We have isolated and characterized a new ultraviolet B (UV-B)-resistant mutant, uvi4 (UV-B-insensitive 4), of Arabidopsis. The fresh weight (FW) of uvi4 plants grown under supplemental UV-B light was more than twice that of the wild-type. No significant difference was found in their ability to repair the UV-B-induced cyclobutane pyrimidine dimers, or in the amount of UV-B absorptive compounds, both of which are well-known factors that contribute to UV sensitivity. Positional cloning revealed that the UVI4 gene encodes a novel basic protein of unknown function. We found that the hypocotyl cells in uvi4 undergo one extra round of endo-reduplication. The uvi4 mutation also promoted the progression of endo-reduplication during leaf development. The UVI4 gene is expressed mainly in actively dividing cells. In the leaves of PUVI4::GUS plants, the GUS signal disappeared in basipetal fashion as the leaf developed. The total leaf blade area was not different between uvi4 and the wild-type through leaf development, while the average cell area in the adaxial epidermis was considerably larger in uvi4, suggesting that the uvi4 leaves have fewer but larger epidermal cells. These results suggest that UVI4 is necessary for the maintenance of the mitotic state, and the loss of UVI4 function stimulated endo-reduplication. Tetraploid Arabidopsis was hyper-resistant to UV-B compared to diploid Arabidopsis, suggesting that the enhanced polyploidization is responsible for the increased UV-B tolerance of the uvi4 mutant.
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Ultraviolet B (UV-B) light is the most harmful radiation within sunlight for plants. Plants possess various protective mechanisms to cope with this problem. Exposure of plants to UV-B stimulates the accumulation of UV-absorbing compounds to attenuate the damaging solar UV-B radiation. Arabidopsis mutants defective in this response or defective in the flavonoid synthetic pathway are hypersensitive to UV-B light (Kliebenstein et al., 2002; Li et al., 1993). UV-B light that is not shielded by these compounds induces various types of DNA lesions, predominantly cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone dimers [(6–4) photoproducts]. Mutants that are deficient in the mechanism for repairing UV-B-induced DNA lesions show hypersensitivity to UV-B light (Britt et al., 1993; Harlow et al., 1994; Hidema et al., 2000; Jiang et al., 1997; Landry et al., 1997; Liu et al., 2001, 2003; Nakajima et al., 1998). Translesion synthesis (TLS) was recently shown to plays a role in the tolerance mechanism for unrepaired DNA lesions (Sakamoto et al., 2003; Takahashi et al., 2005). Analyses of UV-sensitive mutants have indicated that DNA repair and UV-absorbing compounds are the main factors that determine UV sensitivity.
On the other hand, only a few mutants that are hyper-resistant to UV-B light have been reported. The uvt1 and rcd1-2 mutants showed increased accumulation of UV-absorbing compounds, resulting in increased tolerance to UV-B light (Bieza and Lois, 2001; Fujibe et al., 2004). To see whether there are other unidentified mechanisms for protecting against UV light, we screened Arabidopsis mutants that show hyper-resistance to UV-B light. We succeeded in isolating four mutant lines, named UV-insensitive 1-4 (uvi1-4). We previously reported that the uvi1 mutant has an increased photoreactivation activity for CPDs and dark repair activity for (6–4) photoproducts (Tanaka et al., 2002). Here, we describe analyses of the uvi4 mutant and discuss how the uvi4 plants are hyper-resistant to UV-B light.
Increased UV-B tolerance of the uvi4 mutant
The uvi4 mutant is one of four UV-insensitive mutants isolated from Arabidopsis seeds (ecotype Columbia) mutagenized by a carbon ion beam (Tanaka et al., 2002). In order to characterize this mutant, 10-day-old plants were grown for two weeks under supplemental UV-B light, and the FW of aerial parts was compared in uvi4 and the wild-type. As seen in Figure 1(a,b), the FW of uvi4 plants was 2.1 times higher than that of the wild-type plants at a daily dose of 13 kJ m−2. Similar results were obtained in three independent experiments.
In addition, the uvi4 plants have overbranching trichomes on their leaves and stems. Trichomes with more than five branches were often seen on the uvi4 leaves, but were never found on the wild-type leaves.
