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Emerging Roles and New Paradigms in Signaling

Mechanisms of Plant Cryptochromes

Manish Jangra
2016BS24M
Dept. Of Botany & Plant Physiology
 Contents
Introduction

Cryptochromes

Discovery of cryptochromes

Structure of Cryptochrome (CRY)

Distribution of cryptochromes in plant
species
Mechanism of Cryptochrome Action

Interaction between cryptochrome and hormone signaling

Physiological responses regulated by cryptochromes in plants

Conclusions and future perspectives

 Introduction
There are two terms related to light for
the growth of plants either light
available or not :-
1. Skotomorphogenic(Dark Condition)
2. Photomorphogenesis(Light
Condition)
How do plants perceive light?
Plants have photoreceptors, which are
proteins that are specially designed to
perceive light and transmit signal for
certain biological effects in the plant.
The effect of light on
plant morphology (the form and
structure of something) is
called photomorphogenesis.
Holtan et al., (2011) Plant physiology
Zoratti et al., (2014) Frontiers in Plant Science
 Five Phytochromes
(PHYs): phyA to phyE
that are involved in red
and far-red light
sensing.

Classes of
photoreceptors:-

Three Cryptochromes
Two Phototropins:
(CRYs): cry1, cry2, and
phot1 and phot2;
cry-DASH; seedling
differential growth in a
development and
light gradient.
flowering.
 Cryptochromes: The blue light photoreceptors

First discovery of
gene in 1993
that encoding
for the first blue
light
photoreceptor,
cryptochrome,
was isolated
from A. thaliana
(Ahmad and
Cashmore,
1993).
 Why these receptors are called Cryptochrome?

Two Reasons

First, the blue-light- Second, because the

mediated responses were molecular nature of these
found to be prevalent in photoreceptors was
the lower plants like ferns, cryptic (hidden/
mosses, algae, and fungi, unknown) at that time.
collectively called
cryptogams.
 Discovery of cryptochromes

Cry-DASH can repair

HY4 gene encodes Cross-hybridization
UV-damaged DNA,
cry1, the first blue studies identified the
similar to DNA
light photoreceptor second member, cry2
photolyases
 • CRYs were first discovered in the model dicot plant A.
thaliana by Ahmad & Cashmore(1993) based on their
studies on hy4 mutant, a mutant impaired in blue
light-dependent inhibition of hypocotyl elongation
HY4 gene response, suggesting a defect in the blue-light-
signaling pathway.
encodes cry1,
• The HY4 gene was isolated by gene tagging method
the first blue and was found to encode a protein of 681 amino
light acids, of which the first 500 showed striking sequence
photoreceptor homology with the microbial DNA photolyases.
• The speculation that this HY4 gene, then named
cryptochrome 1 (CRY1), encodes for a functional blue
light photoreceptor was confirmed from
overexpression studies.
 • Approximately three years later, a second member called
cryptochrome 2 (CRY2) was identified by cross-
Cross- hybridization using CRY1 cDNA as a probe (Lin et al., 1996).
hybridization • This new member mapped to fha locus, which was earlier
studies characterized to be late-flowering, thereby indicating its
probable role in controlling flowering time.
identified the • Bioinformatic analysis showed that CRY2 and CRY1 are 51%
second identical in amino acid sequence, and this sequence
member, cry2 similarity is mainly concentrated in the PHR domain of
approximately 490 residues, where CRY1 and CRY2 are 58%
identical.

The expression of these two photoreceptors (cry1 & cry2) is independent of each other
 • After about a decade of the discovery of CRYs, a
new class lacking the C-terminal domain, was
identified in A. thaliana, whose homologues were
Cry-DASH can found in diverse organisms like Drosophila,
repair UV- Arabidopsis, Synechocystis, and Homo, and hence
called cry-DASH or Atcry3 (Brudler et al., 2003)
damaged DNA, • While Atcry1 and Atcry2 are nuclear proteins,
similar to DNA Atcry3 has been found to be localized to
photolyases chloroplasts and mitochondria
• The first clue about the biological role of Atcry3
was provided by studying its crystal structure with
an in situ-repaired CPD substrate
 Structure of Cryptochrome (CRY)

• CRYs are basically flavoproteins, composed of two domains: N-terminal Photolyase

Homology-Related (PHR) domain, and a Cryptochrome C-terminal Extension (CCE)
domain.
• DNA photolyases, however, lack the C-terminal domain characteristically found in
CRYs.
• The PHR domain harbors the binding site of two noncovalently bound
chromophores: pterin or MTHF and FAD.

