1 running title: characterization of wheat ......2008/12/03 · 1 running title: characterization...

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Running title: CHARACTERIZATION OF WHEAT CRYPTOCHROME GENES 1

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Pei Xu1, 2, Yang Xiang1, Huilan Zhu1, Haibin Xu1, Zhengzhi Zhang1, Caiqin Zhang1, 3

Lixia Zhang1 and Zhengqiang Ma1* 4

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1College of Agricultural Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 6

210095, P.R. China 7

2Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, 8

Zhejiang, 310021, P.R. China 9

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*E-mail of corresponding author: [email protected] 11

Tel: 86-025-84396029 12

Fax: 86-025-84396707 13

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Research area: Cell Biology and Signal Transduction 15

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Plant Physiology Preview. Published on December 3, 2008, as DOI:10.1104/pp.108.132217

Copyright 2008 by the American Society of Plant Biologists

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Wheat Cryptochromes: Subcellular Localization and Involvement in 2

Photomorphogenesis and Osmotic Stress Responses 3

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Pei Xu1, 2, Yang Xiang1, Huilan Zhu1, Haibin Xu1, Zhengzhi Zhang1, Caiqin Zhang1, 5

Lixia Zhang1 and Zhengqiang Ma1* 6

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8 1The Applied Plant Genomics Lab, Crop Genomics and Bioinformatics Center & 9

National Key Lab of Crop Genetics and Germplasm Enhancement, Nanjing 10

Agricultural University, Jiangsu 210095, China (P.X., Y.X., H.Z., H.X., Z.Z, C.Z., 11

L.Z., Z.M.); 2Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, 12

Hangzhou 310021, China (P.X.) 13

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This project was partially supported by ‘863’ program (2006AA10A104), NSFC 1

program (30430440 and 30025030), Outstanding Youth Funds of MOE, GCP Project 2

(SP2-1), and ‘111’ project (Bo8025). 3

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*Corresponding author: Dr. ZQ Ma 5

E-mail: [email protected] 6

Fax: 86-025-84396707 7

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ABSTRACT 1

Cryptochromes (CRYs) are blue light receptors important for plant growth and 2

development. Comprehensive information on monocot CRYs is currently only 3

available for rice. We report here the molecular and functional characterization of two 4

CRY genes, TaCRY1a and TaCRY2, from the monocot wheat. The expression of 5

TaCRY1a was most abundant in seedling leaves and barely detected in roots and 6

germinating embryos under normal growth conditions. The expression of TaCRY2 in 7

germinating embryos was equivalent to that in leaves and much higher than the 8

TaCRY1a counterpart. Transition from dark to light slightly affected the expression of 9

TaCRY1a and TaCRY2 in leaves, and red light produced a stronger induction of 10

TaCRY1a. Treatment of seedlings with high salt, PEG and ABA up-regulated TaCRY2 11

in roots and germinating embryos. TaCRY1a displays a light-responsive 12

nucleocytoplasmic shuttling pattern similar to that of Arabidopsis CRY1, contains 13

nuclear localization domains in both the N and C termini and includes information for 14

nuclear export in its N-terminal domain. TaCRY2 was localized to the nucleus in the 15

dark. Expression of TaCRY1a-GFP or TaCRY2-GFP in Arabidopsis conferred a 16

shorter hypocotyl phenotype under blue light. These transgenic Arabidopsis plants 17

showed higher sensitivity to high salt, osmotic stress and ABA treatment during 18

germination and post-germination development, and they displayed altered expression 19

of stress/ABA responsive genes. The primary root growth in transgenic seedlings was 20

less tolerant of ABA. These observations indicate that TaCRY1 and TaCRY2 might be 21

involved in the ABA signaling pathway in addition to their role in primary blue light 22

signal transduction. 23

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5

INTRODUCTION 1

Cryptochromes (CRYs) and photolyases belong to the flavoprotein family, 2

widely distributed in bacteria as well as in eukaryotes (Cashmore et al., 1999; Brudler 3

et al., 2003). The CRYs show sequence similarity to photolyases, which function to 4

repair UV light-damaged DNA, but they do not have DNA repair activity and usually 5

possess a C-terminal extension (Todo, 1999). Plant CRYs are blue, green and UV-A 6

light photoreceptors responsible for photomorphogenesis (Briggs and Huala, 1999), a 7

phenomenon in which growing plants under light leads to chloroplast differentiation, 8

chlorophyll accumulation, leaf expansion and inhibition of stem elongation. Moreover, 9

CRYs are involved in circadian, developmental and adaptive growth regulation of 10

plants (Guo et al., 1998; Mao et al., 2005; Ahmad et al., 2006; Canamero et al., 2006; 11

Danon et al., 2006; Zhao et al., 2007). Plant CRYs were originally divided into two 12

subfamilies: CRY1 and CRY2. They have conserved N-terminal photolyase-related 13

(PHR) domains as well as C-terminal DQXVP-acidic-STAES (DAS) domains, and 14

they are distinguished mainly by their C-terminal extensions (Ahmad and Cashmore, 15

1993; Lin et al., 1996b). Brudler et al. (2003) and Kleine et al. (2003) reported a third 16

CRY subfamily, cryptochrome-DASH, in plants. 17

In Arabidopsis, each CRY subfamily consists of a single member. AtCRY1 is its 18

primary photoreceptor, mediating blue light regulation of seedling de-etiolation and 19

phasing of the circadian clock (Ahmad and Cashmore, 1993; Lin et al., 1996a; Somers 20

et al., 1998). AtCRY2 functions redundantly with AtCRY1 under relatively low light 21

and is a key component in the control of photoperiodic flowering (Guo et al., 1998; 22

Lin et al., 1998). Tomato (Solanum lycopersicum, formerly Lycopersicon 23

lycopersicum) possesses two CRY1 subfamily members, LeCRY1a and LeCRY1b 24

(Perrotta et al., 2001), and garden pea (Pisum sativum) contains two CRY2 subfamily 25

members, PsCRY2a and PsCRY2b (Platten et al., 2005). Monocot species rice (Oryza 26

sativa) has four CRY genes, including OsCRY1a, OsCRY1b, OsCRY2 and OsCRY-27

DASH (Hirose et al., 2006; Zhang et al., 2006). The N- and C-terminal domains of 28

OsCRY1a and OsCRY1b are 7% and 19% different, respectively. Hirose et al. (2006) 29

showed that over-expression of OsCRY1 resulted in enhanced responsiveness to blue 30



6

light, suggesting that OsCRY1 is similar to AtCRY1 in regulating photomorphogenesis. 1

Like AtCRY2, OsCRY2 is involved in the promotion of flowering time in rice (Hirose 2

et al., 2006). Barley (Hordeum vulgare) might have the same CRY gene composition 3

as rice (Perrotta et al. 2001). 4

Expression of CRY genes and turnover of CRY proteins are regulated by inner 5

circadian rhythms, light quality and day length. Transcript levels of CRY genes show a 6

near 24 h oscillation period (Toth et al., 2001; Platten et al., 2005). CRY genes in 7

different plants respond differentially to light induction. In garden pea, blue light is an 8

inhibitor of CRY gene expression (Platten et al., 2005), while it enhances the 9

expression of CRY1 in Brassica napus (Chatterjeeet al., 2006). White light has an 10

inhibitory effect on the expression of OmCRY1 (Okazawa et al., 2005). AtCRY2 11

degrades under short-day conditions in a blue light-dependent manner (Lin et al., 12

1998), and OsCRY2 degrades under either blue or red light conditions (Hirose et al., 13

2006). Little is known about the effects of other environmental cues on the expression 14

of CRY genes and CRY protein stability. 15

C-terminal domains of Arabidopsis and rice cryptochromes govern their 16

signaling activity. Over-expression of a fusion protein containing GUS (β-17

glucuronidase) and the AtCRY1 C-terminus causes a constitutive photomorphogenesis 18

response (Yang et al., 2000; Zhang et al., 2006). AtCRY1 and AtCRY2 both localize 19

to the nucleus (Cashmore et al., 1999; Guo et al., 1999; Kleiner et al., 1999), and the 20

GUS-AtCRY1 C-terminus fusion protein displayed a light-dependant 21

nucleocytoplasmic shuttling (Yang et al., 2000). The subcellular localization of 22

AtCRY2 does not change in response to blue light (Yang et al., 2000). In contrast to 23

the Arabidopsis CRY proteins, OsCRY1 has been found in both nucleus and cytosol, 24

irrespective of light conditions (Matsumoto et al., 2003). In A. capillus-veneris, two of 25

its five CRY family members, AcCRY3 and AcCRY4, localize to the nucleus, and, in 26

the case of AcCRY3, this pattern is regulated by light (Imaizumi et al., 2000). 27

However, sequences associated with the subcellular localization of plant CRY 28

proteins have not been well characterized. 29

All of the main staple crops, including rice, wheat (Triticum aestivum) and 30



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maize (Zea mays), are cereal monocots. Among them, the CRYs of rice are the only 1

group that has been characterized with respect to their sequences and functions in 2

photomorphogenesis (Hirose et al., 2006; Zhang et al., 2006). Besides being critical 3

for plant growth and development, light signals may also be involved in plant 4

responses to various abiotic stresses. For instance, the light quality-dependent CBF 5

gene expression was associated with freezing tolerance in Arabidopsis (Franklin and 6