UV-B absorptive compounds and DNA repair
To examine whether the increased UV-B tolerance of uvi4 is related to increased pigmentation, the amount of UV-B absorptive compounds were compared in uvi4 and the wild-type. As shown in Table 1, UV-B exposure stimulated the accumulation of UV-B absorptive compounds in both uvi4 and the wild-type. The absorbance of the leaf extract at 300 nm in the uvi4 was 5–10% less than that in the wild-type in both +UV and -UV conditions. Similar results were obtained in three independent experiments with different durations and doses of UV-B exposure. These results show that the uvi4 plants do not contain more UV-B absorptive compounds than the wild-type does.
Table 1. Amount of ultraviolet B (UV-B) absorptive compounds
Two-week-old plants were grown with supplemental UV-B light for 3 days, and then UV-B absorptive compounds were extracted from leaves using acidified methanol. Values represent absorbance at 300 nm.
The great majority of the DNA lesions induced by UV-B light are CPDs. To examine the DNA repair ability of uvi4, the induction and reduction of CPDs were measured by ELISA. The amount of CPDs increased linearly with UV-B doses up to 2 kJ m−2, and no difference between uvi4 and the wild-type was found at any dose examined (data not shown). As seen in Figure 2(a), the reduction of CPDs under white light after 2 kJ m−2 of UV-B exposure was not different between uvi4 and the wild-type in two independent experiments. These results show that increased levels of UV-B absorptive compounds or increased repair of CPDs do not contribute to the increased UV-B tolerance of the uvi4 plants.
Positional cloning of UVI4
For segregation analysis and gene mapping, uvi4 was crossed with wild-type Ws ecotype, because the UV sensitivity of the Ws ecotype was most similar to that of Col among five ecotypes tested (data not shown). F1 plants were self-pollinated to produce F2 plants. F3 plants derived from self-pollination of individual F2 plants (i.e. F3 lines) were subjected to a UV sensitivity test. As around a quarter of the F3 lines were insensitive to UV-B light, uvi4 was thought to have a single recessive mutation. During this process, we noticed that the UV-insensitive phenotype was linked to the overbranching trichome phenotype, which is another phenotype of the uvi4 mutant as described above. The F3 lines whose parental F2 plant has overbranching trichomes were relatively tolerant to UV-B light. This fact suggests that the UV-insensitive phenotype and the overbranching trichome phenotype are due to the same genetic lesion. As the trichome phenotype is much easier to distinguish than the UV-insensitive phenotype, we carried out the following positional cloning based on the trichome phenotype.
The trichome phenotype segregated as a single recessive trait in the F2 population [normal:overbranching = 106:29 (approximately 3:1, χ2 = 0.89, P > 0.30)]. By using 400 plants from the F2 population derived from Ws × uvi4, the genetic location of the uvi4 locus was examined by determining linkage between the trichome phenotype and DNA markers as previously described (Bell and Ecker, 1994; Konieczny and Ausubel, 1993). Finally, the uvi4 locus was mapped to the 240 kb region (Figure 3a,b). As the uvi4 mutant was generated by a carbon ion beam that frequently causes large rearrangements of DNA (Shikazono et al., 2001), we tried to identify the mutation by Southern analysis. As shown in Figure 3(c), different band patterns were found in uvi4 and the wild-type when BamHI-digested genomic DNA was hybridized with probe prepared from the T24P15 clone. The 1834 bp BamHI fragment of the wild-type contains the At2g42260 gene. Sequencing in the uvi4 background revealed a deletion of 123 bp in the second exon of this gene (Figure 3d). As shown in Figure 3(e), the breakpoints seem to be rejoined based on the homology of the sequence AGA. Reverse transcriptase-PCR (RT-PCR) showed a shorter transcript in uvi4 corresponding to the deletion size (data not shown).
We found that the polychome (pym) mutant that has been characterized as a trichome mutant by Perazza et al. (1999) is allelic to uvi4, because the overbranching trichome phenotype was not complemented in F1 plants from cross between the uvi4 and pym mutants. We revealed that the pym allele has a single base change of C for T at position 696 in the second exon, and this creates a new stop codon as determined by sequencing the At2g42260 gene in the pym background (Figure 3e). The pym mutant was also insensitive to UV-B light (data not shown). For a complementation test of the uvi4 mutant, cDNA of the At2g42260 (UVI4) gene expressed under the authentic promoter (PUVI4::UVI4cDNA) was introduced into the uvi4 plants. The leaves of PUVI4::UVI4cDNA plants bear normal three-branched trichomes, and trichomes with more than five branches were never found. These facts show that the UV-insensitive and trichome phenotype are both due to the single mutation of UVI4 and are not just closely linked.