MTHF FAD

NH2 Photolyase Homology Region (PHR) C-Terminal Domain COOH

 The role of pterin as
• On blue light exposure, the pterin light-sensing
chromophore acts as a light- chromophore was
revealed from two
harvesting antenna pigment, which observations:
transfers the resulting excitation
energy to the catalytic cofactor, FAD
• The flavin chromophore can exist in
multiple redox states—oxidized First, the Atcry1 Second, DHAP, a
. pterin biosynthesis
(FADox), semiquinone (FADH ), and overexpressing
insect cells were inhibitor, was
fully reduced (FADH2), each having found to be more selectively found to
different absorption characteristics sensitive to light of reduce
• In fact, the C-terminal region of CRY2 380 nm than that cryptochrome
of 450nm. responsivity at 380
is very different from the nm in these cell
corresponding region of CRY1; there cultures.
is only 14% sequence similarity.

Hoang et al., (2008) Molecular Plant

 Distribution of cryptochromes in plant species

CRYs in higher plants

• A. Thaliana and tomato, both of which contains one member of each CRY subfamily:
CRY1, CRY2 and CRY-DASH
• CRY gene family comprising one CRY1 and two CRY2 genes were identified from garden
pea
• In monocots, the first report of CRY characterization came from rice which contains two
CRY1 (OsCRY1a and OsCRY1b) and one CRY2 (OsCRY2) member
• In contrast to rice, only one CRY member, SbCRY2, has been so far reported from
Sorghum
• The CRY gene family in wheat comprises two CRY1 (TaCRY1a and TaCRY1b), one CRY2,
and one CRY-DASH
 Mechanism of Cryptochrome Action
How do CRYs decode light signals to regulate
biological responses?

Faster which precede the

Photochemical slower Biochemical
reactions, reactions
Photochemical reactions: Light-induced changes in the oxidation state of FAD
chromophore

• Among the different redox forms,

only the oxidized flavin absorbs
significant amounts of blue light
(~400–500nm).
• Pterin chromophore, which primarily
functions in light harvesting
• Neutral radical semiquinone
FADH• that characteristically absorbs
green light.
• The photoreduction of oxidized FAD
to the semireduced FADH• triggers a
conformational change of the
cryptochromes and the subsequent
signal transduction
Mishra & Khurana (2017) Critical Reviews
In Plant Sciences
(a) Five possible redox forms of flavins. One is the Flavoquinone which are fully
oxidized. the two different forms of semiquinone radicals: anion radical (e.g. FAD•—)
and neutral radical (e.g. FADH•), and two forms of reduced flavins: protonated
hydroquinone (e.g. FADH2) and anionic hydroquinone (e.g. FADH—) are shown. R:
side groups of flavins.
Liu et al., (2011) Trends Plant Science
Biochemical reactions:- Blue light-induced conformational changes in CRY
structure

• Photoexcited cryptochrome
change conformation to initiate
signal transduction by interacting
with signaling proteins.
• This model depicts cryptochrome
homodimerization via the PHR
domains.
• In the absence of light, the PHR
domain and the C-terminal tail of
the unphosphorylated CRY2 form
a “closed” conformation to
suppress the NC80 motif, an 80-
amino acid sequence located
between the N-terminal PHR
domain and the C-terminal tail of
CRY2.

Liu et al., (2011) Trends Plant Science

Mechanism of Phosphorylation

The photolyase-like cyclic

electron shuttle model of
cryptochrome photoexcitation.
In this model, the resting state
of a cryptochrome contains the
anion radical semiquinone
(FAD•—). Upon photon
absorption, the excited FAD•—
transfers an electron to ATP,
triggering phosphotransfer and
autophosphorylation of the
cryptochrome. The electron is
subsequently transferred back
to flavin to complete the cycle.
The putative locations of
phosphorous group (red circle)
and electron transfer path (red
Liu et al., (2011) Trends arrows) are indicated.
Plant Science
 Phosphorylation of CRYs is a pre-requisite for its activation
• In CRYs too, the formation of signaling active semiquinone state (FADH.) on blue light
exposure is accompanied by the phosphorylation of serine residues in the C-terminal
domain.

Do CRYs possess autophosphorylation activity?

• It has been demonstrated that CRYs can undergo phosphorylation in the presence of
ATP without the addition of a protein kinase, suggesting the occurrence of
autophosphorylation in these blue light photoreceptors

Significance of phosphorylation
• The phosphorylated form of cry2 represents the physiologically active state of the
photoreceptor.
Are CRYs similar to DNA photolyases in the mechanism of action?

