Cryptochromes are blue light photoreceptors in plants that regulate important physiological processes. They were first discovered in Arabidopsis thaliana and are structurally similar to microbial DNA photolyases, containing a photolyase homology region and C-terminal extension. Cryptochromes exist in multiple redox states of their flavin chromophore, and blue light excitation triggers a conformational change involved in signaling. Cryptochromes interact with hormone signaling pathways to control processes like seedling development and flowering time.

1. Emerging Roles and New Paradigms in Signaling
Mechanisms of Plant Cryptochromes
MASTER SEMINAR
ON
Manish Jangra
2016BS24M
Dept. Of Botany & Plant Physiology

2. 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
Contents

3. 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

4. Zoratti et al., (2014) Frontiers in Plant Science

5. Classes of
photoreceptors:-
Five Phytochromes
(PHYs): phyA to phyE
that are involved in red
and far-red light
sensing.
Three Cryptochromes
(CRYs): cry1, cry2, and
cry-DASH; seedling
development and
flowering.
Two Phototropins:
phot1 and phot2;
differential growth in a
light gradient.

6. First discovery of
gene in 1993
that encoding
for the first blue
light
photoreceptor,
cryptochrome,
was isolated
from A. thaliana
(Ahmad and
Cashmore,
1993).
Cryptochromes: The blue/UV-A light photoreceptors

7. Why these receptors are called Cryptochrome?
First, the blue-light-
mediated responses were
found to be prevalent in
the lower plants like ferns,
mosses, algae, and fungi,
collectively called
cryptogams.
Second, because the
molecular nature of these
photoreceptors was
cryptic (hidden/
unknown) at that time.
Two Reasons
• Other blue light photoreceptors in plants that were discovered later include phototropins and
ZTL/FKF1/LKP2 family

8. Discovery of cryptochromes
HY4 gene encodes
cry1, the first blue
light photoreceptor
Cross-hybridization
studies identified the
second member, cry2
Cry-DASH can repair
UV-damaged DNA,
similar to DNA
photolyases
• “The Power of Movement in Plants” – Phototropism(1880)

9. • 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
response, suggesting a defect in the blue-light-
signaling pathway.
• The HY4 gene was isolated by gene tagging method
and was found to encode a protein of 681 amino
acids, of which the first 500 showed striking sequence
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.
HY4 gene
encodes cry1,
the first blue
light
photoreceptor

10. • Approximately three years later, a second member called
cryptochrome 2 (CRY2) was identified by cross-
hybridization using CRY1 cDNA as a probe (Lin et al., 1996).
• This new member mapped to fha locus, which was earlier
characterized to be late-flowering, thereby indicating its
probable role in controlling flowering time.
• Bioinformatic analysis showed that CRY2 and CRY1 are 51%
identical in amino acid sequence, and this sequence
similarity is mainly concentrated in the PHR domain of
approximately 490 residues where CRY1 and CRY2 are 58%
identical.
• It was found that at fluence rate lower than 1 mmol/m2/s,
the CRY2-OE transgenics developed shorter hypocotyl than
CRY1-OE lines, whereas this was exactly opposite under
fluence rate greater than 1 mmol/m2/s.
Cross-
hybridization
studies
identified the
second
member, cry2
The expression of these two photoreceptors (cry1 & cry2) is independent of each other

11. • 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
found in diverse organisms like Drosophila,
Arabidopsis, Synechocystis, and Homo, and hence
called cry-DASH or Atcry3 (Brudler et al., 2003)
• While Atcry1 and Atcry2 are nuclear proteins,
Atcry3 has been found to be localized to
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
Cry-DASH can
repair UV-
damaged DNA,
similar to DNA
photolyases

12. NH2 COOH
MTHF FAD
Photolyase Homology Region (PHR) C-Terminal Domain
• CRYs are basically flavoproteins, composed of two domains: N-terminal Photolyase
Homology-Related (PHR) domain, and a Cryptochrome C-terminal Extension (CCE)
domain.
• CRYs exhibit greatest degree of sequence homology with DNA photolyases,
(Deisenhofer, 2000; Lin and Todo, 2005)
• 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.
Structure of Cryptochrome (CRY)

13. • On blue/UV-A light exposure, the
pterin chromophore acts as a light-
harvesting antenna pigment, which
transfers the resulting excitation
energy to the catalytic cofactor, FAD
• The flavin chromophore can exist in
multiple redox states—oxidized
(FADox), semiquinone (FADH
.
), and
fully reduced (FADH2), each having
different absorption characteristics
• In fact, the C-terminal region of CRY2
is very different from the
corresponding region of CRY1; there
is only 14% sequence identity.
The role of pterin as
light-sensing
chromophore was
revealed from two
observations:
First, the Atcry1
overexpressing
insect cells were
found to be more
sensitive to light of
380 nm than that
of 450nm
Second, DHAP, a
pterin biosynthesis
inhibitor, was
selectively found to
reduce
cryptochrome
responsivity at 380
nm in these cell
cultures
Hoang et al., (2008) Molecular Plant

14. Distribution of cryptochromes in plant species
CRYs in higher plants
• A. thaliana, which contains one member of each CRY subfamily: CRY1, CRY2 and CRY-
DASH
• The next report of CRY1, CRY2 and CRY-DASH characterization came in the subsequent
year from tomato
• 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

