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Algae 2017, 32(3): 235-244https://doi.org/10.4490/algae.2017.32.9.9

Open Access

Research Article

Copyright © 2017 The Korean Society of Phycology 235 http://e-algae.org pISSN: 1226-2617 eISSN: 2093-0860

Isolation and characterization of two phototropins in the freshwater green alga, Spirogyra varians (Streptophyta, Zygnematales)

Ji Woong Lee and Gwang Hoon Kim*

Department of Biology, Kongju National University, Kongju 32588, Korea

Freshwater algae living in shallow waters have evolved various photomovement to stay in the optimum light condition

for survival. Previous action-spectra investigations showed that Spirogyra filaments have phototropic movement in blue

light. To decipher the genetic control of phototropic movement, two phototropin homologues were isolated from Spi-

rogyra varians, and named SvphotA and SvphotB. Both phototropins have similar molecular structure consisted of two

light–oxygen–voltage domains (LOV1, LOV2) and a serine / threonine kinase domain. SvphotA and SvphotB had 48.7%

sequence identity. Phylogenetic analysis showed SvphotA and SvphotB belong to different clades suggesting early diver-

gence, possibly before the divergence of land plants from the Zygnematales. Quantitative PCR and northern blot analysis

showed that SvphotA and SvphotB responded differently to red and blue light. SvphotA was consistently expressed in the

dark and in blue light, while SvphotB was expressed only when the plants were exposed to light. When the filaments were

exposed to red light, SvphotA was significantly downregulated whereas SvphotB was highly upregulated. These results

suggest that the two phototropins may have different roles in the photoresponse in S. varians.

Key Words: blue light; photomovement; phototropin; phylogenetic analysis; qPCR

INTRODUCTION

Plants have evolved various photomovement to protect

themselves against excess energy in strong light and to

optimize photosynthesis efficiency in low light (Banaś et

al. 2012). There are four main families of photoreceptors

in plants to perceive light: red / far-red-absorbing phyto-

chromes, blue / UVA absorbing cryptochromes, Zeitlupe

family proteins, and phototropins (Łabuz et al. 2012). In

terrestrial angiosperms only the blue light receptor, pho-

totropin, provides a fine-tuning mechanism by control-

ling rapid responses and transient movement (Suetsugu

and Wada 2007). Phototropins are also involved in a wide

range of light-dependent responses including phototro-

pism, chloroplast movement, nuclear positioning and

stomata opening (reviewed in Kami et al. 2010).

Higher plants have two different phototropins, phot1

and phot2, that are differentially regulated in chloroplast

movement (Huala et al. 1997, Kasahara et al. 2002). Mo-

lecular characteristics of algal phototropins have also

been reported (Huang et al. 2002). In the green alga Mou-

geotia scalaris, two phototropins, MsPHOTA and Ms-

PHOTB, mediate blue-light-induced chloroplast move-

ment (Suetsugu et al. 2005). Only a single phototropin

was identified in both Volvox carteri and Chlamydomo-

nas reinhardtii (Huang et al. 2002, Prochnik et al. 2010).

The basic signal transduction mechanism is conserved

in algal phototropins although they share only 30-40%

Received July 16, 2017, Accepted September 9, 2017

*Corresponding Author

E-mail: [email protected]: +82-41-850-8504, Fax: +82-41-850-8504

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Com-

mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Algae 2017, 32(3): 235-244

https://doi.org/10.4490/algae.2017.32.9.9 236

ic light, an LED system with blue and red light was used.

The LED system consisted of 400 diodes with a controller

for maintaining the photoperiod and light intensity. Each

LED diode emitted a narrow band of monochromatic

light. Spectral distributions of blue (peak at 470 nm) and

red (peak at 650 nm), were determined with a spectro-

radiometer (Li-1800; Li-COR, Lincoln, NE, USA) in the

range of 300-1,100 nm as described previously (Kim et al.

2005). Photographs and videographs were taken using a

time-lapse recorder fitted onto the inverted microscope.