• Both CRYs and DNA photolyases possess identical chromophores (MTHF and
FAD) and share high sequence and structural similarity.
• Also, the light dependent cyclic flow of electrons in DNA photolyases does not
involve any net loss or gain of electrons.
• One of the striking features in which CRYs are distinct from DNA photolyases is
the enhanced stability of semiquinone state of FAD chromophore (FADH), which
represents the active signaling state of CRYs. On the other hand, DNA
photolyases catalyze light-dependent DNA repair only in the fully reduced form
(FADH2) of the flavin chromophore.
• Another point of distinction between the two is the lack of correspondence in
the absorption and action spectrum of cry, while in DNA photolyases, it matches
quite closely
 Cryptochrome-mediated regulation of gene expression
Two mechanisms of cryptochrome signal transduction:-

Liu et al., (2011) Trends Plant Science

 Other mechanisms of cryptochrome action

Depolarization of anion channels in the plasma membrane

• Observations were made in protoplasts isolated from red light-
adapted maize coleoptile cells that shrank transiently due to
depolarization of anion channels occurs in the plasma
membrane upon a pulse of blue light. The fact that this
response was prevented by the anion channel blocker 5-nitro-2-
(3-phenylpropylamino)-benzoic acid, indicated that probably a
net efflux of anions (mainly Cl¡) leads to shrinking of protoplasts
(Parks et al., 1998; Folta and Spalding, 2001; Folta et al., 2003).
Generation of reactive oxygen species
• The photoreduction cycle of CRYs involves the conversion of
FADH or FADH2 (semi-reduced or reduced state) to FAD
(oxidized state), in dark to complete the cycle. This process
involves the cleavage of molecular oxygen (O2) to form
superoxide and subsequently H2O2, such that under
continuous blue light illumination, CRY activation would lead
to the accumulation of reactive oxygen species (ROS).
 Desensitization of blue light signal

• The light-labile CRY2 protein undergoes rapid degradation through

ubiquitination by COP1 or other E3 ubiquitin ligases in high fluence blue
light. CRY1, being stable in light, uses some other mechanisms for
desensitization of blue light signal, probably involving a dephosphatase
(Shalitin et al., 2002, 2003; Liu et al., 2011).
• The blue light-dependent CRY2 degradation requires the flavin
chromophore.
• Both CRY2 phosphorylation and degradation take place in the nucleus.
• Both the PHR domain and the C-terminal domain are required for the
blue-light-dependent degradation of CRY2 (Ahmad et al., 1998).
 The “green side” of cryptochrome signaling

Hypocotyl
elongation
Anthocyanin
Germination
accumulation.

Physiological
response under
green and blue
light
Stomatal opening Flowered earlier

Mishra & Khurana (2017) Critical Reviews In Plant Sciences

• Moreover, AtCRY2, being light-labile, was found to be degraded under green light as well.
Interaction between cryptochrome and hormone signaling
Auxins & Cytokinins
• Action of Auxin and
Cytokinin in respect
of blue light is
antagonistic to each
other.
• Auxin induce the
elongation of
hypocotyl in respect
of blue light which
cause shortening of
hypocotyl and
Cytokinins(opposite)
induce similar
photomorphogenic
responses, as CRYs.
Kobayashi et al.,
(2012) The Plant Cell
 Gibberellins
• Pea seedlings, when
DF
Ff transferred from
dark to blue light,
exhibit inhibition in
hypocotyl
elongation, which is
accompanied by
reduction in the
amount of bioactive
gibberellin, GA1.
ststrstrststst
HY5 • Mechanism
rt between light
induce
photomorphogenesi
s and Gibberellins is
shown here:

Weller et al., (2009) The

Plant Cell
Brassinosteroids
• BRs are required to
maintain the repression
of photomorphogenesis
in dark (Li et al., 1996).
• Interactions between
light and endogenous
hormones are critical
for plant development.
It has been long
recognized that BR plays
a major role in light-
regulated plant
development. The BR
and light-signaling
pathways for
coordinated regulation
of gene expression and
photomorphogenesis.
Luo et al., (2010) Developmental Cell
 Other hormones

• Ethylene help in apical hook opening or closing in

response to blue light and also regulate cotyledon
expansion phenotype in pea.
• Mutant analysis has demonstrated that AtMYC2 in ABA and
JA signaling acts as a negative regulator of blue light-
mediated photomorphogenesis in Arabidopsis. Moreover,
these atmyc2 seedlings are also insensitive to ABA and JA,
as revealed by seed germination and root growth
retardation assays.
Physiological responses regulated by cryptochromes in plants
Seed dormancy and germination
• Left: Barley grain at
different stages of
germination (photo credit:
Rosemary White, CSIRO
Plant Industry). Right: In
dormant barley grain, BL
enhances the ABA
content to inhibit
germination.
• ABA biosynthetic gene
encoding (NCED), and also
inhibited the expression
of ABA 8’-hydroxylase, an
ABA catabolic enzyme
Barrero et al., (2014) Plant Cell
 De-etiolation responses and Root growth