15. How do CRYs decode light signals to regulate
biological responses?
Faster
Photochemical
reactions,
which precede the
slower Biochemical
reactions
Mechanism of Cryptochrome Action

16. Photochemical reactions: Light-induced changes in the oxidation state of FAD
chromophore
• Among the different redox forms,
only the oxidized flavin and anion
radical semiquinone flavin (FAD•—)
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

17. Photoexcitation of cryptochromes. (a) Five
possible redox forms of flavins. 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. (b) 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 arrows) are
indicated.
Liu et al., (2011) Trends Plant Science

18. • 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.
Biochemical reactions:- Blue light-induced conformational changes in CRY
structure
Liu et al., (2011) Trends Plant Science

19. 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.
• 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.
• Also, the light dependent cyclic flow of electrons in DNA photolyases does not
involve any net loss or gain of electrons.
• 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

20. • 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.
Phosphorylation of CRYs is a pre-requisite for its activation
Do CRYs possess autophosphorylation activity?
• The protein kinase(s) responsible for the complete phosphorylation of Arabidopsis
cryptochrome has not been identified, although multiple protein kinases, including an
AMPK, CKIε, GSK-3β and MAPK.
Significance of phosphorylation
• The phosphorylated form of cry2 represents the physiologically active state of the
photoreceptor.

21. Cryptochrome-mediated regulation of gene expression
• Apart from light-dependent transcriptional regulation, another common mechanism of cryptochrome-
mediated regulation of photomorphogenesis is light-dependent suppression of proteolysis
Liu et al., (2011) Trends Plant Science

22. Two mechanisms of cryptochrome signal transduction:-
• One of the pathways by which CRYs may cause transcriptional activation in response to blue light is
through direct interaction with cryptochrome interacting basic helix-loop-helix (CIB) transcription
factors. An example of this type of regulation is found when CIB1 protein binds to the promoter of
FLOWERING LOCUS T (FT) gene and interacts with cry2 to positively regulate floral initiation in
Arabidopsis
• The other pathway by which CRYs indirectly modulate gene expression is through their interaction
with SUPPRESSOR OF PHYA 1 (SPA1)/CONSTITUTUVE PHOTOMORPHOGENIC 1 (COP1), which
comprise an E3 ubiquitin ligase complex. In dark, the SPA1/ COP1 complex targets the positive
regulators of photomorphogenesis for degradation, while light inactivates the COP1/SPA1 complex
so that HY5 and other light induced transcription factors could accumulate in the nucleus,
triggering photomorphogenic responses.
 The cry1-mediated phase of growth inhibition is marked by the repression of genes
involved in gibberellin and auxin biosynthesis and/or sensitivity levels (Folta et al., 2003)

23. 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

24. 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).
• as determined by Trypan Blue vital stain.

25. 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).

26. Physiological
response under
green and blue
light
Hypocotyl
elongation
Anthocyanin
accumulation.
Flowered earlierStomatal opening
Germination
• Moreover, AtCRY2, being light-labile, was found to be degraded under green light as well.
Mishra & Khurana (2017) Critical Reviews In Plant Sciences
The “green side” of cryptochrome signaling

27. Interaction between cryptochrome and hormone signaling
Auxins & Cytokinins
Kobayashi et al.,
(2012) The Plant Cell
• 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
opposite, Cytokinins
induce similar
photomorphogenic
responses, as CRYs.

28. Mazzella et al., (2014)
Frontiers in Plant Science
• Pea seedlings, when
transferred from
dark to blue light,
exhibit inhibition in
hypocotyl
elongation, which is
accompanied by
reduction in the
amount of bioactive
gibberellin, GA1.
• Mechanism
between light
induce
photomorphogenesi
s and Gibberellins is
shown here:
Gibberellins

29. • 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.
Brassinosteroids
Luo et al., (2010) Developmental Cell

30. • 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.
Other hormones

31. Barrero et al., (2014) Plant Cell
• 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
Seed dormancy and germination
Physiological responses regulated by cryptochromes in plants

32. • 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).
De-etiolation responses and Root growth

33. http://www.pnas.org/content/104/47/18813/F7.expansion.html

34. Inoue & Kinoshita
(2017) Plant Physiology
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),

35. Zhang et al., (2017) Plant Physiology
Photosynthetic reactions
• Indeed, a large number of
photosynthesis-related genes
and proteins, including
components of the light and
dark reactions and of starch
and sucrose biosynthetic
pathways, and
photorespiratory pathway
were found to show high
transcript levels in tomato
CRY2-OE plants (Lopez et al.,
2012).
• This upregulation of
photosynthesis-related genes
and proteins is consistent with
the increased accumulation of
chlorophyll in CRY2-OE plants
(Gilibertoetal.,2005).

36. 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 (Lin,
1996, Lin et al., 1998), which was found to be allelic to the late flowering
mutant, fha (Koornneef et al., 1991). cry2 mutants flowered late in long days
but not in short days (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
Photoperiodic induction of flowering

37. Liu et al., (2008) The Plant Cell

38. Kojima et al., (2011) Journal of Cell
Science
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.
• The effect of light on post-
transcriptional regulation of clock
genes in Arabidopsis.

39. Llorente et al., (2016)
Frontiers in Plant Science
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

40. 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.

41. 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.
• Another 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.