Functional and expression analysis of Arabidopsis SPA genes during seedling photomorphogenesis and adult growth

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1 The Plant Journal (2006) 47, doi: /j X x Functional and expression analysis of Arabidopsis SPA genes during seedling photomorphogenesis and adult growth Kirsten Fittinghoff 1,, Sascha Laubinger 1,, Markus Nixdorf 1,, Petra Fackendahl 1,, Rosalinde-Louise Baumgardt 1,, Alfred Batschauer 2 and Ute Hoecker 1,,* 1 Department of Plant Developmental and Molecular Biology, Geb , University of Düsseldorf, D Düsseldorf, Germany, and 2 FB Biology Plant Physiology, Philipps University, Karl-von-Frisch-Str. 8, D Marburg, Germany Received 6 March 2006; revised 13 April 2006; accepted 19 April * For correspondence (fax þ ; hoeckeru@uni-duesseldorf.de). Present address: Institute of Botany, University of Cologne, D Cologne, Germany. Present address: Max-Planck-Institute of Developmental Biology, Spemannstr , D Tübingen, Germany. Present address: Otsuka Pharma GmbH, Grüneburgweg 102, Frankfurt am Main, Germany. Summary The four members of the Suppressor of phya-105 (SPA) gene family function to inhibit photomorphogenesis in dark- and light-grown seedlings. Additionally, SPA1 SPA4 regulate elongation growth of adult plants. In these processes, SPA2, SPA3 and SPA4 have overlapping but distinct functions. Here, we have further investigated the role of SPA1 which is partially masked by functional redundancy. We show that SPA1 represses not only red, far-red and blue light responses in a PHYA-dependent fashion, but also acts to suppress light signaling in darkness. We demonstrate that deletion-derivatives of SPA1 lacking the complete N-terminus or part of the kinase-like domain retain SPA1 function in light- and dark-grown seedlings, while deletion of the constitutive photomorphogenesis 1 (COP1)-interacting coiled-coil domain eliminates SPA1 activity. This suggests that the coiled-coil domain and the WD-repeat domain of SPA1 are sufficient for SPA1 function. An analysis of spa2 spa3 spa4 triple mutants demonstrates that SPA1, like SPA2, is sufficient for normal etiolation of dark-grown seedlings. In light-grown seedlings and adult plants, in contrast, SPA1 function is divergent from SPA2 function, with SPA1 playing the predominant role. Levels of SPA1, SPA3 and SPA4 transcript are increased by red, far-red and blue light, consistent with a role of these three SPA genes in light-grown seedlings. The abundance of SPA2 mrna, in contrast, is not altered by light. Taken together, the analysis of SPA transcript levels suggests that differences in SPA gene expression patterns contribute to divergence in SPA1 SPA4 function. Keywords: SPA1, light signal transduction, photomorphogenesis, Arabidopsis. Introduction Plants monitor ambient light conditions to adjust their developmental programs to changes in light quality, quantity, direction and day length. In Arabidopsis multiple classes of wavelength-specific photoreceptors have been identified: the red light (R)-absorbing and far-red light (FR)- absorbing phytochromes (phy) and several blue light (B)-responsive photoreceptor families, among those the cryptochromes (cry) and phototropins (phot; Briggs and Spudich, 2005). Two crys (cry1 and cry2) play important roles in seedling de-etiolation and the promotion of flowering (Banerjee and Batschauer, 2005; Lin and Shalitin, 2003). Among the five phytochromes (phya phye), phya and phyb play the most important roles. Phytochrome A mediates responses to continuous FR (FRc) and very low fluences of light and is therefore responsible for seed germination and seedling de-etiolation under these light conditions. Phytochrome B, in contrast, is activated by R and regulates de-etiolation in R and the shade avoidance response (Franklin et al., 2005). Downstream of these photoreceptors, an important negative regulator, COP1 (constitutive photomorphogenesis 1), suppresses light signaling in dark-grown seedlings (Yi and Deng, 2005). cop1 mutants therefore undergo constitutive photomorphogenesis, exhibiting features of light-grown 577 Journal compilation ª 2006 Blackwell Publishing Ltd

2 578 Kirsten Fittinghoff et al. seedlings even in complete darkness. COP1 is an E3 ubiquitin ligase that acts in darkness to ubiquitinate activators of the light response, such as the transcription factors HY5, LAF1 and HFR1, which are subsequently targeted for degradation in the 26S proteasome. In the light, active photoreceptors inhibit the function of COP1, thus HY5, LAF1 and HFR1 are stabilized and regulate transcription of lightresponsive genes (Duek et al., 2004; Jang et al., 2005; Osterlund et al., 2000; Saijo et al., 2003; Seo et al., 2003; Yang et al., 2005b). The COP1 protein contains three structural domains involved in protein protein interactions. An N-terminal RING-finger domain, a coiled-coil structure and a WD-repeat domain which is involved in substrate recognition (Yi and Deng, 2005). COP1 associates with itself and a number of other proteins, and forms high-molecular-mass complexes in vivo (Saijo et al., 2003). Arabidopsis COP1 interacts with the four members of the SPA (suppressor of phya-105) protein family (SPA1 SPA4; Hoecker, 2005). Genetic studies have shown that SPA proteins function redundantly in suppressing photomorphogenesis in dark-grown seedlings. Hence, spa1 spa2 spa3 spa4 quadruple-mutant seedlings exhibit constitutive photomorphogenesis similar to that observed in a cop1 mutant. Adult spa quadruple mutants, like cop1 4 mutants, are very small and dwarfed plants (Laubinger et al., 2004). Mutations in SPA genes are able to synergistically enhance the effect of weak cop1 mutations, thus corroborating the biological relevance of the SPA COP1 interaction (Laubinger et al., 2004; Saijo et al., 2003). Moreover, SPA1 physically interacts with targets of COP1 activity (HY5 and HFR1) and also controls the protein stability of these transcription factors (Jang et al., 2005; Saijo et al., 2003). Taken together, these findings suggest that SPA proteins are co-factors of the ubiquitin ligase COP1. Consistent with this idea, recombinant SPA1 can modulate the E3 ubiquitin ligase activity of COP1 in vitro (Saijo et al., 