Wheat cryptochromes: subcellular localization and involvement in photomorphogenesis and osmotic stress responses.

Cryptochromes (CRYs) are blue light receptors important for plant growth and development. Comprehensive information on monocot CRYs is currently only available for rice (Oryza sativa). We report here the molecular and functional characterization of two CRY genes, TaCRY1a and TaCRY2, from the monocot wheat (Triticum aestivum). The expression of TaCRY1a was most abundant in seedling leaves and barely detected in roots and germinating embryos under normal growth conditions. The expression of TaCRY2 in germinating embryos was equivalent to that in leaves and much higher than the TaCRY1a counterpart. Transition from dark to light slightly affected the expression of TaCRY1a and TaCRY2 in leaves, and red light produced a stronger induction of TaCRY1a. Treatment of seedlings with high salt, polyethylene glycol, and abscisic acid (ABA) up-regulated TaCRY2 in roots and germinating embryos. TaCRY1a displays a light-responsive nucleocytoplasmic shuttling pattern similar to that of Arabidopsis (Arabidopsis thaliana) CRY1, contains nuclear localization domains in both the N and C termini, and includes information for nuclear export in its N-terminal domain. TaCRY2 was localized to the nucleus in the dark. Expression of TaCRY1a-green fluorescent protein or TaCRY2-green fluorescent protein in Arabidopsis conferred a shorter hypocotyl phenotype under blue light. These transgenic Arabidopsis plants showed higher sensitivity to high-salt, osmotic stress, and ABA treatment during germination and postgermination development, and they displayed altered expression of stress/ABA-responsive genes. The primary root growth in transgenic seedlings was less tolerant of ABA. These observations indicate that TaCRY1 and TaCRY2 might be involved in the ABA signaling pathway in addition to their role in primary blue light signal transduction.

Cryptochromes (CRYs) and photolyases belong to the flavoprotein family, widely distributed in bacteria as well as in eukaryotes (Cashmore et al., 1999;Brudler et al., 2003). The CRYs show sequence similarity to photolyases, which function to repair UV light-damaged DNA, but they do not have DNA repair activity and usually possess a C-terminal extension (Todo, 1999). Plant CRYs are blue, green, and UV-A light photoreceptors responsible for photomorphogenesis (Briggs and Huala, 1999), a phenomenon in which growing plants under light leads to chloroplast differentiation, chlorophyll accumulation, leaf expansion, and inhibi-tion of stem elongation. Moreover, CRYs are involved in circadian, developmental, and adaptive growth regulation of plants Mao et al., 2005;Ahmad et al., 2006;Canamero et al., 2006;Danon et al., 2006;Zhao et al., 2007). Plant CRYs were originally divided into two subfamilies: CRY1 and CRY2. They have conserved N-terminal photolyase-related domains as well as C-terminal DQXVP-acidic-STAES (DAS) domains, and they are distinguished mainly by their C-terminal extensions (Ahmad and Cashmore, 1993;Lin et al., 1996b). Brudler et al. (2003) and Kleine et al. (2003) reported a third CRY subfamily, CRY-DASH, in plants.
In Arabidopsis (Arabidopsis thaliana), each CRY subfamily consists of a single member. AtCRY1 is its primary photoreceptor, mediating blue light regulation of seedling deetiolation and phasing of the circadian clock (Ahmad and Cashmore, 1993;Lin et al., 1996a;Somers et al., 1998). AtCRY2 functions redundantly with AtCRY1 under relatively low light and is a key component in the control of photoperiodic flowering Lin et al., 1998). Tomato (Solanum lycopersicum, formerly Lycopersicon esculentum) possesses two CRY1 subfamily members, LeCRY1a and LeCRY1b (Perrotta et al., 2000(Perrotta et al., , 2001, and garden pea (Pisum sativum) contains two CRY2 subfamily members, PsCRY2a and PsCRY2b (Platten et al., 2005). The monocot rice (Oryza sativa) has four CRY genes: OsCRY1a, OsCRY1b, OsCRY2, and OsCRY-DASH (Hirose et al., 2006;Zhang et al., 2006). The N-and C-terminal domains of OsCRY1a and OsCRY1b are 7% and 19% different, respectively. Hirose et al. (2006) showed that overexpression of OsCRY1 resulted in enhanced responsiveness to blue light, suggesting that OsCRY1 is similar to AtCRY1 in regulating photomorphogenesis. Like AtCRY2, OsCRY2 is involved in the promotion of flowering time in rice (Hirose et al., 2006). Barley (Hordeum vulgare) might have the same CRY gene composition as rice (Perrotta et al., 2001).
Expression of CRY genes and turnover of CRY proteins are regulated by inner circadian rhythms, light quality, and daylength. Transcript levels of CRY genes show a nearly 24-h oscillation period (Toth et al., 2001;Platten et al., 2005). CRY genes in different plants respond differentially to light induction. In garden pea, blue light is an inhibitor of CRY gene expression (Platten et al., 2005), while it enhances the expression of CRY1 in Brassica napus (Chatterjee et al., 2006). White light has an inhibitory effect on the expression of Orobanche minor CRY1 (Okazawa et al., 2005). AtCRY2 degrades under short-day conditions in a blue light-dependent manner , and OsCRY2 degrades under either blue or red light conditions (Hirose et al., 2006). Little is known about the effects of other environmental cues on the expression of CRY genes and CRY protein stability.
C-terminal domains of Arabidopsis and rice CRYs govern their signaling activity. Overexpression of a fusion protein containing GUS and the AtCRY1 C terminus causes a constitutive photomorphogenesis response (Yang et al., 2000;Zhang et al., 2006). AtCRY1 and AtCRY2 both localize to the nucleus (Cashmore et al., 1999;Guo et al., 1999;Kleiner et al., 1999), and the GUS-AtCRY1 C terminus fusion protein displayed a light-dependent nucleocytoplasmic shuttling (Yang et al., 2000). The subcellular localization of AtCRY2 does not change in response to blue light (Yang et al., 2000). In contrast to the Arabidopsis CRY proteins, OsCRY1 has been found in both nucleus and cytosol, irrespective of light conditions (Matsumoto et al., 2003). In Adiantum capillus-veneris, two of the five CRY family members, AcCRY3 and AcCRY4, localize to the nucleus, and, in the case of AcCRY3, this pattern is regulated by light (Imaizumi et al., 2000). However, sequences associated with the subcellular localization of plant CRY proteins have not been well characterized.
All of the main staple crops, including rice, wheat (Triticum aestivum), and maize (Zea mays), are cereal monocots. Among them, the CRYs of rice are the only group that has been characterized with respect to their sequences and functions in photomorphogenesis (Hirose et al., 2006;Zhang et al., 2006). Besides being critical for plant growth and development, light signals may also be involved in plant responses to various abiotic stresses. For instance, the light quality-dependent CBF gene expression was associated with freezing tolerance in Arabidopsis (Franklin and Whitelam, 2007). Currently, blue light receptors of model plants have been well characterized regarding their involvement in photomorphogenesis, but little is known about their roles in stress responses.
In this study, two CRY genes, TaCRY1a and TaCRY2, from hexaploid wheat were characterized. We found that TaCRY1a exhibits a subcellular localization mechanism akin to that of AtCRY1 and that both TaCRY1a and TaCRY2 were involved in osmotic stress/abscisic acid (ABA) responses in addition to their roles in the primary light signal transduction pathway.

Gene Organizations of TaCRY1a and TaCRY2 and Their Primary Protein Structures
By mining wheat dbESTs, we obtained TaCRY1a and TaCRY2 contigs with the full open reading frames. We then isolated the full-length cDNA sequences (EF601539 and EF601541) through reverse transcription (RT)-PCR and the genomic DNA (EF601540 and EF601542) encompassing the coding sequences. The coding region of TaCRY1a has three introns, and that of TaCRY2 has four (Fig. 1). In addition, TaCRY1a contains an intron in its 3# untranslated region (UTR). Based on 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 UTRs, 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, and AB103094 (OsCRY1a, OsCRY1b, and OsCRY2 cDNA); AF545572 and AY835380 (SbCRY2 cDNA and genomic sequence); AY508972 and AY508974 (PsCRY2a and PsCRY2b cDNA); and AY508973 and AY508975 (PsCRY2a and PsCRY2b genomic sequences). CRY genomic sequences of Arabidopsis and rice were extracted from the published genome sequences. the structural correspondence between TaCRY1a and TaCRY2, the last two exons of TaCRY2 might have evolved from a 1,047-bp intron insertion within the fourth exon of TaCRY1a. Considerable variation in length and composition exists in the corresponding introns of these two genes. The CRY gene structure was well conserved among monocots and dicots, although the size of particular introns in monocots appeared larger than those of their dicot counterparts (Fig. 1). The CRY1 genes of Arabidopsis, rice, and wheat, and SbCRY2 of sorghum (Sorghum bicolor; Xie et al., 2005), all have a characteristic intron in the 3# UTR.
TaCRY1a cDNA encodes a 700-amino acid polypeptide with 82% and 78% identity to OsCRY1a (BAB70686) and OsCRY1b (BAB70688), respectively, and 46% identity to OsCRY2 (BAC56984). TaCRY2 cDNA encodes a 650-amino acid polypeptide with 78% identity to OsCRY2 and less than 46% identity to OsCRY1a and OsCRY1b. TaCRY1a and TaCRY2, each with an N-terminal photolyase-related domain and a C-terminal DAS domain typical of CRYs, have 60% overall similarity and 30% similarity in their C-terminal regions (Fig. 4). Both polypeptides are highly hydrophilic, although the regions of TaCRY2 from amino acid 65 to 100 and from amino acid 141 to 176 and that of TaCRY1a from amino acid 155 to 190 are hydrophobic. TaCRY2 possesses a putative monopartite nuclear localization signal (NLS) peptide, PISRKRS (AA576-AA582). However, we did not find canonical NLS signals in TaCRY1a or nuclear export signals (NES) in either TaCRY1a or TaCRY2.
Alignment of CRY polypeptide sequences from plants, including Arabidopsis, rice, tomato, and wheat, showed that besides the C termini, the N-terminal regions of CRYs corresponding to the hydrophobic domains mentioned above are also highly variable (Fig. 2). The CRY2 proteins showed greater variation than the CRY1 proteins among species and in dicots compared with monocots. Within individual CRY subfamilies, the monocot proteins are generally larger than their dicot counterparts. This length difference mainly occurs in the C termini and at the very ends of the N termini. A few amino acid substitutions are present within the most conserved N-terminal domains that can distinguish monocots from dicots or individual CRY subfamilies from each other (Fig. 2). The C-terminal STAESS domain previously characterized in dicots is less conserved in monocots, but the amino acid sequences following this domain are better conserved. The plant CRYs compared have perfectly conserved DQXVP domains in their C termini, except for TaCRY2, as determined in this study.

Subcellular Localization of TaCRY1a-GFP and TaCRY2-GFP
To determine the subcellular localization patterns of TaCRY1a and TaCRY2, GFP fluorescence signals were examined in onion (Allium cepa) epidermal cells transiently transformed with TaCRY1a-GFP and TaCRY2-GFP and in root cells of transgenic Arabidopsis plants. Similar fluorescence distributions in cells were obtained for the respective constructs in both types of transformations (Fig. 3). In the dark, for the TaCRY2-GFP construct, intense fluorescence signals appeared in nuclei, whereas only background signals were detected in the other cellular compartments. TaCRY1a-GFP showed a similar dark-associated nuclear accumulation in the transiently transformed onion epidermal cells and transgenic Arabidopsis cells. Accumulation of this fusion protein also appeared along the inner cell wall, suggesting that part of the translational product is associated with the plasma membrane. This is consistent with the observation of Ahmad et al. (1998) regarding AtCRY1. Once transferred from dark to blue light, TaCRY2-GFP fusion proteins disappeared, and only background signals lingered. TaCRY1a-GFP fusion proteins, in contrast, emptied into the cytoplasm. The GFP-only control transformants showed a demonstrably light-insensitive fluorescence distribution. These results suggest that TaCRY1a is a light-dependent nucleocytoplasmic shuttling protein and that TaCRY2 is a nuclear protein that degrades when exposed to light.

TaCRY1a Domains Conditioning Nuclear Import and Export
To determine which TaCRY1a domains are responsible for its nuclear localization and export, a series of fusion constructs was made by ligating segments of TaCRY1a coding DNA sequence to GFP. As shown in Figures 4A and 5A, the fusion protein made with the N-terminal segment of AA1-AA492 (TaCNT1a-GFP) displayed a light-dependent nucleocytoplasmic shuttling pattern similar to the whole TaCRY1a sequence. However, the fusion protein made with the C-terminal segment of AA493-AA700 (GFP-TaCCT1a) exhibited light-independent nuclear accumulation (Figs. 4E and 5C). Therefore, sequence information necessary for nuclear targeting exists in both AA1-AA492 and AA493-AA700, but that required for nuclear export only exists in the N-terminal segment.
We noted that deletion of the first 260 amino acids of the 492-amino acid N terminus completely abolished the nuclear targeting function (Figs. 4G and 5D), suggesting that the AA1-AA260 region is responsible for nuclear targeting. Consistent with this, the GFP fusion with the AA1-AA260 segment accumulated in the nucleus in the dark (Fig. 4B). Similar to the GFP fusions with full-length TaCRY1a and with the 492amino acid N terminus, this fusion product translocated into the cytoplasm upon exposure to blue light. Thus, the AA1-AA260 segment carries sufficient information for both nuclear import and export. The GFP fusion with the AA136-AA260 segment is smaller than 45 kD, which allows free diffusion between cellular compartments (Dingwall and Laskey, 1986). We noted that this fusion product distributed through- . This result indicates that the nuclear export signal lies in AA136-AA260. Fusion constructs that accumulated in the nucleus in the dark but did not contain the AA136-AA260 segment were all retained in the nucleus under light conditions.
To identify the C-terminal region responsible for nuclear targeting, transient GFP fusion protein assays were conducted in onion cells for the regions of AA261-AA577, AA261-AA492, and AA578-AA700. Only the first fusion protein showed nuclear accumulation (Fig. 4, F and H). Since the AA261-AA492 segment does not carry nuclear import sequence in- 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 asterisks at the top. The position corresponding to D493 in TaCRY1a was set as the demarcation between N and C termini. Black triangles indicate the starting and ending points of the TaCRY1a segments used to produce GFP fusion proteins. White triangles indicate amino acid substitutions distinguishing monocots from dicots or individual CRY subfamilies from each other. Black 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 amino acid 74 to 92, amino acid 246 to 248, and amino acid 550 to 552 in TaCRY1a. The hydrophobic stretches in the N terminus of TaCRY1a and the C terminus of AtCRY1 are underlined. formation, the nuclear targeting signal in the C terminus was thus restricted to the AA493-AA577 region.
Cellular distribution of the GFP fusion with the AA1-AA135 segment did not respond to light changes (data not shown). Because of its small size (,45 kD), we could not exclude the possibility of free diffusion and thus could not tell whether or not it carries sequence signals for nuclear targeting. However, this segment seemed essential for nuclear export of the whole protein, since when the C-terminal domain with the putative nuclear targeting region was present, fusion products without the AA1-AA135 segment showed constitutive nuclear accumulation, even in the presence of the AA136-AA260 segment (Fig. 4D).

TaCRY1a and TaCRY2 Showed Different Expression Patterns across Tissue Types and in Response to Osmotic Stress
We performed RT-PCR assays of TaCRY1a and TaCRY2 expression in seedling leaves and roots, spikes, and germinating embryos (Fig. 6A). TaCRY1a transcripts were detected mainly in leaves and at low levels in roots and germinating embryos. Compared with TaCRY1a, TaCRY2 was expressed less efficiently in leaves and spikes but more strongly in roots and germinating embryos, especially in the latter tissue.
To investigate the effects of light quality on the expression of TaCRY1a and TaCRY2, we placed etio- . 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 for the AA1-AA492 segment, which was fused to the N terminus of GFP, all others were fused to the C terminus of GFP.
lated seedlings under continuous white, red, or blue light for 48 h after transferring them from the dark. The transcripts of both genes increased slightly after the transfer, although TaCRY1a was expressed more efficiently under red light (Fig. 6A).
To investigate the effects of osmotic stress on the expression of TaCRY1a and TaCRY2, RT-PCR was performed for seedling tissues treated with 20% polyethylene glycol (PEG)-6000 or 250 mM NaCl. As compared with the mock treatment, the expression level of TaCRY1a in germinating embryos as well as in seedling leaves and roots was not affected by either treatment after 12 h ( Fig. 6B; data not shown). However, these treatments repressed TaCRY2 expression in leaves to a certain extent and strongly up-regulated TaCRY2 in seedling roots and germinating embryos. As shown by real-time PCR for a span of 28 h of NaCl treatment, TaCRY2 transcripts increased steadily in roots beginning 12 h after treatment (Fig. 6C). This experiment also indicated that TaCRY1a is salt inducible when the treatments last for over 24 h. These expression pattern changes were far more significant than the fluctuations observed in the water mock treatment and thus did not correspond to the circadian rhythm.
Since TaCRY2 was induced by osmotic stress in seedling roots and germinating embryos, we examined the effects of applying the phytohormones GA 3 , indole-3-acetic acid (IAA), and ABA on its expression. Of these, only ABA, the stress hormone, increased the transcription level of TaCRY2 (Fig. 7). In the same tissues, the expression state of TaCRY1a was not altered (data not shown).
Overexpression of TaCRY1a and TaCRY2 Caused Susceptibility to ABA and Osmotic Stress To understand the response of transgenic Arabidopsis lines overexpressing TaCRY1a and TaCRY2 to osmotic stress/ABA, we investigated two homozygous TaCRY1a-GFP transformants (C1-L4-2 and C1-L7-4) and two homozygous TaCRY2-GFP transformants (C2-L2-2 and C2-L7-3), all derived from independent transformation events. The TaCRY1a transformants carry a single-copy transgene, and the TaCRY2 transformants carry multiple copies (data not shown). These lines had a considerable level of expression for  transgenes (Fig. 8A) and endogenous CRY genes (data not shown), and they showed no obvious morphological differences from the wild type when grown for 4 d in the dark, but they had comparatively shorter hypocotyls with deeper color when grown for 4 d in blue light (Fig. 8B). This is typical of lines with overexpressed CRY genes (Lin et al., 1996aMatsumoto et al., 2003). The rate of hypocotyl elongation inhibition ranged from 24% (C1-L4-2) to 11% (C2-L7-3), relative to the wild type.
To determine the effects of ABA and osmotic stress treatments on seed germination of these transgenic lines, the germination assay was conducted on filter papers soaked with 120 mM NaCl, 300 mM mannitol, or 0.3 mM ABA. As compared with the water control, all three treatments slowed the pace of germination and reduced the germinating rate (Fig. 9A). The transgenic lines were consistently more severely affected than the wild type, despite some between-line variations (Fig.  9B). The TaCRY1a transgenic lines seemed to be more sensitive to ABA and salinity stress than the TaCRY2 transgenic lines. At day 7 of ABA treatment, all transgenic lines displayed significantly reduced proportions of seedlings with fully opened cotyledons as compared with wild-type lines (Fig. 9C), indicating an arrest in postgermination development in the transgenic lines. The mannitol treatment induced similar effects on cotyledon opening. Cotyledon opening was not investigated in the NaCl treatment because all seedlings, whether wild type or transgenic, had died by day 7.
The primary root growth of these transgenic line seedlings showed an elevated sensitivity to ABA. When seedlings were grown on agar plates supplemented with 10 mM ABA for 2 d, inhibition of root growth was noted for all lines, including the wild-type control; however, the primary roots of transgenic lines had an even smaller relative growth rate (Fig. 10). When pots with transgenic Arabidopsis plants were supplied with 250 mM NaCl, plant wilting was noted at day 9. At day 11, the transgenic lines started dying, while the wildtype seedlings were still vigorous (Fig. 11).

Expressions of Two Stress/ABA-Responsive Genes in Transgenic Seedlings
We investigated the transcript levels of two ABA/ stress-responsive genes, RD29A and ADH1, in T2 seedlings of the above-mentioned transgenic lines C1-L4 and C2-L2 after exposure to 300 mM mannitol and ABA treatments (Fig. 12). ADH1 encodes an alcohol dehydrogenase and is inducible by dehydration, ABA, and low temperature (de Bruxelles et al., 1996). RD29A encodes a LEA-like protective protein and is inducible by dehydration, salt, ABA, and low temperature (Yamaguchi-Shinozaki and Shinozaki, 1994). In the mock treatment, both genes were consistently expressed at slightly lower levels in the TaCRY1a-GFP transgenic line as compared with the wild type, whereas the expression pattern was unaltered in the TaCRY2-GFP transgenic line. As expected, exogenous application of ABA and mannitol enhanced the expression of these genes in the wild type. ABA induction of RD29A in the transgenic line C1-L4 was not different from that seen in the wild type, but its induction in the transgenic line C2-L2 was only half  that of the wild type. Relative to the wild type, RD29A was less effectively induced by the mannitol treatment in both transgenic lines, especially in C1-L4, in which it was almost unresponsive to the osmotic stress. Interestingly, the ABA induction level of ADH1 dropped off by approximately 40% and approximately 20% in C1-L4 and C2-L2, respectively, but after the mannitol treatment the induction level was identical to that of the wild type in the former line and increased by approximately 2-fold in the latter (Fig. 12). Conley et al. (1999) reported that less efficient expression of the ADH gene led to a lower seed germination rate in Arabidopsis. The change of ADH1 gene expression level in the two transgenic lines was consistent with the germination rate variation (Fig. 9B).

Structural Conservation and Variation of Monocot and Dicot CRY Proteins
Plant CRY genes fall into three subfamilies: CRY1, CRY2, and CRY-DASH (Perrotta et al., 2000(Perrotta et al., , 2001Kleine et al., 2003;Platten et al., 2005), although individual species may have different CRY family members. The dicot CRY genes have been well characterized, but the monocot CRY genes are still poorly understood. In this work, we show that wheat CRY1a and CRY2 genes share conserved sequences with their rice counterparts and that the intron/exon organization of these two genes is well conserved among plants. Some conserved domains/motifs previously identified in dicot CRYs, such as TGYP and DQXVP, are present in wheat and other monocot CRYs, implying their common origin and functional conservation. Transgenic analysis suggested that TaCRY1a and TaCRY2 are similar to Arabidopsis and rice CRY genes in modulating photomorphogenesis. However, differentiation of CRY proteins between monocots and dicots is noticeable. The C terminal STAESS motif of dicot CRY proteins that is important for CRY phosphorylation is less conserved in monocot CRY proteins, even though they retain an S and E residue-rich feature. For CRY2 proteins, there are a few residue substitutions between monocots and dicots involving acidic-to-basic or basicto-acidic alterations (Fig. 2). These structural changes might lead to evolution of gene function.

Sequence Characteristics of the TaCRY1a Domains Required for Nuclear Import and Export
Nuclear import of proteins usually involves NLS recognition by importins and translocation through 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 25°C ambient temperature of seeds sown on filter papers saturated with water, 0.3 mM ABA, 120 mM NaCl, or 300 mM mannitol. The photographs were taken 3 d after sowing. B, Mean germination rate curve from day 0 to day 4 after sowing. C, Proportion of seedlings with healthy, opened cotyledons at 7 d after sowing.
the nuclear pore complex (Jiang et al., 1998). We have shown that TaCRY2 is a nucleus-localized protein and has a C-terminal monopartite NLS. TaCRY1a is a lightdependent nucleocytoplasmic shuttling protein that does not have known NLS and NES signals. However, its N-terminal AA1-AA260 segment and C-terminal AA493-AA577 segment are sufficient for nuclear targeting. This is similar to OsCRY1, as both its 213-amino acid N terminus and 235-amino acid C terminus can modulate its nuclear accumulation (Matsumoto et al., 2003). The N-terminal domains of TaCRY1a and the bona fide OsCRY1a are highly conserved (Fig. 2). More and more sequences bearing no classic NLS have been associated with nuclear import of proteins, for example, the M9 domain of the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein (Siomi and Dreyfuss, 1995) and the KNS nuclear shuttling domain of the hnRNP K protein (Michael et al., 1997). These findings indicate that multiple strategies exist for mediating protein nuclear targeting.
At the sequence level, the NLS is usually either a short stretch of several basic amino acids (monopartite NLS; Kalderon et al., 1984) or it consists of two separated clusters of basic residues (bipartite NLS; Robbins et al., 1991). Within the two segments mentioned above, there is a 19-amino acid peptide, KHLDASLRRLGATRLVTRR, with seven basic amino acid residues (underlined) from amino acid 74 to 92, a basic RKK stretch from amino acid 246 to 248, and a basic RRR stretch from amino acid 550 to 552 (Fig. 2). These sequence characteristics, especially those in the N-terminal domain, are conserved across plant CRY1 proteins. Nevertheless, further work is required to clarify their roles in mediating nuclear targeting.
The NES of TaCRY1a is present in the AA136-AA260 segment of the N-terminal region. This is unlike OsCRY1, which does not have a nuclear export function (Matsumoto et al., 2003), and AcCRY3 and AtCRY1, which carry nuclear export signals in their C-terminal domains (Imaizumi et al., 2000;Yang et al., 2000). Compared with NLS, NES signals are even less well defined. The best characterized NES are the Leu-rich domains in PKI and Rev (Wen et al., 1995). Significantly, we noted three Leu dimers within every 32-to 35-amino acid span of the AA136-AA260 corresponding domains of all compared CRY proteins (Fig. 2). However, this conserved feature is not the same as that reported by Wen et al. (1995) and might not be essential for nuclear export, since AcCRY4 (BAA88423) cannot direct export to the cytoplasm despite having these conserved Leu residues (Imaizumi et al., 2000). A few reports documented non-Leu-rich NES domains, for example, the Pro-and Gln-rich NES in human pUL69 protein (Lischka et al., 2001) and the M9 domain of hnRNP A1 and the KNS domain of hnRNP K (Michael et al., 1995(Michael et al., , 1997. In the human NFAT transcription factor, the NES sequence is IVAAINALTT (Klemm et al., 1997). Wen et al. (1995) demonstrated that hydrophobic residues are critical for a NES. Consistent with this finding, the AA136-AA260 region of TaCRY1a is hydrophobic and has a Pro-rich core motif (PPAPMLPP) between amino acids 178 and 185. The Pro-rich feature has been associated with nuclear export mediation in other studies (Lee et al., 2001;Lischka et al., 2001). In the C-terminal AtCRY1 region that contains a NES, we identified the hydrophobic stretch AMIPEFNIRI (amino acids 603-614). Within AcCRY3 (BAA32809) Figure 10. Relative root growth of Col-0 and the transgenic Arabidopsis lines treated with 10 mM ABA for 2 d, estimated as the percentage of primary root length on the ABA plate relative to that on the control plate. Shown are the results from one representative experiment. and its C terminus, which could modulate a lightdependent intracellular localization pattern (Imaizumi et al., 2000), we found a classical NES-like peptide, LKQSLIQLDISLRSL, at amino acids 55 to 69 and hydrophobic stretches in the C-terminal region. The C termini of AtCRY2, TaCRY1a, and TaCRY2 have no predicted hydrophobic domains. Like the GFP-TaCRY1a C terminus fusion, the AtCRY2 C terminus-GUS fusion constitutively accumulates in the nucleus (Yang et al., 2000). However, even though these short hydrophobic domains are associated with NES, other sequence motifs or structural characteristics may be required for nuclear export. For example, the AcCRY4 C terminus has a hydrophobic domain but it does not mediate nuclear export (Imaizumi et al., 2000). As shown in this study, nuclear export of GFP constructs with C-terminal segments of TaCRY1a was lost in the absence of the first 135 amino acids of the N terminus. AtCRY1 and OsCRY1 constitutively form homodimers through their N termini Zhang et al., 2006). This is possibly also true for TaCRY1a, because its N terminus is highly similar to that of OsCRY1a. Sang et al. (2005) demonstrated that deletion of the AA1-AA99 region of AtCRY1, which corresponds to the first 105 amino acids of the TaCRY1a N terminus, abolished the dimerization. This dimerization might be required for nuclear export of TaCRY1a when its C-terminal region is present. The GFP-TaCRY1a segment constructs without the highly hydrophilic C-terminal region might exist in different conformations and thus might not require the N terminus-mediated dimerization for nuclear export.
Subcellular localization of CRY proteins is regulated in a complex manner. Besides the NLS and NES signals, other factors, such as partner binding, multimerization (Gaits and Russell, 1999;Stommel et al., 1999), phosphorylation (Jensen et al., 1998), and prenylation (Rodriguez-Concepcion et al., 1999), may also affect nuclear import and export. In Arabidopsis, nucleocytoplasmic shuttling of AtCRY1 may be facilitated by C-terminal binding of COP1, a NLS-and NES-containing downstream repressor of photomor-phogenesis (von Arnim and Deng, 1994;Wang et al., 2001).

Tissue-Specific and Light-Inducible Expression of TaCRY1a and TaCRY2
TaCRY1a and TaCRY2 were expressed at relatively high levels in seedling leaves. TaCRY1a transcript levels were negligible in roots and germinating embryos. In comparison with the TaCRY1a transcript, there was more TaCRY2 transcript in these two tissues, especially in germinating embryos. Differences in CRY family member expression profiles may imply functional diversification, which has been suggested in a few previous studies. In Arabidopsis, CRY1 mainly functions in deetiolation (Lin et al., 1996a), while CRY2 plays a role in the regulation of photoperiodic flowering  and the mediation of lightdependent phenylpropanoid metabolism in the roots (Hemm et al., 2004). Canamero et al. (2006) reported that both AtCRY1 and AtCRY2 are involved in the blue light induction of primary root elongation, but in an antagonistic manner. Our findings suggested that TaCRY2 expression might be important in nongreen tissues such as germinating embryos and roots.
The expression of TaCRY2 in seedling leaves seemed to be light independent, but TaCRY1a was red light inducible. Imaizumi et al. (2000) reported red light induction of AcCRY3 expression in A. capillus-veneris. These light-inducible regulatory patterns are contradictory to those of the pea CRY genes PsCRY1 and PsCRY2, as lower levels of those transcripts were detected in seedlings grown with light compared with those kept in complete darkness (Platten et al., 2005). In other reports, Brassica napus CRY1 is blue light inducible (Chatterjee et al., 2006); the expression of Arabidopsis and rice CRY genes was light independent at the transcriptional level, but AtCRY2, OsCRY1a, and OsCRY2 were under light-dependent translational and/or posttranslational control (Ahmad and Cashmore, 1993;Lin et al., 1998;Hirose et al., 2006). Therefore, light regulation of CRY gene expression is not identical in different kinds of plant species, an observation that may be related to their growth habits or growth environments.

Involvement of CRY Genes in Osmotic and Other Stress Responses
Expression of both TaCRY2 and TaCRY1a is induced by osmotic stress, even though their induction patterns are different. We found that overexpressing TaCRY genes in Arabidopsis resulted in decreased seed germination, impaired cotyledon opening, and less tolerance to ABA and high salinity at the vegetative growth stage. These findings suggest that overexpressing CRY genes compromises plant resistance to osmotic stress and ABA. Consistent with this, Mao et al. (2005) reported that, due to increased stomatal opening, Arabidopsis plants overexpressing myc-AtCRY1 and myc-AtCRY2 are more sensitive to drought. In other reports, Phee et al. (2007) identified stress/defenserelated proteins that were differentially expressed between wild-type Arabidopsis and the hy4 (cry1) mutant, among which the TIR-NBS-LRR class proteins were down-regulated and the jacalin lectin family members were up-regulated in the hy4 line. Danon et al. (2006) documented that AtCRY1 is required for singlet oxygen-induced programmed cell death. Thus, plant CRY genes are associated with abiotic/biotic stress signaling in several ways.
Overexpression of TaCRY2 in Arabidopsis did not affect the expression of RD29A and ADH1 under normal conditions. In agreement with this, germination and postgermination development were not altered in the corresponding transgenic lines used in this study. There was only a slight reduction in the expression of these genes in the TaCRY1a transgenic lines. In contrast, these marker genes had significantly altered expression patterns in both transgenic lines under the applied osmotic stress and ABA treatments. Overexpressing TaCRY1a severely compromised the osmotic stress induction of RD29A, but it did not influence the ABA induction pattern. Overexpressing TaCRY2 attenuated the induction of RD29A by both osmotic stress and ABA. Phenotypically, these transgenic lines were more sensitive to treatments of stress factors, such as supplying high levels of mannitol and NaCl. Up-regulation of RD29A has been positively associated with osmotic stress tolerance in Arabidopsis (Umezawa et al., 2004). Our results clearly suggest that overexpressing CRY genes weakens the regulatory roles of stress-responsive genes in stress tolerance and that the ways in which TaCRY1a and TaCRY2 interact with stress/ABA signals are complicated.
The ABA-and stress-inducible gene ADH1 showed different expression patterns in the transgenic lines when treated with 300 mM mannitol and with 0.3 mM ABA. This was unexpected, as osmotic stress was presumed to increase the ABA levels in cells (Zeevaart and Creelman, 1998) and thus should have affected ADH1 in a way similar to the ABA treatment. However, as de Bruxelles et al. (1996) have demonstrated, ADH1 could respond to osmotic stress by an ABAindependent pathway. The unaltered or increased expression of ADH1 in the transgenic lines after the mannitol treatment suggests that overexpressing CRY genes does not affect this ABA-independent osmotic stress-response pathway. The more severely repressed RD29A induction and the less induced ADH1 expression observed under osmotic stress in the TaCRY1a transgenic line as compared with the TaCRY2 transgenic line may explain the higher sensitivity of TaCRY1a transgenic lines to osmotic stress.

CONCLUSION
Monocots, in particular the grass crops, provide the staple foods for human beings. Because of the close relationship between light and the growth and development of plants, studies on light receptors have great agronomical importance. We showed that TaCRY1a and TaCRY2 of wheat share conserved structures and functions with other CRY genes. TaCRY1a and TaCRY2 are differentially expressed in tissues/organs, making further investigation of their functional diversification more intriguing. Like AtCRY1, TaCRY1a is a lightdependent nucleocytoplasmic shuttling protein. It carries at least two atypical NLS, one at the N terminus and one at the C terminus, and a NES within the AA136-AA260 region. This arrangement is different from that of AtCRY1. We demonstrated that TaCRY1a and TaCRY2 are also involved in abiotic stress responses, some of which are related to ABA signaling. However, their involvement may employ different mechanisms. Thus, questions still remain regarding the cross talk between light signaling and stress adaptation. A systematic expression profiling of the genes involved in ABA, osmotic stress, and CRY signal transduction pathways is necessary to elucidate the interaction network between CRY and osmosis/ABA signals.
Adult wheat plants were grown in a field in Nanjing, China, in the normal growing season. Spikes 2 d after anthesis were harvested for RNA extraction.

Stress and Hormone Treatments
After germination in petri dishes, seedlings were grown at room temperature with 16 h of light daily. Six-day-old seedlings were transferred to petri dishes with 250 mM NaCl (Nanjing Chemical Reagent) or 20% PEG-6000 (Amersco) for osmotic stress treatments. For hormone treatments, the procedures were the same as for the stress treatments except that the chemicals were replaced with 10 mm ABA, GA 3 , or IAA (Sigma). Tissues were harvested 12 h after the transfer.
In the time course treatment with 250 mM NaCl, plants were given 12 h light per day. The root tissues were harvested at 4,8,12,16,20,24, and 28 h after the transfer. To prepare germinating embryos, seeds imbibed for 15 h were placed at room temperature in petri dishes fitted with filter papers that were soaked in 250 mM NaCl, 20% PEG-6000, or 10 mM ABA, GA 3 , or IAA for 12 h. The embryos were then separated from endosperms by hand. Distilled water treatments were used as the mock controls.
For light quality treatment, the etiolated seedlings grown in the dark for 6 d were divided into four groups. One group was kept in the dark while the remaining groups were placed under continuous white, red, or blue light for 48 h. The white light was generated from a white fluorescent tube. The blue or red light (2.7 or 0.7 W s 21 m 22 , respectively) was generated by filtering the fluorescent tube with a blue or red filter (model M34; Sea-Gull, Ltd.).

DNA and RNA Isolation
Genomic DNA was extracted according to the procedures described by Ma and Sorrells (1995). RNA was extracted using the Trizol reagent (Invitrogen) following the manufacturer's protocol and quantified with Ultrospec 2100 pro (Amersham Pharmacia). The RNA was checked for DNA contamination by direct PCR amplification of the wheat b-tubulin gene TaTUB using the extract as the template. The primers used were 5#-CGTGGTGATGTTGTGCCA-AAG-3# and 5#-ACTTCTTCATAGTCCTTCTCCAGG-3#.
The first-strand cDNA was synthesized using the Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. The PCR was performed in a 25-mL mixture containing approximately 5 ng of template, 5 pmol of each primer, 5 nmol of each of the deoxynucleoside triphosphates, 37.3 nmol of MgCl 2 , 0.5 units of rTaq DNA polymerase (Takara), and 13 PCR buffer supplied with the enzyme. The thermal cycle profile was 94°C for 3 min, 36 cycles of 94°C for 30 s, 58°C for 40 s, and 72°C for 2.5 min, and a final extension of 5 min at 72°C.
The same sets of primers were used to amplify genomic DNA. Included in the 25-mL PCR were 40 ng of genomic DNA, 0.2 M D-(+)-trehalose (Sigma) as the PCR enhancer, 0.5 units of Ex-Taq (Takara), and other PCR components in the same concentrations as described above. The thermal cycling conditions were 94°C for 4 min, 36 cycles of 94°C for 40 s, 58°C for 45 s, and 72°C for 5 min, and a final extension of 5 min at 72°C. The PCR products were separated with 1% agarose gels.
DNA from the target bands excised from the gels was purified and ligated into the pUC19-based TA cloning vector pX-T. Positive clones from transformation of competent JM109 cells were sequenced by Shanghai Invitrogen Biotechnology.
The exon/intron organization of CRY1a and CRY2 was determined by comparing the corresponding cDNA and genomic DNA sequences. Scanning for NLS was carried out by WoLF PSORT (http://psort.nibb.ac.jp/), and prediction of NES was carried out with the prediction server in the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/NetNES). Other bioinformatic analyses of the sequences were conducted with Macvector 9.0 software (Accelrys).

Constructs
Inserts were amplified using pfu DNA polymerase (Dingguo Biotech) with primers containing an XbaI or XhoI restriction site. The restricted amplicons were inserted into the CaMV35S-EGFP-NOS sequence of a pUC19-based expression vector to generate cauliflower mosaic virus 35S promoter-driven GFP fusion constructs. GFP is a 27-kD spontaneously fluorescent protein, allowing direct visualization of the fusion proteins (Scott et al., 1999). After ligation and transformation into JM109 competent cells, these constructs were confirmed by sequencing.

Transient Expression in Onion Epidermal Cells
Using the PDS-1000 particle delivery system (Bio-Rad), the fusion constructs were introduced through 1.1-mm tungsten particles into the epidermal cells of onion (Allium cepa) bulb scales placed on Murashige and Skoog (MS) agar plates in the procedure recommended by the manufacturer. The bombardment parameters were 1,100 pounds per square inch of pressure, 85 mm from the macrocarrier to samples, and 28 inches Hg vacuum. Bombarded onion epidermis was incubated at 25°C in the dark for 16 h, or in the dark for 4 h followed by 12 h of blue light (2.7 W s 21 m 22 ).

Microscopy
GFP fluorescent signals were examined with a confocal laser-scanning microscope (TCS NT; Leica) in the 488-nm excitation wavelength. Cellular structure was visualized using bright-field optics.

Arabidopsis Transformation and GFP Signal Assay of Root Cells
Inserts were amplified using pfu DNA polymerase (Dingguo Biotech) with primers containing the XbaI/EcoRI or XbaI/BglII restriction sites. After restriction digestion, the amplicons were inserted into a modified GFP-containing pBI121 expression vector to generate cauliflower mosaic virus 35S promoter-driven GFP fusion constructs. Arabidopsis ecotype Col-0 was transformed with these constructs using the floral dip method (Martinez-Trujillo et al., 2004) with Agrobacterium tumefaciens strain LBA4404.
Harvested seeds were screened on MS plates (pH 5.8) containing 50 mg L 21 kanamycin. Putative transgenic plants were verified by PCR and Southernblot hybridization. T2 transgenic seeds were germinated and grown on sterile MS plates in darkness for 3 d or in darkness for 2 d followed by irradiation with white light 1 d before observation. Roots were put under glass coverslips in phosphate-buffered saline (137 mM NaCl, 1.4 mM KH 2 PO 4 , 4.3 mM Na 2 HPO 4 , and 2.7 mM KCl, pH 7.4) and then observed in the 488-nm excitation wavelength with the confocal laser-scanning microscope. Threedimensional rendering was created from 20 optical sections acquired through continuous scanning.

RT-PCR
Semiquantitative RT-PCR was carried out in 12.5-mL reactions with approximately 2.5 ng of template. The PCR profile was 94°C for 3 min, 20 to 30 cycles of 94°C for 20 s, 58°C for 30 s, and 72°C for 30 s, and a 5-min extension at 72°C. Primer sequences used were 5#-CTCAGACGAGCCACCCATTG-3# and 5#-CCCCACCTTCTCTCCCAGTC-3# for TaCRY1a and 5#-GAACTGAAG-GGCACAAATAAACAGACC-3# and 5#-ATGAGAGTGGGGTGCAGGAA-GATCC-3# for TaCRY2, all designed based on 3# end sequences. TaTUB cDNA amplification was used as the external control. Five microliters from the PCR product of each reaction was electrophoresed on a 1.5% agarose gel and viewed under UV light after standard staining with ethidium bromide.

Hypocotyl Measurements
Seeds were sown on MS agar plates with 5% Suc (pH 5.8). The plates were placed in a 4°C refrigerator for 2 d and were then placed at room temperature for 4 d under either dark or blue light (2.7 W s 21 m 22 ). Hypocotyl length was measured for 15 seedlings of each sample. The experiments were repeated three times.

Germination Assay
Seeds were sown on filter papers in petri dishes soaked with distilled water, 120 mM NaCl, 300 mM mannitol (osmotic stress), or 0.3 mM ABA. One hundred fifty seeds on three plates were sown for each line. After stratification at 4°C for 2 d, the plates were moved to room temperature with 12 h of light per day. Germination (emergence of radicals) was scored daily until day 5 after sowing.

Root Growth Inhibition Assay
Seeds were sown on vertically placed MS agar plates for 4 d. Fifteen phenotypically uniform seedlings were retained, and half of these were carefully transferred onto a MS agar plate supplemented with 10 mM ABA. Root length was measured 2 d after the transfer. The experiment was repeated three times.

Salt Tolerance Assay
Seeds were sown on MS plates. After 6 d of growth, they were transferred into pots filled with a mixture of soil and vermiculite and given 16 h of light per day and 25°C ambient temperature. Twenty days after the transfer, the plants were irrigated with saline water containing 250 mM NaCl every 2 d.
The sequences described in this article have been submitted to the GenBank data library under accession numbers EF601539 to EF601542.