DNA and RNA extraction and cDNA synthesis

Genomic DNA of S. varians was extracted using i-ge-

nomic Plant DNA mini kit (iNtRON Biotechnology Inc.,

Seongnam, Korea). Total RNA was isolated using an acid

guanidium thiocyanate–phenol–chloroform extraction

method (Han et al. 2012, 2015). Genomic DNA contami-

nation was eliminated by on-column digestion with treat-

ment of RNase-free DNase I using the RNeasy plant RNA

extraction kit (QIAGEN, Hilden, Germany). The RNA con-

centration was determined spectrophotometrically and

its integrity was assessed by 1.2% agarose-formaldehyde

gel electrophoresis. First strand cDNAs were synthesized

using Accuscript High Fidelity 1st strand cDNA kit (Agi-

lent Technologies, Santa Clara, CA, USA) with random

primers on a polymerase chain reaction (PCR) apparatus

(Bio-Rad, Hercules, CA, USA) following the manufactur-

er’s protocol.

Cloning of Svphots and exon-intron structure analysis

Primers for the amplification of Svphot genes (Supple-

mentary Table S1) were designed based on the partial

phototropin sequences obtained from our S. varians

transcriptome data in combination with previously re-

ported algal phototropin sequences in NCBI Genbank

(Han and Kim 2013). For the PCR amplifications and

identifications, we used the cDNA library and AccuPrime

Pfx SuperMix (Life Technologies, Grand Island, NY, USA).

The PCR program was an initial denaturation at 95°C for

3 min, 35 cycles at 95°C for 30 s, 55°C for 30 s, and 68°C for

3 min, followed by 68°C for 10 min. The Svphots was iden-

tified by sequence analysis and cloned in TOPcloner TA

cloning vector (Enzynomics, Daejeon, Korea). Genomic

PCR was carried out to analyze intron arrangement in

each phototropin gene. Full length sequences of Svpho-

tA and SvphotB were registered in NCBI (accession No.

MF422121, MF422122).

sequence identity with higher plant phototropins (Ki-

anianmomeni and Hallmann 2014). It has been shown

that chloroplast movement was restored when the pho-

totropin of Chlamydomonas was expressed in Arabidop-

sis phot1-phot2-deficient mutant (Onodera et al. 2005).

Expression profiles of phototropin genes differ consid-

erably in various organs of higher plants (Ma et al. 2005).

Arabidopsis phot1 and phot2 genes show different expres-

sion patterns in seedlings and mature leaves (Jarillo et al.

2001, Kagawa et al. 2001). The expression of phot1 and

phot2 genes is affected by blue light (Kang et al. 2008),

red light (Kagawa et al. 2001) and UVA (Jarillo et al. 2001)

in different stages of etiolated seedlings. Similar expres-

sion changes were observed in rice phototropins (Jain et

al. 2007). Light not only activates phototropins, but also

affects the level of their expression. In Arabidopsis seed-

lings, phot1 is downregulated and phot2 is upregulated by

light (Łabuz et al. 2012).

Previous action-spectra studies suggested that a blue-

light photoreceptor is involved in Spirogyra filament

movement with an optimum wavelength at 470 nm (Kim

et al. 2005). In this study, we isolated and characterized

two phototropin homologues involved in phototropic

movement in Spirogyra varians.

MATERIALS AND METHODS

Plant material and inhibition experiment

Spirogyra varians was collected from a shallow pond in

Kongju, Korea (36°20′34″ N, 127°12′28″ E). Collected sam-

ples were cleaned and cultured in DP11 medium (Kloch-

kova et al. 2016) at 20°C under 20 µmol photons m−2 s−1 (12

L : 12 D). A phototropin inhibitor K-252a (Sigma, St. Louis,

MO, USA) was used for inhibition experiments. A stock

solution of K-252a (1 mM) was prepared using DMSO and

stored at -20°C. Final concentration 0.1 µM was obtained

by diluting the stock solution in DP11 medium. S. vari-

ans filaments were incubated with K-252a for 3 h then

washed with DP11 medium at least three times for recov-

ery of inhibition.

Observation of photomovement

To observe photomovement, S. varians filaments were

fragmented into 5-10 cells pieces and observed in a mi-

cro-container (0.5 × 1.2 × 1.2 cm) placed on the stage of

an inverted microscope with the monochromatic light

sources placed at the side of stage. For the monochromat-

Lee and Kim Phototropins in Spirogyra

237 http://e-algae.org

were calculated based on 1,000 bootstrap replicates. The

Ostreococcus tauri PHOT gene was used as an outgroup.