UVI4 encodes a novel basic protein
The predicted UVI4 gene encodes a novel basic protein of 259 amino acids with an isoelectric point of 11.48 (Figure 4). The 123 bp deletion of the uvi4 locus causes the loss of 41 amino acids near the C terminus. The nonsense mutation in the pym locus results in loss of almost all the latter half of the protein. A BLAST search found a putative Arabidopsis homologue, At3g57860 (named UVI4-like), that shares 62% homology with UVI4 at the amino acid level. The At3g57860 protein was annotated as having a ferredoxin hydrogenase activity, although it has not been experimentally characterized so far. A pair of rice hypothetical proteins has low homology with UVI4. As seen in Figure 4, the C-terminal region and the middle region seem to be conserved among these proteins. As there is no other protein that shows significant homology with UVI4, UVI4 is likely to be a plant-specific protein. As Ds transposon-tagged lines in the UVI4-like gene do not show an overbranching trichome phenotype (data not shown), the UVI4 and UVI4-like genes have different roles, at least in trichome development.
Expression of UVI4 is developmentally regulated
The tissue specificity of UVI4 expression was analyzed using the GUS reporter gene under the control of the promoter region of UVI4 (PUVI4::GUS). Eleven independent transgenic lines were observed. Although two of them showed weak expression, a similar expression pattern was observed with all the lines. As seen in Figure 5(a), the PUVI4::GUS construct was strongly expressed in young leaves and vascular tissues. The GUS activity in leaf cells decreased in basipetal fashion as the leaf developed. The GUS signal in vascular tissues also gradually decreased, but, in some leaves, a weak signal remained after maturation of the leaves. The protruded young trichomes showed weak GUS activity but the mature trichomes had no signal (not shown). Strong GUS activity was also observed in the root tip, especially in the central cylinder, and the GUS signal became weaker in the cells that were more distant from the root tip (Figure 5b). It has been reported that, as they are distant from the root tip, outer cell layers have left the cell cycle and become differentiated, whereas the cells in the central cylinder remain in the mitotic state (Beeckman et al., 2001). These facts suggest that the UVI4 gene may be necessary for maintenance of the mitotic state. As shown in Figure 5(c), semi-quantitative RT-PCR analysis showed that the expression level of UVI4 decreased as the leaf developed. The expression level of mitotic cyclin CYCB2;2 decreased in a similar manner, suggesting the role of UVI4 in cell proliferation. The UVI4-like gene showed a similar expression pattern. These results show that the UVI4 expression is developmentally regulated and its expression level decreases with the cessation of cell proliferation.
uvi4 mutation promotes the progression of endo-reduplication
Most plants, including Arabidopsis, undergo endo-reduplication in their final differentiation step, in which cells increase their genomic DNA content without dividing (Edgar and Orr-Weaver, 2001; Larkins et al., 2001; Traas et al., 1998). As a direct correlation between the ploidy level and the number of trichome branches has been reported in Arabidopsis (Hülskamp et al., 1994), we investigated the ploidy level of uvi4 plants. Hypocotyls of 7-day-old seedlings of uvi4 and the wild-type were collected and analyzed by a flow cytometer. As seen in Figure 6A(a), four peaks up to 16C were observed in the wild-type, where the peaks higher than 2C result from endo-reduplication. The uvi4 hypocotyls had a higher proportion of 16C cells, and also had a 32C peak that was absent in wild-type hypocotyls [Figure 6A(b)]. This phenotype was complemented in the PUVI4::UVI4cDNA transgenic plants [Figure 6A(c)]. These results show that the uvi4 mutation results in one additional round of endo-reduplication in hypocotyl cells. It is known that hypocotyl cells grown in darkness undertake one more round of endo-reduplication due to the release of light-dependent suppression (Gendreau et al., 1998). This is also true in the uvi4 mutant. As shown in Figure 6A(d), the dark-grown uvi4 hypocotyls showed one more round of endo-reduplication up to 64C. Therefore, the enhanced endo-reduplication caused by the uvi4 mutation is unrelated to light perception.
