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First published online December 3, 2008; 10.1104/pp.108.132217 Plant Physiology 149:760-774 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Wheat Cryptochromes: Subcellular Localization and Involvement in Photomorphogenesis and Osmotic Stress Responses1,[OA]Applied Plant Genomics Laboratory, Crop Genomics and Bioinformatics Center and National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Jiangsu 210095, China (P.X., Y.X., H.Z., H.X., Z.Z., C.Z., L.Z., Z.M.); and Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China (P.X.)
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 inhibition of stem elongation. Moreover, CRYs are involved in circadian, developmental, and adaptive growth regulation of plants (Guo et al., 1998; 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
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
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
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 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 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
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)
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 492-amino 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 throughout the entire cell, similar to the GFP-only control, in the dark and emptied into the cytoplasm under blue light (Figs. 4C and 5B). 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 information, 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).
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 etiolated 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 GA3, 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., 1996a
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 µM 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 µM 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 wild-type 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
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
Nuclear import of proteins usually involves NLS recognition by importins and translocation through the nuclear pore complex (Jiang et al., 1998
At the sequence level, the NLS is usually either a short stretch of several basic amino acids (monopartite NLS; Kalderon et al., 1984
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
Wen et al. (1995)
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
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
The expression of TaCRY2 in seedling leaves seemed to be light independent, but TaCRY1a was red light inducible. Imaizumi et al. (2000)
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)
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
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 µM ABA. This was unexpected, as osmotic stress was presumed to increase the ABA levels in cells (Zeevaart and Creelman, 1998
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 light-dependent 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.
Plant Materials The plant lines used in this study included common wheat (Triticum aestivum) cultivar ‘Sumai No. 3’ (2n = 6x = 42, genome = AABBDD), nulli-tetrasomic lines of common wheat cultivar ‘Chinese Spring’, and Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0). 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.
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 µm ABA, GA3, 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 µM ABA, GA3, 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–1 m–2, respectively) was generated by filtering the fluorescent tube with a blue or red filter (model M34; Sea-Gull, Ltd.).
Genomic DNA was extracted according to the procedures described by Ma and Sorrells (1995)
The amino acid sequences of barley (Hordeum vulgare) CRY1a (ABB13330) and rice (Oryza sativa) CRY2 (BAC56984) were queried against wheat dbESTs (580,000 ESTs, extracted from GenBank dbESTs of the 154th release, 2006) by tBLASTn. Twenty EST hits for CRY1a and 15 for CRY2 were identified based on more than 90% identity for CRY1a and more than 85% identity for CRY2 in a greater than 33-amino acid overlap. After removing possible vector sequence contamination, contigs were assembled with the parameter settings of greater than 40-bp overlap and greater than 95% identity. One 2,719-bp CRY1a contig and one 2,375-bp CRY2 contig were obtained. The primers used for RT-PCR were as follows: 5'-CCAAAATCAAGAAACCCTGGCAACTCTG-3' (sense primer) and 5'-CGTCACTCTCCAACTCCCTACACAAT-3' (antisense primer) for CRY1; 5'-CTGCTCGACGTAATGCTCGTGAGAG-3' (sense primer) and 5'-ATGAGAGTGGGGTGCAGGAAGATCC-3' (antisense primer) for CRY2, all designed based on the 5' and 3' UTR sequences of the contigs. 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-µL mixture containing approximately 5 ng of template, 5 pmol of each primer, 5 nmol of each of the deoxynucleoside triphosphates, 37.3 nmol of MgCl2, 0.5 units of rTaq DNA polymerase (Takara), and 1x 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-µL 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
Transient Expression in Onion Epidermal Cells
Microscopy
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 Harvested seeds were screened on MS plates (pH 5.8) containing 50 mg L–1 kanamycin. Putative transgenic plants were verified by PCR and Southern-blot 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 KH2PO4, 4.3 mM Na2HPO4, and 2.7 mM KCl, pH 7.4) and then observed in the 488-nm excitation wavelength with the confocal laser-scanning microscope. Three-dimensional rendering was created from 20 optical sections acquired through continuous scanning.
Semiquantitative RT-PCR was carried out in 12.5-µL 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'-GAACTGAAGGGCACAAATAAACAGACC-3' and 5'-ATGAGAGTGGGGTGCAGGAAGATCC-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.
Quantitative RT-PCR amplifications for Arabidopsis genes were performed on a Bio-Rad iCYCLER iQ5 real-time PCR instrument in 25-µL reactions containing approximately 5 ng of template, 12.5 µL of SYBR-Green PCR Mastermix (Toyoba), and 10 pmol of each primer. Reactions for transgenic line analysis were made in duplicate; the others were made in triplicate. Gene-specific primers for Arabidopsis genes actin2, RD29A, and ADH1 were 5'-TGGTGATGGTGTGTCT-3' and 5'-ACTGAGCACAATGTTAC-3', 5'-ATCACTTGGCTCCACTGTTGTTC-3' and 5'-ACAAAACACACATAAACATCCAAAGT-3', and 5'-TCCACGTATCTTCGGCCATG-3' and 5'-TAGCACCTTCTGCAGCGCC-3', respectively. Cycle threshold values for each target gene were normalized according to those obtained in the corresponding reactions for Arabidopsis gene or wheat β-tubulin gene. Relative expression was estimated using the 2–
Hypocotyl Measurements
Germination Assay
Root Growth Inhibition Assay
Salt Tolerance Assay The sequences described in this article have been submitted to the GenBank data library under accession numbers EF601539 to EF601542.
We thank Dr. Zhigang Xu of Nanjing Agricultural University for kindly supplying the light equipment. Received November 6, 2008; accepted November 28, 2008; published December 3, 2008.
1 This work was supported by the 863 program (grant no. 2006AA10A104), the programs of the National Science Foundation of China (grant nos. 30430440 and 30025030), the Outstanding Youth Fund of the Ministry of Education, the Generation Challenging Program (grant no. SP2–1), and the 111 project (grant no. Bo8025).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Zhengqiang Ma (zqm2{at}njau.edu.cn).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.132217 * Corresponding author; e-mail zqm2{at}njau.edu.cn.
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