The sequences and their accession number in Genbank

are given in Supplementary Table S2.

Quantitative PCR and northern blot analysis

Total RNA was extracted from S. varians over a time

course incubation in various light conditions. cDNA

was synthesized using the protocol described previous-

ly (Shim et al. 2016). Real-time PCR was performed us-

ing QuantiSpeed SYBR Hi-Rox Kit (PhileKorea, Daejeon,

Korea) in a StepOnePlus Real-Time PCR System (Applied

Biosystems, Foster City, CA, USA). cDNA samples were

amplified in triplicate from the same RNA preparation.

The final reaction volume was 50 µL and included 10 µL

of diluted cDNA, 25 µL of QuantiSpeed SYBR Hi-Rox mix

Phylogenetic analysis

Multiple align of sequences was performed among

our Svphots and other phototropin genes from the NCBI

database using MUSCLE (Edgar 2004) in Geneious (ver. 7.1.5, Biomatters, Auckland, NZ, USA). The conserved

region from the light–oxygen–voltage (LOV) 2 domain

to partial serine / threonine kinase domain was used for

phylogenetic analysis. The tree was generated using these

amino acid sequences with Bayesian phylogenetic analy-

sis (MrBayes 3.2.6) (Huelsenbeck and Ronquist 2001) us-

ing a Blosum substitution model, 2,000,000 generations,

subsampling frequency every 400 generations, and a

burn-in length of 200,000 generations. Maximum likeli-

hood analysis used RAxML 7.2.8 (Stamatakis 2014) using

a WAG + gamma model with a method described previ-

ously (Diehl et al. 2017). Bootstrap support values (%)

6 h

0 h

3 h

0 h

4 h

1 h

6 h

1 h

5 h

3 h

6 h

A

B

K-252a 0.1 μM(Blue 15 μmol m-2 s-1)

Wash out

Fig. 1. Phototropic movement in Spirogyra varians. (A) Distal ends of the fragmented filaments curve towards light direction. (B) Inhibition of phototropic movement with K-252a treatment. Filaments began to curve towards light after wash out. White arrows indicate direction of light. Scale bars represent: A, 150 µm; B, 100 µm.

Algae 2017, 32(3): 235-244


ans first strand cDNA using the DIG probe Synthesis Kit

(Roche Diagnostics, Berlin, Germany). Northern blot was

performed as described previously (Han and Kim 2013).

RESULTS

When fragments of S. varians filaments were exposed

to unilateral blue light each distal end of the filament

began to curve towards the light in 1 h (Fig. 1A). A pho-

totropin inhibitor, K-252a, blocked this movement, but

and 500 nM of each primer. The primers used for quan-

titative PCR (qPCR) are shown in Supplementary Table

S1. The reaction protocol was set first on 95°C for 5 min

and then 40 cycles of 95°C for 15 s, and 60°C for 20 s. Ex-

pression levels were then calculated against the house-

keeping gene GAPDH as a calibrator using the delta-delta

Ct method (Livak and Schmittgen 2001). qPCR result

was confirmed using northern blot analysis. The prim-

ers used for probes are shown in Supplementary Table

S1. The DNA probe was directly amplified and labeled

with DIG-dUTP by PCR of the Svphot genes from S. vari-

Fig. 2. Secondary structure of Spirogyra varians phototropins. (A) SvphotA and SvphotB have different sequence lengths, but the number and order of introns are the same. Introns whose positions are equivalent in each gene are connected with thin lines. (B) Amino acid sequences were well conserved in light–oxygen–voltage domains (LOVs) and kinase domains.

A

B

Serine / Threonine kinase

LOV2

LOV1



compared with other organisms, SvphotA showed higher

sequence identity than SvphotB to the genes compared

(Table 1). All flavin mononucleotide (FMN) and FMN-

interacting sites in the LOV1 and LOV2 domains are well

conserved in SvphotA and SvphotB (Fig. 3). Phylogenetic

analysis showed that SvphotA and SvphotB belonged to

distantly related subclades. SvphotA nested within the

Charophyte lineage, while SvphotB was sister to the cha-

rophytes and land plants, but with poor support (Fig. 4).

