above discussion it is evident that IPS1 functions as non-cleavable target mimic of
miR399 to sequester of miR399 which in turn attenuates miR399-mediated repression of
PHO2. Thus the increased expression of IPS1 under phosphate starvation appears to counter
balance the effect of increased miR399 accumulation under the same condition, resulting in
balance of pho2 (UCB24) expression and phosphate uptake (Rymarquis et al., 2008). The
inhibition of miRNA activity by an endogenous non cleavable ncRNA target has been termed
as target mimicry.

5.2. lncRNA as Molecular cargo for protein re-localization

The nodulin gene Enod40 is a plant gene that participates in the regulation of symbiotic
interaction between leguminous plant and soil bacteria. Enod40 gene first identified in
soybean and Medicago sativa spp.Varia (Yang, et al., 1993). Enod40 is rapidly induced by
rhizobia in the root pericycle and in the dividing cortical cells of the nodule primordial during
the symbiotic interaction. Transgenic approach confirmed role of Enod40 in nodulation.
Enod40 transcript lacks long open reading frames, but encode two short peptides (12 and 24
amino acid residues in soybean; 13 and 27 amino acid residue in Medicago truncatula).). But
later the uncovserved activity of one of the two short peptide and highly stable RNA
secondary structure. More importantly Enod40 has been shown to directly interact with
MtRBP1 (Medicago truncatula RNA binding protein 1) and play a role in relocalization of
MtRBP1 from nuclear speckles into cytoplasmic granules during nodulation in M. truncatula.
This re-localization suggesting that Enod40 RNA rather than Enod40 encoded short peptides
is important for MtRBP1 re-localization. Thus lncRNA Enod40 is part of the
nucleocytoplasmic trafficking machinery (Yamashita et al., 1998). Example of lncRNAs in
plants and their role is summarized in Table 1.
Table 1: Diverse functions of lncRNAs (Source- Quan et al., 2015)

LncRNA Biological function Biological processes regulated by

lncRNAs in plants
IPS1 competes with PHO2 to interact with Phosphate balance; example-
miR399 and act as miRNA target mimic Arabidopsis, Rice, Tomato, M.
truncatula
Enod40 Encodes two short peptides and an Sucrose utilization in nodules and
ncRNA with secondary structure nodulation, example-M. truncatula
COOLAIR Functions with other vernalization- Vernalization; example-Arabidopsis
related elements in regulating FLC
expression
COLDAIR Recruits PRC2 to silence in vernalization Vernalization, example- Arabidopsis
response
ASL Regulated by AtRRP6L to maintain the Vernalization, example- Arabidopsis
level of H3K4me3
H1D1 Act as positive regulator in photo photo morphogenesis
morphogenesis
LDMAR Regulates photoperiod sensitive male Photoperiod-sensitive male sterility in
sterility Rice
 5.3. lncRNAs and regulation of flowering in Arabidopsis
5.3.1. Genetic regulation of flowering in Arabidopsis
Plants undergo a series of phase transitions during their life-cycle in response to environment
and endogenous factors. The transition from vegetative to reproductive growth, which
happens under tight genetic control, is one of the most fundamental developmental steps.
Arabidopsis thaliana is a small flowering plant of Brassicaceae family. Flowers are 2mm
long and self-pollinate. The small size of its genome (around 140 Mb) and shorter lifecycle is
advantageous for research. Such advantages have made Arabidopsis a model organism for
studies cellular and molecular biology of flowering plants. Genetic and molecular analysis of
flowering time mutant in Arabidopsis has established a current model in which five major
genetically defined pathways regulate the transition from the vegetative to reproductive phase
(Srikanth and Schmid, 2011; Gul Khan et al., 2014). (Please refer figure 2).

Figure 2. Five independent pathways involved in flowering in Arabidopsis have been

characterized e.g., vernalization, photoperiod, autonomous, gibberellin, and aging pathway.
The function of the autonomous and vernalization pathways is to repress the activity of
FLC, a repressor of flowering, via chromatin modifications of the FLC locus. The targets of
repression by FLC are FT and SOC1, also known as floral pathway integrators, which are
positively regulated by the photoperiod pathway [source- (Gul Khan et al., 2014)].
 The photoperiod pathway regulates flowering time based on day length and light
intensity. The vernalization pathway mediates the response to low temperature over long
periods, enabling plant to sense and remember winter has come and gone. Autonomous
pathway act independently from the photoperiod and vernalization pathway, and share a few
same targets with the vernalization pathway. The gibberellins pathway defines a requirement
for gibberellic acid, primarily under unfavorable condition for flowering (Srikanth and
Schmid, 2011). Aging pathways (miR156 and miR172 dependent pathway) are independent
of environment cues and directed by internal program (Wang et al., 2009). Above mentioned
five pathways together constitute a complex gene network including CONSTANS (CO),
FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1),
Flowering time control protein FCA, FVE, and FLOWERING LOCUS C (FLC) are regulated
by extensive DNA methylation, histone modifications, small RNA-mediated chromatin
silencing, and alternative splicing mechanisms (Turck et al., 2008).