Subsequently, we investigated changes in the ploidy level during leaf development. From 8 to 22 days after seed sowing, the first leaf pair of uvi4 and the wild-type plants were harvested and analyzed (Figure 6B). In the wild-type, 2C cells steeply decreased and reached a constant level after day 16. The proportion of 4C cells rapidly increased and peaked at day 12, and declined thereafter. Following this, the 8C cells started to increase. A small fraction of 16C cells was observed after day 16. After day 16, the ploidy level was fairly constant. In the uvi4 mutant, the ploidy distribution was similar to that of the wild-type at day 8. The proportion of 2C cells steeply decreased, and the fraction of 4C cells started to increase as was observed in the wild-type. However, the fraction of 4C cells at day 12 was smaller than that in the wild-type (52.5 ± 0.6% in uvi4, 63.8 ± 1.5% in the wild-type). Corresponding to this reduction, 8C and 16C cells started to increase earlier than they did in the wild-type. The ploidy distribution in uvi4 reached a constant level after day 18. At the latest time point analyzed, the uvi4 mutant had a higher proportion of 16C and 32C cells as well as a lower proportion of 4C cells as compared to the wild-type. Similar results were obtained in two independent experiments. These results show that the uvi4 mutation promotes progression of the endo-reduplication cycle.
In endo-reduplicated cells, cell size is known to be generally proportional to the amount of nuclear DNA. To examine the number of cells and their size in uvi4 leaves, we investigated changes in the total leaf blade area and the size of adaxial epidermal cells during leaf development. As shown in Figure 7(b), the average size of adaxial epidermal cells was larger in uvi4. During the period examined, the total leaf size was not significantly different between uvi4 and the wild-type (Figure 7a). Taken together, these results indicate that the uvi4 mutant has a lesser number of cells in the adaxial epidermis.
Ploidy level affects UV-B sensitivity
In order to examine the possibility that the increased ploidy level in uvi4 is responsible for the increased tolerance to UV-B light, we compared the UV sensitivity between tetraploid Arabidopsis and its parental diploid line. The tetraploid line showed higher tolerance than the diploid line (Figure 1c). The FW of tetraploid plants was 2.6 times higher than that of diploid plants at a daily dose of 17 kJ m−2. The photoreactivation activity for CPDs and the amount of UV-B-absorbing compounds were not different between the tetraploid and diploid lines (Figure 2b, Table 1). The tetraploid line undergoes the same number of endo-reduplication rounds as the diploid line. Therefore, hypocotyls of the tetraploid line contained 4C to 32C cells (data not shown). These results support the view that the promoted polyploidization is probably responsible for the increased tolerance of the uvi4 plants to UV-B light.
To our knowledge, only three UV-tolerant mutants have been reported in Arabidopsis. The uvt1 and rcd1-2 mutants are tolerant to UV-B light due to an increased accumulation of UV-absorbing compounds, and the uvi1 mutant is tolerant to UV-B light due to an increased DNA repair activity (Bieza and Lois, 2001; Fujibe et al., 2004; Tanaka et al., 2002). These results agree with the fact that UV-absorbing compounds and the repair of DNA damage are important contributors to UV tolerance of plants. However, in the uvi4 mutant, the amount of UV-B-absorbing compounds was not higher than that in the wild-type, and the repair activity for CPDs was not different from that in the wild-type. We propose that the uvi4 mutant is tolerant to UV-B light due to enhanced endo-reduplication. Consistent with this hypothesis, the tetraploid Arabidopsis was more UV-B-tolerant than the diploid Arabidopsis (Figure 1c), and this was not due to either a difference in UV-B-absorbing compounds (Table 1) or a difference in repair activity for CPDs (Figure 2b). The adaxial epidermis is thought to be the most important for protecting plants from UV light, because it protects the lower palisade layers in which the greatest amount of photosynthesis occur. The average cell area in the adaxial epidermis of uvi4 was larger than that of the wild-type. As there is a good correlation between epidermal cell size and ploidy level in Arabidopsis (Melaragno et al., 1993), this result clearly shows the increased ploidy level in the adaxial epidermis of uvi4. This paper shows that the ploidy level and endo-reduplication are important factors involved in protecting plants from UV light.
Although endo-reduplication is widespread in plants, its physiological role is poorly understood. It has been proposed that endo-reduplication is an effective strategy of cell growth because cell size is generally proportional to the amount of nuclear DNA. Simultaneously, endo-reduplication may be necessary to achieve transcriptional and metabolic activities that are proportional to the increased cell size. Furthermore, endo-reduplication is thought to be important for the differentiation processes such as those that occur during trichome and fruit development (Edgar and Orr-Weaver, 2001; Larkins et al., 2001; Traas et al., 1998).