Two phototropins of S. varians responded differently

to blue and red light (Fig. 5). SvphotA was consistently

expressed in blue light, but was dramatically downregu-

lated in red light. When filaments were transferred to

dark condition, the expression of SvphotA was rapidly re-

covered (Fig. 5A). SvphotB was expressed only when the

filaments were exposed to light. The expression reached

a peak in 3 h after light exposure, and gradually reduced

the movement was recovered as soon as the inhibitor was

washed out (Fig. 1B). Full-length cDNA sequences of two

phototropin homologues of S. varians were obtained and

named SvphotA and SvphotB (Fig. 2). Two phototropins

had well conserved LOV1, LOV2 and serine / threonine

kinase domains (Fig. 2A), although the sizes were smaller

than those of higher plants. The protein structure of Sv-

photA and SvphotB was different, especially in the serine

/ threonine kinase domain (Fig. 2B). The two phototro-

pins showed quite low sequence similarity (48.7% in ami-

no acid sequence and 54.2% in cDNA sequence), but the

number of introns were the same (Fig. 2A). The genomic

structure of SvphotA and SvphotB comprised 21 exons

and 20 introns (Fig. 2A). When we compared the positions

of introns in each gene the exon-intron structures were

identical between SvphotA and SvphotB (Fig. 2A).

When the sequences of LOV1 and LOV2 domains were

LOV2

LOV1

Fig. 3. Alignment of light–oxygen–voltage (LOV) 1 and LOV2 domains of phototropins isolated from plants and algae. All flavin mononucleotide (FMN) and FMN-interacting sites are well conserved in SvphotA and SvphotB. The site for FMN adduct formation is marked by an arrow, and the FMN-interacting sites are shown in red.

Algae 2017, 32(3): 235-244


Table 1. Amino acid sequence % identity of LOV domains among chosen phototropin genes in algae and higher plants

Species NameLOV1 LOV2

SvPHOTA SvPHOTB SvPHOTA SvPHOTB

Charophyte Spirogyra varians PHOTA - 63.4 - 67.6PHOTB 63.4 - 67.6 -

Land plant Arabidopsis thaliana PHOT1 59.2 64.8 71.8 64.8PHOT2 69.0 62.0 76.1 66.2

Physcomitrella patens PHOT1A 67.6 70.4 78.9 62.0PHOT2B 62.0 66.2 74.7 62.0

Charophyte Cylindrocystis cushleckae PHOTA 77.5 60.6 83.1 63.4PHOTB 76.1 57.8 81.7 66.2

Mougeotia scalaris PHOTA 77.5 63.4 81.7 62.0PHOTB 69.0 57.8 77.5 64.8

Mesotaenium caldariorum PHOTA 76.1 60.6 83.1 62.0Desmidium aptogonum PHOT 71.8 63.4 84.5 69.0Klebsormidium flaccidum PHOT 67.6 66.2 73.2 66.2

Chlorophyte Chlamydomonas reinhardtii PHOT 54.9 50.7 56.3 50.7Volvox carteri PHOT 53.5 52.1 62.0 49.3Ostreococcus tauri PHOT 52.1 50.7 62.0 47.9

LOV, light–oxygen–voltage domains.

Fig. 4. Phylogenetic tree of phototropins from Spirogyra varians and other plant and algae. A majority consensus phylogeny of 20 phototropins from green algae and land plants was reconstructed by Bayesian inference analysis of an alignment of amino acid sequences (Supplementary Fig. S1). The Ostreococcus tauri PHOT sequence was used as the outgroup. Posterior probabilities are indicated at the nodes. RAxML bootstrap percentages and posterior probabilities are indicated at the nodes. Bootstrap percentage values <50% are not shown.



this study. Our inhibition experiments indicated the in-

volvement of phototropins in blue-light movement. Phy-

logenetic analyses showed two different phototrophins in

S. varians, SvphotA is closely related to phototropins from

other zygnematalecean algae, while SvphotB is more dis-

tantly related, and could be due to a duplication before

the split of land plants and zygnematalecean algae. The

two phototropins from S. varians showed different ex-

pression patterns suggesting that SvphotA and SvphotB

may have different roles in photomovement of S. varians.