REGULATION OF FLOWERING BY PHOTOPERIOD PATHWAY

Photoperiod is one of the most important environmental factors affecting floral

transition. The genes CRYPTOCHROME2 (CRY2), GIGANTEA (GI), FT, CO, and FWA have
major regulatory roles in the photoperiod pathway.GI increases CO mRNA abundance. FWA
and FT may interfere with pathways downstream of CO (Lin et al., 2007).

FWA encode a homeodomain containing TF required during flowering. CRY2,

CRY1, PHYA (PHYTOCHROME A) contribute to the stabilization of CO protein in late
afternoon whereas PHYB (PHYTOCHROME B) destabilize CO in the morning. CO
transcription is under circadian regulation through GI and Cycling DOF factor 1(CDF1)
activity (Turck et al., 2008). The circadian clock and light signaling tightly control CO
protein activity throughout the day in the companion cells of the leaf phloem. The amount of
FT mRNA, which is directly induced by the transcriptional activator CO protein, strongly
influences the timing of flowering. FT protein encodes a 20-kD protein of the PEBP/RKIP
(for phosphatidyl ethanolamine binding protein/Raf kinase inhibitor protein) family, major
component of florigen, a floral inducing substrate (Kobayashi et al., 1999). FT forms a
complex with FLOWERING LOCUS D (FD) at shoot apical meristems and initiates
flowering by activating floral meristem identity gene such as AP1 and other floral promoter
such as SOC1 (Michaels., 2009).
REGULATION OF FLOWERING BY VERNALIZATION
Vernalization is defined as the process by which exposure of plants to the cold of
winter causes them to accelerate flowering. Two dominant genes, FRIGIDA (FRI) and FLC
are involved in this pathway.FRI encodes a coiled coil protein that activates the expression of
FLC. FLC encode a MADS- box transcription factor and acts as dominant repressor of
flowering through its control of FT and SOC1. FLC block the transcriptional activation of
SOC1 and FT by directly interacting with CArG boxes of their respective chromatin
(Helliwell et al., 2006). Another major regulator of flowering in response to temperatures is
SHORTVEGETATIVE PHASE (SVP). ChiP analysis of FLC and SVP showed binding sites in
both the flowering integrator FT and SOC1.VIN3, a chromatin remodeling plant
homeodomain (PHD) finger protein that increases acetylation level. VIN3 gets activated by
long cold periods and establishes the vernalization response by promoting histone
deacetylation at the FLC locus and effectively repressing FLC expression (Sung and
Amasino, 2004). VRN1 encodes DNA binding protein and VRN2 encodes nuclear protein.
Additionally, VRN1 and VRN2 are also involved in the maintenance of FLC repression
(Gendall et al., 2001).

AUTONOMOUS PATHWAY OF FLOWERING REGULATION

The autonomous pathway was identified via a group of mutants that were late
flowering under all photoperiods and were highly responsive to vernalization. Their late-
flowering phenotype was overcome by vernalization. These mutants included
LUMINIDEPENDENS (LD), FCA, FY, FPA, FLOWERINGLOCUS D (FD), FVE, FLOWERING
LOCUS K (FLK), and REF6 (RELATIVE OF EARLY FLOWERING 6) (Michaels and Amasino,
1999). So, vernalization and the autonomous pathway have been considered to function in parallel.
LD is expressed primarily in regions of cell proliferation and encodes a nuclear protein that regulates
LEAFY expression. FD and FVE participate in the deacetylation of FLC chromatin. Three proteins,
FCA, FPA, and FLK, contain RNA binding domains, and one FY, is a member of a cleavage and
polyadenylation specificity factor (CPSF) RNA 3' processing complex. FCA interact with FY
through its C-terminal WW domain (Marquardt et al., 2006). Abscisic acid (ABA) binds FCA
and blocks this interaction. WW domain require for FCA autoregulation. Both FCA and FPA
function in autonomous pathway to control FLC mRNA accumulation and flowering. FD encodes
a bZIP protein which is required for the function of FT. FLK encodes an RNA binding protein
with k homology motifs and regulates the autonomous flowering pathway via FLC. RFL6
demethylates H3K27me3 and H3K27me2.
GA PATHWAY: A HORMONAL CONTROL OF FLOWERING

Three related genes with key roles in GA signaling, GIBBERELLIC ACID

INSENSITIVE (GAI), REPRESSOR OF GA1-3 (RGA), RGA-LIKE 1(RGL1), have
overlapping functions (Mutasa-Gottgens and Hedden, 2009). All are members of GRAS
family, contain highly conserved central (VHIID), C-terminal (RVER) region and N-
terminal DELLA region. RGL1 plays role in GA response in inflorescence whereas RGA,
RGL1 are less important. GA increases the transcriptional activity of floral meristem LFY and
promotes flowering.