Although there are few reports describing the relationship between ploidy and radiation sensitivity in plants, polyploid cells are believed to be more resistant to irradiation because they have an increased gene copy number. In addition, as the onset of endo-reduplication coincides with the loss of mitotic activity, cells undergoing endo-reduplication do not need to segregate their chromosomes via mitosis that may causes genetic loss. Wangenheim et al. (1995) reported that, in principle, polyploidy can exert a radioprotective effect in plants. They pointed out that this effect is evident in terminally differentiated tissues but not in mitotically active cells because the differentiation enhancement dominates the effects of genetic lesions in mitotically active cells. Polyploidization by endo-reduplication might be advantageous for protection from UV light, especially in tissues that are terminally differentiated but that continue to grow. In fact, the uvi4 leaves were less withered than the wild-type leaves at higher UV-B daily doses.
We examined the UV sensitivity based on the FW of the aerial parts of the plants. Despite the reduction in cell number in the uvi4 mutant, the unexposed uvi4 plants had similar FW to wild-type plants (Figure 1b). This is probably due to the enhanced endo-reduplication because the endo-reduplicated cells generally expand according to their ploidy level. As a preliminary result, when 10-day-old seedlings were grown under supplemental UV light for 4 days, the progression of endo-reduplication was inhibited in both uvi4 and wild-type leaves, although the uvi4 mutant still showed a slightly higher ploidy level compared to the wild-type. Thus, UV light causes general growth inhibition in both uvi4 and wild-type. The increased UV tolerance of uvi4 plants is not due to the stimulation of cell division or endo-reduplication by UV light, but is due to the increased proportion of the cells with higher ploidy level.
The number of DNA replication rounds is tightly controlled in hypocotyl cells (Gendreau et al., 1998). As the tetraploid line undergoes the same number of endocycles as the diploid line, it has been thought that the cells do not sense the DNA amount but the number of DNA replication rounds. The hypocotyl cells of uvi4 undergo one extra round of endo-reduplication (Figure 6A), indicating that the UVI4 gene is working as a suppressor of endo-reduplication in hypocotyl cells.
Vlieghe et al. (2005) reported that a T-DNA insertion in DEL1 (DP-E2F-like1) resulted in an increased ploidy level without affecting cell division. DEL1 transcripts were detected exclusively in mitotically dividing cells, and the ectopic expression of DEL1 reduced endo-reduplication. It has been reported that over-expression of both DPa and E2Fa induced ectopic cell division in mitotic cells and extra DNA replication in endo-reduplicating cells (De Veylder et al., 2002). Over-expression of DEL1 in the DPa-E2Fa over-expressor inhibited only the endo-reduplication phenotype (Vlieghe et al., 2005). From these facts, they concluded that DEL1 is an inhibitor of the endocycle, which preserves the mitotic state of proliferating cells by suppressing the transcription of genes that are required for cells to enter the endocycle. Similar to DEL1, UVI4 is expressed mainly in mitotically active cells, and the loss of UVI4 function resulted in increased ploidy level. However, UVI4 has different molecular function to DEL1 because cell number is reduced in the uvi4 mutant. Furthermore, we think the UVI4 do not play a major role in the transition from the mitotic cycle to the endo-reduplication cycle for the following reasons.
During development of the first leaf pair, the fraction of 2C cells steeply decreased as endo-reduplication proceeded (Figure 6B). The proportion of 2C cells is thought to represent the proportion of cells that do not endo-reduplicate. Boudolf et al. (2004) reported that the cyclin-dependent kinase CDKB1;1 is required to suppress endo-reduplication. In transgenic plants that over-express dominant-negative CDKB1;1, the fraction of 2C cells decreased faster than they did in the wild-type and the ploidy level was increased compared to the wild-type. If UVI4 is involved in the control of the transition from mitosis to endocycle, the population of 2C cells should decrease faster in uvi4 than in the wild-type. The repeated experiments showed that the proportion of 2C cells was surprisingly similar between uvi4 and the wild-type throughout leaf development, indicating that similar fractions of cells entered the endo-reduplication cycle at a given time point. Although, at present, we cannot exclude the possibility that specific phenotypes are masked in the uvi4 mutant by the redundant function of the UVI4-like gene, our results suggest that the UVI4 does not play a major role in the mitosis-to-endocycle transition. The most plausible explanation for the UVI4 function is that the UVI4 is necessary for maintenance of the mitotic state, and the loss of UVI4 function results in the reduction of cell number and also stimulates endo-reduplication.
