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Plant, Cell and Environment (2015) 38, 411–422

doi: 10.1111/pce.12395

Original Article

Glycosyltransferase-like protein ABI8/ELD1/KOB1 promotes Arabidopsis hypocotyl elongation through regulating cellulose biosynthesis Xin Wang1,2, Yanjun Jing1, Baocai Zhang3, Yihua Zhou3 & Rongcheng Lin1 1

Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China, 2University of the Chinese Academy of Sciences, Beijing 100049, China and 3State Key Laboratory of Plant Genomics and National Plant Gene Research Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

ABSTRACT Seedling de-etiolation (photomorphogenesis) is an important light-regulated developmental process in plants. Here, we showed that disruption of the gene encoding a glycosyltransferase-like protein, ABA INSENSITIVE 8 (ABI8)/ELONGATION EFFECTIVE 1 (ELD1)/KOBITO1 (KOB1), caused short-hypocotyl elongation under all light conditions examined and even in darkness. We found that the ABI8 transcript level was down-regulated by light in a phytochrome A-dependent manner. Furthermore, light destabilized ABI8 protein via the 26S proteasome degradation pathway. We showed that ABI8 promoted the expression of genes involved in cell elongation and cellulose synthesis. Consistently, the cellulose content was reduced in the abi8 mutants and application of 2, 6-dichlorobenzonitrile (an inhibitor of cellulose biosynthesis) mimicked the abi8 mutant phenotype. Moreover, we found that phytochrome and cryptochrome photoreceptors negatively, whereas CONSTITUTIVE PHOTOMORPHOGENIC 1 positively, regulated cellulose synthesis. We also showed that ELONGATED HYPOCOTYL 5 directly bound to the promoters of ABI8 and several cellulose synthesis genes and repressed their expression in light conditions. Taken together, our study reveals that ABI8 functions as a negative factor in light inhibition of hypocotyl elongation through modulating cellulose biosynthesis. Key-words: cellulose synthesis; light; photomorphogenesis.

INTRODUCTION Light plays crucial roles in various aspects of plant growth and development, including early seedling de-etiolation (also known as photomorphogenesis). Seedling development of Arabidopsis thaliana has been used as a model system to study the molecular mechanism regulating light signalling in higher plants. Under light conditions, Arabidopsis undergoes photomorphogenesis, displaying short hypocotyls and open cotyledons with functional chloroplasts. In contrast, darkgrown plants exhibit long hypocotyls and closed cotyledons Correspondence: R. Lin. E-mail: [email protected] © 2014 John Wiley & Sons Ltd

and develop etioplasts, in a process designated as etiolation or skotomorphogenesis (von Arnim & Deng 1996; Arsovski et al. 2012). To respond and adapt to their ambient light environment, plants have evolved a complicated network of photoreceptors and numerous downstream signalling factors (Chen et al. 2004). Plants contain two major types of photoreceptors, namely phytochromes (sensing 600–750 nm or red and far-red light) and cryptochromes (sensing 320– 500 nm or blue and UV-A light), which perceive light signals to govern the photomorphogenic response (Briggs & Olney 2001). Among the Arabidopsis phytochromes (phys), phyA primarily regulates the high irradiance response to continuous far-red light, while phyB plays a dominant role in mediating responses under continuous red light (Li et al. 2011). Cryptochrome 1 (cry1) is a major photoreceptor for high intensities of blue light, whereas cry2 primarily responds to low-intensity blue light (Lin 2002). Accumulating studies have identified a large number of signalling components downstream of the photoreceptors and some of these components have been extensively characterized (Chen et al. 2004; Jiao et al. 2007). COP1 (for CONSTITUTIVE PHOTOMORPHOGENIC 1) is a negative regulator in the light signalling pathway (Deng et al. 1992; Lau & Deng 2010). Seedlings of the cop1 mutant exhibit constitutively short hypocotyls in dark and light conditions and open cotyledons in the dark (Deng et al. 1991). COP1 encodes a RING-type E3 ubiquitin ligase that interacts with positive factors of the light signalling pathway and mediates their degradation via the 26S proteasome pathway (Lau & Deng 2012). ELONGATED HYPOCOTYL 5 (HY5) is a master transcription factor that relays signals from COP1 to reprogram the activity of downstream genes (Oyama et al. 1997; Lee et al. 2007). HY5 is targeted by COP1 for degradation in the dark and is stabilized after light exposure (Hardtke et al. 2000; Osterlund et al. 2000). PHYTOCHROME INTERACTING FACTORs (PIFs), which negatively regulate phytochrome signalling, are another important type of transcription factor. PIFs encode basic helix-loop-helix proteins that are phosphorylated and degraded by light in a phy-dependent manner (Shen et al. 2005; Al-Sady et al. 2006; Henriques et al. 2009). Multiple PIF 411