Our inhibition experiment using K-252a showed that

phototropic movement in S. varians is mediated by pho-

totropins. K-252a is an alkaloid isolated from a soil fun-

gus which has been used to block phototropin-mediated

response in higher plant as it suppresses phosphoryla-

tion of phototropin by inhibiting its serine / threonine

kinase domain (Inoue et al. 2005). Although it is possible

that K-252a may affect some other kinase activity which

results in inhibition of movement, phototropin is still

the most probable candidate that mediates phototropic

movement in Spirogyra. Another blue-light photorecep-

tor, cryptochrome, does not have a kinase domain, and is

not blocked with K-252a treatment. Two chromophores

in cryptochromes have different absorption wavelength;

pterin at 380 nm and flavin (in the form of FAD) at 450

nm (Song et al. 2006, Hoang et al. 2008). Previous ac-

tion spectra studies showed that phototropic movement

in Spirogyra did not occur in UV light and the optimum

wavelength of phototropic movement was 470 nm, which

is higher than that of cryptochromes (Kim et al. 2005).

These results support our hypothesis that the phototro-

pins isolated in this study mediate phototropic move-

ment in S. varians.

Phototropic movement in Spirogyra is different from

higher plant because the movement is unsteady and

often interrupted by other movements such as gliding

movement or gravitational movement (Kim et al. 2005).

The phototropic movement of Spirogyra was first report-

ed by Tanaka et al. (1986). They reported that phototropic

movement occurred only at certain times of the day, and

the movement might be relatively inferior to gravitational

movements because a typical and steady phototropic cur-

vature was not observed in Spirogyra (Ojima and Tanaka

1970, Tanaka et al. 1986). In a previous study, we showed

that there is no diurnal rhythm in the photomovement of

Spirogyra, and confirmed that mucilage secretion is not

the cause of movement (Kim et al. 2005). Further stud-

ies on the downstream signaling of these phototropins

are necessary to understand how these genes regulate the

movement.

over the time course (Fig. 5B). Red light also induced up-

regulation of SvphotB although blue light induced greater

expression (Fig. 5B). Northern blot analysis showed simi-

lar results. A significant amount of SvphotA was expressed

consistently regardless of light condition, while SvphotB

transcript was much weaker and higher levels seen tran-

siently after exposure to light (Supplementary Fig. S2).

DISCUSSION

Light is an important environmental cue, and photore-

ceptors play a pivotal role in controlling light-dependent

movement. The filament movement in Spirogyra has been

observed for more than a century (Hofmeister 1874), but

the photoreceptors are characterized for the first time in

D 12 h Dark 6 h

0

10

20

30

40

50

60

70

0 10 30 60 180 360 420 540 720

Rela

tive

exp

ress

ion

leve

ls(F

old

chan

ge)

Time (min)

Blue light

Red light

0 10 30 60 180 360 420 540 720

Time (min)

D 12 h

SvphotA

SvphotB

Light (15 μmol m-2 s-1) 6 h

Light (15 μmol m-2 s-1) 6 h

Dark 6 h

-30

-25

-20

-15

-10

-5

0

5

Rela

tive

exp

ress

ion

leve

ls(F

old

chan

ge)

Blue light

Red light

A

B

Fig. 5. Quantitative polymerase chain reaction results of SvphotA and SvphotB expression in blue and red light after 12 h preincubation in dark. (A) SvphotA expression did not change in blue light and dark, but was significantly reduced in red light. The reduced expression in red light was recovered as soon as the filaments were moved to the dark. (B) SvphotB expression significantly increased after the filaments were exposed to both blue and red light. SvphotB expression was greater in blue light.

Algae 2017, 32(3): 235-244


It is possible that phototropic movement may enhance

conjugation chance in Spirogyra because the filaments

will align parallel to each other during this movement.

Many questions still remain on the role of these iso-

forms of phototropin in S. varians. What are the signal

transduction pathways of phototropin action? What are

the mechanical effectors for the movement? And what are

the components of the downstream signaling pathways?

Functional genomic studies using model expression sys-

tems will help to answer some of the above questions.

SUPPLEMENTARY MATERIALS

Supplementary Table S1. List of primers used in this

study (http://www.e-algae.org).

Supplementary Table S2. Genbank accession number

of sequences of phototropin used for phylogenetic analy-

ses (http://www.e-algae.org).

Supplementary Fig. S1. Alignment of phototropin ami-

no acid sequences used in phylogenetic analysis (http://

www.e-algae.org).