AGING PATHWAY

Recent studies indicate that miR156 and miR172 play key roles in vegetative
phase change, with both targeting DNA-binding transcription factors. In Arabidopsis
miRNA172 promotes photoperiodic flowering independent of CO. miRNA156 is highly
abundant in the juvenile stage decreases at adult stage, while miRNA172 has an opposite
expression pattern. Overexpression of miRNA156 negatively regulates several SQUAMOSA
PROMOTER BINDING PROTEIN-LIKE (SPL) genes that delays both the juvenile to adult
and adult to reproductive phase transition. The SPL3/4/5 genes have overlapping functions in
the regulation of vegetative phase transition and floral induction in Arabidopsis, while
miRNA156 is responsible for the temporal change in SPL3 expression during vegetative
development (Wu and Poethig, 2006).

Vegetative meristem (VM)- inflorescence meristem (IM) transition is directly

promoted by SOC1-AGL24 (AGAMOU LIKE 24) complex. IM gives rise to floral meristem
(FM). SOC1 and AGL24 dimer activates LFY expression on the flank of the developing IM.
Later AP1 and LFY up-regulate each other in the feedback loop that functions to maintain
floral meristem identity.

The developmental changes that occur in the FM were confined in the ABCE model,
which postulate that four regulatory function (A, B, C and E) work combinatorial for the
proper organ formation in each whorl. A class of protein AP1 and AP2 confer sepal identity
in the first whorl. Their activity overlaps in the second whorl with the B class protein AP3
and PISTILATA (PI), resulting in petal identity. Stamens are formed in third whorl due to
combine activity of the B class proteins together with C class protein AG (AGAMOUS). E
class protein SEPALLATA3 (SEP3) plays a crucial role in carpel development in fourth whorl
(Pose et al., 2012).
 5.3.2. Role of lncRNAs in flowering

A set of lncRNAs plays crucial role in regulating the expression of FLC. Several
modification of histone 3, such as the methylation of H3K4 and H3K27affect FLC
expression. In FLC chromatin, H3K4 methylation leads to an active chromatin state, which is
required for FLC expression (Letswaart et al., 2012). H3K27me3 is also required for FLC
silencing (Heo and Sung, 2011). Two classes of lncRNA are transcribed from FLC locus;
participate in epigenetic silencing of FLC.

The first class is COLD INDUCED LONG ANTISENSE INTRAGENIC RNA

(COOLAIR) antisense lncRNAs, which transcribe from the 3′ end of FLC (please refer figure
3). COOLAIR is alternatively spliced and alternatively polyadenylated with two major classes
in warm, the proximal polyadenylated in the six intron of FLC class I(ASI) and the distal
polyadenylated class II in the promoter region of FLC (overlapping the sense promoter). The
two different isoforms affect H3K4 demethylation to silence FLC (Quan et al., 2015).

Figure 3: The FLC locus expresses multiple types of transcripts. COOLAIR is non coding
transcript that fully encompasses FLC in antisense direction; it is alternatively polyadenylated
with a proximal poly A site in sense intron 6 and distal poly A site in sense promoter region.
COLDAIR form within intron 1 of FLC in the sense direction is a cap but non-polyadenylated
LncRNA [Source-(Letswaart et al., 2012)].

5.3.2.1. Action of COOLAIR in Autonomous Pathway

The autonomous pathway consists of seven known genes FCA, FLOWERING LOCUS
D (FD), FPA, FVE, FY, LD and FLK. FCA plays a major role in the promotion of transition
of vegetative meristem to reproductive development by decreasing FLC mRNA level.
FD and FVE participate in the deacetylation of FLC chromatin. FCA and FY are
important for RNA 3′ processing by associated with two canonical 3 processing factors,
CstF64 and CstF77. FCA and FY function to promote the use of the proximal site, and FCA
associates with FLC chromatin in this region.FCA interacts with COOLAIR through its
RRMs (RNA recognition motif) bringing that transcript to the RNA 3′- processing machinery
through the interaction with FY. FPA another RRM protein that acts independently of FY
(Baurle and Dean, 2008), also promotes use of the proximal site (Hornyik et al., 2012). The
proximal poly A site then trigger the FD demethylation to reduce the levels of dimethylated
histone H3K4me2 in the body of FLC , leading to a repressed chromatin state and reduce
sense and antisense expression resulting in a positive feedback mechanism that reinforces
proximal polyadenylation and low expression of FLC (Letswaart et al., 2012) (figure 4).