2003; Seo et al., 2003). The functional relevance of these effects of SPA1 observed in vitro remains to be shown. The SPA proteins contain three characteristic domains: an N-terminal serine/threonine kinase-like motif followed by a coiled-coil structure and a C-terminal WD-repeat domain which is also found in COP1. Sequence similarity among SPA proteins is very high in the respective WDrepeat domains but rather low when comparing the N-terminal domains (Hoecker et al., 1999; Laubinger and Hoecker, 2003). For SPA1 it was shown that its coiled-coil domain interacts with the coiled-coil domain of COP1 (Hoecker and Quail, 2001; Saijo et al., 2003). The WDrepeat domain of SPA1 is involved in binding HY5 and HFR1 (Saijo et al., 2003; Yang et al., 2005a). SPA1 and SPA2 are constitutively nuclear proteins and are therefore localized in the same cellular compartment as active COP1 (Hoecker et al., 1999; Laubinger et al., 2004; Yi and Deng, 2005). Unlike COP1, which is also found in humans, SPA proteins are plant-specific. In order to adjust to the light environment it might be an evolutionary advantage that the genome of Arabidopsis contains more than one SPA gene. Analysis of various single, double and triple spa mutant combinations has revealed that the four SPA genes have overlapping but distinct functions in regulating plant development (Laubinger et al., 2004). Due to functional redundancy, mutations in individual SPA genes (i.e. single mutants) allow wild-type elongation growth of dark-grown seedlings and adult plants. When spa1, spa3 or spa4 single-mutant seedlings are grown in R or FR, they exhibit exaggerated de-etiolation in a PHYAdependent manner, while spa2 mutants show no visible defect. This indicates that SPA1, SPA3 and SPA4 are of particular importance for normal seedling development in the light. An analysis of several spa triple mutants that thus contain only one functional SPA gene allowed us to further investigate the individual contributions of the four SPA genes. These results show that SPA2 primarily regulates seedling development in darkness and has little function in light-grown seedlings or adult plants. SPA3 and SPA4, in contrast, predominantly regulate elongation growth in adult plants because triple mutants that only contain a functional SPA3 or SPA4 gene, respectively, grow similarly to adult plants (Hoecker et al., 1998; Laubinger and Hoecker, 2003; Laubinger et al., 2004). Triple mutants that contain only functional SPA1 have not as yet been analyzed. Thus, due to functional redundancy, the involvement of SPA1 in the control of plant growth and development is unknown. Here, we have generated and analyzed spa2 spa3 spa4 triple mutants that contain only a functional SPA1 gene. We also investigated spa1 single mutants grown in blue light, a light condition that had not been characterized in detail before. Second, we have conducted a structure/function analysis of the SPA1 protein to determine which domains are important for SPA1 function. Third, we have addressed the question of whether the partially distinct functions of the four SPA genes throughout development result from differences in SPA gene expression patterns. Results SPA1 inhibits photomorphogenesis in blue light in a PHYAdependent manner It was shown previously that spa1 mutants exhibit enhanced responsiveness to R and FR (Baumgardt et al., 2002; Hoecker and Quail, 2001; Hoecker et al., 1998; Zhou et al., 2002). Also, we demonstrated that spa3 and spa4 mutants show increased responses to R, FR and B (Laubinger and Hoecker, 2003). To investigate whether SPA1 also modulates B signaling, we analyzed the hypocotyl length of spa1 mutant and wild-type seedlings under various fluence rates of continuous B (Bc). Under all fluence rates tested, spa1 mutants

3 Functional and expression analysis of Arabidopsis SPA genes 579 (a) (b) (c) Figure 1. Effects of blue light on the spa1 mutant phenotype and on SPA1 transcript accumulation. (a) Hypocotyl length of wild-type (WT), spa1-2, phya-101 and spa1 phya double-mutant seedlings grown in Bc of various fluence rates. Error bars denote one standard error of the mean. (b) Total RNA gel blot analysis (top) and quantification (bottom) of SPA1 transcript levels in seedlings that were transferred from darkness to 5 lmol m )2 sec )1 Bc for 0 24 h. Transcript levels were normalized by rehybridization with an 18S rrna-specific probe. (c) Quantification of SPA1 transcript accumulation in wild-type (WT), phya, cry1 cry2 and phya cry1 cry2 mutant seedlings that were transferred from darkness to 5 lmol m )2 sec )1 Bc for 0 24 h. SPA1 mrna levels were normalized to 18S rrna levels. exhibited significantly shorter hypocotyls than the wild type (Figure 1a), indicating that SPA1 inhibits responses to Bc. Because previous results demonstrated that spa1 mutations increase the responsiveness to R and FR in a PHYAdependent manner (Hoecker et al., 1998), we investigated the relationship between PHYA and SPA1 in regulating B signaling. When grown in various fluence rates of Bc, spa1 phya double mutants were indistinguishable from phya mutants (Figure 1a), indicating that enhanced responsiveness of spa1 mutants to B was fully dependent on a functional PHYA gene. cry1, cry2 and phya act redundantly to increase SPA1 transcript levels in blue light Previous results showed that R and FR increase SPA1 transcript levels in a fashion that is not solely dependent on PHYA (Hoecker et al., 1999). To investigate possible regulation of SPA1 mrna levels by B, we conducted an RNA gel blot analysis of wild-type seedlings grown in the dark or exposed to B. Continuous B increased the abundance of SPA1 mrna by approximately 5 10-fold when compared with the dark control (Figure 1b). The abundance of SPA1 mrna was not affected within the first 30 min of Bc irradiation, but a full response occurred between 30 and 60 min of Bc treatment (Figure 1b). This B response of SPA1 is similar to the effect of Rc and FRc on SPA1 transcript levels (Hoecker et al., 1999). Blue light can be perceived by cry1, cry2 and phya (Neff and Chory, 1998; Poppe et al., 1998). To investigate which of these photoreceptors are responsible for the increased abundance of SPA1 mrna in Bc-irradiated seedlings, we determined SPA1 transcript levels in the wild type, in phya mutants, in cry1 cry2 double mutants and in phya cry1 cry2 triple mutants exposed to low or high fluence rates of Bc. SPA1 mrna levels in phya mutant seedlings exposed to high Bc fluence rates (5 lmol m )2 sec )1 ) were similar to those in the wild type (Figure 1c). cry1 cry2 mutants accumulated lower amounts of SPA1 mrna than the wild type, but they retained a clear B response within the first 2 h of Bc treatment. Only in phya cry1 cry2 triple mutants was the B response of SPA1 lost (Figure 1c). Thus, the photoreceptors cry1, cry2 and phya act redundantly to increase SPA1

