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First published online July 17, 2003; 10.1104/pp.103.024703 Plant Physiology 133:203-217 (2003) © 2003 American Society of Plant Biologists Members of the Arabidopsis-SKP1-like Gene Family Exhibit a Variety of Expression Patterns and May Play Diverse Roles in Arabidopsis1Department of Biology and the Huck Institute for Life Sciences, 313 Wartik Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802 (D.Z., W.N., B.F., T.H., M.G.P., H.M.); and Institute of Crop Breeding and Cultivation, the Chinese Academy of Agricultural Sciences, Beijing 100081, China (T.H.)
Ubiquitin-mediated proteolysis by the proteasome is a critical regulatory mechanism controlling many biological processes. In particular, SKP1, cullin/CDC53, F-box protein (SCF) complexes play important roles in selecting substrates for proteolysis by facilitating the ligation of ubiquitin to specific proteins. In plants, SCF complexes have been found to regulate auxin responses and jasmonate signaling and may be involved in several other processes, such as flower development, circadian clock, and gibberellin signaling. Although 21 Skp1-related genes, called Arabidopsis-SKP1-like (ASK), have been uncovered in the Arabidopsis genome, ASK1 is the only gene that has been analyzed genetically. As a first step toward understanding their functions, we tested for expression of 20 ASK genes using reverse transcription-polymerase chain reaction experiments. Also, we examined the expression patterns of 11 ASK genes by in situ hybridizations. The ASK genes exhibit a spectrum of expression levels and patterns, with a large subset showing expression in the flower and/or fruit. In addition, the ASK genes that have similar sequences tend to have similar expression patterns. On the basis of the expression results, we selectively suppressed the expression of a few ASK genes using RNA interference. Compared with the ask1 mutant, the strong ASK1 RNA interference (RNAi) line exhibited similar or enhanced phenotypes in both vegetative and floral development, whereas ASK11 RNAi plants had normal vegetative growth but mild defects in flower development. The diverse expression patterns and distinct defects observed in RNAi plants suggest that the ASK gene family may collectively perform a range of functions and may regulate different developmental and physiological processes.
Selective proteolysis of proteins has been recognized as a very important mechanism for regulating many cellular events (Hershko and Ciechanover, 1998  ; Zheng et al., 2002). A major pathway for controlled protein destruction is the ubiquitin-mediated proteolysis by the proteasome (Hershko and Ciechanover, 1998; del Pozo and Estelle, 2000). Three types of enzymes (E1, E2, and E3) are responsible for ubiquitination of proteins. E1 is known as the ubiquitin-activating enzyme, E2 is the ubiquitin-conjugating enzyme, and E3 is the ubiquitin-protein ligase, which acts as a factor for substrate recognition (Koepp et al., 1999; Pickart, 2001). Both E1 and E2 are less specific than E3. The SKP1, cullin/CDC53, F-box protein (SCF) complexes are the largest and best studied family of E3 ubiquitin-protein ligases and are known to control cell cycle regulation, signal transduction, transcription, and other biological events (Bai et al., 1996; Hershko and Ciechanover, 1998; Schulman et al., 2000; Shen et al., 2002; Zheng et al., 2002).
Among the subunits of the SCF complex, SKP1 acts as an adapter that links cullin to one of the F-box proteins, which are highly variable (Willems et al., 1999
Recent studies suggest that plants make extensive use of SCF complexes to regulate multiple biological processes. Several SCF complexes have been characterized in Arabidopsis. For example, the SCFTIR1 complex regulates auxin response (Ruegger et al., 1998
In both yeast and human, there is only one known functional SKP1 gene (Yu et al., 1998
The Arabidopsis genome contains 21 predicted ASK genes (Arabidopsis Genome Initiative, 2000
Expression Analysis by RT-PCR For the convenience of description and discussion, we divide the genes into several groups based on their sequence similarity: group 1, ASK1 and ASK2; group 2, ASK3 and ASK4; group 3, ASK5 and ASK6; group 4, ASK7 through ASK10; group 5, ASK11 and ASK12; group 6, ASK13; group 7, ASK14 through ASK19; and group 8, ASK20 and ASK21 (Fig. 1). Although the ASK genes share higher levels of amino acid sequence similarity within each group than between groups, the levels of similarity within different groups are not uniform.