Figure 4. COOLAIR and Autonomous pathway, A) In the absence of FCA, COOLAIR

transcripts are polyadenylated under the influence of FY, CstF64 andCstF77 at distal poly (A)
site. This is associated with high H3K4me3 levels in the body of the FLC gene and with high
level of functional FLC mRNA and COOLAIR expression. B) FCA promotes alternative
polyadenylation of COOLAIR through targeting FY and CstF activity to proximal poly (A)
site. This event is likely to trigger FD-dependent demethylation in the body of FLC, leading
to a transcriptional repressed state and low levels of functional FLC mRNA [source-
(Letswaart et al., 2012)].
 5.3.2.2. ACTION OF COOLAIR IN VERNALIZATION PATHWAY

A second pathway represses FLC expression by COOLAIR is vernalization pathway.

Vernalization is a process whereby flowering is accelerated by prolonged cold and it ensures
that plants align their flowering with spring. Prolonged cold represses FLC transcriptionally
and induces epigenetic silencing that is mediated by a polycomb (PcG) silencing complex
including proteins containing a plant homeodomain (PHD). A core polycomb repressive
complex 2 (PRC2) associated with FLC chromatin independent of temperature. The
expression of COOLAIR increases earlier than vernalization markers VIN3
(VERNALIZATION INSENSITIVE 3). Cold induces more accumulation of ASI than ASII.
COOLAIR is physically associated with genomic DNA in the FLC locus i.e., enriched over
the whole FLC but concentrate in two discrete regions of FLC genomic DNA the nucleation
region and 3' region of the gene/ COOLAIR promoter. Most of COOLAIR induced in cold is
in the OFF chromatin fraction. ON-chromatin COOLAIR is required for FLC regulation.

The nucleation region is where the localized increase of H3K27me3 and decrease of
H3H36me3 occur. This has a vital role in accelerating transcriptional repression of FLC
during cold exposure. The 3' region of the gene /COOLAIR promoter has been shown to
contain R-loop (DNA-RNA hybrid) that suppresses COOLAIR transcription.

In addition AtNDX a homeodomain protein associated with ssDNA rather dsDNA non-
sequence specifically in vitro and localize to a heterochromatic region in the COOLAIR
promoter in vivo. ssDNA was detected in vivo as part of RNA-DNA hybrid or R-loop that
cover the COOLAIR promoter. R-loop stabilization mediated by AtNDX inhibits COOLAIR
transcript transcription, which in turn modify FLC expression. Use of multiple T-DNA
insertion lines across the FLC COOLAIR showed that COOLAIR is not required for initial
repression of FLC: instead promoter and first axon of FLC gene sufficient to initiate FLC
repression during vernalization. After return to warm temperature, sequence in intron 1 of
FLC is required to maintain a repressed chromatin state in this region (where H3K27me3
accumulate). COOLAIR regulates H3K36me3 which has important effect on chromatin states,
but COOLAIR and H3K27me3 function independently, consistent with the antagonistic
interaction between H3K27me3 and H3K36me3 (Please refer figure 5). FLC down regulation
and silencing in the cold is therefore a consequence of parallel activities that result in H3K27
trimethylation and H3K36me3 demethylation (Csorba et al., 2014).
Figure 5. COOLAIR in vernalization pathway, COOLAIR negatively affects H3K36me3
methylation status and function independently of PHD-PRC2 mediated H3K27me3 to
promote FLC silencing during vernalization. [Source - (Csorba et al., 2014)]