4 580 Kirsten Fittinghoff et al. (a) (d) (b) (e) (c) Figure 2. SPA1 protein abundance is increased by light. (a) Diagram of the SPA1::SPA1-HA cassette introduced into the spa1-3 mutant. Black rectangles represent the coding region, open rectangles are non-coding exons, lines are SPA1 5 regulatory sequences, introns or SPA1 3 regulatory sequences, respectively. (b) Hypocotyl length of FRc-grown (0.2 lmol m )2 sec )1 ) seedlings of the wild type, spa1-3 mutant and two independent transgenic spa1-3 SPA1::SPA1-HA lines. Error bars denote one standard error of the mean. (c) Anthocyanin content of FRc-grown (0.3 lmol m )2 sec )1 ) seedlings. Genotypes and error bars are as in (b). (d) a-hemagglutinin antibodies recognize the SPA1-HA protein. Western blot analysis of protein extracts isolated from etiolated seedlings exposed to FRc (0.4 lmol m )2 sec )1 ) for 2 h. The signal produced by a tubulin-specific antibody was used as a loading control. Genotypes are as in (b). (e) Light increases SPA1 protein abundance. Total protein was isolated from transgenic seedlings of line 26 that had been grown in darkness for 4 days and subsequently transferred to 0.4 lmol m )2 sec )1 FRc, 10 lmol m )2 sec )1 Rc or 2 lmol m )2 sec )1 Bc for 0 24 h. Protein extracts were used for Western blot analysis using a-ha antibodies and a tubulin-specific antibody as a loading control. Similar results were obtained with the transgenic line 11 (data not shown). transcript levels under these conditions. Under lower fluence rates of Bc (0.3 lmol m )2 sec )1 ), a similar functional redundancy among the three photoreceptors was observed, except that under these conditions phya predominated and an effect of cry1 cry2 mutations was only detected when PHYA was mutated also (Figure S1). Taken together, these results show that three photoreceptors mediate the Bc-induced increase of SPA1 transcript levels. Hence, because this increase is not mediated specifically by phya, the light-induced increase in the abundance of SPA1 mrna cannot be the sole mechanism by which SPA1 participates in phya-mediated B signaling. Light increases SPA1 protein levels To test whether the amount of SPA1 mrna correlates with the abundance of SPA1 protein, we analyzed the amount of SPA1 protein in dark- and light-grown seedlings. To this end, we constructed an SPA1 expression cassette (SPA1::SPA1- HA) that contained the entire transcribed region of SPA1 and the cdna sequence of a triple influenza hemagglutinin (HA)- tag under the control of the endogenous SPA1 regulatory sequences (2241 base pairs upstream of the SPA1 start codon and 1026 base pairs downstream of the SPA1 stop codon; Figure 2a). We introduced the SPA1::SPA1-HA construct into the spa1-3 mutant and analyzed de-etiolation and anthocyanin content in FRc in multiple independent transgenic lines. spa1-3/spa1::spa1-ha transgenic seedlings were indistinguishable from wild-type seedlings (Figure 2b,c), indicating that SPA1::SPA1-HA completely rescued the mutant phenotype of spa1-3 seedlings. Hence, the SPA1-HA protein is functional. When examining protein extracts, an HA-specific antibody recognized a specific protein of the expected molecular

5 Functional and expression analysis of Arabidopsis SPA genes 581 mass of SPA1-HA in spa1-3/spa1::spa1-ha seedlings (Figure 2d). To investigate the effect of light on the abundance of SPA1-HA protein, we grew spa1-3/spa1::spa1-ha seedlings for 4 days in darkness and then transferred the seedlings to FRc, Rc or Bc for 0, 2, 4 or 24 h, respectively. The abundance of SPA1-HA was increased about eightfold within 2 h of light treatment when compared with darkness (Figure 2e). All three light qualities tested caused a similar increase in levels of SPA1-HA protein. These results show that the SPA1 protein is more abundant in light-exposed seedlings than in dark-grown seedlings. Hence, levels of SPA1 protein closely correlated with SPA1 transcript abundance. the spa triple mutant in some but not all experiments (Figure 3c). Hence, SPA1 function in the spa2 spa3 spa4 triple mutant is not significantly distorted by feedback regulation. Taken together, these results show that, among the four SPA genes, SPA1 is sufficient for normal seedling development in darkness and under low fluence rates of light. At high light fluence rates SPA1 is not sufficient for normal photomorphogenesis. However, it is clearly a very important repressor of light signaling, as the contribution of SPA1 is similar to the contributions of SPA2, SPA3 and SPA4 together. SPA1 is the main regulator of seedling de-etiolation We have shown here and previously that spa1 mutants are hypersensitive to R, FR and B, but do not show a mutant phenotype in darkness (Hoecker and Quail, 2001; Hoecker et al., 1998; Figure 1a). However, because spa1 spa2 spa3 spa4 quadruple-mutant seedlings undergo constitutive photomorphogenesis in darkness (Laubinger et al., 2004), a SPA1 function in dark-grown seedlings might be masked by functional redundancy with other SPA proteins. To investigate the contribution of SPA1, we compared the phenotypes of wild type, spa quadruple mutants, spa2 spa3 spa4 triple mutants (that contain only functional SPA1) and spa1 mutants. All mutants were generated in the same genetic background (Columbia). While dark-grown spa quadruple mutants exhibited constitutive photomorphogenesis, spa2 spa3 spa4 seedlings etiolated normally and were indistinguishable from wild-type seedlings (Figure 3a). This indicates that SPA1 does not only contribute but is even sufficient for normal repression of photomorphogenesis