To learn whether predicted ASK genes are expressed during normal development and to estimate the relative abundance of ASK messages in different organs, we performed RT-PCR using gene specific primers (Table I). To control for specificity, PCR products from genomic DNA and cDNA templates were sequenced directly, and their sequences match known sequences of the corresponding genes (except ASK9), other than polymorphisms between the Landsberg erecta (Ler) and Columbia (Col) ecotypes. PCR using primers matching the Col ASK9 gene yielded products that were most similar to ASK8. Thus, ASK9 expression was not further analyzed. Expression of the 20 ASK genes was tested using RT-PCR, and the expression of 18 genes was detected in one or more part(s) of plants grown under normal conditions (Fig. 2; Table II). However, the sequence of the ASK15 RT-PCR product matched the known ASK15 genomic sequence, including a predicted 50-bp intron, suggesting that ASK15 may be a pseudogene. The expression of ASK6 and ASK19 was not detected. As a control for RNA extraction and RT-PCR for different tissues, we detected uniformly strong RT-PCR products in all RNA samples for the APT1 gene encoding adenine phosphoribosyltransferase (Moffatt et al., 1994
RT-PCR produced nearly uniformly strong bands for ASK1 and ASK2 in all tissues tested: young seedlings, roots, leaves, floral stems, inflorescences, and siliques. ASK3 seems highly expressed in the silique. ASK4 expression seems to be at a higher level in inflorescence and siliques than in seedlings, roots, and stems. For group 4, the expression of ASK7 and ASK8 was detected only in the silique, whereas ASK10 expression was found in all tissues tested. In group 5, ASK11 and ASK12 had similar expression patterns, with perhaps a slightly higher level in the inflorescence than in other tissues. In group 3, ASK5 is expressed in the inflorescence, as confirmed by in situ hybridization (see below). ASK13 expression was detected as a stronger PCR band for the inflorescence than those for the seedling and silique. In group 7, ASK14 expression was only detected in the inflorescence. ASK15 expression was difficult to detect after one round of PCR; a second PCR using nested primers found it to be expressed in the seedling and leaf (Fig. 2). ASK16 and ASK17 expressions were mainly detected in the silique (Fig. 2). For ASK18, ASK20, and ASK21, similar levels of expression were detected in all tissues examined (Fig. 2).
The expression patterns of 11 ASK genes were further characterized using RNA in situ hybridization experiments; these 11 genes were chosen in part based on their amino acid sequence similarities. ASK1 was included as a positive control; moreover, we wanted to compare ASK1 and ASK2 expression patterns because they have very similar sequences and RT-PCR results. ASK3 and ASK4 are highly similar to each other and, to a lesser extent, to ASK1 and ASK2. ASK14, ASK15, ASK18, and ASK19 are relatively similar in sequence, but differ in expression based on RT-PCR experiments. Finally, ASK5, ASK9, and ASK11 were chosen as representatives of three other distinct groups.
To obtain gene-specific probes, we used 3'-untranslated regions (UTRs) of the ASK genes. Previous in situ hybridization experiments indicated that probes of 400 bps or longer do not cross-hybridize to related sequence with 83% or less nucleotide sequence identity (Yanofsky et al., 1990
The ASK1 and ASK2 mRNAs were detected in all major organs examined, although ASK2 expression was at lower levels than those of ASK1 (Fig. 3). Both ASK1 and ASK2 mRNAs accumulate weakly in the cortical layer and epidermis of the root (Fig. 3, A and B) but are not detectable in the vascular tissue. In the young stem, ASK1 and ASK2 are expressed uniformly in the pith and vascular bundle (Fig. 3, C and D). No expression was observed for either ASK1 or ASK2 in the mature stem (data not shown). In the leaf, ASK1 is uniformly expressed at a higher level than that of ASK2 (Fig. 3, E and F). In the silique, ASK1 is expressed throughout the valve and developing seed (Fig. 3G), again at levels higher than those of ASK2 (Fig. 3H). ASK1 expression is strong in the inflorescence meristem (IM) and young floral bud (Fig. 3I), at high levels in all floral organ primordia; furthermore, it is expressed at a high level in the male meiocytes in a flower at approximately stage 9, when meiosis occurs (Fig. 3K). ASK1 expression remained high in the pollen grains (Fig. 3M). Compared with ASK1, ASK2 is expressed in the IM and flower at lower levels (Fig. 3J). ASK2 signal is also present in the male meiocytes and pollen grains (Fig. 3, L and N). These in situ hybridization results indicate that ASK1 and ASK2 have very similar expression patterns.
The ASK3 and ASK4 genes also have similar expression patterns (Fig. 4). In the root, young stem, and young leaf, the signals of ASK3 (Fig. 4, A, C, and E) and ASK4 (Fig. 4, B, D, and F) are very low if detectable at all. Both ASK3 and ASK4 have high levels of expression in the silique (Fig. 4, G and H); ASK4 is very strongly expressed in the valve, septum, and developing seed (Fig. 4H). Weak signals of ASK3 were also detected in the inflorescence (Fig. 4I). No expression of ASK3 was detected in the IM, and ASK3 mRNA in the flower is mostly restricted to the sepal and pedicel (Fig. 4, I and K). ASK4 expression was detected throughout the inflorescence at a higher level in the IM than in the young flower (Fig. 4, J and L). Although ASK3 expression was not detected in pollen grains (Fig. 4M), ASK4 (Fig. 4N) is expressed in the pollen grains, but at a lower level than those of ASK1 and ASK2 (Fig. 3, M and N).