5.3.2.3. Action of COLDAIR in vernalization pathway

Vernalization induces the expression of COLDAIR, but COLDAIR expression peaks later
than COOLAIR expression. COLDAIR is in the sense direction relative to FLC mRNA
transcription. It contains a 5′ cap structure. The approximate size of COLDAIR is 1100 bases
long. Cold induces the accumulation of RNA Pol II which in turn is responsible for
COLDAIR transcription. COLDAIR is required to establish a silenced state that is stable
chromatin at FLC through its interaction with PRC2. Protein components of PRC2 are
CURLY LEAF (CLF), SWINGER (SWN) and VERNALIZATION 2 (VRN2). The CLF and
SWN bind to COLDAIR through CXC domain. Other FLC related floral repressor genes
including FLOWERING LOCUS M/MADS AFFECTING FLOWERING (MAF1), MAF2 and
MAF3 are also suppressed in response to vernalization. Deletion of the vernalization response
element (VRE) in the intron of FLC impairs vernalization-mediated FLC repression,
describing that transcription start site of COLDAIR is within 5′ region of VRE (Castaings et
al., 2014). COLDAIR is necessary for the activity of PRC2 to be directed to FLC chromatin.
There is a basal level of CLF occupancy at FLC before vernalization and the level of
occupancy increases after vernalization also increases the level of H3K27me3 at FLC
chromatin. In addition vernalization results in the stable reduction of the levels of histone H3
lysine 4 trimethylation a histone mark associate with active chromatin at FLC chromatin.
Thus the lncRNA COLDAIR mainly plays a role in the recruitment of PRC2 to FLC
chromatin to establish the stable silencing of FLC by vernalization (Heo and Sung, 2011)
(Please refer figure 6).
Figure 6. COLDAIR regulates FLC in a PRC2 associated histone modification. Top)
COLDAIR is induced by cold treatment. Mid) COLDAIR recruit PRC2 complex to the FLC.
Bottom) PRC2 complex deposit H3K27me3 on FLC. [source (Zhang et al., 2013)].

5.3.2.4. CUL3 mediated FRI degradation modulates flowering by COLDAIR

Arabidopsis thaliana requires cold stress or vernalization to initiate flowering via FRIGIDA
(FRI). FRI encodes a coiled coil protein that activates the expression of FLC. In Arabidopsis
proteasome mediated FRI degradation regulates flowering during vernalization. FRI directly
interacts with the BTB protein LIGHT RESPONSE BTB1 (LRB1) and LRB2 as well as
CULLIN3 (CUL3) ubiquitin E3 ligases leading to proteosomal degradation of FRI during
vernalization (Christians et al.,2012). The degradation of FRI is accompanied by an increase
in the levels of COLDAIR, which reduce the level of histone H3K4 trimethylation in FLC
chromatin to promote flowering. Cold induced WRKY34 transcription factor bind to the W-
box in the promoter region of CUL3A to modulate CUL3A expression (please refer figure 7).
Deficiency of WRKY34 suppressed CUL3A transcription to enhance FRI protein stability
and led to late flowering after vernalization. Conversely over-expression of WRK34
promoted FRI degradation and early flowering through inducing CUL3A accumulation (Hu
et al., 2014).
Figure 7. Cold stress activates WRKY34 accumulation and potentiates its binding to the
CUL3A promoter region to enhance its accumulation and FRI protein degradation. Loss of
the FRI scaffold impairs the interaction of its partner protein with FLC and suppresses FLC
transcription [SOURCE- (Hu et al., 2014)].

6. Identification of new LncRNA for flowering regulation

Recent work in Arabidopsis thaliana identified a novel antisense transcript termed ASL
(Antisense long), which is transcribed from the FLC locus in wild type plant.ALS has the
same promoter as COOLAIR AS I and ASII, and the transcript of ASL overlaps with the 5′
region of ASI and ASII: however, ASL differ from ASI and ASII in the sense ASL is caped,
non-polyadenylated where as ASI and ASII which are polyadenylated (please refer figure
8). ASL is transcribed by RNA Pol II and alternative splicing produces two isoform of ASL,
2236 and 2536 nucleotide long. Mutations of two Arabidopsis genes encoding nuclear
exosome components AtRRP6L1 and AtRRP6L2, cause de-repression of the main flowering
repressor FLC and thus delay flowering in early flowering Arabidopsis. RRP6 (ribosomal
RNA processing protein 6) is a subunit of exosome, a nuclear complex with 3′to 5′
exoribonuclease activity (Quan et al., 2015).
Figure 8. COOLAIR and ASL transcripts are transcribed in the antisense direction. AS I and
AS II, two antisense transcripts of COOLAIR, are transcribed in the antisense direction
relative toFLC mRNA. Red boxes correspond to AS I and II exons, and dotted red lines
correspond tothe spliced regions of AS I and II. AS I has a proximal poly A in the sixth intron
of FLC, andAS II has a distal poly A that overlaps with the promoter region of FLC. ASL is
an antisense LncRNA with two isoforms produced by alternative spicing. The two red bold
lines indicate the two isoforms of ASL. They transcribe from the same promoter as
COOLAIR and span the first intron of FLC [SOURCE (Quan et al., 2015)].