in the dark. Also when grown under low fluence rates of Rc, FRc or Bc, spa2 spa3 spa4 triple-mutant seedlings exhibited a hypocotyl length similar to wild-type seedlings (Figure 3a). spa1 single mutants, in contrast, showed a significantly reduced hypocotyl length and fully expanded cotyledons when compared to wild-type seedlings (Figure 3a, upper photos). Hence, SPA1 is necessary and sufficient to allow normal seedling development under these conditions. At all higher fluence rates tested, spa2 spa3 spa4 mutants exhibited shorter hypocotyls than the wild type (Figure 3a,b). When compared with spa1 mutants, the hypocotyl length of spa2 spa3 spa4 mutants was similar (in FRc and Bc) or slightly shorter (in Rc; Figure 3a,b). To test whether SPA1 gene function in the spa2 spa3 spa4 mutant is altered due to positive feedback regulation, we compared the abundance of SPA1 mrna between the wild type and the spa2 spa3 spa4 mutant. Under all growth conditions and developmental stages tested, the SPA1 mrna abundance in spa2 spa3 spa4 mutants was very similar to that in the wild type. In dark-grown seedlings, we observed slightly increased SPA1 transcript accumulation in The two homologous genes, SPA1 and SPA2, have evolved different functions To investigate the reason for the emphasized function of SPA1, we compared the function of SPA1 with the function of the most closely related SPA gene, SPA2. SPA1 and SPA2 derived from a direct duplication event in the Arabidopsis genome (Simillion et al., 2002). To test whether SPA1 and SPA2 have similar functions in regulating plant development we analyzed the wild type, spa2 spa3 spa4 mutants (that contain only functional SPA1), spa1 spa3 spa4 mutants (that contain only functional SPA2) and spa3 spa4 mutants (that contain SPA1 and SPA2). Dark-grown spa2 spa3 spa4 and spa1 spa3 spa4 triplemutant seedlings were indistinguishable from wild-type seedlings (Figure 4a), indicating that both SPA1 and SPA2 are, on their own, sufficient to suppress photomorphogenesis in the dark. Hence, SPA1 and SPA2 have similar functions in dark-grown seedlings. In the light, these two triple mutants behaved very differently. spa1 spa3 spa4 mutants exhibited extremely short hypocotyls, even under low fluence rates of light (Figure 4a,b; Laubinger et al., 2004). spa2 spa3 spa4 mutants, in contrast, were much taller and only slightly shorter than wild-type seedlings (Figure 4a,b). This shows that in light-grown seedlings, the contribution of SPA1 is much larger than that of SPA2. In agreement with this finding, spa2 spa3 spa4 mutants were indistinguishable from spa3 spa4 mutants, indicating that loss of SPA2 function in a spa3 spa4 mutant background has no visible effect (Figure 4a,b). Previously, we have shown that SPA genes also control elongation growth of adult plants, with adult spa quadruple mutants being extremely small and dwarfed (rosette size approximately 0.5 cm; Laubinger et al., 2004). We therefore compared the contributions of SPA1 and SPA2 to this response. spa1 spa3 spa4 triple mutants (that contain only functional SPA2) were more dwarfed than spa2 spa3 spa4 mutants (that contain only functional SPA1), indicating that the contribution of SPA1 is greater than that of SPA2 (Figure 4c,d). Consistent with this observation, loss of SPA2 function in a spa3 spa4 mutant background did not

6 582 Kirsten Fittinghoff et al. (a) (b) (c) Figure 3. SPA1 is sufficient for normal etiolation in darkness and under low light fluence rates. (a) Visual phenotypes of wild-type, spa1-7 and spa2 spa3 spa4 mutant seedlings grown in the dark or in two different fluence rates of FRc, Rc or Bc. For comparison, a dark-grown spa quadruple-mutant seedling is shown as well. Low fluence rates (upper figures) were 0.05 lmol m )2 sec )1 for FRc, 1 lmol m )2 sec )1 for Rc and 0.5 lmol m )2 sec )1 for Bc. High fluence rates (lower figures) were 0.4 lmol m )2 sec )1 for FRc, 10 lmol m )2 sec )1 for Rc and 2 lmol m )2 sec )1 for Bc. (b) Hypocotyl length of wild-type, spa1-7 and spa2 spa3 spa4 mutant seedlings grown in FRc, Rc and Bc of various fluence rates. Error bars denote one standard error of the mean. (c) Representative images of RNA gel blot analyses and quantification of SPA1 transcript levels in the wild type and the spa2 spa3 spa4 mutants harvested at different developmental stages. Blots were hybridized with a SPA1-specific probe and, as a loading control, with a 18S rrna probe. alter adult growth, whereas additional loss of SPA1 led to further dwarfism (Figure 4c,d). Taken together, these results show that SPA1 and SPA2 are both equally sufficient for normal development of darkgrown seedlings. In light-grown seedlings and adult plants, in contrast, SPA1 function is clearly divergent from SPA2 function, with SPA1 playing the predominant role. Light increases SPA3 and SPA4, but not SPA2 transcript levels To further examine the partially distinct functions of the SPA genes in light- and dark-grown seedlings, we investigated whether SPA1, SPA2, SPA3 and SPA4 are differently regulated by light. Figure 5(a) (c) shows that SPA2 mrna

7 Functional and expression analysis of Arabidopsis SPA genes 583 (a) (b) (c) (d) Figure 4. The two most closely related genes SPA1 and SPA2 have similar functions in dark-grown seedlings, but have evolved distinct functions in light-grown seedlings and adult plants. (a) Visual phenotype of wild-type, spa3 spa4, spa2 spa3 spa4 and spa1 spa3 spa4 mutant seedlings grown in the dark or in different fluence rates of FRc, Rc or Bc. For comparison, a dark-grown spa quadruple-mutant seedling is shown as well. Low fluence rates (upper figures) were 0.05 lmol m )2 sec )1 for FRc, 1 lmol m )2 sec )1 for Rc and 0.5 lmol m )2 sec )1 for Bc. High fluence rates (lower figures) were 0.4 lmol m )2 sec )1 for FRc, 10 lmol m )2 sec )1 for Rc and 2 lmol m )2 sec )1 for Bc. (b) Hypocotyl length of seedlings grown in FRc, Rc or Bc of various fluence rates. Genotypes are as in (a). Error bars denote one standard error of the mean. (c) Visual phenotype of 26-day-old adult plants. Genotypes are as in (a). (d) Petiole length and total leaf length of 26-day-old plants. Genotypes are as in (a). Error bars denote one standard error of the mean.