Although the ASK14, ASK15, ASK18, and ASK19 genes are similar in sequence, they have different expression patterns (Fig. 5). In the root, there is no detectable expression of ASK14 and ASK19 (Fig. 5, A and S), and signals of ASK15 and ASK18 are barely detectable (Fig. 5, G and M). ASK15 and ASK18 are highly expressed in the pith and vascular bundle in the stem, with the levels of ASK15 greater than that of ASK18 (Fig. 5, H and N). In contrast, little or no expression was seen for ASK14 or ASK19 in either young or mature stem (Fig. 5, B and T; data not shown). Their expression in leaf varied in levels from least to greatest: ASK14, ASK19, ASK15, and ASK18 (Fig. 5, C, U, I, and O). Barely detectable expression of ASK14 was observed in the silique (Fig. 5D). In the silique, ASK15 and ASK18 mRNAs are higher in inner epidermis of the valve than elsewhere (Fig. 5, J and P), and ASK19 signals are detectable in the valve and seed coat (Fig. 5V). ASK14, ASK15, ASK18, and ASK19 are all expressed in the inflorescence but with different spatial patterns. There is no expression of ASK14 in the IM or pollen grains (Fig. 5E; data not shown). However, ASK14 is expressed in the male meiocytes and even more in the tetrads (Fig. 5F; data not shown). The expression of ASK15 and ASK18 was found in the pedicel of young buds, with lower expression elsewhere in the flower (Fig. 5, K, Q, and R). ASK15 is also expressed in the inner epidermis of the carpel and pedicel in the mature flower (Fig. 5L). The level of ASK19 expression is moderate and nonspecific in the IM and flower (Fig. 5W). In the mature flower, ASK19 expression was seen in the sepal, petal, and filament of the stamen (Fig. 5X).
Our results showed that the expression patterns of ASK5 and ASK11 were different (Fig. 6). The expression of ASK5 was not found in the root, stem, and leaf (Fig. 6, A-C), was barely detectable in the silique (Fig. 6D), and was observed at a very low level in the inflorescence except for the IM (Fig. 6E). In the young bud, the ASK5 mRNA is restricted to the sepal and pedicel (Fig. 6F). ASK11 has a very weak expression in the root, stem, leaf, and silique (Fig. 6, M-P). However, ASK11 has a slightly higher expression in the IM and young bud, particularly in the stamen (Fig. 6Q). We also detected the expression of ASK11 in the pollen grains (Fig. 6R). As a negative control, sense probes detected very low levels of signals that matched backgrounds seen with our experimental probes (e.g. Fig. 6, S-X; data not shown). Because the ASK9 UTR used for probe synthesis has high identity to UTRs of ASK7, ASK8, and ASK10, the signals detected here probably represent the combined expression of all four genes. The ASK9-hybridizing signal was detected in every tissue, very weakly in root (Fig. 6G), at moderate levels in the pith and vascular bundle of the young stem and in the leaf blade (Fig. 6, H and I), and at moderate levels in the valve and seed coat in silique (Fig. 6J). Signals are also present in the inflorescence except for the IM (Fig. 6K) and are especially high in the pedicel of the young flower (Fig. 6L).
To begin investigating the function of ASK genes, we selectively suppressed the expression of a few ASK genes using double-stranded RNA interference (RNAi) method. ASK1 was chosen as a positive control because the ask1-1 null mutant phenotypes have been described. In addition, we also wanted to know whether ASK1 shares redundant functions with ASK2, or possibly other ASK genes, by comparing RNAi plants with the ask1-1 mutant. ASK11 was chosen to test the hypothesis that it plays some specific roles, because it has a more restricted expression pattern.
We generated approximately 200 independent ASK1 RNAi lines; more than 80% of the transgenic lines showed some degrees of floral defects similar to those of the ask1-1 mutant, including reduced fertility, indicating that the suppression is very efficient. ASK1 RNAi lines with a weak phenotype usually had normal vegetative growth, normal stature, mild defects in flower development, and normal or partially reduced fertility. In contrast, lines with a strong phenotype had abnormal vegetative growth, short stature, and severe defects in flower development, and were usually male sterile. A strong (ASK1-Line 3) line and relative weak (ASK1-Line 11 and 14) lines were further examined.