• The development of seedlings in light is marked by de-etiolation responses that

constitute: Inhibition of hypocotyl elongation and stimulation of cotyledon
expansion.
 In case of hypocotyl growth inhibition, caused primarily by a reduction in cell
length and not by a reduction in cell number (Gendreau et al., 1997).
 Cell expansion in cotyledons As in the case of hypocotyl growth inhibition,
CRY1 stimulates cotyledon expansion in both high and low irradiance,
whereas the role of CRY2 is limited to low irradiance (Blum et al., 1994; Lin et
al., 1998).
• In contrast to the inhibition of hypocotyl elongation, blue light induces the
promotion of root growth in a CRY1-dependent manner.
• The cryptochrome-mediated root growth is also regulated at the level of cell
elongation and not number (Canamero et al., 2006).
http://www.pnas.org/content/104/47/18813/F7.expansion.html
Stomatal opening
• The observation that
cry1cry2 double
mutant displayed an
increased drought
tolerance (Mao et al.,
2005) led to a finding
that CRYs also
contribute to blue-
light stimulation of
stomatal opening.
• Although phototropins
are the major
photoreceptor
regulating this process
(Shimazaki et al.,
2007),

Inoue & Kinoshita

(2017) Plant Physiology
 Photosynthesis
Blue light
• Indeed, a large number of
photosynthesis-related genes
and proteins, including
crys Photoreceptors components of the light and
dark reactions and of starch
and sucrose biosynthetic
pathways, and
COP1 Negative regulator photorespiratory pathway
were found to show high
transcript levels in tomato
CRY2-OE plants (Lopez et al.,
2012).
HY5 • This upregulation of
Positive regulator photosynthesis-related genes
and proteins is consistent with
BBX23 the increased accumulation of
chlorophyll in CRY2-OE plants
HY5 (Gilibertoetal.,2005).
Photosynthesis
related gene Zhang et al., (2017) Plant Physiology
 Photoperiodic induction of flowering

1. Role of CRY2 in promoting flowering

• The first indication of the involvement of CRYs in the regulation of floral
induction was obtained through analysis of Arabidopsis cry2 mutant, which
was found to be allelic to the late flowering mutant, fha (Koornneef et al.,
1991). cry2 mutants flowered late in blue light but not in absence of light
(Guo et al., 1998; El-Assal et al., 2001, 2003)
2. Role of CRY1 in inducing flowering
• To investigate the contributions of cry1 and cry2 in regulating flowering time,
cry1cry2 double mutants were generated. The cry1cry2 double mutant
showed delayed flowering than the wild type or the cry1 and cry2 monogenic
mutants under monochromatic blue light (Bagnal et al., 1996). These
observations suggested that CRY2 acts redundantly with CRY1 in promoting
flowering induction
Dark Condition Blue Light

Liu et al., (2008) The Plant Cell

Circadian rhythms
• Light signals are perceived
and transduced to the central
oscillator via specialized
photoreceptors like
cryptochromes and
phytochromes
• In fact, the genes encoding for
cryptochrome photoreceptors
are also regulated by circadian
clock at the level of RNA
abundance.
• A characteristic feature of
plant circadian clock is the
shortening of period length
with increasing intensity of
light.

McClung (2006) The Plant Cell

Antioxidant content in tomato fruits
• The role of CRYs in
affecting the antioxidant
content in tomato was
reported by Giliberto et
al. (2005), who found
that the LeCRY2-OE
transgenics had 1.5- to
2-fold higher lyopene
content at the red ripe
stage.
• Light signaling
components involved in
the regulation of tomato
fruit pigmentation and
ripening

Llorente et al., (2016)

Frontiers in Plant Science
 Leaf senescence

• The process of leaf senescence is regulated by both developmental

program and environmental conditions like light (Quirino et al., 2000;
Lim et al., 2007).

Stress Response
• In addition to various growth and developmental processes, light also
plays a role in regulating stress responses by activating several
defense genes and regulation of cell death response.
 Conclusions and future perspectives
• Apart from promoting anthocyanin accumulation in light-grown seedlings, the synthesis
of other plant pigments like chlorophyll and carotenoids in leaves, and lycopene in fruits
is also enhanced by cryptochrome photoreceptors
• The recent reports of CRYs in algae represent a connecting link between DNA
photolyases and plant CRYs, and could provide more information on the evolutionary
history of Photolyase/Cryptochrome superfamily.
• Interesting field of research would be to explore the different photoreceptors present in
plants growing at high altitudes, where thinner atmosphere means more intense
sunlight and a light spectrum distinct from other places
• Since CRYs regulate two of the most important agronomic traits—hypocotyl/stem
elongation and promotion of flowering time—it could be a promising target for crop
improvement programs.
• Finally, another fascinating area of research would be to determine whether the CRY-
dependent increased cotyledon expansion, in any way, affects the overall yield of a
plant; an area which has been largely unexplored.