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factors (including PIF1, 3, 4 and 5) play partially redundant roles in promoting skotomorphogenesis (Leivar et al. 2008; Shin et al. 2009; Leivar & Quail 2011). Together, COP1, HY5 and PIF proteins are proposed to constitute the key elements of the light signalling pathway (Lau & Deng 2010). Although other mediators, such as transcription factors, kinases and phosphatases, that either positively or negatively regulate the light signalling pathway, have been identified, the mechanisms underlying the actions of these regulators remain to be fully understood. More importantly, increasing evidence indicates that light signalling is often integrated with other signalling pathways to regulate a common set of responses in plants, thus increasing the complexity of the regulatory network (Alabadi & Blazquez 2009; Seo et al. 2009; Lau & Deng 2010; Leivar & Monte 2014). Studies from our laboratory and others suggest that ABA INSENSITIVE 5 (ABI5) is a regulator of the light response, and that light and ABA signalling can interact (Chen et al. 2008; Tang et al. 2013). To further investigate whether other ABI genes are involved in light signalling, we focused on ABI8 in this study. ABI8, which is also known as ELONGATION DEFECTIVE 1 (ELD1) and KOBITO1 (KOB1), encodes a glycosyltransferase-like protein in Arabidopsis previously implicated in cellulose biosynthesis and hypocotyl elongation (Cheng et al. 2000; Pagant et al. 2002; Brocard-Gifford et al. 2004; Kong et al. 2012). However, its mechanism of action remains unclear. Our study demonstrates that ABI8/ELD1/ KOB1 defines a negative regulator of photomorphogenesis and provides insight into how cellulose synthesis is modulated in response to the ambient light environment.

MATERIALS AND METHODS Plant materials and growth conditions The abi8-1 (Salk_087345), abi8-2 (CS904266), kob1-3, cop1-4, phyA-211, phyB-9, cry1-304, hy5-215, pif1-2, pif3-1 and pifq mutants were derived from the A. thaliana Columbia (Col) ecotype (Reed et al. 1993, 1994; McNellis et al. 1994; Oyama et al. 1997; Mockler et al. 1999; Huq et al. 2004; Leivar et al. 2008; Kong et al. 2012; Chen et al. 2013).The T-DNA insertion mutants were confirmed by PCR genotyping and the insertion site was verified by sequencing. Double mutants were generated by genetic crossing and homozygous lines were used in these experiments. After sterilization, seeds were sown onto Murashige and Skoog (MS) medium containing 1% sucrose and 0.8% agar and were incubated at 4 °C in darkness for 3 d, followed by irradiation for 9 h with white light to promote uniform germination. Far-red (12 μmol m−2 s−1), red (20 μmol m−2 s−1) and blue (14 μmol m−2 s−1) light were supplied by light-emitting diode light sources, and white light (60 μmol m−2 s−1) was supplied by cool white fluorescent lamps.

50 μm MG132 [in 0.5% dimethyl sulfoxide (DMSO)] or DMSO alone under white light for the periods of time indicated in the text. For 2,6-dichlorobenzonitrile (DCB) treatment, seedlings were grown in MS medium supplemented with various concentrations of DCB or Mock (DMSO) for 5 d.