ALS may be responsible for maintenance of H3K27me3, as ASL span the first intron
of FLC and overlap with COLDAIR, which function in establishing and sustaining the level
of H3K27me3. AtRRP6L1 physically associated with ASL transcript and directly interacts
with the FLC locus. AtRRP6L mutations affect the expression of known FLC regulatory
antisense RNAs ASI and ASII and cause an increase in H3K4me3 at FLC. AtRRP6Ls might
participate in multiple FLC silencing pathways by regulating diverse antisense RNAs derived
from the FLC locus (Shin and Chekanova, 2014).
 Vernalization pathway Autonomous pathway

FCA,CstF64, CstF 77

COLDAIR COOLAIR-Genomic DNA at FLC Proximal 3′processing

CLF, SWN of COOLAIR

COLDAIR-PRC2 RNA-DNA Hybrid(R-loop) FY

VRN2 FD demethylation

H3K27 increase H3K36me3 decrease

Reduced dimethylated H3K4me2

FLC Silencing FLC Silencing

FLC Silencing

Vegetative SOC1, FT Flowering

Figure 9. Schematic diagram showing involvement of lncRNAs in FLC silencing. In

autonomous pathway, FCA and FY interact with CstF64 and CstF77and process 3'end of
COOLAIR. It triggers FD demethylation and silencing of FLC by reducing H3K4me2. In
vernalization pathway, COOLAIR physically associates with FLC forming RNA-DNA
hybrid R-loop. This results in decrease of H3K36me3 leading to FLC silencing.
Alternatively, COLDAIR binds with CLF and SWN protein components of PRC2, and VRN2
to increase H3K27me3 level at FLC chromatin. FLC silencing de-represses flowering
integrator FT and SOC1 which activate floral meristem gene and initiate flowering.

7. Concluding Remarks

The advancement of sequencing technology has significantly contributed the function and
action of long noncoding RNAs in FLC repression pathways. The recent studies have
indicated that two classes of lncRNAs transcribed from FLC, COOLAIR and COLDAIR
participate in epigenetic silencing of FLC. Repression of FLC in turn activates SOC1 and FT
and promotes flowering (Schematic summary of molecular circuit of vernalization process is
depicted above in Figure 9). The biochemical function of FRI is poorly understood. Also
mechanism by which FCA regulates FLC is not known. How use of proximal poly (A) site
stimulates FD activity remains unclear. Arabidopsis genome (125mbp) has 25,498 genes and
about 70% non-annotated protein coding region. So far 13,000 transcripts with description of
lncRNAs have been reported in PLncDB. Further studies of lncRNAs in plants will require
high throughput technique such as chromatin isolation by RNA purification chiRP (to study
epigenetic regulation by lncRNA), Photo-inducible ribonucleoside enhanced-cross linking
and immunoprecipitation (PAR-CLIP) (for identification of various targets of lncRNAs).
Complete elucidation of flowering pathway will have wider commercial and agricultural
application as the environmental and seasonal barrier for flowering could be overcome with
molecular tool.

With respect to flowering-time control, subtle changes in timing have large implications
for seed yield and thus reproductive success. One might see many environmental inputs
influencing the antisense-mediated regulation of FLC in specific genotypes, and also find
extensive variation in this mechanism in natural populations adapted to very different
climates. The technological knowledge gathered using Arabidopsis as a model system for
vernalization molecular circuit analysis can be useful in understanding and manipulating
vernalization process for molecular breeding in economically important crops such as apple
and sugar-beets. Dissection of the complexity of antisense-mediated regulation of FLC and
other important plant targets, aided by concepts from many other systems, will be key areas
for future investigations.

8. References

Aung, K., Lin, S., Wu, C., Huang, Y., Su, C., & Chiou, T. (2006). pho2, a phosphate overaccumulator, is
caused by a nonsense mutation in a microRNA399 target gene. Plant Physiology , 141, 1000-1011.

Baurle, I., & Dean, C. (2008). Differential interactions of the autonomous pathway RRM proteins and
chromatin regulators in the silencing of Arabidopsis targets. PLoS ONE , 3,1-9.

Buske, F. A., Mattick, J., & Bailey, T. (2011). Potential in vivo roles of nucleic acid triple-helices. RNA
Biology , 8, 427-439.

Carrieri, C., Cimatti, L., Zucchelli, S., Beugnet, A., Biagioli, M., Fedele, S., et al. (2012). Long non-
coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature , 491,
454-457.

Castaings, L., Bergonzi, S., Albani, M., Kemi, U., Savolainen, O., & Coupland, G. (2014). Evolutionary
conservation of cold-induced antisense RNAs of FLOWERING LOCUS C in Arabidopsis thaliana
perennial relatives. Nature Communications ,5, 1-9.
Christians, M., Gingerich, D., Hua, Z., Lauer, T., & Vierstra, R. (2012). The light-response BTB1 and
BTB2 proteins assemble nuclear ubiquitin ligases that modify phytochrome B and D signaling in
Arabidopsis. Plant Physiology , 160, 118–134.

Chu, C., Qu, K., Zhong, F., Artandi, S., & Chang, H. (2011). Genomic maps of long noncoding RNA
occupancy reveal principles of RNA-chromatin interactions. Molecular Cell , 44, 667-678.

Csorba, T., Questa, J., Sun, Q., & Dean, C. (2014). Antisense COOLAIR mediates the coordinated
switching of chromatin states at FLC during vernalization. PANAS , 111, 16160-16165.

Djupedal, I., & Ekwall, K. (2009). Epigenetics: heterochromatin meets RNAi. Cell Research , 19, 282–
295.

Do Toit, A. (2013). Non-coding RNA: RNA stability control by Pol II. Nature Reviews Molecular Cell
Biology , 14, 128-129.

Fatica, A., & Bozzoni, I. (2014). Long non-coding RNAs: new players in cell differentiation and
development. Nature Review Genetics , 15, 7-21.

Gendall, A., Levy, Y., Wilson, A., & Dean, C. (2001). The VERNALIZATION 2 gene mediates the
epigenetic regulation of vernalization in Arabidopsis. Cell , 107, 525–536.

Gul Khan, M. R., Ai, X. Y., & Zhang, J. Z. (2014). Genetic regulation of flowering time in annual and
perennial plant. WIREs RNA , 5 (3), 347-359.

Helliwell, C., Wood, C., Robertson, M., James Peacock, W., & Dennis, E. (2006). The Arabidopsis FLC
protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight
protein complex. Plant Journal , 46, 183–192.

Heo, J., & Sung, S. (2011). Vernalization-Mediated Epigenetic Silencing by a Long Intronic Noncoding
RNA. Science , 331, 76-79.

Hornyik, C., Terzi, L. C., & Simpson, G. G. (2012). The Spen Family Protein FPA Controls Alternative
Cleavage and Polyadenylation of RNA. Developmental Cell , 18, 203–213.

Hu, X., Kong, X., Wang, C., Ma, L., Zhao, J., Wei, J., et al. (2014). Proteasome-Mediated Degradation
of FRIGIDA Modulates Flowering Time in Arabidopsis during Vernalization. The Plant Cell , 26, 4763-
4781.

Kobayashi, Y., Kaya, H., Goto, K., KoIwabuchi, M., & Araki, T. (1999). A pair of related genes with
antagonistic roles inmediating flowering signals. Science , 286, 1960–1962.

Letswaart, R., Wu, Z., & Dean, C. (2012). Flowering time control:another window to the connection
between antisense RNA and chromatin. Trends in Genetics , 28, 445-453.

Lin, M.-K., Belanger, H., Lee, Y.-J., Varkonyi-Gasic, E., Taoka, K.-I., Miura, E., et al. (2007). FLOWERING
LOCUS T protein may act as the long-distance florigenic signal in the cucurbits. Plant cell , 19, 1488–
1506.

Ma, L., Bajic, V., & Zhang, Z. (2013). On the classification of long non-coding RNAs. RNA Biology , 10,
925-933.

Marquardt, S., Boss, P., Hadfield, J., & Dean, C. (2006). Additional targets of the Arabidopsis
autonomous pathwaymembers, FCA and FY. Journal Of Experimenatal Botany , 57, 3379–3386.
Mercer, T. R., & Mattick, J. S. (2013). Structure and function of long noncoding RNAs in Epigenetic
regulation. Nature Structural & Molecular Biology , 20, 300-307.

Michaels, S. (2009). Flowering time regulation produces much fruit. Current opinion in plant biology ,
12, 75–80.

Michaels, S., & Amasino, R. (1999). FLOWERING LOCUS C encodes a novel MADS domain protein that
acts as a repressor of flowering. Plant cell , 11, 949–956.

Moghe, G. D., Lehti-Shiu, M. D., Seddon, A. E., Yin, S., Chen, S., & Juntawong, P. (2013).
Characteristics and Significance of Intergenic Polyadenylated RNA Transcription in Arabidopsis. Plant
Physiology , 161, 210–224.

Moseley, M. L., Zu, T., Ikeda, Y., Gao, W., Mosemiller, A. K., Daughters, R. S., et al. (2006).
Bidirectional expression of CUG and CAG expantion transcripts and intranuclear polyglutamine
inclusions in spininocerebellar ataxia type 8. Nature Genetics , 38, 758-769.

Mutasa-Gottgens, E., & Hedden, P. (2009). Gibberellin as a factor in floral regulatory networks.
Jouranal of Experimental Botany , 60, 1979-1989.