During vegetative development, we observed a slight reduction of leaf size and plant stature in the ask1-1 mutant, similar to previous findings (Fig. 7, B and F; Zhao et al., 1999
The ask1-1 mutant flowers had a slightly reduced number of petals, staminoid petals, and short filaments (Fig. 7J; Zhao et al., 1999
ASK11 is almost identical to ASK12 in the coding region; in addition, our RT-PCR results for ASK11 and ASK12 were very similar. Therefore, ASK11 and ASK12 may play very similar roles and might be functionally redundant. Consequently, the phenotype observed in ASK11 RNAi plants, if any, might be the result of a reduction of both ASK11 and ASK12 functions. Approximately 100 independent transgenic ASK11 RNAi lines were generated, and no obvious phenotype was observed during vegetative development. All transgenic lines had normal fertility (Fig. 7Q). In other words, none of the ASK11 RNAi plants had vegetative or fertility defects that were observed in typical ASK1 RNAi plants. However, we observed slight defects in early flowers during reproductive development, although more severe phenotypes were observed in the flowers and inflorescences at late stages. Flowers of ASK11 RNAi plants had normal sepals, petals, and carpels. The only defects were petaloid or fused stamens in the third whorl of about 15% of the first 15 flowers (Fig. 7, R and S). Occasionally filament or abnormal anthers were also found in the transgenic plants, whereas other stamens in the same flower still had normal length and produced viable pollen (Fig. 7T). The phenotypes of the ASK11 RNAi plants differ from the ASK1 RNAi plants in that even weak ASK1 RNAi plants exhibit defects in both second and third whorls, as do the ask1-1 mutant flowers. Comparison of the RT-PCR results between the control APT1 gene and various ASK genes suggested that the expression of ASK11 and ASK12 was slightly reduced in the transgenic plants. In addition, ASK1 expression was reduced slightly, but ASK2, ASK4, and ASK18 expression was close to normal (Fig. 8D).
ASK Genes Exhibit Several Different Expression Patterns
Our RT-PCR and RNA in situ hybridization experiments indicate that nearly all of the ASK genes predicted from genomic sequences are expressed at different levels and exhibit a variety of spatial patterns. In addition, highly similar genes within each of several groups tend to have similar expression levels and patterns, although members of some groups show different expression patterns. In particular, ASK1 and ASK2 are both highly expressed in all of the tissues that were tested. In situ hybridization experiments further indicate that these two genes share very similar spatial patterns of expression throughout the plant, suggesting that they may be functionally redundant. This is further supported by the results that both ASK1 and ASK2 proteins interact with one of several F-box proteins in vitro and/or in yeast two-hybrid assays (Gray et al., 1999 In contrast with the above ASK genes, those in groups 3, 6, 7, and 8 exhibit greater divergence in sequence and/or in expression pattern, suggesting that they may have evolved to have different functions and/or lost their original functions. As mentioned above, no ASK6 expression was detected. This and the fact that its sequence predicts a truncated protein lacking parts of both the highly conserved N- and C-terminal domains strongly suggest that it is a pseudogene. In addition, ASK15 mRNA was found to contain a predicted 50-bp intron, making it a possible pseudogene. A moderate expression level of ASK15 and ASK19 was detected from our in situ experiments, whereas RT-PCR results suggest that they are expressed at very low levels. One explanation for this is that the tissues used for those two experiments were not identical; alternatively, the primers for ASK19 RT-PCR may be inefficient.
Our results clearly indicate that some ASK genes are widely expressed, suggesting that they may have pleiotropic functions, whereas others have more restricted expression and perhaps specific functions. Sequence divergence and expression differences between some groups of ASK genes also suggest that genes of these different groups may have evolved to have distinct functions. Yeast two-hybrid results showed that ASK1, ASK2, ASK11, and ASK19 can interact with about half or more of the 23 F-box proteins tested, and ASK4 interacts with several F-box proteins, whereas ASK5, ASK16, and ASK18 can interact only with a few of the F-box proteins (Gagne et al., 2002
Our results show that most of the ASK genes are either expressed in all tissues tested or are preferentially expressed in the inflorescence and/or fruit. This is consistent with the fact that flowers and fruits are more complex structures than roots or leaves. It is possible that reproductive development and physiology require many regulatory proteolytic pathways, not only those found in vegetative tissues, but also those specific to reproductive structures. For example, the expression of ASK1, ASK2, ASK4, and ASK11 in the shoot apical meristem suggests that they are involved in regulating cellular events in the meristem. Their expression in the pollen at relatively high levels suggests that they may also play a role in pollen development. In addition, the fact that ASK15 and ASK18 were expressed in the pedicel of young flowers suggests roles during pedicel development. ASK1 is known to regulate male meiosis; the detection of ASK2 expression in meiotic cells suggests that it may also be involved in male meiosis. Furthermore, the ASK14 mRNA is present at a high level in the microspores but is absent in the pollen grains, suggesting that the ASK14 gene may function in the newly formed microspores.