Measurement of hypocotyl length and cotyledon angle, and scoring of green cotyledons For hypocotyl length and cotyledon angle measurements, seedlings of different genotypes were grown side by side on the same plate, and at least three independent plates were used in all experiments. Seedlings were then photographed and their hypocotyl lengths and cotyledon angle were measured using Image-J software (http://rsb.info.nih.gov/ij). For seedling greening analysis, dark-grown seedlings were transferred to continuous white light for 1 d, and the percentage of dark-green cotyledons in 50 to 80 seedlings of each genotype was determined.

Determination of anthocyanin content The anthocyanin content was measured using a spectrophotometer according to Fankhauser & Casal (2004). The relative amount of anthocyanin per seedling was calculated by subtracting the A657 from A530.

Cellulose measurement The cellulose content was measured using a modified method as described (Zhang et al. 2012). Briefly, 5-day-old Arabidopsis seedlings were ground to a fine powder using a Tissue Lyser II (Qiagen, Valencia, CA, USA) and the powder was used to prepare de-starched alcohol-insoluble residues (AIR) as described previously (Harholt et al. 2006). Two milligrams of each de-starched AIR sample was hydrolysed in 2 m trifluoroacetic acid (TFA) at 121 °C for 90 min. The deposit obtained after TFA treatment was hydrolysed in Updegraff reagent (acetic acid/nitric acid/water, 8:1:2 v/v) and used in an anthrone assay to quantify the cellulose content (Updegraff 1969).

Gene expression analysis by quantitative RT-PCR Plant total RNA was extracted using an RNAprep Pure Plant Kit (Tiangen, Beijing, China), and first-strand cDNA was synthesized with oligo (dT) using Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Real-time PCR was carried out using the SYBR Premix ExTaq Kit (Takara, Kyoto, Japan) following the manufacturer’s instructions. The expression levels were normalized to the expression of a ubiquitin (UBQ1) gene. The PCR primer sets are listed in Supporting Information Table S1.

Chemical treatment

Plasmid construction

For MG132 proteasome inhibitor treatment, 5-day-old etiolated seedlings were transferred to solutions containing

To construct the ABI8p::GUS reporter vector, a fragment spanning the region 2 kb upstream of the ATG start site of © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

ABI8 regulates hypocotyl elongation ABI8 was amplified by PCR, and cloned into the pEASY vector (TransGen, Beijing, China), resulting in pEASYABI8p. The pEASY-ABI8p plasmid was digested with SalI and BamHI to release the ABI8 promoter, which was then ligated into pBI101 digested with the same enzymes to generate ABI8p::GUS. To obtain the ABI8 coding sequence, first-strand cDNA was reverse transcribed using oligo (dT) primer from total RNA extracted from Col wild-type seedlings. The ABI8 sequence was amplified using High Fidelity Pfu DNA Polymerase (Invitrogen) and cloned into pEASY, resulting in pEASY-ABI8. The pEASY-ABI8 plasmid was digested with XbaI and SalI to release the ABI8 coding sequence, which was then ligated into modified pCAMBIA1302 (http:// www.cambia.org/daisy/cambia/585) digested with the same enzymes, generating 35S::ABI8-GFP. All amplified fragments were validated by sequencing.

Plant transformation The binary vectors were electroporated into Agrobacterium tumefaciens strain GV3101 and then transferred into Col wild-type or abi8 plants via the floral dip method (Clough & Bent 1998). Transgenic plants were selected on MS plates in the presence of 50 mg L−1 kanamycin (for 35S::ABI8-GFP) or hygromycin (for ABI8p::GUS) and homozygous lines in the T3 generation were used in this study.

GUS histochemical staining Seedlings of the ABI8p::GUS transgenic line were harvested and incubated overnight in 0.1 m sodium phosphate buffer containing 50 mm K3Fe(CN)6, 50 mm K4Fe(CN)6 and 1 mm 5-bromo-4-chloro-3-indolyl-β-D-glucuronideat 37 °C. GUS expression was examined under a dissecting microscope and images were captured by a digital camera (Olympus, Tokyo, Japan).