Whitelam, 2007). Currently, blue light receptors of model plants have been well 7

characterized regarding their involvement in photomorphogenesis, but little is known 8

about their roles in stress responses. 9

In the present study, two CRY genes, TaCRY1a and TaCRY2, from hexaploid 10

wheat were characterized. We found that TaCRY1a exhibits a subcellular localization 11

mechanism akin to that of OsCRY1 and AtCRY1, and that both TaCRY1a and TaCRY2 12

were involved in osmotic stress/ABA responses in addition to their roles in the 13

primary light signal transduction pathway. 14

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RESULTS 16

Gene Organizations of TaCRY1a and TaCRY2 and Their Primary Protein 17

Structures 18

By mining wheat dbESTs, we obtained TaCRY1a and TaCRY2 contigs with the 19

full ORFs. We then isolated the full-length cDNA sequences (EF601539 and 20

EF601541) through RT-PCR and the genomic DNA (EF601540 and EF601542) 21

encompassing the coding sequences. The coding region of TaCRY1a has three introns, 22

and that of TaCRY2 has four (Fig. 1). In addition, TaCRY1a contains an intron in its 3’ 23

untranslated region (UTR). Based on the structural correspondence between TaCRY1a 24

and TaCRY2, the last two exons of TaCRY2 might have evolved from a 1047 bp intron 25

insertion within the fourth exon of TaCRY1a. Considerable variation in length and 26

composition exists in the corresponding introns of these two genes. The CRY gene 27

structure was well conserved among monocots and dicots, although the size of 28

particular introns in monocots appeared larger than those of their dicot counterparts 29

(Fig. 1). The CRY1 genes of Arabidopsis, rice and wheat, and SbCRY2 of sorghum 30



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(Xie et al., 2005) all have a characteristic intron in the 3’ UTR. 1

TaCRY1a cDNA encodes a 700 aa polypeptide with 82% and 78% identity to 2

OsCRY1a (BAB70686) and OsCRY1b (BAB70688), respectively, and 46% identity 3

to OsCRY2 (BAC56984). TaCRY2 cDNA encodes a 650 aa polypeptide with 78% 4

identity to OsCRY2 and less than 46% to OsCRY1a and OsCRY1b. TaCRY1a and 5

TaCRY2, each with an N-terminal PHR domain and a C-terminal DAS domain typical 6

of CRYs, have 60% overall similarity and 30% similarity in their C-terminal regions 7

(Fig. 4). Both polypeptides are highly hydrophilic, although the regions of TaCRY2 8

from AA65 to AA100 and from AA141 to AA176 and that of TaCRY1a from AA155 9

to AA190 are hydrophobic. TaCRY2 possesses a putative monopartite nuclear 10

localization signal (NLS) peptide PISRKRS (AA576-AA582). However, we did not 11

find canonical NLS signals in TaCRY1a or nuclear export signals (NES) in either 12

TaCRY1a or TaCRY2. 13

Alignment of CRY polypeptide sequences from plants including Arabidopsis, 14

rice, tomato and wheat showed that besides the C-termini, the N-terminal regions of 15

CRYs corresponding to the hydrophobic domains mentioned above are also highly 16

variable (Fig. 2). The CRY2 proteins showed greater variation than the CRY1 proteins 17

among species and in dicots as compared to monocots. Within individual CRY 18

subfamilies, the monocot proteins are generally larger than their dicot counterparts. 19

This length difference mainly occurs in the C-termini and at the very ends of the N-20

termini. A few amino acid substitutions are present within the most conserved N-21

terminal domains that can distinguish monocots from dicots or individual CRY 22

subfamilies from each other (Fig. 2). The C-terminal STAESS domain previously 23

characterized in dicots is less conserved in monocots, but the amino acid sequences 24

following this domain are better conserved. The plant CRYs compared have perfectly 25

conserved DQXVP domains in their C-termini, except for TaCRY2 as determined in 26

this study. 27

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Subcellular Localization of TaCRY1a-GFP and TaCRY2-GFP 29

To determine the subcellular localization patterns of TaCRY1a and TaCRY2, 30



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GFP fluorescence signals were examined in onion epidermal cells transiently 1

transformed with TaCRY1a-GFP and TaCRY2-GFP and in root cells of transgenic 2

Arabidopsis plants. Similar fluorescence distributions in cells were obtained for the 3

respective constructs in both types of transformations (Fig. 3). In the dark, for the 4

TaCRY2-GFP construct, intense fluorescence signals appeared in nuclei, whereas only 5

background signals were detected in the other cellular compartments. TaCRY1a-GFP 6

showed a similar dark-associated nuclear accumulation in the transiently transformed 7

onion epidermal cells and transgenic Arabidopsis cells. Accumulation of this fusion 8

protein also appeared along the inner cell wall, suggesting that part of the translational 9

product is associated with the plasma membrane. This is consistent with the 10

observation of Ahmad et al. (1998) regarding AtCRY1. Once transferred from dark to 11

blue light, TaCRY2-GFP fusion proteins disappeared, and only background signals 12

lingered. TaCRY1a-GFP fusion proteins, in contrast, emptied into the cytoplasm. The 13

GFP-only control transformants showed a demonstrably light-insensitive fluorescence 14

distribution. These results suggest that TaCRY1a is a light-dependent 15

nucleocytoplasmic shuttling protein and that TaCRY2 is a nuclear protein that 16

degrades when exposed to light. 17

18

TaCRY1a Domains Conditioning Nuclear Import and Export 19

To determine which TaCRY1a domains are responsible for its nuclear 20

localization and export, a series of fusion constructs was made by ligating segments of 21

TaCRY1a coding DNA sequence to GFP. As shown in Fig. 4A and 5A, the fusion 22

protein made with the N-terminal segment of AA1-AA492 (TaCNT1a-GFP) displayed 23

a light-dependent nucleocytoplasmic shuttling pattern similar to the whole TaCRY1a 24

sequence. However, the fusion protein made with the C-terminal segment of AA493-25

AA700 (GFP-TaCCT1a) exhibited light-independent nuclear accumulation (Fig. 4E, 26

5C). Therefore, sequence information necessary for nuclear targeting exists in both 27

AA1-AA492 and AA493-AA700, but that required for nuclear export only exists in 28

the N-terminal segment. 29

We noted that deletion of the first 260 aa of the 492 aa N-terminus completely 30



10

abolished the nuclear targeting function (Fig. 4G, 5D), suggesting that the AA1-1

AA260 region is responsible for nuclear targeting. Consistent with this, the GFP 2

fusion with the AA1-AA260 segment accumulated in the nucleus in the dark (Fig. 4B). 3

Similar to the GFP fusions with full length TaCRY1a and with the 492 aa N-terminus, 4

this fusion product translocated into the cytoplasm upon exposure to blue light. Thus, 5

the AA1-AA260 segment carries sufficient information for both nuclear import and 6

export. The GFP fusion with the AA136-AA260 segment is smaller than 45kDa, 7

which allows free diffusion between cellular compartments (Dingwall and Laskey, 8

1986). We noted that this fusion product distributed throughout the entire cell, similar 9

to the GFP-only control, in the dark and emptied into the cytoplasm under the blue 10

light (Fig. 4C, 5B). This result indicates that the nuclear export signal lies in AA136-11

AA260. Fusion constructs that accumulated in the nucleus in the dark but did not 12

contain the AA136-AA260 segment were all retained in the nucleus under light 13

conditions. 14

To identify the C-terminal region responsible for nuclear targeting, transient GFP 15

fusion protein assays were conducted in onion cells for the regions of AA261-AA577, 16

AA261-492 and AA578-AA700. Only the first fusion protein showed nuclear 17

accumulation (Fig. 4F, H). Since the AA261-AA492 segment does not carry nuclear 18

import sequence information, the nuclear targeting signal in the C-terminus was thus 19

restricted to the AA493-AA577 region. 20

Cellular distribution of the GFP fusion with the AA1-AA135 segment did not 21

respond to light changes (data not shown). Because of its small size (<45kDa), we 22

could not exclude the possibility of free diffusion, and thus could not tell whether or 23

not it carries sequence signals for nuclear targeting. However, this segment seemed 24

essential for nuclear export of the whole protein, since when the C-terminal domain 25

with the putative nuclear targeting region was present, fusion products without the 26

AA1-AA135 segment showed constitutive nuclear accumulation, even in the presence 27

of the AA136-AA260 segment (Fig. 4D). 28

29

TaCRY1a and TaCRY2 Showed Different Expression Patterns across Tissue Types 30



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and in Response to Osmotic Stress 1

We performed RT-PCR assays of TaCRY1a and TaCRY2 expression in seedling 2

leaves and roots, spikes and germinating embryos (Fig. 6A). TaCRY1a transcripts 3

were detected mainly in leaves and at low levels in roots and germinating embryos. 4

Compared with TaCRY1a, TaCRY2 was expressed less efficiently in leaves and spikes, 5

but more strongly in roots and germinating embryos, especially in the latter tissue. 6

To investigate the effects of light quality on the expression of TaCRY1a and 7

TaCRY2, we placed etiolated seedlings under continuous white, red or blue light for 8

48 h after transferring them from the dark. The transcripts of both genes slightly 9

increased after the transfer, though TaCRY1a was expressed more efficiently under red 10

light (Fig. 6A). 11

To investigate effects of osmotic stress on the expression of TaCRY1a and 12

TaCRY2, RT-PCR was performed for seedling tissues treated with 20% PEG-6000 or 13

250mM NaCl. As compared to the mock treatment, the expression level of TaCRY1a 14

in germinating embryos as well as in seedling leaves and roots was not affected by 15

either treatment after 12 h (Fig. 6B, and data not shown). However, these treatments 16

repressed TaCRY2 expression in leaves to a certain extent and strongly up-regulated 17