The expression of most ASK genes was detected in the silique, suggesting that they play important roles during embryo, seed, or fruit development. However, the details of the expression patterns were different. For example, the ASK4 gene is expressed at high levels in several tissues, including the valve, septum, and developing seed, but ASK1 expression is higher in the embryo than other parts. The signals of ASK3 and ASK19 were more restricted to the seed coat, whereas, most interestingly, the ASK15 and ASK18 mRNAs are mostly present in the inner epidermis of the valve. Therefore, different ASK genes may have distinct functions in the development of seed and/or fruit. Recently, a mutation in the AtCUL1 gene was found to cause an arrest of embryo and endosperm development before either the first or second mitosis of the zygote/embryo (Shen et al., 2002
ASK1 is strongly expressed in all tissues tested, consistent with previous reports on its expression (Porat et al., 1998 On the other hand, the relatively weak vegetative and floral phenotypes observed in the ask1-1 null mutant indicate that other ASK genes also play a role and may share redundant functions with ASK1; one likely candidate is the ASK2 gene, as suggested by its expression pattern (see above). This is supported by the enhanced phenotypes in the strong ASK1 RNAi line together with a reduction in expression of both ASK1 and ASK2 and perhaps that of other ASK genes.
The dwarf phenotype and the dark-green leaves of the strong ASK1 RNAi transgenic plant are similar to those found in GA-deficient or -insensitive mutants (Koornneef and van der Veen, 1980
Previous studies in our lab have demonstrated that ASK1 is required for the regulation of B function of the ABC model in flower development through interacting with an F-box protein UFO (Zhao et al., 1999
ASK11 and ASK12 are extremely similar in both sequence and expression patterns, suggesting that they have similar functions. ASK11 was able to interact with a similar set of F-box proteins as ASK1 and ASK2 in yeast two-hybrid assays (Gagne et al., 2002 Although the expression of ASK11 and ASK12 was not eliminated in the RNAi lines, weaker RT-PCR bands were reproducibly obtained from the RNAi lines than from the wild-type plants. Because additional RT-PCR cycles were needed to detect expression, the RT-PCR results suggest a reduction of ASK11/12 expression from already-low wild-type levels. It is possible that the RNAi construct with the 35S promoter was not able to completely eliminate weak expression. However, because the reduction was observed after an increased number of PCR cycles, the interpretation of a role of ASK11/12 in stamen development must be regarded as tentative. More definitive understanding of the function of these genes will require the analysis of mutations in these genes. We have also noticed that ASK1 expression seems to be reduced slightly in the ASK11 RNAi plants, suggesting that the reduction of ASK1 might have contributed to the observed phenotypes. Nevertheless, the ASK11 RNAi flowers were different from flowers of ASK1 RNAi transgenic lines, supporting a possible distinct role for ASK11 (ASK12) in flower development.
Clearly, the ASK gene family contains a spectrum of actively expressed members that exhibit a variety of patterns. The encoded proteins can potentially form a multitude of SCF complexes that regulate plant development and physiology at different stages. It has been shown that SKP1 may form a complex lacking cullin, and results from C. elegans indicate that some SKP1 homolog do not interact with cullins (Yamanaka et al., 2002 ). Therefore, some ASK proteins could function by forming complexes different from SCF. In summary, ASK genes may play a wide range of important regulatory roles, and future studies using reverse genetics, biochemistry, and cell biology promise to uncover additional functions for this important regulatory gene family.
Plant Materials and Growth Conditions Arabidopsis plants were of the Ler ecotype. Except otherwise noted, they were grown on Metro-Mix 360 (Scotts-Sierra Horticultural Products, Maryville, OH) at 22°C (16 h of light and 8 h of dark).
For RT-PCR experiments, RNA was isolated from 3- to 4-d-old seedlings, roots of 2-week-old plants, leaves of 3- to 4-week-old plants, inflorescence stems and inflorescences of 5-week-old plants, and 2- to 10-d-after-pollination siliques. Total RNA was isolated using the RNeasy mini kit (Qiagen USA, Valencia, CA) and was treated with DNase I (Invitrogen, Carlsbad, CA). One microgram of RNA from different tissues was reverse transcribed into cDNA with oligo(dT) (16-mer) using Super Script II reverse transcriptase (Invitrogen). A fraction (about 1/20) of the first strand cDNAs was used as a template for PCR with gene-specific primers (Table I). PCR was carried out under standard conditions using 10 pmol of each primer and 27 (for APT1 gene) or 35 cycles (for most ASK genes) of 94°C for 30 s, 56°C to 66°C for 40 s, and 72°C for 60 s. A second PCR of 12 cycles (for ASK11 and ASK15) or 20 cycles (for ASK12) was performed with the same or nested primers (Table I). Control PCRs without reverse transcriptase did not produce any PCR bands. Fifty microliters of PCR products was separated on 1.5% (w/v) agarose gels containing ethidium bromide and visualized by UV light.
Samples of root, leaf, inflorescence stem, inflorescence, and silique were isolated from 3- or 4 week-old plants and were immediately fixed in an formaldehyde-acetic acid fixative. RNA in situ hybridizations with radioactive probes were performed as previously described (Drews et al., 1991
The RNAi vector pRR2222 was kindly provided by Dr. Ramesh Raina (Pennsylvania State University). It was derived from another RNAi vector pFGC1008 (kindly provided by Dr. Carolyn Napoli [University of Arizona, Tucson]) by replacing the
The ASK11 RNAi vector was constructed in a similar way. The ASK11 cDNA was amplified from inflorescence of Ler ecotype using primers oMC 611 (5'-CCTCCTCCACAAGGAACACACAATG-3') and oMC612 (5'-GCTAGTTAGGGTTTTGATTCATGGG-3') and cloned into the pCRII-TOPO vector, resulting the plasmid pMC2569. The cloned ASK11 cDNA was then digested with XhoI and SpeI and cloned in the sense orientation into the XhoI and AvrII sites of pMC2560, a vector modified from pRR2222, yielding pMC2594. The pMC2560 plasmid was derived from pRR2222 by replacing the fragment flanked by XhoI and AscI restriction enzyme sites with the 3'-UTR of ASK1 gene amplified by oMC438 (Table III) and oMC577, the latter of which contains a same sequence with oMC439 (Table III) plus AvrII and AscI sites. The antisense ASK11 fragment was cloned into pMC2594 in the same way as that for ASK1, yielding pMC2600 (Fig. 9B). All constructs described above were confirmed by sequencing and were introduced into Arabidopsis plants by Agrobacterium tumefaciens-mediated in planta transformation (Clough and Bent, 1998 To detect the expression of ASK genes in the transgenic plant, RT-PCR was carried out, with 25 cycles for ASK1 and ASK2, 28 cycles for ASK4, and 25 cycles followed by another 20 cycles for ASK11 and ASK12, and 35 cycles for ASK18. For each reaction, three replications were performed. Primers for ASK1 were oMC529 (describe above) and oMC799 (5'-GAGTAAGAAACATTGGTTCTTG-3'); primers for other genes are shown in Table I.
We thank E. Risseeuw and W.L. Crosby for kindly providing the ASK1, ASK2, and ASK4 cDNA clones and R. Raina for the generous gift of the pRR2222 vector. We also thank H. Kong for providing the ASK phylogenetic tree shown in Figure 1 and Y. Hu and R. Walsh for technical assistance with tissue section and in situ hybridization. In addition, we thank A. Omeis and J. Wang for plant care. We are grateful for helpful comments from C. Hendrix, W. Li, L.M. Zahn, G. Wang, H. Kong, and L. Timofejeva. Received April 2, 2003; returned for revision April 25, 2003; accepted May 29, 2003.
1 This work was supported by the National Science Foundation (grant nos. MCB-9896340 and MCB-0092075 to H.M.), by the National Institutes of Health (grant no. RO1 GM63871 to H.M.), and by Funds from the Department of Biology and the Huck Institute for Life Sciences at the Pennsylvania State University. T.H. was supported by China Scholarship Council and National Natural Science Foundation of China. 
2 These authors contributed equally to this paper. * Corresponding author; e-mail hxm16{at}psu.edu; fax 814-863-1357.
Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796-815[CrossRef][Medline] Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge SJ (1996) SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86: 263-274[CrossRef][Web of Science][Medline] Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743[CrossRef][Web of Science][Medline]
del Pozo JC, Boniotti MB, Gutierrez C (2002) Arabidopsis E2Fc functions in cell division and is degraded by the ubiquitin-SCF(AtSKP2) pathway in response to light. Plant Cell 14: 3057-3071 del Pozo JC, Estelle M (2000) F-box proteins and protein degradation: an emerging theme in cellular regulation. Plant Mol Biol 44: 123-128[CrossRef][Web of Science][Medline]
Dieterle M, Zhou YC, Schafer E, Funk M, Kretsch T (2001) EID1, an F-box protein involved in phytochrome A-specific light signaling. Genes Dev 15: 939-944 Drews GN, Bowman JL, Meyerowitz EM (1991) Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product. Cell 65: 991-1002[CrossRef][Web of Science][Medline] Farras R, Ferrando A, Jasik J, Kleinow T, Okresz L, Tiburcio A, Salchert K, del Pozo C, Schell J, Koncz C (2001) SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase. EMBO J 20: 2742-2756[CrossRef][Web of Science][Medline] Flanagan CA, Ma H (1994) Spatially and temporally regulated expression of the MADS-box gene AGL2 in wild-type and mutant Arabidopsis flowers. Plant Mol Biol 26: 581-595[CrossRef][Web of Science][Medline]
Gagne JM, Downes BP, Shiu SH, Durski AM, Vierstra RD (2002) The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc Natl Acad Sci USA 99: 11519-11524
Gray WM, del Pozo JC, Walker L, Hobbie L, Risseeuw E, Banks T, Crosby WL, Yang M, Ma H, Estelle M (1999) Identification of an SCF ubiquitinligase complex required for auxin response in Arabidopsis thaliana. Genes Dev 13: 1678-1691 Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M (2001) Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature 414: 271-276[CrossRef][Medline] Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67: 425-479[CrossRef][Web of Science][Medline] Ingram GC, Goodrich J, Wilkinson MD, Simon R, Haughn GW, Coen ES (1995) Parallels between UNUSUAL FLORAL ORGANS and FIMBRIATA, genes controlling flower development in Arabidopsis and Antirrhinum. Plant Cell 7: 1501-1510[Abstract]
Kamura T, Koepp DM, Conrad MN, Skowyra D, Moreland RJ, Iliopoulos O, Lane WS, Kaelin WG Jr, Elledge SJ, Conaway RC et al. (1999) Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science 284: 657-661
Kim HS, Delaney TP (2002) Arabidopsis SON1 is an F-box protein that regulates a novel induced defense response independent of both salicylic acid and systemic acquired resistance. Plant Cell 14: 1469-1482 Kipreos ET, Pagano M (2000) The F-box protein family. Genome Biol 1: REVIEWS3002.1-3002.7 Koepp DM, Harper JW, Elledge SJ (1999) How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell 97: 431-434[CrossRef][Web of Science][Medline] Koornneef M, Elgersma A, Hanhart CJ, van Loenen-Martinet EP, van Rign L, Zeevaart JAD (1985) A gibberellin insensitive mutant of Arabidopsis thaliana. Physiol Plant 65: 33-39[CrossRef] Koornneef M, van der Veen JH (1980) Induction and analysis of gibberellin sensitive mutants in Arabidopsis thaliana (L.) Hehyn. Theor Appl Genet 58: 257-263[CrossRef][Web of Science]
Kuroda H, Takahashi N, Shimada H, Seki M, Shinozaki K, Matsui M (2002) Classification and expression analysis of Arabidopsis F-box-containing protein genes. Plant Cell Physiol 43: 1073-1085 Levin JZ, Meyerowitz EM (1995) UFO: an Arabidopsis gene involved in both floral meristem and floral organ development. Plant Cell 7: 529-548[Abstract]
Ma H, Yanofsky MF, Meyerowitz EM (1991) AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes. Genes Dev 5: 484-495
McGinnis KM, Thomas SG, Soule JD, Strader LC, Zale JM, Sun TP, Steber CM (2003) The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase. Plant Cell 15: 1120-1130 Mizoguchi T, Coupland G (2000) ZEITLUPE and FKF1: novel connections between flowering time and circadian clock control. Trends Plant Sci 5: 409-411[Medline] Moffatt BA, McWhinnie EA, Agarwal SK, Schaff DA (1994) The adenine phosphoribosyltransferase-encoding gene of Arabidopsis thaliana. Gene 143: 211-216[CrossRef][Web of Science][Medline] Nayak S, Santiago FE, Jin H, Lin D, Schedl T, Kipreos ET (2002) The Caenorhabditis elegans Skp1-related gene family: diverse functions in cell proliferation, morphogenesis, and meiosis. Curr Biol 12: 277-287[CrossRef][Web of Science][Medline] Olszewski N, Sun TP, Gubler F (2002) Gibberellin signaling: biosynthesis, catabolism, and response pathways. Plant Cell Suppl 14: S61-S80 Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503-533[CrossRef][Web of Science][Medline] Porat R, Lu P, O'Neill SD (1998) Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells. Planta 204: 345-351[CrossRef][Web of Science][Medline]
Ruegger M, Dewey E, Gray WM, Hobbie L, Turner J, Estelle M (1998) The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast grr1p. Genes Dev 12: 198-207 Samach A, Klenz JE, Kohalmi SE, Risseeuw E, Haughn GW, Crosby WL (1999) The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem. Plant J 20: 433-445[CrossRef][Web of Science][Medline]
Sasaki A, Itoh H, Gomi K, Ueguchi-Tanaka M, Ishiyama K, Kobayashi M, Jeong DH, An G, Kitano H, Ashikari M et al. (2003) Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant. Science 299: 1896-1898 Schulman BA, Carrano AC, Jeffrey PD, Bowen Z, Kinnucan ER, Finnin MS, Elledge SJ, Harper JW, Pagano M, Pavletich NP (2000) Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex. Nature 408: 381-386[CrossRef][Medline]
Schwechheimer C, Serino G, Callis J, Crosby WL, Lyapina S, Deshaies RJ, Gray WM, Estelle M, Deng XW (2001) Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response. Science 292: 1379-1382
Seol JH, Feldman RM, Zachariae W, Shevchenko A, Correll CC, Lyapina S, Chi Y, Galova M, Claypool J, Sandmeyer S et al. (1999) Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev 13: 1614-1626
Shen WH, Parmentier Y, Hellmann H, Lechner E, Dong A, Masson J, Granier F, Lepiniec L, Estelle M, Genschik P (2002) Null mutation of AtCUL1 causes arrest in early embryogenesis in Arabidopsis. Mol Biol Cell 13: 1916-1928 Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW (1997) F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91: 209-219[CrossRef][Web of Science][Medline] Somers DE, Schultz TF, Milnamow M, Kay SA (2000) ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis. Cell 101: 319-329[CrossRef][Web of Science][Medline]
Steber CM, Cooney SE, McCourt P (1998) Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1 in Arabidopsis thaliana. Genetics 149: 509-521 Strohmaier H, Spruck CH, Kaiser P, Won KA, Sangfelt O, Reed SI (2001) Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413: 316-322[CrossRef][Medline]
Willems AR, Goh T, Taylor L, Chernushevich I, Shevchenko A, Tyers M (1999) SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis. Philos Trans R Soc Lond B Biol Sci 354: 1533-1550 Winston JT, Koepp DM, Zhu C, Elledge SJ, Harper JW (1999) A family of mammalian F-box proteins. Curr Biol 9: 1180-1182[CrossRef][Web of Science][Medline]
Woo HR, Chung KM, Park JH, Oh SA, Ahn T, Hong SH, Jang SK, Nam HG (2001) ORE9, an F-box protein that regulates leaf senescence in Arabidopsis. Plant Cell 13: 1779-1790
Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280: 1091-1094
Xu L, Liu F, Lechner E, Genschik P, Crosby WL, Ma H, Peng W, Huang D, Xie D (2002) The SCFCOI1 ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. Plant Cell 14: 1919-1935 Yamanaka A, Yada M, Imaki H, Koga M, Ohshima Y, Nakayama KI (2002) Multiple Skp1-related proteins in Caenorhabditis elegans: diverse patterns of interaction with Cullins and F-Box proteins. Curr Biol 12: 267-275[CrossRef][Web of Science][Medline]
Yang M, Hu Y, Lodhi M, McCombie WR, Ma H (1999) The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation. Proc Natl Acad Sci USA 96: 11416-11421 Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann KA, MeyerowitzEM (1990) The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346: 35-39[CrossRef][Medline]
Yu ZK, Gervais JL, Zhang H (1998) Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins. Proc Natl Acad Sci USA 95: 11324-11329 Zhang H, Kobayashi R, Galaktionov K, Beach D (1995) p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell 82: 915-925[CrossRef][Web of Science][Medline] Zhao D, Yang M, Solava J, Ma H (1999) The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis. Dev Genet 25: 209-223[CrossRef][Web of Science][Medline] Zhao D, Yu Q, Chen M, Ma H (2001) The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis. Development 128: 2735-2746 Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C, Koepp DM, Elledge SJ, Pagano M et al. (2002) Structure of the Cul1-Rbx1-Skp1-F box Skp2 SCF ubiquitin ligase complex. Nature 416: 703-709[CrossRef][Medline] This article has been cited by other articles:
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ABSTRACT
RESULTS
-glucuronidase fragment flanked by AscI and BamHI restriction sites in pFGC1008 with the first intron of the Arabidopsis GPA1 gene. To generate the ASK1 RNAi construct, the ASK1 coding region was amplified from the ASK1 cDNA with primers oMC529 (5'-TCACTAGTGAGCTCATAACCATGTCTGCGAAGAA-3') and oMC 530 (5'-TCGGATCCGGCGCGCCGATAGTCATGATTCATGAAG-3'; an AscI site is underlined). The PCR product was cloned into the pCRII-TOPO vector (Invitrogen), resulting in the pMC2550 plasmid. The cloned ASK1 cDNA was then digested with XhoI (in ASK1 coding region) and AscI (introduced by oMC530) and was cloned into the XhoI and AscI sites of pRR2222 in the sense orientation. Finally, pMC2550 was digested with BamHI (introduced by oMC530) and XbaI (from pCRII-TOPO) and cloned in the antisense orientation into the BamHI and SpeI site of the pRR2222-derived plasmid with the sense ASK1 fragment to produce pMC2570 (