Plant lines and growth conditions
The uvi4 mutant was isolated from an M2 population of Arabidopsis seeds (ecotype Columbia) mutagenized by a carbon ion beam as previously described (Tanaka et al., 2002). The pym mutant was kindly provided by Dr Jean-Marc Bonneville (Université J. Fourier, France). The pym mutant was isolated from Landsberg erecta (Ler) seeds mutagenized with EMS and has been characterized as a trichome mutant (Perazza et al., 1999). A tetraploid line of Columbia ecotype (4n; CS5131) and its parental diploid line (2n; CS3176) were provided by the Arabidopsis Biological Resource Center (ABRC). The 2n line was used as a control for the 4n line. An Arabidopsis line (PST15307) in which At3g57860 (UVI4-like) was disrupted by a Ds insertion was provided by the RIKEN Bioresource Center (Ito et al., 2002; Kuromori et al., 2004). Plants not otherwise described were grown in pots containing Metro-Mix 350 (Scotts-Sierra Horticultural Products) in a growth room at 23°C under a 16/8 h photoperiod with 3000–4000 lux of white fluorescent light.
Quantitative plant growth measurements
To examine their sensitivity to UV-B light, 10-day-old plants grown in a pot were transferred to a growth chamber equipped with a UV-B light source. UV-B light was supplied by a UV lamp cassette (type CSL-30B, COSMO BIO, Tokyo) that radiates at wavelengths above 280 nm with a high peak at 312 nm. The UV-B dose rate was measured with a UV-B radiometer (CSV-312, COSMO BIO) whose filter transmits UV-B radiation with a peak at 313 nm and has a half bandwidth of 12 nm. Plants were exposed to UV-B light for 10 h in the middle of the light period. Two weeks after transfer, the FW of aerial parts of the plants was measured.
Measurement of UV-B-absorptive compounds
Two-week-old plants were grown with supplemental UV-B light (daily dose = 9 kJ m−2) for 3 days. Several plants were collected and extracted with 90% methanol/1% HCl at a concentration of 10 mg FW per ml. The extract was cleared by centrifugation at 20 000 g for 10 min. The absorbance of the supernatant was measured at 300 nm using a photometer (model DU60, Beckman Instruments, Fullerton, CA, USA).
Seven-day-old seedlings that had been vertically grown on agar plates in 16 h light/8 h dark conditions were exposed to UV-B at a dose of 2 kJ m−2 in the dark condition. The seedlings (around 100 seedlings for each data point) were collected and frozen in liquid nitrogen immediately or after 1, 3 or 5 h incubation in the light condition after UV-B exposure. Genomic DNA was extracted using a DNeasy Plant Mini Kit (QIAGEN, Tokyo, Japan). A 50 μl aliquot of the extracted DNA at a concentration of 0.02 μg ml−1 was placed in each well. CPDs were detected with specific antibodies (TDM-2) as previously described (Tanaka et al., 2002). For each sample, the mean value of three wells was calculated and the background was subtracted.
For segregation analysis and gene mapping, uvi4 was crossed with wild-type Ws ecotype. More than 20 F3 lines were tested for segregation of UV-insensitive and trichome phenotypes. As the UV-insensitive and trichome phenotypes were thought to be linked, the uvi4 locus was mapped based on the trichome phenotype using CAPS and SSLP markers (Bell and Ecker, 1994; Konieczny and Ausubel, 1993). More than 400 F2 plants with overbranching trichomes that were obtained from a cross between Ws × uvi4 were used for a gene mapping. Ten new CAPS and SSLP markers were created between the Col and Ws ecotypes. Rearrangement of DNA was examined by a Southern blotting analysis. Genomic DNA was digested by BamHI and blotted onto a nylon membrane (Roche Diagnostics GmbH, Manheim, Germany). A DIG-labeled probe was prepared from BAC DNA. The membrane was hybridized and washed according to the manufacturer's instructions. Signal was detected with a CDP-star (Roche Diagnostics GmbH) using an IS8000 digital imaging system (Alpha Innotech, San Leandro, CA, USA).