TaCRY2 in seedling roots and germinating embryos. As shown by real-time PCR for a 18

span of 28 h of NaCl treatment, TaCRY2 transcripts increased steadily in roots 19

beginning 12 h after treatment (Fig. 6C). This experiment also indicated that TaCRY1a 20

is salt inducible when the treatments last for over 24 h. These expression pattern 21

changes were far more significant than the fluctuations observed in the water-mock 22

treatment and thus did not correspond to the circadian rhythm. 23

Since TaCRY2 was induced by osmotic stress in seedling roots and germinating 24

embryos, we examined the effects of applying the phytohormones GA3, IAA and ABA 25

on its expression. Of these, only ABA, the stress hormone, increased the transcription 26

level of TaCRY2 (Fig. 7). In the same tissues, the expression state of TaCRY1a was 27

not altered (data not shown). 28

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Over-Expression of TaCRY1a and TaCRY2 Caused Susceptibility to ABA and 30



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Osmotic Stress 1

To understand the response of transgenic Arabidopsis lines over-expressing 2

TaCRY1a and TaCRY2 to osmotic stress/ABA, we investigated two homozygous 3

TaCRY1a-GFP transformants (C1-L4-2, C1-L7-4) and two homozygous TaCRY2-4

GFP transformants (C2-L2-2, C2-L7-3), all derived from independent transformation 5

events. The TaCRY1a transformants carry a single copy transgene, and the TaCRY2 6

transformants carry multiple copies (data not shown). These lines had a considerable 7

level of expression for transgenes (Fig. 8A) and endogenous CRY genes (data not 8

shown), and they showed no obvious morphological differences from the wild type 9

when grown for four days in the dark, but they had comparatively shorter hypocotyls 10

with deeper color when grown for four days in blue light (Fig. 8B). This is typical of 11

lines with over-expressed CRY genes (Lin et al., 1996a, 1998; Matsumoto et al., 2003). 12

The rate of hypocotyl elongation inhibition ranged from 24% (C1-L4-2) to 11% (C2-13

L7-3), relative to the wild type. 14

To determine the effects of ABA and osmotic stress treatments on seed 15

germination of these transgenic lines, the germination assay was conducted on filter 16

papers soaked with 120mM NaCl, 300mM mannitol or 0.3μM ABA. As compared 17

with the water control, all three treatments slowed the pace of germination and 18

reduced the germinating rate (Fig. 9A). The transgenic lines were consistently more 19

severely affected than the wild type, despite some between-line variation (Fig. 9B). 20

The TaCRY1a transgenic lines seemed to be more sensitive to ABA and salinity stress 21

than the TaCRY2 transgenic lines. At day 7 of ABA treatment, all transgenic lines 22

displayed significantly reduced proportions of seedlings with fully opened cotyledons 23

as compared to wild type lines (Fig. 9C), indicating an arrest in post-germination 24

development in the transgenic lines. The mannitol treatment induced similar effects 25

on cotyledon opening. Cotyledon opening was not investigated in the NaCl treatment 26

because all seedlings, no matter whether wild type or transgenic, had died by day 7. 27

The primary root growth of these transgenic line seedlings showed an elevated 28

sensitivity to ABA. When seedlings were grown in agar plates supplemented with 29

10μM ABA for two days, inhibition of root growth was noted for all lines, including 30



13

the wild type control; however, the primary roots of transgenic lines had an even 1

smaller relative growth rate (Fig. 10). When pots with transgenic Arabidopsis plants 2

were supplied with 250mM NaCl, plant wilting was noted at day 9. At day 11, the 3

transgenic lines started dying, while the wild type seedlings were still vigorous (Fig. 4

11). 5

6

Expressions of Two Stress/ABA Responsive Genes in Transgenic Seedlings 7

We investigated the transcript levels of two ABA/stress responsive genes, 8

RD29A and ADH1, in T2 seedlings of the above-mentioned transgenic lines C1-L4 and 9

C2-L2 after exposure to 300 mM mannitol and ABA treatments (Fig. 12). ADH1 10

encodes an alcohol dehydrogenase and is inducible by dehydration, ABA and low 11

temperature (de Bruxelles et al., 1996). RD29A encodes a LEA-like protective protein 12

and is inducible by dehydration, salt, ABA and low temperature (Yamaguchi-13

Shinozaki and Shinozaki, 1994). In the mock treatment, both genes were consistently 14

expressed at slightly lower levels in the TaCRY1a-GFP transgenic line as compared to 15

wild type, whereas the expression pattern was unaltered in the TaCRY2-GFP 16

transgenic line. As expected, exogenous application of ABA and mannitol enhanced 17

the expression of these genes in the wild type. ABA induction of RD29A in the 18

transgenic line C1-L4 was not different from that seen in the wild type, but its 19

induction in the transgenic line C2-L2 was only half that of wild type. Relative to the 20

wild type, RD29A was less effectively induced by the mannitol treatment in both 21

transgenic lines, especially in C1-L4, in which it was almost unresponsive to the 22

osmotic stress. 23

Interestingly, the ABA induction level of ADH1 dropped off by ~40% and ~20% 24

in C1-L4 and C2-L2, respectively, but after the mannitol treatment the induction level 25

was identical to wild type in the former line and increased by ~2-fold in the latter (Fig. 26

12). Conley et al. (1999) reported that less efficient expression of the ADH gene led to 27

lower seed germination rate in Arabidopsis. The change of ADH1 gene expression 28

level in the two transgenic lines was consistent with the germination rate variation 29

(Fig. 9B). 30



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1

Discussion 2

Structural Conservation and Variation of Monocot and Dicot CRY proteins 3

Plant CRY genes fall into three subfamilies: CRY1, CRY2 and CRY-DASH 4

(Perrotta et al., 2001; Kleine et al., 2003; Platten et al., 2005), although individual 5

species may have different CRY family members. The dicot CRY genes have been 6

well characterized, but the monocot CRY genes are still poorly understood. In the 7

present work, we show that wheat CRY1a and CRY2 genes share conserved sequences 8

with their rice counterparts and that the intron/exon organization of these two genes is 9

well conserved among plants. Some conserved domains/motifs previously identified 10

in dicot CRYs, for instance, TGYP and DQXVP, are present in wheat and other 11

monocot CRYs, implying their common origin and functional conservation. 12

Transgenic analysis suggested that TaCRY1a and TaCRY2 are similar to Arabidopsis 13

and rice CRY genes in modulating photomorphogenesis. However, differentiation of 14

CRY proteins between monocots and dicots is noticeable. The C-terminal STAESS 15

motif of dicot CRY proteins that is important for CRY phosphorylation is less 16

conserved in monocot CRY proteins even though they retain an S and E residue-rich 17

feature. For CRY2 proteins, there are a few residue substitutions between monocots 18

and dicots involving acidic to basic or basic to acidic alterations (Fig. 2). These 19

structural changes might lead to evolution of gene function. 20

21

Sequence Characteristics of the TaCRY1a Domains Required for Nuclear Import 22

and Export 23

Nuclear import of proteins usually involves NLS recognition by importins and 24

translocation through the nuclear pore complex (Jiang et al., 1998). We have shown 25

that TaCRY2 is a nuclear-localized protein and has a C-terminal monopartite NLS. 26

TaCRY1a is a light-dependent nucleocytoplasmic shuttling protein that does not have 27

known NLS and NES signals. However, its N-terminal AA1-AA260 segment and C-28

terminal AA493-AA577 segment are sufficient for nuclear targeting. This is similar to 29

OsCRY1, as both its 213 aa N-terminus and 235 aa C-terminus can modulate its 30



15

nuclear accumulation (Matsumoto et al. 2003). The N-terminal domains of TaCRY1a 1

and the bona fide OsCRY1a are highly conserved (Fig. 2). More and more sequences 2

bearing no classic NLS have been associated with nuclear import of proteins, for 3

example, the M9 domain of the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 4

protein (Siomi and Dreyfuss, 1995) and the KNS nuclear shuttling domain of the 5

hnRNP K protein (Michael et al., 1997). These findings indicate that multiple 6

strategies exist for mediating protein nuclear targeting. 7

At the sequence level, the NLS is usually either a short stretch of several basic 8

amino acids (monopartite NLS) (Kalderon et al., 1984), or it consists of two separated 9

clusters of basic residues (bipartite NLS) (Robbins et al., 1991). Within the two 10

segments mentioned above, there is a 19 amino acid peptide 11

KHLDASLRRLGATRLVTRR with seven basic amino acid residues (underlined) 12

from AA74 to AA92, a basic RKK stretch from AA246 to AA248 and a basic RRR 13

stretch from AA550 to AA552 (Fig. 2). These sequence characteristics, especially 14

those in the N-terminal domain, are conserved across plant CRY1 proteins. 15

Nevertheless, further work is required to clarify their roles in mediating nuclear 16

targeting. 17

The NES of TaCRY1a is present in the AA136-AA260 segment of the N-18

terminal region. This is unlike OsCRY1, which does not have a nuclear export 19

function (Matsumoto et al. 2003), and AcCRY3 and AtCRY1, which carry nuclear 20

export signals in their C-terminal domains (Imaizumi et al., 2000; Yang et al., 2000). 21

Compared to NLS, NES signals are even less well defined. The best-characterized 22

NES are the leucine-rich domains in PKI and Rev (Wen et al., 1995). Significantly, 23

we noted three leucine dimers within every 32-35 AA span of the AA136-AA260 24

corresponding domains of all compared CRY proteins (Fig. 2). However, this 25

conserved feature is not the same as that reported in Wen et al. (1995) and might not 26

be essential for nuclear export since AcCRY4 (BAA88423) cannot direct export to the 27

cytoplasm despite having these conserved leucine residues (Imaizumi et al., 2000). A 28

few reports documented non-leucine-rich NES domains, for example, the proline and 29

glutamine-rich NES in human pUL69 protein (Lischka et al., 2001), the M9 domain 30



16

of hnRNP A1 and the KNS domain of hnRNP K (Michael et al., 1995, 1997). In the 1

human NFAT transcription factor, the NES sequence is IVAAINALTT (Klemm et al., 2

1997). 3

Wen et al. (1995) demonstrated that hydrophobic residues are critical for an 4

NES. Consistent with this finding, the AA136-AA260 region of TaCRY1a is 5

hydrophobic and has a proline-rich core motif (PPAPMLPP) between AA178 and 6

AA185. The proline-rich feature has been associated with nuclear export mediation in 7

other studies (Lee et al., 2001; Lischka et al., 2001). In the C-terminal AtCRY1 region 8

that contains an NES, we identified the hydrophobic stretch AMIPEFNIRI (AA603-9

614). Within AcCRY3 (BAA32809) and its C-terminus, which could modulate a light-10

dependent intracellular localization pattern (Imaizumi et al., 2000), we found a 11

classical NES-like peptide LKQSLIQLDISLRSL at AA55-AA69 and hydrophobic 12

stretches in the C-terminal region. The C-termini of AtCRY2, TaCRY1a and TaCRY2 13

have no predicted hydrophobic domains. Like the GFP-TaCRY1a C-terminus fusion, 14

the C-terminus AtCRY2-GUS fusion constitutively accumulates in the nucleus (Yang 15

et al., 2000). However, even though these short hydrophobic domains are associated 16

with NES, other sequence motifs or structural characteristics may be required for 17

nuclear export. For example, the AcCRY4 C-terminus has a hydrophobic domain, but 18

it did not mediate nuclear export (Imaizumi et al., 2000). As shown in this study, 19

nuclear export of GFP constructs with C-terminal segments of TaCRY1a was lost in 20

the absence of the first 135 aa of the N-terminus. AtCRY1 and OsCRY1 constitutively 21

form homodimers through their N-termini (Sang et al. 2005; Zhang et al. 2006). This 22

is possibly also true for TaCRY1a because its N-terminus is highly similar to that of 23

OsCRY1a. Sang et al. (2005) demonstrated that deletion of the AA1-AA99 region of 24

AtCRY1, which corresponds to the first 105 aa of the TaCRY1a N-terminus, 25

abolished the dimerization. This dimerization might be required for nuclear export of 26

TaCRY1a when its C-terminal region is present. The GFP-TaCRY1a segment 27

constructs without the highly hydrophilic C-terminal region might exist in different 28

conformations and thus might not require the N-terminus-mediated dimerization for 29

nuclear export. 30



17

Subcellular localization of CRY proteins is regulated in a complex manner. 1

Besides the NLS and NES signals, other factors, such as partner binding, 2

multimerization (Gaits et al., 1999; Stommel et al., 1999), phosphorylation (Jensen et 3

al., 1998) and prenylation (Rodriguez-Concepcion et al. 1999) may also affect nuclear 4

import and export. In Arabidopsis, nucleocytoplasmic shuttling of AtCRY1 may be 5

facilitated by C-terminal binding of COP1, an NLS- and NES-containing downstream 6

repressor of photomorphogenesis (von Arnim and Deng, 1994; Wang et al., 2001). 7

8

Tissue-Specific and Light-Inducible Expression of TaCRY1a and TaCRY2 9

TaCRY1a and TaCRY2 were expressed at a relatively high level in seedling 10

leaves. TaCRY1a transcript levels were negligible in roots and germinating embryos. 11

In comparison with the TaCRY1a transcript, there was more TaCRY2 transcript in 12

these two tissues, especially in germinating embryos. Differences in CRY family 13

member expression profiles may imply functional diversification, which has been 14

suggested in a few previous studies. In Arabidopsis, CRY1 mainly functions in de-15

etiolation (Lin et al., 1996a), while CRY2 plays a role in the regulation of 16

photoperiodic flowering (Guo et al., 1998) and the mediation of light-dependent 17

phenylpropanoid metabolism in the roots (Hemm et al., 2004). Canamero et al. (2006) 18

reported that both AtCRY1 and AtCRY2 are involved in the blue light induction of 19

primary root elongation, but in an antagonistic manner. Our findings suggested that 20

TaCRY2 expression might be important in non-green tissues such as germinating 21

embryos and roots. 22

The expression of TaCRY2 in seedling leaves seemed to be light independent, 23

but TaCRY1a was red light inducible. Imaizumi et al. (2000) reported red light 24

induction of AcCRY3 expression in Adiantum capillus-veneris. These light-inducible 25

regulatory patterns are contradictory to those of pea CRY genes PsCRY1 and PsCRY2, 26

as lower levels of those transcripts were detected in seedlings grown with light 27

compared to those kept in complete darkness (Platten et al., 2005). In other reports, 28

Brassica napus CRY1 is blue light inducible (Chatterjee et al. 2006); the expression of 29

Arabidopsis and rice CRY genes was light independent at the transcriptional level, but 30



18

AtCRY2, OsCRY1a and OsCRY2 were under light-dependent translational and/or 1

post-translational control (Ahmad and Cashmore, 1993; Lin et al., 1998; Hirose et al., 2

2006). Therefore, light regulation of CRY gene expression is not identical in different 3

kinds of plant species, an observation that may be related to their growth habits or 4

growth environments. 5

6

Involvement of CRY Genes in Osmotic and Other Stress Responses 7

Expression of both TaCRY2 and TaCRY1a is induced by osmotic stress, even 8

though their induction patterns are different. We found that over-expressing TaCRY 9

genes in Arabidopsis resulted in decreased seed germination, impaired cotyledon 10

opening and less tolerance to ABA and high salinity at the vegetative growth stage. 11

These findings suggest that over-expressing CRY genes compromises plant resistance 12

to osmotic stress and ABA. Consistent with this, Mao et al. (2005) reported that, due 13

to increased stomatal opening, Arabidopsis over-expressing myc-AtCRY1 or myc-14

AtCRY2 are more sensitive to drought. In other reports, Phee et al. (2007) identified 15

stress/defense-related proteins that were differentially expressed between wild type 16

Arabidopsis and the hy4 (cry1) mutant, among which the TIR-NBS-LRR class 17

proteins were down-regulated and the jacalin lectin family members were up-18

regulated in the hy4 line. Danon et al. (2006) documented that AtCRY1 is required for 19

singlet oxygen-induced programmed cell death. Thus, plant CRY genes are associated 20

with abiotic/biotic stress signaling in several ways. 21

Over-expression of TaCRY2 in Arabidopsis did not affect the expression of 22

RD29A and ADH1 under normal conditions. In agreement with this, germination and 23

post-germination development were not altered in the corresponding transgenic lines 24

used in this study. There was only a slight reduction in the expression of these genes 25

in the TaCRY1a transgenic lines. In contrast, these marker genes had significantly 26

altered expression patterns in both transgenic lines under the applied osmotic stress 27

and ABA treatments. Over-expressing TaCRY1a severely compromised the osmotic 28

stress induction of RD29A, but it did not influence the ABA induction pattern. Over-29

expressing TaCRY2 attenuated the induction of RD29A by both osmotic stress and 30



19

ABA. Phenotypically, these transgenic lines were more sensitive to treatments of 1

stress factors, such as supplying high levels of mannitol and NaCl. Up-regulation of 2

RD29A has been positively associated with osmotic stress tolerance in Arabidopsis 3

(Umezawa et al., 2004). Our results clearly suggest that over-expressing CRY genes 4

weakens the regulatory roles of stress-responsive genes in stress tolerance and that the 5

ways by which TaCRY1a and TaCRY2 interact with stress/ABA signals are 6

complicated. 7

The ABA and stress-inducible gene ADH1 showed different expression patterns 8

in the transgenic lines when treated with 300mM mannitol and with 0.3μM ABA. 9

This was unexpected, as osmotic stress was presumed to increase the ABA levels in 10

cells (Zeevaart and Creelman, 1998) and thus should have affected ADH1 in a way 11

similar to the ABA treatment. However, as de Bruxelles et al. (1996) have 12

demonstrated, ADH1 could respond to osmotic stress by an ABA-independent 13

pathway. The unaltered or increased expression of ADH1 in the transgenic lines after 14

the mannitol treatment suggests that over-expressing CRY genes does not affect this 15

ABA-independent osmotic stress response pathway. The more severely repressed 16

RD29A induction and less induced ADH1 expression observed under osmotic stress in 17

the TaCRY1a transgenic line as compared to the TaCRY2 transgenic line may explain 18

the higher sensitivity of TaCRY1a transgenic lines to osmotic stress. 19

20

Concluding Remarks 21

Monocots, in particular, the grass crops, provide the staple foods for human 22

beings. Because of the close relationship between light and the growth and 23

development of plants, studies on light receptors have great agronomical importance. 24

We showed that TaCRY1a and TaCRY2 of wheat share conserved structures and 25

functions with other CRY genes. TaCRY1a and TaCRY2 are differentially expressed in 26

tissues/organs, making further investigation of their functional diversification more 27

intriguing. Like ATCRY1, TaCRY1a is a light-dependent nucleocytoplasmic shuttling 28

protein. It carries at least two atypical NLS, one at the N-terminus and one at the C-29

terminus, and an NES within the AA136-AA260 region. This arrangement is different 30



20

from that of AtCRY1. We demonstrated that TaCRY1a and TaCRY2 are also involved 1

in abiotic stress responses, some of which are related to ABA signaling. However, 2

their involvement may employ different mechanisms. Thus, questions still remain 3

regarding the crosstalk between light signaling and stress adaptation. A systematic 4

expression profiling of the genes involved in ABA, osmotic stress and cryptochrome 5

signal transduction pathways is necessary to elucidate the interaction network 6

between CRY and osmosis/ABA signals. 7

8

MATERIALS AND METHODS 9

Plant Materials 10

The plant lines used in this study included common wheat cultivar ‘Sumai No. 11

3’ (Triticum aestivum, 2n=6x=42, genome=AABBDD), nulli-tetrasomic lines of 12

common wheat cultivar ‘Chinese Spring’ (CS) and Arabidopsis ecotype Columbia-0 13

(Col-0). 14

Adult wheat plants were grown in a field in Nanjing, China in the normal 15

growing season. Spikes two days after anthesis were harvested for RNA extraction. 16

17

Stress and hormone treatments 18

After germination in petri dishes, seedlings were grown at room temperature 19

(RT) with 16 h light daily. Six-day-old seedlings were transferred to petri dishes with 20

250mM NaCl (Nanjing Chemical Reagent Co., Ltd, China) or 20% (g/v) PEG-6000 21

(Amersco, USA) for osmotic stress treatments. For hormone treatments, the 22

procedures were the same as the stress treatments except that the chemicals were 23

replaced with 10µm of ABA, GA3 or IAA (Sigma, USA). Tissues were harvested 12 h 24

after the transfer. 25

In the time-course treatment with 250mM NaCl, plants were given 12 h light 26

per day. The root tissues were harvested at 4, 8, 12, 16, 20, 24 and 28 h after the 27

transfer. To prepare germinating embryos, seeds imbibed for 15 h were placed at RT 28

in petri dishes fitted with filter papers that were soaked in either 250mM NaCl, 20% 29

(g/v) PEG-6000, 10µM ABA, GA3 or IAA for 12 h. The embryos were then separated 30



21

from endosperms by hand. Distilled water treatments were used as the mock controls. 1

For light quality treatment, the etiolated seedlings grown in the dark for six days 2

were divided into four groups. One group was kept in the dark while the remaining 3

groups were placed under continuous white, red or blue light for 48 h. The white light 4

was generated from a white fluorescent tube. The blue or red light (2.7W⋅s-1⋅m-2, 5

0.7W⋅s-1⋅m-2, respectively) was generated by filtering the fluorescent tube with a blue 6

or red filter (Model M34, Sea-gull Ltd, Shanghai, China). 7

8

DNA and RNA Isolation 9

Genomic DNA was extracted according to the procedures described by Ma and 10

Sorrells (1995). RNA was extracted using the Trizol reagent (Invitrogen, USA) 11

following the manufacturer’s protocol and quantified with Ultrospec 2100 pro 12

(Amersham Pharmacia, England). The RNA was checked for DNA contamination by 13

direct PCR amplification of the wheat β-tubulin gene using the extract as the template. 14

The primers used were 5'-CGTGGTGATGTTGTGCCAAAG-3' and 5'-15

ACTTCTTCATAGTCCTTCTCCAGG-3'. 16

17

DNA Amplification, Cloning and Sequence Analysis 18

The amino acid sequences of barley CRY1a (ABB13330) and rice CRY2 19

(BAC56984) were queried, respectively, against wheat dbESTs (580,000 ESTs, 20

extracted from GenBank dbESTs of the 154th release, 2006) by tBLASTn. Twenty 21

EST hits for CRY1a and 15 for CRY2 were identified based on >90% identity for 22

CRY1a and >85% identity for CRY2 in a >33 aa overlap. After removing possible 23

vector sequence contamination, contigs were assembled with the parameter settings 24

of >40bp overlap and >95% identity. One 2719bp CRY1a contig and one 2375bp 25

CRY2 contig were obtained. The primers used for RT-PCR were as follows: 5’-26

CCAAAATCAAGAAACCCTGGCAACTCTG-3’ (sense primer) and 5’- 27

CGTCACTCTCCAACTCCCTACACAAT-3’ (antisense primer) for CRY1; 5’- 28

CTGCTCGACGTAATGCTCGTGAGAG-3’ (sense primer) and 5’- 29

ATGAGAGTGGGGTGCAGGAAGATCC-3’ (antisense primer) for CRY2, all 30



22

designed based on the 5’ and 3’ UTR sequences of the contigs. 1

The first-strand cDNA was synthesized using the M-MLV reverse transcriptase 2

(Promega, USA) according to the manufacturer’s instructions. The PCR reaction was 3

performed in a 25 µl mixture containing ~5 ng template, 5 pmol of each primer, 5 4

nmol of each of the dNTPs, 37.3 nmol MgCl2, 0.5 U rTaq DNA polymerase (Takara, 5

Japan) and 1x PCR buffer supplied with the enzyme. The thermal cycle profile was 6

94ºC for 3 min, 36 cycles of 94ºC for 30 s, 58ºC for 40 s, 72 ºC for 2.5 min and a final 7

extension of 5 min at 72ºC. 8

The same sets of primers were used to amplify genomic DNA. Included in the 9

25 µl PCR reaction were 40 ng genomic DNA, 0.2 M D (+)-trehalose (Sigma, USA) 10

as the PCR enhancer, 0.5 U Ex-Taq (Takara, Japan), and other PCR components in the 11

same concentrations as described above. The thermal cycling conditions were 94ºC 12

for 4 min, 36 cycles of 94ºC for 40 s, 58 ºC for 45 s, 72 ºC for 5 min and a final 13

extension of 5 min at 72ºC. The PCR products were separated with 1% agarose gels. 14

DNA from the target bands excised from the gels was purified and ligated into 15

the pUC19-based TA cloning vector pX-T. Positive clones from transformation of 16

competent JM109 cells were sequenced in Shanghai Invitrogen Biotechnology Ltd. 17

Corp. (Shanghai, China). 18

The exon/intron organization of CRY1a and CRY2 was determined by 19

comparing the corresponding cDNA and genomic DNA sequences. Scanning for NLS 20

was carried out by WoLF PSORT (http://psort.nibb.ac.jp/), and prediction of NES was 21

carried out with the CBS prediction server (http://www.cbs.dtu.dk/services/NetNES). 22

Other bioinformatic analyses of the sequences were conducted with software 23

Macvector 9.0 (Accelrys, Oxford, USA). 24

25

Transient Assays 26

Constructs 27

Inserts were amplified using pfu DNA polymerase (Dingguo Biotech, China) 28

with primers containing an XbaI or XhoI restriction site. The restricted amplicons 29

were inserted into the CaMV35S-EGFP-NOS sequence of a pUC19-based expression 30



23

vector to generate CaMV35S promoter-driven GFP fusion constructs. GFP is a 27-1

kDa spontaneously fluorescent protein, allowing direct visualization of the fusion 2

proteins (Scott et al., 1999). After ligation and transformation into JM109 competent 3

cells, these constructs were confirmed by sequencing. 4

5

Transient Expression in Onion Epidermal Cells 6

Using the PDS-1000 particle delivery system (Bio-Rad, USA), the fusion 7

constructs were introduced through 1.1μm tungsten particles into the epidermal cells 8

of onion bulb scales placed on Murashige and Skoog (MS) agar plates in the 9

procedure recommended by the manufacturer. The bombardment parameters were 10

1100-psi pressure, 85 mm from the macrocarrier to samples, and 28 inch Hg vacuum. 11

Bombarded onion epidermis was incubated at 25°C in the dark for 16 h, or in the dark 12

for 4 h followed by 12 h of blue light (2.7 W⋅s-1⋅m-2). 13

14

Microscopy 15

GFP fluorescent signals were examined with a confocal laser-scanning 16

microscope (TCS NT, Leica, Germany) in the 488nm-exciting wavelength. Cellular 17

structure was visualized using bright-field optics. 18

19

Arabidopsis Transformation and GFP Signal Assay of Root Cells 20

Inserts were amplified using pfu DNA polymerase (Dingguo Biotech, China) 21

with primers containing the XbaI/EcoRI or XbaI/BglII restriction sites. After 22

restriction digestion, the amplicons were inserted into a modified GFP-containing 23

pBI121 expression vector to generate CaMV35S promoter-driven GFP fusion 24

constructs. Arabidopsis thaliana ecotype Col-0 was transformed with these constructs 25

using the floral dip method (Martinez-Trujillo et al., 2004) with Agrobacterium 26

tumerfaciens strain LBA4404. 27

Harvested seeds were screened on MS plates (pH=5.8) containing 50 mg/L 28

kanamycin. Putative transgenic plants were verified by PCR and Southern 29

hybridization. T2 transgenic seeds were germinated and grown in sterile MS plates in 30



24

darkness for three days or in darkness for two days followed by irradiation with white 1

light one day before observation. Roots were put under glass coverslips in PBS buffer 2

(137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.4) and then 3

observed in the 488nm-exciting wavelength with the confocal laser-scanning 4

microscope. Three-dimensional rendering was created from 20 optical sections 5

acquired through continuous scanning. 6

7

RT-PCR 8

sqRT-PCR was carried out in 12.5 µl reactions with ~2.5 ng template. The PCR 9

profile was 94ºC for 3 min, 20-30 cycles of 94ºC for 20 s, 58ºC for 30 s, 72ºC for 30 s, 10

and a 5 min extension at 72ºC. Primer sequences used were 5’-11

CTCAGACGAGCCACCCATTG-3’ and 5’-CCCCACCTTCTCTCCCAGTC-3’ for 12

TaCRY1a, and 5’-GAACTGAAGGGCACAAATAAACAGACC-3’ and 5’-13

ATGAGAGTGGGGTGCAGGAAGATCC-3’ for TaCRY2, all designed based on 3’ 14

end sequences. β-tubulin cDNA amplification was used as the external control. Five 15

microliters from the PCR product of each reaction was electrophoresed in a 1.5% 16

agarose gel and viewed under UV light after standard staining with ethidium bromide. 17

qRT-PCR amplifications for Arabidopsis genes were performed on a Bio-Rad 18

iCYCLER iQ5 real time PCR instrument (Bio-Rad, USA), in 25 μl reactions 19

containing ~5 ng template, 12.5 μl SYBR-Green PCR Mastermix (Toyoba, Japan) and 20

10 pmol of each primer. Reactions for transgenic line analysis were made in duplicate; 21

the others were made in triplicate. Gene-specific primers for Arabidopsis genes ACT2, 22

RD29A, and ADH1 were 5’-TGGTGATGGTGTGTCT-3’ and 5’-23

ACTGAGCACAATGTTAC-3’, 5’-ATCACTTGGCTCCACTGTTGTTC-3’ and 5’-24

ACAAAACACACATAAACATCCAAAGT-3’, and 5’-25

TCCACGTATCTTCGGCCATG-3’ and 5’-TAGCACCTTCTGCAGCGCC-3’, 26

respectively. Ct values (cycle threshold) for each target gene were normalized 27

according to those obtained in the corresponding reactions for Arabidopsis actin2 28

(ACT2) gene or wheat β-tubulin gene. Relative expression was estimated using the 2-29

ΔΔCT method (Livak and Schmittgen 2001), by referring to the normalized Ct value 30



25

obtained for Col-0 without any treatment, or for seedlings grown under the non-1

stressed condition at Zeitgeber time 0 (light-on, 8:30 am). 2

3

Transgenic phenotyping 4

Hypocotyl measurements 5

Seeds were sown on MS agar plates with 5% sucrose (pH=5.8). The plates were 6

placed in a 4°C refrigerator for two days and were then placed at RT for four days 7

either under dark or blue light (2.7 W⋅s-1⋅m-2). Hypocotyl length was measured for 15 8

seedlings of each sample. The experiments were repeated three times. 9

10

Germination Assay 11

Seeds were sown on filter papers in petri dishes soaked with distilled water, 12

120mM NaCl, 300mM mannitol (osmotic stress), or 0.3µM ABA. One hundred fifty 13

seeds in three plates were sown for each line. After stratification at 4°C for two days, 14

the plates were moved to RT with 12 h light per day. Germination (emergence of 15

radicals) was scored daily until the fifth day after sowing. 16

17

Root Growth Inhibition Assay 18

Seeds were sown in vertically placed MS agar plates for four days. Fifteen 19

phenotypically uniform seedlings were retained and half of these were carefully 20

transferred onto a MS agar plate supplemented with 10μM ABA. Root length was 21

measured two days after the transfer. The experiment was repeated three times. 22

23

Salt tolerance assay 24

Seeds were sown on MS plates. After six days of growth, they were transferred into 25

pots filled with mixture of soil and vermiculite, given 16 h light per day and 25°C 26

ambient temperature. Twenty days after the transfer, the plants were irrigated with 27

saline water containing 250mM NaCl every two days. 28

29

The sequences described in this article have been submitted to the GenBank data 30



26

library under the accession numbers EF601539-EF601542. 1

2

3

ACKNOWLEDGMENTS 4

We thank Dr. Zhigang Xu of Nanjing Agricultural University for kindly supplying the 5

light equipment. 6

7

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Figure legends 1

2

Figure 1. CRY gene structures of several dicots and monocots drawn according to 3

alignments of cDNA sequences with the corresponding genomic DNA sequences. 4

Exons are shown as rectangles hatched with diagonal lines, except those in un-5

translated regions, which are shown as rectangles filled with dots. Lines between the 6

exons represent intron positions. The sequences other than the wheat sequences have 7

the following GenBank accession numbers: S66907 and NM_179257 (AtCRY1 and 8

AtCRY2 cDNA); AB073546, AB073547, AB103094 (OsCRY1a, OsCRY1b, OsCRY2 9

cDNA); AF545572 and AY835380 (SbCRY2 cDNA and genomic sequence); 10

AY508972 and AY508974 (PsCRY2a and PsCRY2b cDNA), AY508973 and 11

AY508975 (PsCRY2a and PsCRY2b genomic sequences). CRY genomic sequences of 12

Arabidopsis and rice were extracted from the published genome sequences. 13

14

Figure 2. Primary structure comparison of wheat, rice, Arabidopsis and tomato CRYs. 15

Boxed are the conserved TGYP, DQXVP and STAESS domains. The predicted NLS 16

of TaCRY2 is indicated with an asterisk at the top. The position corresponding to 17

D493 in TaCRY1a was set as the demarcation between N and C-termini. Vertical solid 18

triangles indicate the starting or ending points of the TaCRY1a segments used to 19

produce GFP fusion proteins. Vertical empty triangles indicate amino acid 20

substitutions distinguishing monocots from dicots or individual CRY subfamilies from 21

each other. Solid dots signify basic-to-acid or acid-to-basic amino acid substitutions in 22

CRY2 between monocots and dicots. Oval boxes signify the basic amino acid 23

stretches from AA74 to AA92, AA246-AA248 and AA550-AA552 in TaCRY1a. The 24

hydrophobic stretches in the N-terminus of TaCRY1a and the C-terminus of AtCRY1 25

are underlined. 26

27

Figure 3. Subcellular localization of TaCRY1a-GFP and TaCRY2-GFP fusion 28

proteins in transiently transformed onion epidermal cells and transgenic Arabidopsis 29

root cells. The top and middle panels are onion epidermal cells and the bottom panel 30



33

represents Arabidopsis root tip cells. 1

2

Figure 4. Subcellular localization assay of TaCRY1a segment-GFP fusion proteins in 3

transiently transformed onion epidermal cells. The fused TaCRY1a segments are 4

indicated on the left. Except the AA1-AA492 segment, which was fused to the N-5

terminus of GFP, all others were fused to the C-terminus of GFP. 6

7

Figure 5. Subcellular localization of TaCRY1a segment-GFP fusion proteins in 8

transgenic Arabidopsis root cells. The fused TaCRY1a segments are indicated on the 9

left. 10

11

Figure 6. Semi-quantitative RT-PCR (sqRT-PCR) or real time RT-PCR (qRT-PCR) of 12

TaCRY1a and TaCRY2. A, Expression levels in different tissues (top) and in leaves 13

given different types of light (bottom). RT&LF: seedling roots and leaves; SP: spikes; 14

EB: germinating embryos; DK: dark; BL: blue light; RL: red light; WL: white light. B, 15

Expression levels in seedling leaves (top), roots (middle) and germinating embryos 16

(bottom) after 12 h NaCl or PEG treatments. TUB transcripts were amplified for 20-17

23 cycles, while TaCRY1a and TaCRY2 were amplified for 25-26 cycles. C, 18

Expression levels in seedling roots throughout the course of a 28 h NaCl treatment 19

with daily 12 h continuous white light. Each data point indicates the relative 20

expression means from three replicates and the standard deviation (SD) (vertical bar). 21

See ‘Materials and Methods’ for details. 22

23

Figure 7. sqRT-PCR of TaCRY2 in germinating embryos (top) and seedling roots 24

(bottom) treated with IAA, GA3 or ABA for 12 h. TUB and TaCRY2 transcripts were 25

amplified for 21 and 25 PCR cycles, respectively. 26

27

Figure 8. Characterization of TaCRY1a-GFP and TaCRY2-GFP transgenic 28

Arabidopsis lines. A, Relative expression of the transgenes in seedlings of Col-0, two 29

TaCRY1a-GFP lines (C1-L2-4 and C1-L7-4) and two TaCRY2-GFP lines (C2-L2-2 30



34

and C2-L7-3). B, Phenotypes of seedlings grown at 25ºC ambient temperature under 1

dark and blue light. Photographs were taken four days after sowing. Bar=30mm. 2

3

Figure 9. Seed germination and cotyledon opening of Col-0 and the transgenic 4

Arabidopsis lines in response to NaCl, ABA or mannitol treatment. A, Germination at 5

25ºC ambient temperature of seeds sown on filter papers saturated with water, 0.3μM 6

ABA, 120mM NaCl or 300mM mannitol. The photographs were taken three days 7

after sowing. B, Mean germination rate curve from day 0 to day 4 after sowing. C, 8

Proportion of seedlings with healthy, opened cotyledons seven days after sowing. 9

10

Figure 10. Relative root growth of Col-0 and the transgenic Arabidopsis lines treated 11

with 10μM ABA for two days, estimated as the percentage of primary root length on 12

the ABA plate relative to that on the control plate. Shown is the result from one 13

representative experiment. 14

15

Figure 11. The response of plants of Col-0 and the transgenic Arabidopsis lines to 16

irrigation of 250mM NaCl into the soil every two days. 17

18

Figure 12. Expression of RD29A and ADH1 in Col-0 and the transgenic Arabidopsis 19

lines under various stress treatments. Total RNA was extracted from 15-day-old T2 20

kanamycin-resistant seedlings grown on MS plates (control), or grown on the MS 21

plates for twelve days and then transferred to MS plates supplemented with 300mM 22

mannitol, 10μM ABA for three days of growth. Error bars indicate SD. 23

24



Figure 1

Figure 1. CRY gene structures of several dicots and monocots drawn according to

alignments of cDNA sequences with the corresponding genomic DNA sequences. Exons

are shown as rectangles hatched with diagonal lines, except those in un-translated

regions, which are shown as rectangles filled with dots. Lines between the exons

represent intron positions. The sequences other than the wheat sequences have the

following GenBank accession numbers: S66907 and NM_179257 (AtCRY1 and AtCRY2

cDNA); AB073546, AB073547, AB103094 (OsCRY1a, OsCRY1b, OsCRY2 cDNA);

AF545572 and AY835380 (SbCRY2 cDNA and genomic sequence); AY508972 and

AY508974 (PsCRY2a and PsCRY2b cDNA), AY508973 and AY508975 (PsCRY2a and

PsCRY2b genomic sequences). CRY genomic sequences of Arabidopsis and rice were

extracted from the published genome sequences.