Supplementary Fig. S2. Northern blot analysis on ex-

pression of SvphotA and SvphotB in blue (A) and red (B)

light. The filaments were pre-incubated in the dark for 12

h prior to exposure to red and blue light (http://www.e-

algae.org).

ACKNOWLEDGEMENTS

This research was supported by Korea Institute of

Planning and Evaluation for Technology in Food, Agri-

culture, Forestry and Fisheries (IPET) through Golden

Seed Project, funded by Ministry of Agriculture, Food

and Rural Affairs (MAFRA) (213008-05-1-SB810), Minis-

try of Oceans and Fisheries (MOF) and also sponsored

by National Research Foundation of Korea Grant (NRF-

2015M1A5A1041804) funded to GHK.

REFERENCES

Banaś, A. K., Aggarwal, C., Łabuz, J., Sztatelman, O. & Gabryś, H. 2012. Blue light signalling in chloroplast movements.

J. Exp. Bot. 63:1559-1574.

Diehl, N., Kim, G. H. & Zuccarello, G. C. 2017. A pathogen of

New Zealand Pyropia plicata (Bangiales, Rhodophyta),

Pythium porphyrae (Oomycota). Algae 32:29-39.

Edgar, R. C. 2004. MUSCLE: multiple sequence alignment

The two phototropins in S. varians shared low se-

quence similarity compared to phototropin isoforms in

higher plants and other freshwater algae. Higher plants

have two phototropins, phot1 and phot2, which are dif-

ferentially regulated in many cellular processes (Huala et

al. 1997, Kasahara et al. 2002). Phototropins are unique

to Viridiplantae, and this gene family expanded through

duplication within various land plant lineages (Li et al.

2015). Algal phototropins are also involved in various

cellular processes, but little is known for their isoforms

(Kianianmomeni and Hallmann 2014). The two photo-

tropins in S. varians may regulate phototropic movement

differentially like phot1 and phot2 in higher plants. It is

also possible that SvphotA and SvphotB are involved in

different cellular events.

The phylogeny of SvphotA and SvphotB is interesting.

Existing functional data for phototropins, suggest a his-

tory of repeated gene duplications followed by parallel

functional divergences (Li et al. 2015). Phylogenetic anal-

ysis between phototropins in Mougeotia scalaris isoforms

(Li et al. 2015) suggested a close phylogenetic relation-

ship, and both mediate blue-light-induced chloroplast

movement (Suetsugu et al. 2005). It has been shown that

algal phototropins can substitute higher plant phototro-

pins when they were expressed in phototropin-deficient

mutant although they share only 30-40% of sequence

identity with higher plant phototropins (Onodera et al.

2005, Kianianmomeni and Hallmann 2014). The relation-

ship between SvphotA and SvphotB implies that the two

phototropins diverged early in Streptophyte history and

therefore they may be functionally divergent.

Our data showed that the intron structure is highly

conserved between SvphotA and SvphotB. The positions

of equivalent 20 introns in each gene were identical in

two phototropins. Intron shuffling is common in photo-

tropins of same species (Kagawa et al. 2004). It has been

proposed that intron shuffling contributed to function-

al divergence of photoreceptors such as neochrome in

Mougeotia (Suetsugu et al. 2005). The conserved exon-

intron structure in SvphotA and SvphotB is rarely seen in

any other groups of phototropins.

What is the other function of phototropins in Spirogy-

ra? Phototropin in Chlamydomonas reinhardtii regulates

multiple steps in sexual reproduction (Huang and Beck

2003). Overexpression of phototropin in C. reinhardtii

altered the sensitivity of chemotaxis during sexual dif-

ferentiation (Ermilova et al. 2004). Phototropin is also

involved in photosynthesis in C. reinhardtii by regulating

transcription of genes encoding enzymes responsible for

chlorophyll and carotenoid biosynthesis (Im et al. 2006).



Jarillo, J. A., Gabrys, H., Capel, J., Alonso, J. M., Ecker, J. R.

& Cashmore, A. R. 2001. Phototropin-related NPL1 con-

trols chloroplast relocation induced by blue light. Na-

ture 410:952-954.

Kagawa, T., Kasahara, M., Abe, T., Yoshida, S. & Wada, M.

2004. Function analysis of phototropin2 using fern mu-

tants deficient in blue light-induced chloroplast avoid-

ance movement. Plant Cell Physiol. 45:416-426.

Kagawa, T., Sakai, T., Suetsugu, N., Oikawa, K., Ishiguro, S.,

Kato, T., Tabata, S., Okada, K. & Wada, M. 2001. Arabi-

dopsis NPL1: a phototropin homolog controlling the

chloroplast high-light avoidance response. Science

291:2138-2141.

Kami, C., Lorrain, S., Hornitschek, P. & Fankhauser, C. 2010.

Light-regulated plant growth and development. Curr.

Top. Dev. Biol. 91:29-66.

Kang, B., Grancher, N., Koyffmann, V., Lardemer, D., Burney,

S. & Ahmad, M. 2008. Multiple interactions between

cryptochrome and phototropin blue-light signaling

pathways in Arabidopsis thaliana. Planta 227:1091-1099.

Kasahara, M., Kagawa, T., Oikawa, K., Suetsugu, N., Miyao,

M. & Wada, M. 2002. Chloroplast avoidance movement

reduces photodamage in plants. Nature 420:829-832.

Kianianmomeni, A. & Hallmann, A. 2014. Algal photorecep-

tors: in vivo functions and potential applications. Planta

239:1-26.

Kim, G. H., Yoon, M. & Klotchkova, T. A. 2005. A moving mat:

phototaxis in the filamentous green algae Spirogyra

(Chlorophyta, Zygnemataceae). J. Phycol. 41:232-237.

Klochkova, T. A., Jung, S. & Kim, G. H. 2016. Host range and

salinity tolerance of Pythium porphyrae may indicate its

terrestrial origin. J. Appl. Phycol. 29:371-379.

Łabuz, J., Sztatelman, O., Banaś, A. K. & Gabryś, H. 2012. The

expression of phototropins in Arabidopsis leaves: devel-

opmental and light regulation. J. Exp. Bot. 63:1763-1771.

Li, F. W., Rothfels, C. J., Melkonian, M., Villarreal, J. C., Ste-

venson, D. W., Graham, S. W., Wong, G. K., Mathews, S. &

Pryer, K. M. 2015. The origin and evolution of phototro-

pins. Front. Plant Sci. 6:637.

Livak, K. J. & Schmittgen, T. D. 2001. Analysis of relative gene

expression data using real-time quantitative PCR and

the 2−ΔΔCT method. Methods 25:402-408.

Ma, L., Sun, N., Liu, X., Jiao, Y., Zhao, H. & Deng, X. W. 2005.

Organ-specific expression of Arabidopsis genome dur-

ing development. Plant Physiol. 138:80-91.

Ojima, S. & Tanaka, K. 1970. Studies on the growth and de-

velopment in Spirogyra. I. Diurnal movement of the fila-

ments. Sci. Rep. Hirosaki Univ. 17:15-26.

Onodera, A., Kong, S. G., Doi, M., Shimazaki, K., Christie, J.,

Mochizuki, N. & Nagatani, A. 2005. Phototropin from

with high accuracy and high throughput. Nucleic Acids

Res. 32:1792-1797.

Ermilova, E. V., Zalutskaya, Z. M., Huang, K. & Beck, C. F. 2004.

Phototropin plays a crucial role in controlling changes

in chemotaxis during the initial phase of the sexual life

cycle in Chlamydomonas. Planta 219:420-427.

Han, J. H., Klochkova, T. A., Han, J. W., Shim, J. & Kim, G. H.

2015. Transcriptome analysis of the short-term photo-

synthetic sea slug Placida dendritica. Algae 30:303-312.

Han, J. W. & Kim, G. H. 2013. An ELIP-like gene in the fresh-

water green alga, Spirogyra varians (Zygnematales), is

regulated by cold stress and CO2 influx. J. Appl. Phycol.

25:1297-1307.

Han, J. W., Yoon, K. S., Jung, M. G., Chah, K. -H. & Kim, G. H.

2012. Molecular characterization of a lectin, BPL-4, from

the marine green alga Bryopsis plumosa (Chlorophyta).

Algae 27:55-62.

Hoang, N., Bouly, J. P. & Ahmad, M. 2008. Evidence of a light-

sensing role for folate in Arabidopsis cryptochrome

blue-light receptors. Mol. Plant 1:68-74.

Hofmeister, W. 1874. Uber die Bewegungen der Faden der

Spirogyra princeps. Nat. Wurttemb 30:211-226.

Huala, E., Oeller, P. W., Liscum, E., Han, I. S., Larsen, E. &

Briggs, W. R. 1997. Arabidopsis NPH1: a protein ki-

nase with a putative redox-sensing domain. Science

278:2120-2123.

Huang, K. & Beck, C. F. 2003. Phototropin is the blue-light re-

ceptor that controls multiple steps in the sexual life cy-

cle of the green alga Chlamydomonas reinhardtii. Proc.

Natl. Acad. Sci. U. S. A. 100:6269-6274.

Huang, K., Merkle, T. & Beck, C. F. 2002. Isolation and char-

acterization of a Chlamydomonas gene that encodes a

putative blue‐light photoreceptor of the phototropin

family. Physiol. Plant. 115:613-622.

Huelsenbeck, J. P. & Ronquist, F. 2001. MRBAYES: Bayesian

inference of phylogenetic trees. Bioinformatics 17:754-

755.

Im, C. S., Eberhard, S., Huang, K., Beck, C. F. & Grossman, A.

R. 2006. Phototropin involvement in the expression of

genes encoding chlorophyll and carotenoid biosynthe-

sis enzymes and LHC apoproteins in Chlamydomonas

reinhardtii. Plant J. 48:1-16.

Inoue, S., Kinoshita, T. & Shimazaki, K. 2005. Possible in-

volvement of phototropins in leaf movement of kidney

bean in response to blue light. Plant Physiol. 138:1994-

2004.

Jain, M., Sharma, P., Tyagi, S. B., Tyagi, A. K. & Khurana, J. P.

2007. Light regulation and differential tissue-specific ex-

pression of phototropin homologues from rice (Oryza

sativa ssp. indica). Plant Sci. 172:164-171.

Algae 2017, 32(3): 235-244


troscopic characterization of cryptochrome 3 from Ara-

bidopsis thaliana. J. Photochem. Photobiol. B. 85:1-16.

Stamatakis, A. 2014. RAxML version 8: a tool for phylogenetic

analysis and post-analysis of large phylogenies. Bioin-

formatics 30:1312-1313.

Suetsugu, N., Mittmann, F., Wagner, G., Hughes, J. & Wada,

M. 2005. A chimeric photoreceptor gene, NEOCHROME,

has arisen twice during plant evolution. Proc. Natl. Acad.

Sci. U. S. A. 102:13705-13709.

Suetsugu, N. & Wada, M. 2007. Phytochrome‐dependent

photomovement responses mediated by phototropin

family proteins in cryptogam plants. Photochem. Pho-

tobiol. 83:87-93.

Tanaka, K., Kitazawa, S., Sasaki, T., Ooshima, N. & Yamada,

T. 1986. Studies on the growth and development in Spi-

rogyra. VI. Phototropic response of Spirogyra filaments.

Planta 167:19-25.

Chlamydomonas reinhardtii is functional in Arabidopsis

thaliana. Plant Cell Physiol. 46:367-374.

Prochnik, S. E., Umen, J., Nedelcu, A. M., Hallmann, A., Mill-

er, S. M., Nishii, I., Ferris, P., Kuo, A., Mitros, T., Fritz-Lay-

lin, L. K., Hellsten, U., Chapman, J., Simakov, O., Rens-

ing, S. A., Terry, A., Pangilinan, J., Kapitonov, V., Jurka,

J., Salamov, A., Shapiro, H., Schmutz, J., Grimwood, J.,

Lindquist, E., Lucas, S., Grigoriev, I. V., Schmitt, R., Kirk,

D. & Rokhsar, D. S. 2010. Genomic analysis of organismal

complexity in the multicellular green alga Volvox carteri.

Science 329:223-226.

Shim, J., Shim, E., Kim, G. H., Han, J. W. & Zuccarello, G. C.

2016. Keeping house: evaluation of housekeeping genes

for real-time PCR in the red alga, Bostrychia moritziana

(Florideophyceae). Algae 31:167-174.

Song, S. H., Dick, B., Penzkofer, A., Pokorny, R., Batschauer, A.

& Essen, L. O. 2006. Absorption and fluorescence spec-

isolation and characterization of two phototropins in the ... · s1. the dna probe was directly...

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