8 584 Kirsten Fittinghoff et al. (a) (b) (c) Figure 5. SPA3 and SPA4 mrna levels, but not SPA2 transcript levels, are increased by light. Total RNA gel blot analysis and quantification of SPA2, SPA3 and SPA4 transcript accumulation in 4-day-old dark-grown seedlings (wild type RLD) transferred to (a) 3 lmol m )2 sec )1 FRc, (b) 30 lmol m )2 sec )1 Rc or (c) 5 lmol m )2 sec )1 Bc for 0 24 h. SPA2, SPA3 and SPA4 signals were normalized to 18S rrna levels after phosphorimager quantification. abundance was not altered by light. This contrasts the behavior of SPA1 transcript levels that are strongly increased by light (Hoecker et al., 1999; Figure 1b). Thus, the lack of light induction of SPA2 correlates with the lack of SPA2 function in light-grown seedlings. The abundance of SPA3 and of SPA4 mrnas strongly increased after irradiation with FR, R or B (Figure 5a c). This increase was most predominant 2 h after the initiation of light treatment for all light qualities tested (Figure 5a c). Hence, light regulation of SPA3 and SPA4 is similar to that of SPA1. These findings correlate with the observations reported here and previously that SPA1, SPA3 and SPA4 regulate seedling development in the light (Hoecker et al., 1998; Laubinger and Hoecker, 2003). Expression of SPA3 and SPA4 in photoreceptor mutants To determine which photoreceptors mediate the lightinduction of SPA3 and SPA4 mrnas, we analyzed the levels of SPA3 and SPA4 mrnas in photoreceptor mutants. In these experiments, the regulation of SPA3 and SPA4 was very similar. Induction of SPA3 and SPA4 mrnas by FRc was completely abolished in a phya mutant (Figure S2). This is in agreement with the knowledge that phya is the only receptor capable of perceiving FRc (Casal et al., 1997; Neff et al., 2000). In Rc, the levels of SPA3 and SPA4 mrnas were not strongly affected by a phya mutation but were strongly decreased in phyb mutants when compared with the wild type. Induction of SPA3 and SPA4 by Rc was almost fully lost in phya phyb double mutants (Figure S2). This indicates that Rc regulates SPA3 and SPA4 transcript levels primarily through the photoreceptor phyb and secondarily through phya. In Bc, the regulation of SPA3 and SPA4 transcript levels was complex and resembled that of SPA1. In low Bc fluence rates, induction of SPA3 and SPA4 transcripts by B was abolished in phya mutants and phya cry1 cry2 mutants, but not in cry1 cry2 mutants. Under high Bc fluence rates, only phya cry1 cry2 triple mutants completely lacked a B-induced increase in SPA3 and SPA4 mrna levels. Thus, B induction of SPA3 and SPA4 transcript levels depends on phya, cry1 and cry2 (Figure S3, data not shown). Comparison of SPA transcript levels at different developmental stages Our analysis of SPA transcript levels suggests that differential expression of the four SPA genes may at least contribute to the partially distinct functions among SPA genes. To further test this idea, we subsequently conducted a quantitative comparison of SPA transcript levels during development. To this end, we performed RNA gel blot hybridizations using poly(a)þrna (for details see Experimental procedures). In dark-grown seedlings, the levels of the four SPA mrnas were very similar (Figure 6a,b): only the amount of SPA2 transcript was consistently slightly higher when compared to the transcript levels of SPA1, SPA3 and SPA4. In seedlings grown in Rc for 3 days, SPA1, SPA3 and SPA4

9 Functional and expression analysis of Arabidopsis SPA genes 585 (a) (b) Figure 6. Analysis of SPA1-SPA4 transcript levels during development. (a) A comparative poly(a)þrna gel blot analysis of SPA1, SPA2, SPA3 and SPA4 mrna levels in seedlings grown in darkness or in 30 lmol m )2 sec )1 Rc for 3 days and in 4-week-old adult plants. SPA mrnas were detected with SPA-specific probes (for details see Experimental procedures). For normalization, blots were reprobed with a UBIQUITIN10 (UBQ10)-specific probe. (b) Quantification of the SPA transcript levels shown in (a). transcript levels showed the expected light induction, while SPA2 mrna levels were unchanged when compared with the amount found in dark-grown seedlings. When directly comparing the levels of the four transcripts in Rc, differences were small, suggesting that the partially distinct functions of SPA1 SPA4 are unlikely to be caused by differences in the transcript levels per se. In the leaves of adult plants, the mrna levels of SPA1, SPA3 and SPA4 were higher than the amount of SPA2 transcript, and SPA3 was the most abundant SPA mrna. These results show that the SPA transcript levels are differentially regulated in the adult plant. It was noticeable that SPA2 transcript levels were unchanged throughout the developmental stages tested. Thus, the comparatively low transcript levels of SPA2 in adult plants correlate with the observed minor function of SPA2 in controlling adult elongation growth. The kinase-like domain is not essential for SPA1 function The transcript analysis indicates that the different expression patterns of SPAs probably contribute to the partially distinct functions of the four SPA genes but do not fully explain the differences among SPA functions. It is therefore possible that differences in SPA protein sequences add to the functional diversity of SPAs. At least for SPA1 it was shown that the WD-repeat domain is essential for SPA1 function and that it is involved in binding HY5 and HFR1 (Hoecker et al., 1999; Saijo et al., 2003; Yang et al., 2005a), whereas the coiled-coil domain of SPA1 supports interaction with COP1 and HFR1 (Hoecker and Quail, 2001; Saijo et al., 2003; Yang et al., 2005a). Most sequence divergence among the four SPA proteins is found in their respective N-termini which include a serine/threonine kinase-like motif. The in vivo functions of the kinase-like and coiled-coil domains of SPA1 have not yet been characterized. We therefore conducted a structure function analysis of the SPA1 protein to test whether these domains are important for SPA1 function. To this end, we generated SPA1 deletion derivatives (Figure 7a) that lacked most of the N-terminus (DN), a smaller part of the N-terminus (Dkin, which contains highest sequence similarity to serine/threonine kinase subdomains and high sequence homology among the four SPA proteins, Laubinger and Hoecker, 2003), or the predicted coiled-coil domain (Dcc; Hoecker et al., 1999). In all deletion derivatives at least one of the two putative nuclear localization sequences (NLS) of SPA1 was retained. To try to express these mutated SPA proteins at native levels and under normal regulation, the respective cdnas were placed under the control of endogenous 5 and 3 sequences of SPA1. An insertion of a sequence encoding a 3xHA tag allowed detection of the fusion protein in transgenic plants. To test whether these deletion-derivatives have SPA1 function, we transformed these constructs into the spa1-3 mutant. Expression of these constructs was confirmed by Western blot analysis using anti-ha antibodies (Figure 7e,f). Figure 7(b) and (d) shows that Dkin and DN fully complemented the spa1 mutant phenotype. When grown under low fluence rates of FRc, spa1 mutant seedlings showed enhanced de-etiolation, whereas transgenic spa1 mutants that carried DN, Dkin or the full-length SPA1 construct exhibited a phenotype similar to that of wild-type seedlings. Hence, the C-terminus of SPA1 comprising the coiled-coil and WD-repeat domains was sufficient to rescue the spa1 mutant phenotype in FRc-grown seedlings. SPA1 function in dark-grown seedlings was revealed through the analysis of spa triple mutants (Figure 4). To investigate whether these deletion-derivatives of SPA1 were also able to fulfill SPA1 function in darkness, we transformed the respective constructs into a spa triple mutant that undergoes constitutive photomorphogenesis in darkness (spa1 spa2 spa3; Laubinger et al., 2004). Figure 7(c) shows that both N-terminal deletion derivatives of SPA1 restored etiolation in dark-grown transgenic seedlings. Hence, the coiled-coil and WD-repeat domains of SPA1 were also sufficient to allow SPA1 function in dark-grown seedlings. Moreover, these experiments show that the predicted NLS located in the N-terminus of SPA1 is not essential for SPA1

10 586 Kirsten Fittinghoff et al. (a) (b) (c) (d) (e) (f) Figure 7. The N-terminal domain of SPA1 is not required for SPA1 function, whereas the coiled-coil domain is essential. (a) Schematic representation of full-length SPA1 (FL SPA1) and three SPA1 deletion mutants tagged with 3xHA. All constructs are expressed under the control of endogenous SPA1 regulatory elements. (b, d) Visual phenotypes (b) and hypocotyl length (d) of wild-type (WT), spa1-3 and transgenic spa1-3 seedlings that were transformed with FL SPA1 or SPA1 deletion constructs shown in (a). For each construct, two independent transgenic lines are shown. Seedlings were grown in 0.3 lmol m )2 sec )1 FRc for 3 days. Error bars in (d) denote one standard error of the mean. (c) Visual phenotype of dark-grown wild-type (WT), spa1 spa2 spa3 and transgenic spa1 spa2 spa3 seedlings containing FL SPA1, DN ordkin deletion derivates, respectively. (e) Immunoblot analysis of transgenic spa1-3 seedlings transformed with Dcc. For immunodetection the membrane was incubated with an a-ha antibody and subsequently rehybridized with an a-tubulin antibody. (f) Immunoblot analysis of transgenic spa1-3 lines transformed with FL SPA1, DNorDkin deletion derivates. The membrane was treated as in (e). Sizes of molecular weight standards are shown to the right. function. We thus predict that the second NLS motif (KKKKASK) is sufficient for proper nuclear localization of SPA1 (Hoecker et al., 1999). However, it remains to be proven whether nuclear localization is indeed required for SPA1 function. Deletion of the coiled-coil domain of SPA1, in contrast, eliminated any detectable SPA1 activity (Figure 7b). Of 39 transgenic lines examined, none showed any complementation of the spa1-3 mutant phenotype. We randomly selected six transgenic lines for Western blot analysis using anti-ha antibodies. At least four of these lines expressed considerable amounts of Dcc-SPA1 protein, indicating that the lack of complementation was not caused by a lack of Dcc-SPA1 expression and/or stability (Figure 7e). Hence, we conclude that a SPA1 protein lacking the coiled-coil domain is non-functional. Because the coiled-coil domain of SPA1 is essential for the interaction with COP1 (Hoecker and Quail, 2001; Saijo et al., 2003), these findings further support the biological relevance of the SPA1/COP1 interaction. Discussion The four members of the SPA protein family are negative regulators of light signaling that function somewhat redundantly to control seedling development and elongation growth of adult plants. Similar to mutants of the cop/det/fus class, spa1 spa2 spa3 spa4 quadruple mutants exhibit constitutive photomorphogenesis at the seedling stage and severe dwarfism as adult plants (Laubinger et al., 2004). SPA proteins act in concert with the E3 ubiquitin ligase COP1 which ubiquitinates activators of the light response, such as the transcription factors HY5, LAF1 and HFR1 (Hoecker, 2005; Yi and Deng, 2005). Through the analysis of spa mutants, we have previously demonstrated that SPA2, SPA3 and SPA4 exhibit overlapping but distinct roles throughout plant development (Laubinger and Hoecker, 2003; Laubinger et al., 2004). Here, we have examined the contribution of the SPA1 gene in regulating responses and, moreover, have conducted a structure function analysis of the thus far uncharacterized N-terminal domains of SPA1. Also, our

11 Functional and expression analysis of Arabidopsis SPA genes 587 Figure 8. Relative contributions of the four SPA genes to the control of seedling and adult growth. SPA proteins are thought to act in concert with the ubiquitin ligase COP1 which ubiquitinates activators of photomorphogenesis. The sizes of the SPA proteins in the scheme reflect the relative contributions of the four SPA genes to suppression of photomorphogenesis in darkness, inhibition of overstimulation in light-grown seedlings and regulation of elongation growth in the adult plant. The model is derived from data presented here and in Laubinger et al. (2004). transcript analyses suggest that differences in the expression patterns of the four SPA genes contribute to the partially distinct functions of SPA1 SPA4. SPA1 is the main regulator of seedling development Our analysis of spa2 spa3 spa4 triple mutants which carry only a functional SPA1 gene has demonstrated that SPA1 is a potent repressor of photomorphogenesis in dark-grown seedlings. The SPA1 gene is sufficient to fully suppress the constitutive photomorphogenesis observed in spa quadruple mutants (Figure 4a). In this regard, SPA1 behaves similarly to SPA2 (Figure 4a), but differently from SPA3 and SPA4 which on their own cannot support wild-type development in darkness (Laubinger et al., 2004; Figures 7c and 8). In light-grown seedlings, SPA1 is clearly the most important regulator when compared with SPA2, SPA3 and SPA4. In low light fluence rates (Rc, FRc, Bc), the SPA1 gene is the only SPA gene that is sufficient for normal seedling deetiolation. When seedlings are grown in intermediate to high light fluence rates, SPA1 also provides the largest contribution among the four SPA genes, but it is not sufficient to fully prevent overstimulation of photomorphogenesis by light (Figures 3a,b, 8; Laubinger et al., 2004). Hence, taken together, these results show that SPA1 is the primary regulator of seedling de-etiolation in Arabidopsis. Levels of SPA1 protein are increased by light (Figure 2e), which is probably due to an increase in SPA1 transcript levels (Figure 1b; Hoecker et al., 1999). It is interesting that despite this light-induced increase in SPA1 protein abundance, SPA1 is not sufficient for normal seedling development in the light. In dark-grown seedlings, in contrast, where levels of SPA1 protein are much reduced, SPA1 is nevertheless sufficient to allow normal skotomorphogenesis. Thus, the requirement for SPA1 protein appears to be much higher in light-grown seedlings than in dark-grown seedlings, which is consistent with the finding that spa1 mutants show a visible defect only in the light and not in darkness (Figure 1a; Hoecker et al., 1998). The reasons for this phenomenon are as yet unknown. It is unlikely that lightdependent differences in SPA1 subcellular localization contribute to SPA1 function because SPA1 is constitutively nuclear-localized (Hoecker et al., 1999). We can propose three possible reasons for how SPA1 acts in the light. First, it is plausible that in the light higher amounts of SPA1 are necessary to counteract the light-induced inactivation of COP1 and thus to retain sufficient COP1 activity. The idea that COP1 function is more limiting in the light than in darkness is also supported by experiments with the cop1 eid6 missense mutant which shows normal dark development but extreme hypersensitivity to light (Dieterle et al., 2003). Light inactivates COP1 through two processes, a relatively slow one involving nuclear exclusion of COP1 and a more rapid one probably involving direct interaction of COP1 with the photoreceptors (Yi and Deng, 2005). Because light very rapidly increases the abundance of SPA1 protein and, moreover, because spa1 mutants show defects quickly after exposure to light (Hoecker and Quail, 2001), we hypothesize that the primary function of SPA1 in light-grown seedlings is not to balance the activity of decreasing amounts of active COP1 in the nucleus but rather to repress very early light signaling events in concert with active COP1 or to interfere with rapid inactivation of COP1. A second possibility is that SPA1 function is more limiting in light-grown seedlings because SPA1, like COP1, might be involved in the degradation of light-activated photoreceptors (phya and/or cry2; Seo et al., 2004; Shalitin et al., 2002). Though total phya levels were not altered in spa1 mutants (Hoecker et al., 1998), an effect of spa1 mutations on nuclear-localized phya may have eluded detection. Consistent with this idea, many aspects of the spa1 mutant phenotype in light-grown seedlings are dependent on PHYA (Figure 1a; Baumgardt et al., 2002; Hoecker et al., 1998). However, there are also facets of the spa1 mutant phenotype that are independent of PHYA (Parks et al., 2001). Third, it is possible that higher amounts of SPA1 might be necessary in light-grown seedlings, due to a possibly higher abundance and/or a lower affinity of substrate proteins when compared with darkgrown seedlings. The four SPA genes are differentially expressed Our functional analyses of various spa mutants uncovered the partially distinct functions of the four SPA genes in regulating the growth of seedlings and adult plants (Figure 8). We subsequently addressed the question of whether the divergence in SPA gene functions might be caused by differences in SPA gene expression patterns. As a whole, our SPA transcript analyses suggest that some, but not all, divergence in SPA function might be a result of distinct SPA expression.

12 588 Kirsten Fittinghoff et al. In adult plants, SPA transcript abundance correlated with SPA function. The primary regulators (SPA3, SPA4 and SPA1) were more highly expressed than the very minor regulator SPA2. This suggests that the distinct contributions of the four SPA genes in adult plants might, at least in part, be due to different SPA expression levels. In light-grown seedlings, we did not find any great quantitative differences among the four SPA transcripts. Hence, the distinct functions of SPA1 SPA4 are unlikely to be caused by differences in their transcript levels per se. However, it is noticeable that the mrna levels of the predominant regulators (SPA1, SPA3 and SPA4) are all increased by light (Figures 1, 5; Hoecker et al., 1999). SPA2, in contrast, which provides no or only a very minor contribution in the light, is not induced by light. This correlation suggests that light induction of SPA may be important for a significant role in light-grown seedlings. It is possible that a rapid increase in SPA abundance is necessary to adequately prevent overstimulation when seedlings are exposed to light. This idea is also in agreement with our finding discussed above that SPA gene function is more limiting in light-grown seedlings than in dark-grown seedlings. In dark-grown seedlings, SPA1 and SPA2 are equally important in suppressing photomorphogenesis (Figure 4a), indicating that the functions of SPA1 and SPA2 are conserved. SPA3 and SPA4, in contrast, contribute to this response to a much lower extent (Figure 8; Laubinger et al., 2004). We did not find a clear correlation between SPA function and SPA transcript levels, suggesting that other factors determine differences in SPA function. Here it is evident that the two primary regulators (SPA1 and SPA2) are also the most closely related by protein sequence, whereas SPA3 and SPA4 are more divergent from SPA1 and SPA2 (Laubinger and Hoecker, 2003). Hence, differences in SPA protein sequence might contribute significantly to specificities in SPA function. In the future, experiments swapping SPA promoters and coding regions could be helpful for directly testing this idea. Interestingly, the expression of other factors (COP1, DET1) that are responsible for degradation of light-signaling intermediates is constitutive and thus not regulated by light (Deng et al., 1992; Pepper et al., 1994). Nevertheless, a change in the abundance of COP1-complex components, such as the SPA proteins, in response to an exogenous signal could alter the activity of the complex as a whole. Thus, the regulation of SPA expression could be crucial in the adjustment of plant growth and development to changes in the light environment. The serine/threonine kinase-like domain of SPA1 is not required for SPA1 function The biochemical function(s) of SPA1 are thus far unknown. The finding that SPA1 contains a functionally essential WDrepeat domain that can interact with the degradation substrates HY5 and HFR1 suggests that SPA1 aids COP1 in substrate recruitment. An attractive hypothesis is that SPA1 might phosphorylate substrates prior to COP1-dependent ubiquitination. Substrate phosphorylation is a common mechanism regulating ubiquitination (Fang and Weissman, 2004), and at least for HFR1 it has been reported that phosphorylated HFR1 is preferentially degraded in a COP1- dependent fashion (Duek et al., 2004). The results of our structure/function analysis of SPA1 do not support this hypothesis. The N-terminal part of SPA1 including the serine/threonine kinase-like domain was not required to allow restoration of a wild-type phenotype in a spa1 mutant or a spa1 spa2 spa3 mutant (Figure 7). A similar observation was independently made by Yang and Wang (2006). This indicates that SPA1 function in light- as well as darkgrown seedlings does not fully depend on the kinase-like domain. Rather, it appears that a protein comprising only the coiled-coil and WD-repeat domains of SPA1 has full SPA1 activity. However, the N-terminus probably contributes to normal SPA1 function because it is involved in destabilizing the SPA1 protein (Yang and Wang, 2006). Moreover, we cannot fully exclude the possibility that the N-terminal domain in SPA proteins has an essential function that is masked by redundancy. If the function of the SPA1 N-terminal domain is not limiting for the function of the whole SPA1 protein, the SPA2, SPA3 and SPA4 proteins might sufficiently compensate for the lack of the N-terminal domain of SPA1. Such a scenario is, for example, conceivable if more than one SPA protein is part of a COP1 complex. Our results show, though, that even in this scenario the WD-repeat and coiled-coil domains are much more critical for SPA1 function than the N- terminal region of SPA1. We consider functional redundancy of the SPA N-termini less likely, because a SPA1 deletion protein lacking the N-terminus (DN) was also capable of fully complementing a spa1 spa2 spa3 triple mutant that only contains one wild-type SPA gene (Figure 7c). However, to unequivocally prove that the SPA1 N- terminus is indeed dispensable for SPA1 function, we need to await the isolation of a quadruple spa null mutant that is confirmed to produce no SPA1, SPA2, SPA3 or SPA4 protein. Multiple pieces of evidence suggest that SPA proteins function together with COP1. However, it is thus far unknown whether SPA proteins can also act independently of COP1. Our finding that a SPA1 deletion derivative lacking the COP1-interacting coiled-coil domain is devoid of any detectable SPA1 activity suggests that an interaction of SPA1 with COP1 (and potentially other interaction partners) is essential for SPA1 function. This idea is also supported by evidence showing co-fractionation of SPA1 and COP1 in high-molecular-mass complexes (Saijo et al., 2003).

13 Functional and expression analysis of Arabidopsis SPA genes 589 Experimental procedures Plant material, light sources and growth conditions The mutants spa1-2, spa1-3, phya-101, phyb-1, spa1-2 phya-101, phya phyb (all RLD), spa2-1, spa3-1, spa4-1, spa3-1 spa4-1 (all Col) and spa1-3 spa2-1 spa3-1 were described previously (Hoecker et al., 1998, 1999; Laubinger and Hoecker, 2003; Laubinger et al., 2004). The spa1-7 allele (Col) was obtained from the SALK T-DNA collection (Alonso et al., 2003). spa1-7 was confirmed to carry a T-DNA insertion in the third exon at position 2638 bp after the ATG in the SPA1 gene. spa1-7 mutant seedlings are hypersensitive to light in a similar way to seedlings of the previously described five spa1 mutant alleles (Hoecker et al., 1998). The phya cry1 cry2 mutant (phya-201 hy4-1 fha-1, Ler) was described in Mazzella et al. (2001). Gene numbers are At2g46340 for SPA1, At4g11110 for SPA2, At3g for SPA3 and At1g53090 for SPA4. To generate spa1-7 spa3-1 spa4-1 and spa2-1 spa3-1 spa4-1 triple mutants (Col), spa3-1 spa4-1 plants were crossed with spa1-7 or spa2-1 plants, respectively, and resulting F 2 plants exhibiting a short hypocotyl in 0.5 lmol m )2 sec )1 Rc were selected and transferred to soil. The genotype at the respective three SPA loci was determined using molecular markers that can distinguish between mutant and wild-type alleles (Laubinger and Hoecker, 2003; Laubinger et al., 2004). SPA1 wild-type alleles were identified using the primers 5 - CCAGTGCCTTGTTTGTACCAAC-3 and 5 -GGTCCCCACTTCTTAT- TGTCCC-3 and spa1-7 mutant alleles were identified using the latter primer and LBB1 (Alonso et al., 2003). Plants of the desired triple-mutant genotypes were allowed to set seed and the molecular genotype was confirmed in the F 3 generation. F 3 or derived F 4 seed was used for the experiments. The spa quadruple mutant used here was obtained by crossing spa1-7 spa2-1 with spa3-1 spa4-1 plants followed by phenotypic and molecular selection as described above. LED light sources, seedling and adult growth conditions and the determination of hypocotyl length, anthocyanin levels and adult traits were described previously (Laubinger et al., 2004). For transcript analyses using etiolated seedlings, seedlings were grown in darkness for 3 days and subsequently transferred to the indicated light conditions. Construction and analysis of transgenic SPA1::SPA1-HA and SPA1 deletion lines The SPA1::SPA1-HA construct contains 2241 bp of the 5 sequence of SPA1, thespa1 gene with introns up to, but not including, the Stop codon, a 3xHA tag followed by a Stop codon and 1026 bp of the putative 3 -untranslated region (UTR) of SPA1. Detailed information on its construction is provided online as Appendix S1. The construct was transferred into spa1-3 mutant plants by Agrobacterium-mediated transformation. All 28 independent transgenic lines showed full complementation of the spa1 mutant phenotype in FRc (0.3 lmol m )2 sec )1 ). Of these, two lines were propagated by selfing to generate homozygous, single-insertion transgenic lines (line 11 and line 26). Construction of SPA1 deletion constructs is described in Appendix S1. Constructs were transformed into the spa1-3 and spa1-3 spa2-1 spa3-1 mutants and 32 to 56 independent transgenic lines each were generated. For spa1-3 lines carrying full length SPA1, DN or Dkin, respectively, two spa1-3 complementing lines were propagated by selfing to obtain homozygous single-insertion lines. RNA isolation and RNA gel blot analyses To analyze light regulation of SPA transcript levels, total RNA was isolated using the RNeasy plant mini kit (Qiagen, Hilden, Germany) from 4-day-old dark-grown seedlings that had been transferred to the indicated light conditions for 0 30 h. Five to fifteen micrograms of total RNA was separated by standard glyoxal gel electrophoresis and blotted onto nylon membranes. Membranes were hybridized with SPA1-, SPA2-, SPA3- orspa4-specific, 32 P- labeled probes comprising the complete respective open reading frame (ORF). Hybridization and washing procedures were described in Laubinger and Hoecker (2003). Exposure to phosphoimager plates was carried out for at least 4 days. Signals of SPA1, SPA2, SPA3 or SPA4, respectively, were normalized to the signal of 18S rrna. For comparative SPA transcript analysis (Figure 6), we used poly(a)þrna rather than total RNA because the separation behavior during electrophoresis varied among the four SPA transcripts when rrna is present. First, total RNA was isolated from at least 5 g of tissue by standard phenol/chloroform extraction followed by lithium chloride precipitation. This total RNA was subsequently used for poly(a)þ isolation with the oligotex mrna midi kit (Qiagen). One microgram of poly(a)þrna was separated, blotted and hybridized as described above. For normalization, a UBIQUITIN10 (UBQ10)-specific probe was synthesized by PCR using the primers 5 -CTG TTA TGC TTA AGA AGT TCA ATG T-3 and 5 -GAA ACA TAG TAG AAC ACT TAT TCA TC-3. This probe was used to rehybridize the membranes. SPA signals were normalized with the respective UBQ10 signals and the obtained ratio was further divided by a factor that corrected for differences in probe size (SPA1, 3.09 kb; SPA2, 3.11 kb; SPA3, 2.54 kb; SPA4, 2.39 kb). All experiments were repeated at least twice. Protein isolation and Western blotting Seedlings were ground in liquid nitrogen, resuspended in protein extraction buffer [150 mm NaCl; 50 mm 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), ph 7.5; 1 mm EDTA; 10 mm NaF; 25 mm b-glycerophosphate; 2 mm sodium orthovanadate; 0.1% (v.v) Tween-20; 10% (v.v) glycerol, 1 mm DTT; 1 mm phenylmethyl sulfonyl fluoride (PMSF); 2x Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany)] and clarified by centrifugation. After determination of the protein concentration using Bradford reagent (Bio-Rad Protein Assay, Bio-Rad, Munich, Germany), lg of total protein was separated by SDS-PAGE and blotted onto nitrocellulose membranes. Hemagglutinin-tagged proteins were detected with anti-ha monoclonal antibodies (Roche). A tubulin-specific antibody (Sigma, Taufkirchen, Germany) was used as a loading control. Chemiluminescene visualization was carried out with the ECL plus Western Blot Detection kit (GE Health Care, Freiburg, Germany). Acknowledgements We thank Alexander Maier and Sabine Link for excellent technical assistance, and Wilhelm Rogmann and the greenhouse staff for expert care of our plants. We are grateful to Jorge Casal for the gift of phya cry1 cry2 seed, to Margaret Ahmad and Alfred Batschauer for providing cry1 cry2 seed and to Y. Wada for the gift of the 3xHA plasmid. We thank the Nottingham Arabidopsis Stock Centre for providing spa1-7 mutant seed. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 590 to UH).