PsCRY2b

PsCRY2a

AtCRY2

OsCRY2

TaCRY2

TaCRY1a

OsCRY1a

OsCRY1b

SbCRY2

AtCRY1

500bp



1

Figure 2

TaCRY1aOsCRY1aOsCRY1bAtCRY1LeCRY1aLeCRY1bTaCRY2OsCRY2AtCRY2LeCRY2

1 84

1 85

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1 78

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250 334

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D S A N T S L L S P Y L H F G E L S V R K V F H Q I R M K Q L T W S N E S N G D G E E G C S L F L R S I G L R E Y S R R L A F N H P C S H E K P L L A H L R F F P W V V ND S A S T S L L S P Y L H F G E L S V R K V F H Q V R M K Q L M W S N E G N H A G D E S C V L F L R S I G L R E Y S R Y L T F N H P C S L E K P L L A H L R F F P W V V DD S A S T S L L S P Y L H F G E L S V R K V F H L V R M K Q L V W S N E G N R A A E E S C T L F L R S I G L R E Y S R Y L S F N H P C S H E K P L L A H L R F F P W V I ND S A T T S F L S P H L H F G E V S V R K V F H L V R I K Q V A W A N E G N E A G E E S V N L F L K S I G L R E Y S R Y I S F N H P Y S H E R P L L G H L K F F P W A V DD S A T T S F L S P H L H F G E V S V R K V F H F V R I K Q V L W A N E G N K A G E E S V N L F L K S I G L R E Y S R Y M S F N H P Y S H E R P L L G H L R Y F P W V V DD S A T T S F L S P C L H F G E V S V R K V F H R I R T K Q T L W A N E G N K A G E E S V N L F L K S I G L R E F S R Y M S F Y H P Y S H E R P L L G Q L K Y F P W L V DA G T T T S L S S P Y L H Y G E V S V R K I Y Q L V R V Q Q I K W E N E G K S G A G E S D N L F L L S I G L R E Y S R Y L C F N F P F T R E R S L L G N L K H Y P W R A DE G A T T S L L S P Y L H F G E V S V R K V Y Q L V R M Q Q I K W E N E G T S E A E E S I H F F M R S I G L R E Y S R Y L C F N F P F T H E K S L L G N L K H Y P W K V DV G N S T S L L S P Y L H F G E I S V R H V F Q C A R M K Q I I W A R D K N S E G E E S A D L F L R G I G L R E Y S R Y I C F N F P F T H E Q S L L S H L R F F P W D A DG G N S T S L L S P Y L H F G E V S V R K V F N S V R L K Q I L W T K E G N S V G K D S A T I Y L R A I G L R E Y S R Y I C F N F P F T H E R S L L N N L R F F P W N A DD S A . T S L L S P Y L H F G E V S V R K V F H V R K Q . . W N E G N . G E E S . L F L R S I G L R E Y S R Y . F N H P . S H E . P L L . H L R F F P W . V D


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410 494

407 491

L D R I D N P Q F E G Y K F D P Y G E Y V R R W L P E L A R L P T E W I H H P W D A P E S V L R A A G I E L G S N Y P L P I V E L D E A K S R L Q D A L S E M W E L E A AL D R I D N P Q L E G Y K F D P H G E Y V R R W L P E L A R L P T E W I H H P W D A P E S V L Q A A G I E L G S N Y P L P I V E L D A A K T R L Q D A L S E M W E L E A AL D R I D N P Q L E G Y K F D P H G E Y V R R W L P E L A R L P T E W I H H P W D A P A S V L Q A A G V E L G S N Y P L P I V G L D A A N A R L Q E A L S E M W Q L E A AF D R I D N P Q F E G Y K F D P N G E Y V R R W L P E L S R L P T D W I H H P W N A P E S V L Q A A G I E L G S N Y P L P I V G L D E A K A R L H E A L S Q M W Q L E A AL D R I D N P Q F V G Y K C D P H G E Y V R R W L P E L A R L P T E W I H H P W N A P E S V L E A A G I E L G S N Y P L P I V E I D S A K V R L E Q A L S Q M W Q N D A AF L G I D N P Q F E G Y K F D P N G E Y V R R W L P E L A R L P T E W I H H P W D A P E S V L Q A A G I E L G S N Y P F P I V E I V A A K E R L E E A L S Q M W Q L E A AL G R L D N P E V Q G Q K Y D P D G E Y V R T W I P E L A R M P G E W I H H P W D A P S S I L E V A G V E L G F N Y P M P I V E L H T A R E C L D D A I S T M W Q L D T AL S R L D N P E V Q G Q K Y D P D G V Y V R T W I P E L A R M P T E W I H H P W D A P S C I L E V A G V E L G F N Y P K P I V D L H I A R E C L D D S I S T M W Q L D T AL D R L D N P A L Q G A K Y D P E G E Y I R Q W L P E L A R L P T E W I H H P W D A P L T V L K A S G V E L G T N Y A K P I V D I D T A R E L L A K A I S R T R E A Q I ML E R L D N P E V Q G F N Y D P E G E Y V R H W L P E L A R M P A E W I H H P W D A P L N V L K A A G V E L G M N Y P N P I I D V D V A R D R L M Q A I I I M R E K E A AL D R I D N P Q . G Y K . D P G E Y V R R W L P E L A R L P T E W I H H P W D A P S V L A A G . E L G S N Y P P I V . L D A . R L . A L S M W Q L E A A


505 570

507 575

506 573

498 561

491 556

491 561

495 550

495 553

495 548

492 556

S R A E I E N G M E E G L G - D S S D E P - - - - - - - P I A F - P Q E L Q H - M E V D R A - - - - T I H T P A T A G - R R R A D Q M V P S I T S S L V R A - - - - E T ES R A A M E N G M E E G L G - D S S D V P - - - - - - - P I A F - P P E L Q - - M E V D R A P A Q P T V H G P T T A - G R R R E D Q M V P S M T S S L V R A - - - - E T ES R A A M D N G M E E G L G - D S S E V P - - - - - - - P I E F - P R E L Q - - M E V D R E P A R V T A N V L T T A - - R R R E D Q M V P T M T S S L N R A - - - - E T ES R A A I E N G S E E G L G - D S A E V E E A - - - - - P I E F - P R D - - I T M E E T - E P T R L N - - - P N - - - - R R Y E D Q M V P S I T S S L I R P - - - - E E DA R A A I E N G M E E G H G - D S A D S P I A F - - - - P - - - - - Q A M H - - M E M D H E P V R N N - - - P V I V T V R R Y E D Q M V P S M T S S L F R - A - - - E D EA R S A I E N G M E E G H G - D S T D E F V - - - - - - P I A F - P Q A M Q I E M E A N N V P V R N N N - - P T I T A L R R Y G D Q I V P S M S S S F F R - N - - - E D EE K L A E L D G - - E V - V E D N L S H I K S F D V - - P K V V L K E L S - - - - P - - - - - - - - - - - - - H C - - - - - - - D R K V P T D D G R N L E L Q P K E L K GE K L A E L D G - - E V - V E D N L S N I K T F D I - - P K V V L R E T S - - - - P C A L - P I - - - - - - - - - - - - - - - - D Q R V P H A S S K D H N L K S K V L K AI G A A P D - - - - E I - V A D S F E A L G A N T I K E P G L C P S V S S N - - - - - - - - - - - - - - - - - - - - - - - - - - D Q Q V P S A V R Y N G S K R V K P E E EV N T S H A N G T V E V - V F D N S E N V G D S A S I - P K - - - D D V V K G K E P C - - - P - - - - - - - - - - - - S S S S Y D Q R V P S M Q N V G T Y R K R P K P E E

R A A . N G M E E G L G D S . F E P I . F L P Q M E D P R H P A R R E D Q V P S . S S . R K E E


571 643

576 654

574 644

562 627

557 623

562 583

551 599

554 602

549 573

557 592

T E L S A A F E S E V - T - R P E V P S Q V H F - - Q P Q T - R M E V R D E G V S D G T A A R Y N G V - - - - - - - Q Q Q Q Q Y T L H R H R V Q G G I A P S T S E A S S S- - L S A D F D N S M - D S R P E V P S Q V L F - - Q P R M E R E E T V D G G G G G G M V G R S N G G G H Q G Q H - Q Q Q Q H N F Q T T I H R A R G V A P S T S E A S S N- - I S A D F M N S V - D S R A E V P T R V N F E - - P A T E R E E N F R T T A G N - - V A R T N G I H E H N N F - Q Q P Q H R M R N - - - - - - V L A P S V S E A S S GE E S S L N L R N S V G D S R A E V P R N M V N T N Q A Q Q R R A E P A S N - Q V T A M - - - I - P E F N I R I V A E S T E D - - - - - - - - - - - - - - S T A E S S S SE N S - V D I R N S V V E S R A E V P T D I N V A E - V H R R - D T R D Q A V M Q - T A - R T N A T P H F N F - A V G R R N S E D - - - - - - - - - - - - S T A E S S S SE T S - V D I R N S V V D S R A E V P I I S MT N K Q T I C V D V I K A S K M E D T G S I A N S P I S R K R S S S G S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V F N V P S Y S S S V E VS N R S S I C V D M I R S S K M E A T S S V A N S P V S R K R S F C E T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A F H V P S Y S S S A E VE E R - - D M K K S R G F D E R E L F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S T A E S S S SE T K K L N D N K L S Y K N E R I K M Z S N V - D G D L C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S T A E S S S ME S . S . S R E V P . R R E V R N G Q Q Q A P S T S E S S S


644 700

655 710

645 700

628 681

624 679

584 583

600 650

603 651

574 612

593 636

W T G R E - - - G G V V P V W S P P A A S G H S D P Y A A D E T D I S S R - - - - - S Y L D R H P Q Q S H R L M N W N Q L S Q S SW T G R E - - - G G V V P V W S P P A A S G P S D H Y A A D E A D I T S R - - - - - S Y L D R H P - Q S H T L M N W S Q L S Q S LW T G R E - - - G G V V P V W S P P A A S D H S E T F A S D E A D I S S R - - - - - S Y L D R H P - Q S H R L M N W S Q L S Q S LG R R E R S - - G G I V P E W S P - - G Y - - S E Q F P S E E N R I G G G S T T S - S Y L Q N H - - H E - - I L N W R R L S Q T GT - R E R D - - G G V V P T W S P S S S N Y - S D Q Y V G D D N - G I G - T S - S - S Y L Q R H P Q S H Q - L M N W Q R L S Q T G

H S Q N Q H P G G Y L V G S S K Y I Q Q K A - E R N C V G K A E D D D S A D S G T N T S R A S K R P A AH S H I Q D H G G S L V G P S R Y L L Q E A - G R N Y V D E V E D S S T A D S G S S I S - R Q R K - A AS S V F F V S Q S C S L A S E - - G K N L E G I Q D S S D Q I T T S - - L G K N G C K- - K K Q M T V S R N S F S V P R T I T M S H D R K - - - - - S F D D E A S S H V K L Q K E E E I D T

. G G . V P W S P H S . . . D S S Y L H P H R L M N W Q L S Q S

C N

*******



2

Figure 2. Primary structure comparison of wheat, rice, Arabidopsis and tomato CRYs.

Boxed are the conserved TGYP, DQXVP and STAESS domains. The predicted NLS of

TaCRY2 is indicated with an asterisk at the top. The position corresponding to D493 in

TaCRY1a was set as the demarcation between N and C-termini. Vertical solid triangles

indicate the starting or ending points of the TaCRY1a segments used to produce GFP

fusion proteins. Vertical empty triangles indicate amino acid substitutions distinguishing

monocots from dicots or individual CRY subfamilies from each other. Solid dots signify

basic-to-acid or acid-to-basic amino acid substitutions in CRY2 between monocots and

dicots. Oval boxes signify the basic amino acid stretches from AA74 to AA92, AA246-

AA248 and AA550-AA552 in TaCRY1a. The hydrophobic stretches in the N-terminus of

TaCRY1a and the C-terminus of AtCRY1 are underlined.



Figure 3

Figure 3. Subcellular localization of TaCRY1a-GFP and TaCRY2-GFP fusion proteins

in transiently transformed onion epidermal cells and transgenic Arabidopsis root cells.

The top and middle panels are onion epidermal cells and the bottom panel represents

Arabidopsis root tip cells.

GFP control TaCRY1a-GFP TaCRY2-GFP

W

hite

ligh

t

D

ark

B

lue

light

D

ark

48µm

68µm

80µm 40µm

58µm

40µm

112µm

114µm

55µm

95µm

75µm

71µm



Figure 4 Figure 4. Subcellular localization assay of TaCRY1a segment-GFP fusion proteins in

transiently transformed onion epidermal cells. The fused TaCRY1a segments are

indicated on the left. Except the AA1-AA492 segment, which was fused to the N-

terminus of GFP, all others were fused to the C-terminus of GFP.

A

A26

1-49

2

A

A26

1-57

7

A

A49

3-70

0

A

A13

6-70

0

A

A13

6-26

0

A

A1-

260

A

A1-

492

Dark Blue light

64µm

86µm

74µm

78µm

85µm 122µm

112µm

A B C D E F G H

AA

(261

-492

+

(578

-700

)

95µm

123µm

118µm

150µm

55µm

120µm

88µm



Figure 5

Figure 5. Subcellular localization of TaCRY1a segment-GFP fusion proteins in

transgenic Arabidopsis root cells. The fused TaCRY1a segments are indicated on the left.

AA

261-

492

AA

493-

700

AA

136-

260

AA

1-49

2

Dark Blue light

66µm 49µm

52µm 47µm

73µm

80µm

55µm 48µm

A B C D



Figure 6

Figure 6. Semi-quantitative RT-PCR (sqRT-PCR) or real time RT-PCR (qRT-PCR) of

TaCRY1a and TaCRY2. A, Expression levels in different tissues (top) and in leaves given

different types of light (bottom). RT&LF: seedling roots and leaves; SP: spikes; EB:

germinating embryos; DK: dark; BL: blue light; RL: red light; WL: white light. B,

Expression levels in seedling leaves (top), roots (middle) and germinating embryos

(bottom) after 12 h NaCl or PEG treatments. TUB transcripts were amplified for 20-23

cycles, while TaCRY1a and TaCRY2 were amplified for 25-26 cycles. C, Expression

levels in seedling roots throughout the course of a 28 h NaCl treatment with daily 12 h

continuous white light. Each data point indicates the relative expression means from three

replicates and the standard deviation (SD) (vertical bar). See ‘Materials and Methods’ for

details.

A Tissues RT LF SP EB

TaCRY1a TaCRY2

TUB

Light DK BL RL WL

TaCRY1a TaCRY2

TUB

TaCRY2

TUB

TaCRY1a

TaCRY2

TUB

B

TaCRY2

TUB

H2O NaCl PEG

C

Time (ZT)

Rel

ativ

e ex

pres

sion

0 4 8 12 16 20 0 4

16 14 12 10

8 6 4 2

0

TaCRY2, NaCl treated TaCRY2, H2O treated TaCRY1a, NaCl treated TaCRY1a, H2O treated



Figure. 7 Figure 7. sqRT-PCR of TaCRY2 in germinating embryos (top) and seedling roots

(bottom) treated with IAA, GA3 or ABA for 12 h. TUB and TaCRY2 transcripts were

amplified for 21 and 25 PCR cycles, respectively.

H2O GA3 IAA ABA

TaCRY2

TUB

TaCRY2

TUB



Figure 8 Figure 8. Characterization of TaCRY1a-GFP and TaCRY2-GFP transgenic Arabidopsis

lines. A, Relative expression of the transgenes in seedlings of Col-0, two TaCRY1a-GFP

lines (C1-L2-4 and C1-L7-4) and two TaCRY2-GFP lines (C2-L2-2 and C2-L7-3). B,

Phenotypes of seedlings grown at 25ºC ambient temperature under dark and blue light.

Photographs were taken four days after sowing. Bar=30mm.

A

Col-0 C1-L4-2 C1-L7-4 C2-L2-2 C2-L7-3

Rel

ativ

e ex

pres

sion

TaCRY1a TaCRY2

0

20

0

40

0

60

0

80

0

100

0

120

0

140

0

B

Col-0 C1-L4-2 C1-L7-4 C2-L2-2 C2-L7-3

Blu

e lig

ht

D

ark



Figure 9 Figure 9. Seed germination and cotyledon opening of Col-0 and the transgenic

Arabidopsis lines in response to NaCl, ABA or mannitol treatment. A, Germination at

25ºC ambient temperature of seeds sown on filter papers saturated with water, 0.3µM

ABA, 120mM NaCl or 300mM mannitol. The photographs were taken three days after

A

Col-0 C1-L4-2 C1-L7-4 C2-L2-2 C2-L7-3

Water 120mM NaCl 0.3µM ABA 300mM Mannitol

Col

2 3 4 1 1

2 3

4 Col Col

2 3

4 1

1 2

3 4

Col

C

Water treated ABA treated Mannitol treated

Col-0 C1-L4-2 C1-L7-4 C2-L2-2 C2-L7-3

Seed

lings

with

ope

ned

coty

ledo

n (%

)

100

80

60

40

20

0

Ger

min

atio

n (%

)

B 100

80

60

40

20

0 0 1 2 3 0 1 2 3 0 1 2 3 4 0 1 2 3 4 (d)



sowing. B, Mean germination rate curve from day 0 to day 4 after sowing. C, Proportion

of seedlings with healthy, opened cotyledons seven days after sowing.



Figure 10 Figure 10. Relative root growth of Col-0 and the transgenic Arabidopsis lines treated

with 10µM ABA for two days, estimated as the percentage of primary root length on the

ABA plate relative to that on the control plate. Shown is the result from one

representative experiment.

Rel

ativ

e ro

ot g

row

th (

%)

0

20

40

60

Col-0 C1-L4-2 C1-L7-4 C2-L2-2 C2-L7-3



Figure 11 Figure 11. The response of plants of Col-0 and the transgenic Arabidopsis lines to

irrigation of 250mM NaCl into the soil every two days.

1st row

2nd row Day 0 Day 9 Day 11

Order of lines

C1-L4-2 Col-0 C2-L7-3 C2-L2-2 C1-L7-4

C2-L2-2 Col-0 C1-L7-4 C1-L4-2 C2-L7-3



Figure 12

Figure 12. Expression of RD29A and ADH1 in Col-0 and the transgenic Arabidopsis

lines under various stress treatments. Total RNA was extracted from 15-day-old T2

kanamycin-resistant seedlings grown on MS plates (control), or grown on the MS plates

for twelve days and then transferred to MS plates supplemented with 300mM mannitol,

10µM ABA for three days of growth. Error bars indicate SD.

Water ABA Mannitol

Rel

ativ

e ex

pres

sion

RD29A

20

16

12

8

4

0

ADH1

0 2 4 6 8

10 12 14 16

Col-0 C1-L4 C2-L2 Col-0 C1-L4 C2-L2



1 running title: characterization of wheat ......2008/12/03 · 1 running title: characterization...

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