A 1.7 kb 5′ region including the first 39 nucleotides of the UVI4 coding sequence was cloned in the pBI101 plasmid just upstream of the β-glucuronidase coding sequence to create the PUVI4::GUS plasmid. This construct was transferred into Agrobacterium strain GV3101, and then introduced into wild-type Columbia by the floral dip method (Bechtold and Pelletier, 1998). Transgenic plants (T2) were submerged in 90% acetone for 15 min and then in a GUS staining solution containing 1 mm X-Gluc, 3 mm K3Fe (CN)6 and 10 mm EDTA in NaPO4 buffer (pH 7.0). The stained tissues were cleared in 70% ethanol until the GUS stain became clearly visible. For a complementation test, the UVI4 promoter region (−1713 to −1) was cloned between the Sse8387I site and the XbaI site of the pBI121 plasmid, and then the UVI4 cDNA was cloned between the BamHI and SacI sites. The resulting PUVI4::UVI4cDNA construct was introduced into uvi4 plants.
Total RNA was isolated from the first leaf pair of 8–18-day-old plants grown on MS medium containing 2% sucrose and 0.6% gellan gum using an RNeasy Plant Mini Kit (QIAGEN). The RNA was treated with DNaseI according to the manufacturer's instructions. Then, 1 μg of total RNA was reverse transcribed with a BD Power Script RT Kit in a 40 μl total reaction volume according to the manufacturer's instructions (Clontech Laboratories Inc., Mountain View, CA, USA). PCR was performed using 1 μl of cDNA solution, 200 μm of each dNTP, 0.75 U of Takara Ex Taq polymerase (TAKARA BIO Inc, Shiga, Japan) and 1 × Ex Taq Buffer in a 30 μl total volume. The sequences of primers were 5′-AGCGCTCAATCACAAATG-3′ and 5′-AATCACCAGAGGATGATGAA-3′ for UVI4, 5′-CGAGAGGCCTGTGGATTACT-3′ and 5′-CTACGCTTGAATCCCACAGA-3′ for UVI4-like, 5′-AGACAGAACAGGAGAGCATTGG-3′ and 5′-CAATGCAACTAAACCAACAAGC-3′ for CYCB2;2 (At4g35620), and 5′-CTTGCTTTCACCCTTGGTGT-3′ and 5′-TCCCTCGAATCCAGAGATTG-3′ for EF1α (At5g60390). PCR conditions consisted of an initial denaturation at 94°C for 5 min, followed by 40 cycles of 94°C for 15 sec, 58°C for 30 sec and 72°C for 60 sec for UVI4 and UVI4-like, 30 cycles for CYCB2;2 and 24 cycles for EF1α. The PCR products (10 μl aliquots) were separated on agarose gels and visualized by UV excitation of ethidium bromide staining.
Plants were grown on MS medium containing 2% sucrose and 0.6% gellan gum. About 20 hypocotyls were collected from light- or dark-grown 7-day-old seedlings. The first and/or second true leaves were collected from 8–18-day-old plants. Leaves collected from more than three plants were used for each measurement. Ploidy was measured as previously described (Hase et al., 2005).
Analyses of leaf growth
The uvi4 and wild-type plants were grown side by side on MS medium containing 2% sucrose and 0.6% gellan gum. From 8–22 days after seed sowing, the first leaf pair was collected on every second day. The total leaf blade area was measured by Image J version 1.33 (National Institutes of Health, Bethesda, MA, USA) using images captured by a CCD camera mounted on a stereomicroscope. Each data point was based on leaves collected from at least 5 plants. To measure cell area, a replica of the adaxial leaf surface was made using liquid plaster. The replica was removed from the leaf surface and observed under a microscope. Images of the middle of the leaf blade and halfway between the midrib and the leaf margin were captured with a CCD camera. The cell area was measured using Image J software. More than 118 cells from more than five plants were used for each data point.
The authors thank N. Kudo and Y. Yokota for their assistance in the flow cytometric analysis, Jean-Marc Bonneville for kindly providing the pym seeds, C. Suzuki, S. Takahashi and Y. Oono and other members of A. Tanaka's laboratory for their assistance and helpful comments, the ABRC for providing BAC clones and seeds, and the RIKEN Bioresource Center for providing the Ds transposon-tagged line. K.H.T. is supported by the scientist exchange program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT).