The Plant Journal (2015) 81, 183–197

doi: 10.1111/tpj.12721

CLUMSY VEIN, the Arabidopsis DEAH-box Prp16 ortholog, is required for auxin-mediated development Ryuji Tsugeki1,*, Nana Tanaka-Sato1, Nozomi Maruyama1, Shiho Terada1, Mikiko Kojima2, Hitoshi Sakakibara2 and Kiyotaka Okada3,† 1 Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, 2 RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan, and 3 Laboratory of Plant Organ Development, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan Received 6 January 2014; revised 29 October 2014; accepted 31 October 2014; published online 11 November 2014. *For correspondence (e-mail [email protected]). † Present address:National Institutes of Natural Sciences, Toranomon 4-3-13, Minato-ku, Tokyo 105-0001, Japan.

SUMMARY Pre-messenger RNA (pre-mRNA) splicing is essential in eukaryotic cells. In animals and yeasts, the DEAHbox RNA-dependent ATPase Prp16 mediates conformational change of the spliceosome, thereby facilitating pre-mRNA splicing. In yeasts, Prp16 also plays an important role in splicing fidelity. Conversely, PRP16 orthologs in Chlamydomonas reinhardtii and nematode do not have an important role in general pre-mRNA splicing, but are required for gene silencing and sex determination, respectively. Functions of PRP16 orthologs in higher plants have not been described until now. Here we show that the CLUMSY VEIN (CUV) gene encoding the unique Prp16 ortholog in Arabidopsis thaliana facilitates auxin-mediated development including male-gametophyte transmission, apical–basal patterning of embryonic and gynoecium development, stamen development, phyllotactic flower positioning, and vascular development. cuv-1 mutation differentially affects splicing and expression of genes involved in auxin biosynthesis, polar auxin transport, auxin perception and auxin signaling. The cuv-1 mutation does not have an equal influence on pre-mRNA substrates. We propose that Arabidopsis PRP16/CUV differentially facilitates expression of genes, which include genes involved in auxin biosynthesis, transport, perception and signaling, thereby collectively influencing auxin-mediated development. Keywords: pre-mRNA-processing factor 16, vascular formation, organ development, auxin, pre-messenger RNA splicing, Arabidopsis thaliana.

INTRODUCTION Pre-messenger RNA (pre-mRNA) splicing is essential in eukaryotic cells not only for expression of intron-containing genes, but also for its contribution to proteomic diversity (Nilsen and Graveley, 2010) and for remodeling protein– protein interaction networks (Buljan et al., 2012; Ellis et al., 2012). Splicing may also indirectly control the abundance of transcripts (McGlincy and Smith, 2008; Filichkin et al., 2010; Kalyna et al., 2012). It has been considered that the spliceosome interacts nonspecifically with all intron-containing transcripts. However, recent studies have indicated that conserved splicing components can function as splicing regulators. Alteration in the concentrations of certain core components of the spliceosome specifically modulates alternative splicing (Park et al., 2004; Pleiss et al., 2007). In human, mutations © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd

in ubiquitously expressed genes encoding pre-mRNA splicing factors cause highly tissue-specific pathologies with no obvious phenotypes in other cell types (Mordes et al., 2006; Padgett, 2012). In Arabidopsis, evolutionarily conserved splicing factors influence alternative splicing (Sugliani et al., 2010; Ohtani et al., 2013). These facts imply that transcript-specific changes in splicing can occur when the activity of certain core component(s) of the spliceosome is modulated. Here, we describe the CLUMSY VEIN (CUV) gene of Arabidopsis thaliana. It encodes an ortholog of the DEAH-box RNA-dependent ATPase pre-mRNA-processing factor 16 (Prp16), which is conserved in eukaryotic cells. Pre-mRNA splicing proceeds through two sequential catalytic steps: cleavage of the 50 splice site followed by exon 183

184 Ryuji Tsugeki et al. ligation. In yeast and human, Prp16 mediates the firstto-second step transition of splicing (Schwer and Guthrie, 1991; Zhou and Reed, 1998). In yeast, PRP16 is essential for viability (Burgess et al., 1990). However, in Chlamydomonas (Wu-Scharf et al., 2000) and nematode (Puoti and Kimble, 1999), PRP16 orthologs are not generally required for splicing, but are involved in gene silencing and in sex determination, respectively. In higher plants, there have been no reports thus far on the function of Prp16 orthologs. We show here that Arabidopsis cuv mutations influence auxin-mediated development. The cuv-1 mutation differentially affects splicing and expression of genes involved in auxin-mediated development. It appears that the cuv-1 mutation does not have an equal effect on all pre-mRNA splicing events. We propose that CUV differentially facilitates expression of genes including genes involved in auxin biosynthesis, transport, perception and signaling, thereby causing concerted influences on auxin-mediated development. RESULTS

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CUV influences vascular development

Figure 1. cuv-1 is defective in leaf vascular development. (a–c) Leaf-venation pattern. The first or second rosette leaves of 20-day-old wild type (a), cuv-1 (b), and cuv-1/cuv-2 transheterozygote (c). See also Figure S1. (d–f) Cotyledon-venation pattern of wild type (d), cuv-1 (e) and gnomR5 (f). (g–j) Expression of provascular markers J1511–GFP (g, h) and ATHB8pro: GUS (i, j) in wild type (g, i) and cuv-1 (h, j). ATHB8, ATHB8pro:GUS; c-1/c-2, cuv-1/cuv-2 transheterozygote; cot, cotyledon; wt, wild type. Scale bars: 1 mm in (a–f), 20 lm in (g, h) and 50 lm in (i, j).

cuv-1 was identified as a nuclear, recessive mutant showing narrow rosette leaves with altered venation pattern. Whereas the wild-type Arabidopsis leaf has a closed, reticulate venation pattern (Figure 1a), the cuv-1 leaf tends to have an open venation pattern with markedly fewer tertiary and higher-order veins (Figure 1b and Figure S1). The cuv mutant heteroallelic for cuv-1 with cuv-2 (cuv-1/cuv-2) exhibited even more severe defects in leaf-venation pattern (Figure 1c). Expression of provascular markers J1511-GFP (Figure 1g; Tsugeki et al., 2009) and ARABIDOPSIS THALIANA HOMEOBOX 8 (ATHB8) (Figure 1i; Baima et al., 1995) was examined in cuv-1. In cuv-1 leaf primordia, the formation of provascular tissues for secondary veins and of secondary-vein loops was reduced (Figure 1h, j and Table S1). Auxin positively regulates vascular formation. During leaf development, auxin/indole-3-acetic acid-auxin response factor (Aux/IAA-ARF)-dependent transcription from the DR5 promoter is detected at the apical ends of midvein provascular cells (Tsugeki et al., 2009; Figure 2a, c) and in provascular cells (Mattsson et al., 2003; Figure 2c). In cuv-1, the DR5-expression domain was expanded in the apical region of the young leaf primordia (Figure 2b). The isolated epidermal auxin maxima, which are never seen in wild type throughout leaf development (Mattsson et al., 2003; Figure 2a, c), were found

in cuv-1 leaf primordia (Figure 2d). It appears that cuv-1 often failed to form the looped DR5-expression domain for the secondary vein (Figure 2d). DR5 expression and formation of elongated provascular cells were examined for the secondary-vein loop. We considered that the secondary-vein provascular cells were connected to the midvein provascular cells if there was a distal loop of the DR5-expression domain or a distal loop of elongated provascular cells, in which DR5 expression might be decreased below the detection level. The distal connection was formed less in cuv-1 (9 of 23) than in the wild type (14 of 14). In the cotyledon (Figure 1d–f), sepal (Figure S2a, e) and gynoecium (Figure 5i, j), increased formation of veins was detected in cuv-1. In cuv-1 cotyledons, ectopic veins were formed inside the areole (the area completely bounded by veins) (Figure 1e and Table S2). Similar ectopic vein formation occurs when polar auxin transport is compromised (Geldner et al., 2004; Figure 1f). In cuv-1, more xylem strands were formed in the hypocotyl (Figure 2e–g, i–k), leaf petiole (Figure 1a–c), and flower pedicel (Figure S2c, g). Ectopic provascular DR5 expression was detected in the developing gynoecium (Figure 5g, h) and at the base of the sepal vascular tissue and along the vascular tissue in the

Here, Arabidopsis PRP16 was identified as the CUV gene influencing auxin-mediated development. We first describe developmental defects in cuv mutants and then show effects of cuv-1 mutation on splicing and expression of genes involved in auxin-mediated development.

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2015), 81, 183–197

A role of Prp16 in Arabidopsis 185

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Figure 2. cuv-1 is defective in auxin response associated with vascular development in leaf primordia and hypocotyls. (a–d) Expression of DR5pro:GUS in leaf primordia of wild type (a, c) and cuv1 (b, d). (e–g, i–k) Xylem strand formation in hypocotyls of wild type (e–g) and cuv-1 (i–k). Shown in (f, g) and (j, k) are close-up views of (e) for wild type and (i) for cuv-1, respectively. Close-up points are indicated by arrows in (e) and (i). More xylem strands were detected in cuv-1 hypocotyls. (h, l) Expression of BApro:GUS in hypocotyls of wild type (h) and cuv-1 (l). BA, BApro:GUS; DR5, DR5pro:GUS; hyp, hypocotyl; wt, wild type. Scale bars: 50 lm in (a–d), 100 lm in (e, h, i, l), and 20 lm in (f, g, j, k).

pedicel (Figure S2d, h). The auxin-responsive BA promoter (Oono et al., 1998, 2002), whose expression in the hypocotyl vascular tissue was increased by exogenously added IAA (Figure S3), was used to examine auxin response in the cuv-1 hypocotyl vascular tissue. Increased auxin response was observed in the cuv-1 hypocotyl vascular tissue (Figure 2h, l). These results suggest that CUV is required for proper distribution of auxin-response maxima associated with vascular formation in these tissues. Genetic interaction of CUV with AUXIN RESISTANT 2 (AXR2), AXR3 and SOLITARY ROOT (SLR) To further investigate the role of CUV in auxin-mediated development, genetic interaction between the CUV and Aux/IAA genes was analyzed (Figure 3a–h). Aux/IAA genes negatively regulate auxin signaling (Reed, 2001; Leyser, 2002). Gain-of-function mutations of Aux/IAA genes, iaa7/ axr2-1 (Wilson et al., 1990; Masucci and Schiefelbein, 1996; Nagpal et al., 2000), iaa14/slr-1 (Fukaki et al., 2002) and iaa17/axr3-3 (Leyser et al., 1996; Rouse et al., 1998), result in very few root hairs as a low-auxin-response phenotype

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Figure 3. CUV influences root-hair development and auxin response in roots. (a–h) Root tips in wild type (a), axr2-1 (b), axr3-3 (c), slr-1 (d), cuv-1 (e), cuv1 axr2-1 (f), cuv-1 axr3-3 (g) and cuv-1 slr-1 (h). cuv-1 suppressed the roothair phenotype in axr2-1 (12/12), axr3-3 (13/18) and slr-1 (45/45). (i–p) Expression of BApro:GUS in roots of wild type (i–l) and cuv-1 (m–p). Seedlings treated with 0 (i, j, m, n), 10 (k, o), or 100 nM (l, p) IAA were subjected to GUS staining. For better clarity, samples in (j–l, n–p) were GUS stained for a shorter time than those in (i, m). BA, BApro:GUS; wt, wild type. Scale bars: 200 lm in (a–h) and 100 lm in (i– p).

(Figure 3b–d). The cuv-1 mutation suppressed the roothairless phenotype in axr2-1 (12 of 12 cuv-1 axr2-1 examined), slr-1 (45 of 45 cuv-1 slr-1 examined) and axr3-3 (13 of 18 cuv-1 axr3-3 examined) (Figure 3f–h). These results indicate that CUV influences auxin-mediated root-hair outgrowth. In cuv-1, BA expression was increased in the vascular tissue around the root differentiation zone (Figure 3i, m). In response to exogenous application of IAA, BA expression was increased more markedly in cuv-1 (Figure 3n–p) than in wild type (Figure 3j–l). These results further support the idea that CUV influences auxin responses. Expression and subcellular localization of PIN-FORMED (PIN) proteins are altered in cuv-1 roots Efflux-dependent polar auxin transport plays a central role in the formation of auxin gradients and maxima (Petra
sek et al., 2006; Wisniewska et al., 2006). In cuv-1 roots, expression of PIN1 and PIN7 proteins was reduced in their respective expression domains (Figure 4).

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2015), 81, 183–197

186 Ryuji Tsugeki et al.

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Figure 4. Expression of PIN1 and PIN7 proteins is decreased in cuv-1 roots. (a, b) Expression of PIN1pro:PIN1:GFP in wild-type (a) and cuv-1 (b) root tips. (c, d) Expression of PIN7pro:PIN7:GFP in wild-type (c) and cuv-1 (d) root tips. PIN1:GFP, PIN1pro:PIN1:GFP; PIN7:GFP, PIN7pro:PIN7:GFP; wt, wild type. Scale bars: 10 lm in (a, b) and 20 lm in (c, d).

(i) In wild-type endodermis cells, most PIN1 is found on the basal side, with some spreading to the inner-lateral side (Sauer et al., 2006; Figure S4a). Auxin induces relocation of PIN1 to the inner-lateral side (Sauer et al., 2006; Figure S4d). In cuv-1 endodermal cells, PIN1 was localized relatively more on the inner-lateral side (Figure S4b, c). Treatment of cuv-1 with exogenous auxin enhanced the innerlateral localization of PIN1 (Figure S4e). These observations suggest that CUV is required for the expression of PIN1 and PIN7 proteins and for proper subcellular localization of PIN1 in roots. CUV is required for flower development Apical–basal axis-dependent development of the gynoecium is mediated by the apical–basal auxin gradient, with maximum in the apical area of the gynoecium (Nemhauser et al., 2000; Cheng et al., 2006). The cuv-1 gynoecium exhibited an apical–basal patterning defect, having a shorter valve, longer style and longer gynophore (Figure 5a–d). Ectopic auxin-response maxima were detected in the apical domain of the cuv-1 gynoecium (Figure 5g, h). Formation of the interthecal furrow on the anther of stamens was defective in cuv-1 (Figure 5e, f). A similar phenotype was reported for a mutation in the ARF gene (Sessions et al., 1997). The DR5 expression level in the anther was lower in cuv-1 than in wild type (Figure 5g, h). The spiral phyllotaxy for flowers was also disturbed in cuv-1 (Figure S5).

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Figure 5. Flower phenotype in cuv-1. (a-d) Gynoecia in wild type: the left side in (a) and (b); and cuv-1: the right side in (a, c, d). Shown in (b) for wild type) and (c) for cuv-1 are close-up views of the apical and basal domains of wild-type and cuv-1 gynoecia in (a). White and black arrowheads in (b, c) indicate the apical and basal boundaries of the style and gynophore, respectively. (e, f) Adaxial views of wild-type (e) and cuv-1 (f) anthers. In the wild-type anther, two pairs of locules are separated by the interthecal furrow; white arrowheads in (e). In cuv-1, formation of the interthecal furrow is defective (f). (g, h) Expression of DR5pro:GUS in developing flowers of wild type (g) and cuv-1 (h). The white arrow in (h) indicates ectopic auxin-response maxima in the apical domain of the cuv-1 gynoecium. (i, j) Venation pattern of the wild-type (i) and cuv-1 (j) gynoecia. DR5, DR5pro:GUS; wt, wild type. Scale bars: 1 mm in (a–d, i, j), and 200 lm in (e–h).

CUV is required for male-gametophytic transmission and embryonic development Plants homozygous for the cuv T-DNA alleles were not found among the progeny of the heterozygous plants. We therefore analyzed the gametophytic (Table 1) and embryonic phenotype in cuv-2 and cuv-3 (Figure 6). Whereas transmission of the cuv-2 allele through the female gametophyte was normal, cuv-2 transmission through the male

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2015), 81, 183–197

A role of Prp16 in Arabidopsis 187 Table 1 Male gametophyte transmission is defective in cuv-2 Parental genotype

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cuv-3 T-DNA transmission (%)

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46.6 40.9

Whereas transmission of the cuv-2 allele through female gametophytes is normal (49.4%, n = 77), cuv-2 transmission through male gametophytes is significantly reduced (7%, n = 128, P = 2.9 9 1012, chi-squared test). There was no significant reduction in cuv-3 transmission either through female (46.6%, n = 88) or male (40.9%, n = 93) gametophyte (P = 0.44, chi-squared test). All the tested samples were first confirmed to be F1 hybrids from the reciprocal crosses between wild type and cuv heterozygote.

gametophyte was significantly reduced (Table 1). It has been shown that auxin transport has an important role in male-gametophyte transmission (Ding et al., 2012). The cuv-2 and cuv-3 homozygotes were also embryonic lethal, arresting embryonic development around the preglobular stage in cuv-2 (Figure 6d) and heart to early torpedo stages in cuv-3 (Figure 6f). The apical–basal axis-dependent celldivision patterns (Figure 6a, c, e) were disrupted in cuv-2 and cuv-3 embryos (Figure 6b, d, f). In cuv-2 embryos, ectopic expression of DR5 was detected not only in embryonic cells but also in suspensor cells, suggesting that the spatial pattern of auxin response was disrupted in cuv-2 embryos (Figure 6g–i). CUV encodes an ortholog of DEAH-box RNA-dependent ATPase Prp16 The CUV gene (At5g13010) was identified by map-based cloning. Stronger T-DNA insertion alleles cuv-2 and cuv-3 were identified (Figure S6a), and cuv-2 was confirmed to be allelic to cuv-1 (Figure 1c). Introduction of a genomic fragment containing At5g13010 complemented cuv-1 and cuv-2 (Figure S7). CUV encodes a protein of a predicted 1255 amino acids. The deduced amino acid sequence of CUV protein is highly similar to those of the Prp16 proteins conserved in eukaryotes (Figure S8). In animals and yeast, Prp16 facilitates pre-mRNA splicing by mediating structural transitions of the spliceosome during the catalytic steps (Schwer and Guthrie, 1991; Zhou and Reed, 1998). In yeast, Prp16 also mediates kinetic proofreading of splice site usage in the first catalytic step (Burgess and Guthrie, 1993; Koodathingal et al., 2010; Horowitz, 2011; Tseng et al., 2011). CUV contains domains that are highly conserved among Prp16 homologs: the DEAH-box helicase N-terminal domain, helicase C-terminal domain, helicase-associated domain 2 (HA2), and domain of unknown function 1605 (DUF1605) (Figures S6b and S8). The cuv-1 mutation causes substitution of a glycine residue by arginine at the 974th amino acid from the N-terminus, which is located in

the HA2 domain (Figures S6 and S8). The corresponding glycine residue is conserved in 459 out of 463 HA2 sequences in the database, suggesting that this glycine residue is important for HA2 function. It is noteworthy that CUV is the only gene that encodes a Prp16 homolog in Arabidopsis. CUV is a nuclear protein preferentially expressed in developing organs The deduced amino acid sequence of CUV protein includes three potential nuclear localization signals: RHREEHRRDR (144–153), RRRESYRQSDRDYHGEKRRR (172–191), and EKRRRY (187–192) (https://www.predictprotein.org/; Rost et al., 2003). In fact, CUV was specifically localized in the nucleus (Figure 7a). CUV expression was analyzed in CUVpro:CUV:GUS and CUVpro:CUV:GFP plant lines (Figure 7). In embryos (Figure 7b–e), CUV was expressed in both the embryo and suspensor. In seedlings (Figure 7f–k), CUV was strongly expressed in developing leaf primordia, meristematic regions in shoots and roots, and developing vascular tissues. CUV was expressed in floral meristem, developing floral organ primordia and developing ovules (Figure 7l–q). CUV expression was also detected in developing and germinating pollens (Figure 7r–t). In the developing gynoecium, CUV transiently exhibited a graded expression pattern along the apical–basal axis, with highest expression of CUV on the apical side (Figure 7n). We tested whether auxin influences CUV expression. Exogenous application of auxin did not change the amount of CUV mRNA in wild-type seedlings (Figure S9). cuv-1 differentially influences expression of genes involved in auxin biosynthesis, transport, perception and signaling We then investigated whether CUV was involved in expression and splicing of genes. We basically used two types of primer pairs for quantitative RT-PCR (qRT-PCR) analyses:

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2015), 81, 183–197

188 Ryuji Tsugeki et al.

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Figure 6. Embryonic phenotype in cuv-2 and cuv-3. (a–f) Embryos of wild type (a, c, e), cuv-2 (b, d), and cuv-3 (f). The first division plane of the apical cell in (a, b) is marked by a magenta line. In embryos in (c, d), division planes formed earlier and later are marked by magenta and blue lines, respectively. Yellow lines in (d) indicate ectopic vertical divisions in the cuv-2 suspensor. Schematic views of embryos are shown above each panel in (a–d). (g–i) Expression of DR5pro:GFP in wild-type (g) and cuv-2 (h, i) embryos. In cuv-2 embryos, ectopic expression of DR5 was detected in the suspensor and embryos. wt, wild type; DR5, DR5pro:GFP. Scale is the same in (a–f). Scale bars: 20 lm in (f), and 10 lm in (g–i).

one pair that would detect only spliced transcripts and the other that would detect both unspliced and spliced transcripts (Table S5), which are hereafter referred to as total transcripts. We first examined the splicing of ubiquitously expressed genes that are often used as controls for transcriptome analysis: ACT7, TUA4, UBP3, VPS41, AP2M, ATKU70, MAP2B and SAR2 (Figure 8a, Figures S10 and S11). Except for AP2M expression in flowers, there was no statistically significant difference in the amount of spliced or of total transcripts between wild type and cuv-1. Ubiquitously expressed intronless genes were also examined for their expression in cuv-1. The expression of the intronless genes examined was not significantly changed in cuv-1 seedlings or flowers (Figure S12).

Next, we examined transcripts of genes involved in auxin biosynthesis, auxin homeostasis, polar auxin transport, auxin perception and auxin response in cuv-1. Because it is the amounts of spliced transcripts that have an actual impact on development, we first examined the expression of spliced transcripts and then analyzed the total transcripts for genes with significant reduction in levels of spliced transcripts. If pre-mRNA splicing is defective, one possible scenario is that the levels of spliced transcripts are reduced, whereas the levels of the unspliced precursor transcripts are correspondingly increased, so that there is no significant difference in the levels of total transcripts between wild type and cuv-1. However, it must be noted that, as described in the Discussion, even if splicing is impaired, a reduction in levels of spliced transcripts is not necessarily accompanied by a corresponding accumulation of the unspliced transcript. It has been known that unspliced transcripts may be degraded (Bousquet-Antonelli et al., 2000; Mitchell and Tollervey, 2000; Chekanova et al., 2007; Kim et al., 2009; Kalyna et al., 2012). Genes involved in auxin biosynthesis. We analyzed the expression of TAA, TAR and YUC genes, which encode key enzymes in the main auxin biosynthesis pathway in Arabidopsis (Stepanova et al., 2008; Tao et al., 2008; Mashiguchi et al., 2011; Won et al., 2011), as well as that of other genes that have been considered to contribute to auxin biosynthesis (Figure S21). In cuv-1, expression of spliced transcripts for TAA1, TAR2, YUC2, YUC3, YUC6, YUC9 and YUC11 was decreased either in flowers or in both flowers and seedlings (Figures 8b, 9a and Figure S13 and Table S3). We found that expression of total transcripts for TAA1, TAR2, YUC2, YUC3, YUC6 and YUC9 was also decreased when their spliced transcripts were decreased (Figure 9a). Conversely, expression of spliced transcripts for YUC1 and YUC7 and of intronless YUC5 and YUC8 was not significantly changed in cuv-1 (Figure 9b and Figure S14 and Table S3). In our experiments, expression of TAR1 and YUC10 was not detected. Expression of YUC11 was also not detected in seedlings (Figure S13). YUC4 expression is regulated by alternative splicing, which generates two splice variants, YUC4.1 and YUC4.2 (Kriechbaumer et al., 2012; Figure 10a). YUC4.1 mRNA contains four exons. In the YUC4.2 transcript, the fourth exon is replaced by 72 bp of the third intron (Kriechbaumer et al., 2012). In cuv-1, the amount of YUC4.1 mRNA was decreased in seedlings and cauline leaves, and not much changed in flowers (Figure 10b, upper panel). In contrast, the amount of YUC4.2 mRNA was increased in seedlings and cauline leaves (Figure 10b, lower panel). Conversely, the total amount of YUC4 transcripts was not very different between cuv-1 and wild type (Figure 10c). These results suggest that CUV influences alternative splicing of YUC4.

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2015), 81, 183–197

A role of Prp16 in Arabidopsis 189 Figure 7. Nuclear localization of CUV and expression of CUV in embryos, seedlings and developing flowers. (a) Nuclear localization of CUV:GFP. The root epidermis of DAPI-stained CUVpro:CUV:GFP seedlings was examined. GFP and DAPI fluorescence in the same focal point are shown in the upper and middle panels, respectively. The merged image is shown in the bottom panel. Scale bar = 5 lm. (b–e) Expression of CUVpro:CUV:GFP in the embryo. CUV:GFP was expressed in the embryo (emb) and suspensor (sus). Scale bars: 10 lm in (b–d), 20 lm in (e). (f–k) Expression of CUVpro:CUV:GUS and CUVpro:CUV:GFP in seedlings grown for 4 days (4d in f–h), 5 days (5d in j, k) or 7 days (7d in i). cot, cotyledon; hyp, hypocotyl; rt, root; lfp, leaf primordium; sm, shoot apical meristem; stp, stipule. Scale bars: 1 mm in (f), 100 lm in (g–i), 10 lm in (j), 20 lm in (k). (l–t) Expression of CUVpro:CUV:GUS and CUVpro:CUV:GFP in developing floral organs and pollens. The image in (r) is a close-up view of an anther in (p). Shown in (q), (s) and (t) are elongating stamens, pollinated stigma and germinated pollen attached to the stigma in (s), respectively. The white arrow in (t) indicates an emerging pollen tube. fm, floral meristem; gy, gynoecium; pt, petal; sep, sepal; st, stamen. Scale bars: 10 lm in (l), 100 lm in (m, n, p–s), 20 lm in (o), 50 lm in (t).

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Defects in expression of the TAA and YUC genes may change the amount of auxin. We examined the amount of auxin in cuv-1 seedlings. The free IAA level in cuv-1 was not significantly different from that in wild type (Table S4). Conversely, the level of the IAA-amino acid conjugate indole-3-acetyl-aspartic acid (IAA-Asp) was significantly increased in cuv-1 seedlings (Table S4). IAA-Asp is produced by IAA-amido synthetase. None of the genes encoding IAA-amido synthetase (Staswick et al., 2005) exhibited increased expression of spliced transcripts in cuv-1 seedlings (Figure S15 and Table S3).

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Genes involved in polar auxin transport. PIN genes except for PIN5 exhibited a decreased expression of spliced mRNA either in flowers or seedlings or in both flowers and seedlings (Figures 8c, 9 and Figure S16 and Table S3). In cuv-1 flowers, the amounts of spliced and total transcripts of PIN2, PIN3, PIN4, PIN7 and PIN8 were both decreased (Figure 9a and Table S3). In cuv-1 seedlings, the amount of spliced and total transcripts of PIN4 was both decreased (Figure 9a and Table S3). However, for PIN1 and PIN2 transcripts in cuv-1 seedlings, whereas the amount of their spliced transcripts was decreased, their

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2015), 81, 183–197

190 Ryuji Tsugeki et al. Figure 8. The preferential effect of cuv-1 mutation on pre-mRNA splicing. (a) Analysis of the expression and splicing of ACT7, TUA4, UBP3 and VPS41. If not spliced, transcripts include intervening introns: 4th intron for ACT7; 2nd intron for TUA4; 5th intron for UBP3; 18th intron for VPS41. Small white arrowheads indicate bands derived from spliced transcripts. See also Figures S10 and S11 and Table S3. (b) Upper schema represents simplified structure of YUC3 gene. Horizontal bold lines indicate three exons, of which black and grey parts are coding and non-coding regions, respectively. Introns are indicated by bent narrow lines between two adjoining exons. Primers used for RT-PCR analysis are schematically indicated at their annealing sites. Primers F1, R1, F2, R2, F-i1 and R-i2 are YUC3-F2, YUC3-R3, YUC3-F3, YUC3-R2, YUC3-F4 and YUC3-R4 in Table S5, respectively. Lower panels show RTPCR analysis of the expression and splicing of YUC3. If transcripts are not spliced, the primer combinations F1R1 and F2R2 produce bands including the intervening first and second introns, respectively. Using the primer combination F-i1R-i2, DNA is amplified only from the spliced transcript. See also Figure 9 and Table S3. (c) RT-PCR analysis of the expression and splicing of PIN6. The indicated introns beneath each panel are the intervening introns in the amplified DNA, respectively, if not spliced. F, flowers; S, seedlings; c-1, cuv-1; wt, wild type; gDNA, total genomic DNA.

(a)

(b)

(c)

total transcripts were not significantly decreased (Figure 9a and Table S3), suggesting that pre-mRNA splicing of PIN1 and PIN2 transcripts was defective in cuv-1 seedlings. Genes involved in auxin perception and signaling. Expression of genes that encode auxin receptor proteins TIR1, AFB1, AFB2 and AFB3 (Dharmasiri et al., 2005a, b; Kepinski and Leyser, 2005) was examined (Figures 9 and S17 and Table S3). A decrease in expression of spliced mRNA was detected only for AFB1 in seedlings and

flowers, and for AFB2 in seedlings (Figure 9a and Table S3). A decrease in expression of total transcripts was also detected for AFB1 in flowers, but not for AFB1 and AFB2 in seedlings (Figure 9a and Table S3), suggesting that splicing of AFB1 and AFB2 transcripts was defective in cuv-1 seedlings. We examined the expression of Aux/IAA genes: SHY2, AXR2, BDL, IAA13, SLR, AXR3, IAA30 and ARF genes: ETT, MP, NPH4 (Figure 9 and Figures S18–S20 and Table S3). Decreased levels of spliced mRNA were found for SHY2,

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A role of Prp16 in Arabidopsis 191 Figure 9. Expression analysis of genes involved in auxin biosynthesis, transport, perception and response in cuv-1. (a) Expression analysis of genes involved in auxin biosynthesis, transport, perception and response that exhibited reduced expression in cuv-1. Relative expression levels (cuv-1/wt) of spliced transcripts (light grey bars) and total transcripts (dark grey bars) were measured by qRT-PCR. Genes that exhibit a more than twofold decrease in expression of spliced transcripts are marked by hash marks (#). Boxed gene names indicate genes that show that levels of their spliced transcripts are decreased, whereas levels of their total transcripts are not decreased. (b) Relative expression levels (cuv-1/wild type) of genes involved in auxin biosynthesis, transport, perception and response that did not exhibit reduced expression of spliced transcripts in cuv-1. Spliced transcripts were measured by qRT-PCR. In cuv-1, levels of spliced transcripts of PIN5 and YUC2 were increased in flowers and in seedlings, respectively. Expression levels of the genes were normalized to those of ACT7. Primer pairs used are listed in Table S5. Data of qRT-PCR are also shown in Table S3. wt, wild type. Asterisks indicate P-values: *P < 0.05; **P < 0.01; ***P < 0.001.

(a)

(b)

AXR2, IAA13, SLR, AXR3, MP and NPH4 in flowers and for SHY2, IAA13, and SLR in seedlings, but not for BDL, IAA30 or ETT either in flowers or seedlings (Figure 9 and Table S3). A decrease in expression of total transcripts was also detected for AXR2, IAA13, SLR and MP in flowers and for

IAA13 in seedlings, but not for SHY2, AXR3 and NPH4 in flowers and for SHY2 and SLR in seedlings (Figure 9 and Table S3). These facts suggest that cuv-1 is defective in pre-mRNA splicing of transcripts for SHY2, AXR3 and NPH4 in flowers and for SHY2 and SLR in seedlings.

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192 Ryuji Tsugeki et al. Figure 10. RT-PCR analysis of the expression and splicing of YUC4 in cuv-1. (a) Schematic structures of YUC4 splice variants YUC4.1 and YUC4.2. Bold and narrow lines represent exons and introns, respectively. Grey regions of rightmost exons represent 30 noncoding regions. Primers used for RT-PCR are indicated at their annealing sites. Primers 4.1R and 4.2R are specific to YUC4.1 and YUC4.2, respectively. (b) RT-PCR analysis of the expression and splicing of YUC4.1 and YUC4.2. Total RNA isolated from flowers, seedlings and cauline leaves was subjected to RT-PCR analysis using primer combinations specific to YUC4.1 or YUC4.2. (c) RT-PCR analysis of the expression and splicing of YUC4. Primers F and R indicated in (a) were used for RT-PCR analysis. Amplified DNA is derived from transcripts for both YUC4 isoforms, YUC4.1 and YUC4.2. wt, wild type; gDNA, total genomic DNA.

(a)

(b)

(c)

Among 64 genes examined by qRT-PCR in this study, a more than two-fold decrease in expression of mRNA was detected only for YUC2, YUC3, YUC6, YUC9, YUC11, PIN2, PIN4, PIN8, IAA13 and IAA14/SLR (Figure 9a and Table S3). The other genes examined did not exhibit more than a two-fold decrease or increase. These results indicated that the cuv-1 mutation differentially affects expression of genes, which include genes involved in auxin biosynthesis, polar auxin transport and auxin signaling. DISCUSSION Arabidopsis Prp16/CUV is required for auxin-mediated development including male-gametophyte transmission, apical–basal patterning of embryonic and gynoecium development, stamen development, phyllotactic flower positioning, and vascular development. cuv-1 mutation differentially affects expression of genes, which include key genes for auxin biosynthesis and genes involved in auxin transport and signaling. The strongest cuv-2 allele has selective influence on male-gametophyte transmission. In contrast, defective transmission through female gametophyte is a common phenotype among mutants deficient in core spliceosomal components reported so far. The spliceosome is comprised of small nuclear ribonucleoprotein particles

€ hrmann, (snRNPs) and numerous proteins (Will and Lu 2011). Loss-of-function Arabidopsis mutations in genes that encode components of snRNPs, U2 snRNP-specific proteins ATROPOS (ATO)/Prp9 (Moll et al., 2008) and SF3b130 (Aki et al., 2011), U5 snRNP-specific protein GAMETOPHYTIC FACTOR 1 (GFA1)/CLOTHO (CLO)/VAJRA (VAJ)/Snu114 (Coury et al., 2007; Moll et al., 2008; Yagi et al., 2009), and U4/U6 snRNP-specific protein LACHESIS € lz et al., 2012), cause (LIS)/Prp4 (Groß-Hardt et al., 2007; Vo no or less genetic transmission through the female gametophyte. shoot redifferentiation defective 2 (srd2) mutant defective in expression of all the spliceosomal snRNAs (Ohtani and Sugiyama, 2005; Ohtani et al., 2008, 2010) and root initiation defective1 (rid1) mutants deficient in another spliceosomal DEAH-box RNA-dependent ATPase Prp22 (Ohtani et al., 2013) have similar defects in the female gametophyte. Less severe defects in male-gametophyte development are also seen in mutants of ATO, SF3b130, GFA1/CLO/VAJ and RID1, but not in SRD2. These facts indicate that spliceosomal gene mutations do not equally affect on male and female gametophyte development. Sexbiased lethality or transmission incurred by defects in transcriptional machineries has been described in Arabidopsis (Onodera et al., 2008). Loss-of-function mutants defective in essential subunits of RNA polymerases are not able to

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2015), 81, 183–197

A role of Prp16 in Arabidopsis 193 transmit the mutant allele through the female gametophyte, but able to transmit the mutant allele through the male gametophyte. It has been considered that female gametophyte development requires transcription machinery generated de novo in the haploid female gamete, while male gametophytes can utilize transcription machinery derived from diploid pollen mother cells. The similar scenario can be applied for mutants disrupted in essential components of splicing machinery, in which mutant alleles are not transmitted through the female gametophyte, but through the male gametophyte. On the other hand, the cuv-2 mutant exhibits opposite gametophyte phenotypes, which is that paternal transmission of the mutant allele is significantly reduced, whereas the maternal transmission is normal. One possibility is that although CUV is required for splicing both in the female and male gametophyte, CUV mRNA and/or CUV protein derived from the diploid sporophyte are sufficient for splicing in the cuv-2 female gametophyte, but not for splicing in the cuv-2 male gametophyte. Another possibility is that CUV is specifically required for male-gametophyte development, and CUV mRNA and/or CUV protein derived from diploid pollen mother cells are not sufficient for cuv-2 male-gametophyte development. In either case, de novo expression of CUV in the haploid state is very important for male-gametophyte development, but not for female gametophyte development. Disruption of an essential cellular function often leads to defects not only in the gametophyte but also in the embryo (Muralla et al., 2011). Among the aforementioned spliceosomal mutations, lis (Meinke et al., 2008) and srd2 (Ohtani et al., 2008) produce defective embryos, which arrest development around the preglobular stage without noted abnormalities in patterning and cell proliferation. Embryonic lethality of cuv-2 and cuv-3 mutations suggests that CUV also plays an essential role during embryogenesis. However, in cuv-2 and cuv-3, the apical–basal axis-dependent cell-division patterns were disrupted both in the embryo and suspensor. Efflux-dependent auxin gradients are required for apical–basal patterning during embryonic development. cuv-2 embryos, whose phenotype is similar to the pin7 embryo (Friml et al., 2003), exhibit ectopic auxin response both in the embryo and suspensor. These suggest that the auxin-mediated apical–basal axis-dependent development is impaired in embryos of cuv-2 and cuv-3. Thus, CUV may be required for establishment of the apical–basal axis of Arabidopsis. Plant organ development (Benkova
et al., 2003) and vascular development (Mattsson et al., 1999, 2003; Sieburth, 1999) are directed by local auxin gradients associated with auxin maxima, which are defined by local auxin biosynthesis (Cheng et al., 2006) and by dynamic expression and asymmetric subcellular localization of PIN auxin efflux proteins (Petra
sek et al., 2006; Wisniewska et al., 2006). cuv-1

affects auxin-mediated developmental processes and auxin-response patterns in shoots and roots. Both the cuv1 gynoecium and the cuv-2 embryo are defective in apical– basal axis-dependent development and spatial patterns of auxin response. These facts support the idea that CUV influences developmental events mediated by auxin gradients and maxima. An altered leaf-venation pattern was found not only in cuv-1 but also in other spliceosomal mutants deficient in a U4/U6.U5 tri-snRNP-specific protein DEFECTIVELY ORGANIZED TRIBUTARIES 2 (DOT2)/MERISTEM-DEFECTIVE (MDF)/Snu66 (Petricka et al., 2008; Casson et al., 2009), SmD3-b, a core component of spliceosomal snRNPs (Swaraz et al., 2011) and snRNAs (srd2-1; Ohtani et al., 2008). These facts suggest that leaf-vein development requires the action of genes whose expression is vulnerable to genetic lesions of the above splicing factors. In cuv-1, dot2/ mdf-1 (Casson et al., 2009) and srd2-1 (Ohtani et al., 2010), decreased expression of PIN genes was observed, although sets of affected PIN genes were different from one another. In cuv-1 and srd2-1, expression of PIN proteins was also decreased and auxin-response pattern was disrupted. In cuv-1, decreased expression of genes involved in auxin biosynthesis, perception and signaling was observed. These facts raise a possibility that expression of PIN genes may be susceptible to defects in premRNA splicing. Taken together, our data suggest that the altered expression of genes important for auxin homeostasis, perception and signaling at least in part contribute to developmental defects in cuv mutants. To further clarify the role of CUV/Prp16 in plant development, a comprehensive search for genes affected by cuv mutations is required. A comparative study of cuv and other spliceosomal gene mutations would not only clarify roles of splicing factors in plant development, but may also reveal transcript specificity in Arabidopsis, which has been described well in yeast (Pleiss et al., 2007). In cuv-1, some genes exhibit that levels of their spliced transcripts were decreased, whereas levels of their total transcripts were not significantly changed (Figure 9 and Table S3), suggesting that splicing of these genes is defective in cuv-1. In contrast, other genes exhibit that levels of both their spliced and total transcripts were decreased in cuv-1 (Figure 9 and Table S3). We consider two possibilities to explain the phenotype of a reduction in levels of both spliced and total transcripts. The first possibility is that unspliced transcripts may be subjected to degradation. In eukaryotic cells, aberrantly spliced mRNAs such as unspliced pre-mRNAs are rapidly degraded by the mRNA surveillance or nonsense-mediated mRNA decay (NMD) pathway (Bousquet-Antonelli et al., 2000; Mitchell and Tollervey, 2000; Chekanova et al., 2007; Kim et al., 2009; Kalyna et al., 2012). In Arabidopsis, unspliced pre-mRNAs are degraded through the NMD pathway even in wild type

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2015), 81, 183–197

194 Ryuji Tsugeki et al. (Kim et al., 2009), although transcripts with the NMD feature are not necessarily subject to degradation (Kalyna et al., 2012; Marquez et al., 2012). In yeast, many splicing mutants show a strong reduction in mRNA levels without a corresponding accumulation of unspliced pre-mRNAs, which are degraded by the nuclear pre-mRNA turnover pathway (Bousquet-Antonelli et al., 2000). The second possibility is that the expression level is transcriptionally changed due to secondary effects of cuv-1 mutation, leading to a reduction in transcript levels. Combination of these two scenarios is also possible. It has been considered that the spliceosome uses optimal splice sites, which leads to constitutive splicing. Alternative splicing may represent the repression of optimal splice sites and/or the use of suboptimal splice sites (Smith et al., 2008). In the case of YUC4, whether or not the third intron is spliced is probably decisive for the alternative production of the YUC4.1 or YUC4.2 transcript. Retention of the third intron is necessary for the production of the YUC4.2 transcript. In cuv-1, whereas the YUC4.1 transcript is decreased, the YUC4.2 transcript is increased, suggesting that CUV facilitates splicing of the third intron of YUC4 pre-mRNA. Since Prp16 is implicated to function as a guarantor for poor splicing substrates (Horowitz, 2011), it would be intriguing to see if splicing of the third intron of YUC4 is particularly sensitive to cuv mutations. It has been postulated that relatively small differences in conditions, such as tissue-specific or developmentalstage-specific changes in the concentration of splicing factors could alter splice sites, resulting in alternative splicing (Yu et al., 2008). Therefore, modulation of function of splicing factors such as Prp16/CUV could be used as a means for fine tuning of gene expression. One of the features common to transcriptomes for model organisms might be the presence of many more transcript variants than previously thought. In human (Pan et al., 2008; Wang et al., 2008) and Arabidopsis (Marquez et al., 2012), more than 95 and 61% of intron-containing genes are alternatively spliced, respectively. Our study on Arabidopsis Prp16/CUV indicates that a general splicing component could have differential influence on plant development and function as a contributor to transcriptomic diversity in plants. EXPERIMENTAL PROCEDURES Plant materials and growth conditions Arabidopsis thaliana ecotypes C24 and Columbia (Col) were used as wild-type controls. The following mutants were used: cuv-1 (C24), cuv-2 (Col), cuv-3 (WS: Wassilewskija), axr2-1 (Wilson et al., 1990), axr3-3 (Leyser et al., 1996), slr-1 (Fukaki et al., 2002) and gnomR5 (Geldner et al., 2004). cuv-1 was identified in the mutant screening performed as described previously (Tsugeki et al., 2009). cuv-2 (salk_019541, emb3011-2) and cuv-3 (CS24359, emb3011-1) were obtained from Arabidopsis Biological Resource

Center (ABRC, https://www.arabidopsis.org/abrc/). Marker lines used were ATHB8pro:GUS (Baima et al., 1995), BApro:GUS (Oono et al., 1998, 2002), DR5pro:GUS (Ulmasov et al., 1997), DR5pro:GFP (Ottenschla€ger et al., 2003), J1511 (from ABRC; Laplaze et al., 2005), PIN1pro:PIN1:GFP (Benkova
et al., 2003) and PIN7pro:PIN7: GFP. PIN1pro:PIN1:GFP and PIN7pro:PIN7:GFP were constructed from genomic DNA containing PIN1 and PIN7 genes, respectively. T-DNA insertion cuv alleles were characterized at both ends of the insertion. The cuv-2 T-DNA insertion was found in the 13th exon of the CUV gene and accompanied by about an 800-bp duplication encompassing from the 11th exon to 13th exon and with a 65-bp deletion in the 13th intron. The cuv-3 T-DNA insertion was located 322-bp upstream of the initiator ATG in the 50 untranslated region of the putative transcriptional unit of the CUV gene. cuv mutants were backcrossed at least twice. Plants were grown as described previously (Tsugeki et al., 2009).

Phenotypic analyses Venation patterns (Tsugeki et al., 2009) and developing embryos (Tsugeki et al., 1996) were observed as described previously. To test the ability to transmit male and female gametophytes of cuv-2 and cuv-3, reciprocal crosses were made between cuv-2 (Col) or cuv-3 (WS) and wild type (C24). The F1 genotype was confirmed by checking genotyping markers specific to Col, WS and C24, respectively. Confirmed F1 progeny were checked for cuv-2 and cuv-3 transmission, respectively.

GUS staining GUS staining was done as described previously (Tsugeki et al., 2009). GUS staining was performed either at room temperature or at 37°C: DR5pro:GUS, for 16–18 h at room temperature for the aerial tissue of seedlings, 18 h at 37°C for flowers; ATHB8pro:GUS, 1– 2 h at 37°C for the aerial tissue of seedlings; BApro:GUS, 22 h at room temperature or 4 h at 37°C for seedlings; CUVpro:CUV:GUS, 2 h at 37°C for seedlings, 14 h at 37°C for flowers.

Microscopy Images of root hairs were captured using a Leica M420 Macroscope (Leica Microsystems, http://www.leica-microsystems.com/) equipped with a Nikon COOLPIX 990 (Nikon, http://www.nikon. com/). Optical microscopic images were captured using a Zeiss Axioplan 2 microscope equipped with an AxioCam (Carl Zeiss, http:// www.zeiss.com/). GFP fluorescence was obtained using a Zeiss Axioplan 2 microscope equipped with LSM 5 PASCAL (Carl Zeiss). Fluorescence of GFP and DAPI (40 ,6-diamidino-2-phenylindole) was observed as described previously (Tsugeki et al., 2009). Unless described otherwise, in each experiment, images of mutants and wild type were captured under the identical conditions.

Positional cloning, plasmid construction and plant transformation The F2 population between cuv-1 (C24) and Col was used for mapping. An approximately 11-kb genomic fragment containing the At5g13010 gene (from 3-kb upstream from the initiator codon ATG to 1-kb downstream from the stop codon TGA) was cloned into pGW-NB1 (Nakagawa et al., 2007). The resulting construct was able to complement cuv-1. The G3GFP (Kawakami and Watanabe, 1997) or GUS gene was inserted into the aforementioned 11-kb genomic fragment in such a way that translational fusion genes CUV:GFP and CUV:GUS would be expressed under the CUV promoter. CUVpro:CUV:GFP was also confirmed to complement cuv-1 and cuv-2.

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A role of Prp16 in Arabidopsis 195 Auxin treatment Three-day-old seedlings of wild type (Col) were incubated in liquid medium supplemented with 50 lM 1-naphthaleneacetic acid (NAA). Seedlings incubated with NAA for 0, 5 or 24 h were used for RNA isolation. To test the auxin response using BApro:GUS, seedlings grown for 6 days were incubated in liquid medium supplemented with 10 or 100 nM IAA for 6 h.

RNA expression analysis Total RNA was isolated from seedlings, cauline leaves and flowers that included flowers, floral buds and inflorescence meristems by use of an RNeasy Plant Mini kit (Qiagen, http://www.qiagen.com/). Isolated RNA was treated with RNase-free DNase I (Life Technologies, http://www.lifetechnologies.com), subjected to cDNA synthesis, and then used for expression analysis. All the synthesized cDNA were first analyzed for expression of ACT7 by RT-PCR to check the quality of cDNA and to confirm no contamination of genome DNA. Control experiments using samples without reverse transcription were also performed (Figure S10). In quantitative RTPCR (qRT-PCR) analyses, expression levels of target genes were normalized to those of ACT7, and expression levels of ACT7 were normalized to those of UBP3 (Figure S11). qRT-PCR was performed using FastStart Universal SYBR Green Master (Roche Applied Science, http://www.roche-applied-science.com) and analyzed using an Mx3000P QPCR System (Agilent Technology, http:// www.agilent.com/). For each experiment, three independent biological replicates were made. For each biological replicate, three technical replicates of each PCR reaction were also made. Data were statistically analyzed by F-test and by unpaired two-tailed Student’s t-test (P-values: *P < 0.05; **P < 0.01; ***P < 0.001).

ACKNOWLEDGEMENTS We thank the Arabidopsis Biological Resource Center (ABRC) for J1511, cuv-2 (salk_019541) and cuv-3 (CS24359), Hidehiro Fukaki for axr2-1 and slr-1, Tom J. Guilfoyle for DR5pro:GUS, Jim Haseloff for making J1511 available in the ABRC, Ottoline Leyser for axr3€ rgens for gnomR5, Giorgio Morelli for ATHB8pro:GUS, 3, Gerd Ju Tsuyoshi Nakagawa for pGWB-NB1, Yutaka Oono for BApro:GUS, Klaus Palme for DR5pro:GFP, PIN1pro:PIN1:GFP and PIN7pro:PIN7: GFP, Yuichiro Watanabe for G3GFP, Toshiharu Shikanai, Franck Ditengou, William Teale and Elizabeth Nakajima for critically reading the manuscript, and Noritaka Matsumoto, Noriyoshi Yagi and Koichi Toyokura for stimulating discussions. This work was supported in part by a Grant-in-Aid for Scientific Research (24570047 to R.T.) from the Japan Society for the Promotion of Science, by a Grant-in-Aid for Creative Scientific Research (19GS0315 to K.O. and R.T.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, by a grant from the Human Frontier Science Program, and by the Grants for Excellent Graduate Schools program of MEXT, Japan. Hormone analysis was supported by Japan Advanced Plant Science Network.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Analyses of vein complexity of wild-type and cuv-1 rosette leaves. Figure S2. Phenotype of vascular development in cuv-1 floral organs. Figure S3. Exogenously applied IAA increases BApro:GUS expression in the hypocotyl vascular tissue.

Figure S4. Effect of cuv-1 mutation on PIN1 polarity in roots. Figure S5. Phenotype of the inflorescence in cuv-1. Figure S6. Schematic structures of CUV gene and CUV protein. Figure S7. Molecular complementation of cuv-1 and cuv-2 alleles. Figure S8. Comparison of Arabidopsis CUV/Prp16 with Prp16 orthologs in rice, human, Drosophila and yeast. Figure S9. Analysis of auxin inducibility of CUV expression. Figure S10. RT-PCR analysis of expression of ACT7 in wild type and cuv-1. Figure S11. Expression analysis of ubiquitously expressed genes in cuv-1. Figure S12. Expression analysis of intronless genes in cuv-1. Figure S13. RT-PCR analysis of expression and splicing of TAR2, YUC9 and YUC11. Figure S14. RT-PCR analysis of expression and splicing of YUC1 and YUC7. Figure S15. Expression analysis of GH3 transcripts in cuv-1. Figure S16. RT-PCR analysis of expression and splicing of PIN. Figure S17. RT-PCR analysis of expression and splicing of TIR1, AFB1, AFB2 and AFB3. Figure S18. RT-PCR analysis of expression and splicing of IAA3/ SHY2, IAA7/AXR2, IAA13 and IAA30. Figure S19. RT-PCR analysis of expression and splicing of IAA14/ SLR and IAA17/AXR3. Figure S20. RT-PCR analysis of expression and splicing of ARF3/ ETT, ARF5/MP and ARF7/NPH4. Figure S21. Expression analysis for genes that have been considered to contribute to auxin biosynthesis. Table S1. Analysis of secondary veins in wild-type and cuv-1 leaf primordia. Table S2. Analyses of vein complexity of wild-type and cuv-1 cotyledons. Table S3. Data of quantitative RT-PCR. Table S4. Quantification of IAA and IAA-Asp in wild-type and cuv1 seedlings. Table S5. Primers used in RT-PCR.

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