Nie, L., Wu, H.-J., Hsu, J.-M., Chang, S.-S., LaBaff, A. M., L, C.-W., et al. (2012). Long non-coding RNAs:
versatile master regulators of gene expression and crucial players in cancer. American Journal of
Translational Research , 4, 127-150.

Ponting, C. P., Oliver, P. L., & Reik, W. (2009). Evolution and functions of long noncoding RNAs. Cell ,
136, 629-641.

Pose, D., Yant, L., & Schmid, M. (2012). The end of innocence: flowering networks explode in
complexity. Current Opinion in Plant Biology , 15, 45–50.

Prensner, J. R., & Chinnaiyan, A. M. (2011). The emergence of lncRNAs in cancer biology. Cancer
Discovery , 1, 391-407.

Quan, M., Chen, J., & Zha, D. (2015). Exploring the Secrets of Long Noncoding RNAs. International
Journal of Molecular Sciences , 16, 5467-5496.

Reiter, N. J., Chan, C. W., & Mondragon, A. (2011). Emerging structural themes in large RNA
molecules. Current Opinion in Structural Biology , 21, 319-326.

Riordan, D. P., Gerber, A. P., Herschlag, D., Brown, P. O., & Hogans, D. J. (2008). Diverse RNA-binding
proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system.
PLos Biology , 6, 2297-2313.

Rohrig, H., Schmidt, J., Miklashevichs, E., Schell, J., & John, M. (2002). Soybean ENOD40 encodes two
peptides that bind to sucrose synthase. PNAS , 99, 1915-1920.

Rymarquis, L., Kastenmayer, J., Huttenhofer, A., & Green, P. (2008). Diamonds in the rough: mRNA-
like non-coding RNAs. Trends Plant science , 13, 329-334.

Scheibe, M., Butter, F., Hafner, M., Tuschl, T., & Mann, M. (2012). Quantitative mass spectrometry
and PAR-CLIP to identify RNA-protein interactions. Nucleic Acids Research , 40, 9897-9902.

Shin, J., & Chekanova, J. (2014). Arabidopsis RRP6L1 and RRP6L2 function in FLOWERING LOCUS C
silencing via regulation of antisense RNA synthesis. PLos Geneetics , 10, 1-14.
Srikanth, A., & Schmid, M. (2011). Regulation of flowering time: all roads lead to Rome. Cellular and
Molecular Life Sciences , 68, 2013–2037.

Sung, S., & Amasino, R. (2004). Vernalization in Arabidopsis thaliana is mediated by the PHD finger
protein VIN3. Nature , 427, 159–164.

Swiezewski, S., Liu, F., Magusin, A., & Dean, C. (2009). Cold-induced silencing by long antisense
transcripts of an Arabidopsis Polycomb target. . Nature , 462, 799-802.

Turck, F., Fornara, F., & Coupland, G. (2008). Regulation and Identity of Florigen: FLOWERING LOCUS
T Moves Center Stage. The Annual Review of Plant Biology , 59, 573–594.

Wang, J., Czech, B., & Weigel, D. (2009). miRNA156 regulated SPL transcription factors define an
endogenous flowering pathway in Arabidopsis thaliana. Plant Cell , 138, 1231-1243.

Wapinski, O., & Chang, H. Y. (2011). Long noncoding RNAs and human disease. Trends in cell biology ,
21, 354-361.

Wilusz, J., Freier, S., & Spector, D. (2008). 3′ end processing of a long nuclearretained noncoding RNA
yields a tRNA-like cytoplasmic RNA. Cell , 135, 919-932.

Wu, G., & Poethig, R. (2006). Temporal regulation of shoot development in Arabidopsis thaliana by
miR156 and its target SPL3. Development , 133, 3539–3547.

Yamaguchi, A., & Abe, M. (2012). Regulation of reproductive development by non-coding RNA in
Arabidopsis: to flower or not to flower. Journa of Plant Research , 125, 693-704.

Yamashita, A., Watanabe, Y., Nukina, N., & Yamamoto, M. (1998). RNA-assisted nuclear transport of
the meiotic regulator Mei2p in fission yeast. Cell , 95, 115–123.

Yang, W., Katinakis, P., Hendriks, P., Smolders, A., de, V. F., Spee, J., et al. (1993). Characterization of
GmENOD40, a gene showing novel patterns of cell-specific expression during soybean nodule
development. the plant journal , 3, 573–585.

Zhang, J. M. (2013). Plant Long ncRNAs: A New Frontier for Gene Regulatory Control. American
Journal of Plant Sciences , 4, 1038-1045 .

Zhu, Q.-H., & Wang, M.-B. (2012). Molecular Functions of Long Non-Coding RNAs in Plants. Genes , 3,
176-190.