GFP fluorescence observation The fluorescence of 35S::ABI8-GFP transgenic seedlings was visualized using a confocal microscope (Leica, Mannheim, Germany). All images were captured at the same settings.

Protein immunoblotting Seedlings were homogenized in extraction buffer containing 50 mm Tris-HCl, pH7.5, 150 mm NaCl, 10 mm MgCl2, 0.1% Tween 20, 1 mm phenylmethylsulfonyl fluoride and 1× complete protease inhibitor cocktail (Roche, Mannheim, Germany). The extracts were centrifuged at 14 000 × g twice at 4 °C for 10 min each, and the protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). The proteins were separated on 10% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes and blotted with anti-GFP (Clontech, Mountain View, CA, USA) or anti-tubulin (Jing et al. 2013) antibodies. The protein bands were visualized using the standard enhanced chemiluminescence method. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

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ChIP assay The ChIP assay was performed according to a method described previously (Jing et al. 2013). Protein-DNA samples were precipitated with HY5 antibodies or IgG serum and the amount of DNA was quantified by real-time PCR using the primers listed in Supporting Information Table S1. Relative enrichment is expressed as the ratio of DNA amount after ChIP with HY5 antibodies to that with the IgG serum control.

RESULTS Disruption of ABI8 causes short hypocotyls under light conditions and in darkness To investigate the role of ABI8 in light-regulated responses, we obtained three abi8 mutant alleles: two T-DNA insertion lines, namely abi8-1 (Salk_087345) and abi8-2 (CS904266), and one harbouring a point mutation, that is, kob1-3 (Kong et al. 2012; Fig. 1a). The seedlings were grown in MS medium under white light for 5 d and their endogenous ABI8 levels were determined. ABI8 transcript was barely detected in abi8-1 and abi8-2, while the ABI8 level in kob1-3 was almost the same as that in the wild type (Fig. 1b), indicating that abi8-1 and abi8-2 are null alleles. We noticed that the homozygotes of abi8-1 and abi8-2 could germinate and develop small seedlings when sown on MS plates. However, these null mutant plants showed a strong dwarf phenotype and died later when grown in soil, while the kob1-3 plants were dwarf, but able to set a few seeds, as previously reported (Cheng et al. 2000; Pagant et al. 2002). Therefore, in the following experiments, heterozygous mutant seeds were sown on MS medium and the homozygous seedlings were selected for analysis based on their aberrant phenotypes. We then grew Columbia (Col) wild type and abi8 mutants in continuous red, far-red and blue light conditions and in darkness for 5 d. The abi8 mutant seedlings developed much shorter hypocotyls and roots, and cotyledons that were wider open than those of the wild-type plants in all light conditions tested. The phenotypes of abi8-1 and abi8-2 were stronger than those of the kob1-3 allele (Fig. 1c–e). Strikingly, abi8-1 and abi8-2 displayed open cotyledons even in darkness (Fig. 1c). We further examined the expression of COP3/ HOOKLESS 1 (HLS1), which is a regulator of cotyledon development in photomorphogenesis (Hou et al. 1993), and found that the HLS1 mRNA level was drastically reduced in abi8-2 compared with the wild type (Supporting Information Fig. S1), indicating that the open cotyledon of etiolated abi8 is likely not due to a mechanical consequence of the thicker hypocotyls. These results together suggest that loss of ABI8 results in reduced hypocotyl elongation and therefore that ABI8 represses photomorphogenesis and promotes skotomorphogenesis. We also generated transgenic plants overexpressing an ABI8 in-frame fusion with green fluorescence protein (GFP) under the control of the CaMV 35S promoter (35S::ABI8-GFP) in the Col wild-type background. These transgenic seedlings were indistinguishable from those of wild-type plants when grown in darkness (Supporting Information Fig. S2).

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ABI8 expression is repressed by light

Figure 1. Loss-of-function mutants of ABI8 exhibit short-hypocotyl responses. (a) Diagram of ABI8 and the sites altered in the abi8 mutants. Block boxes represent exons and lines between the boxes indicate introns. Triangles denote T-DNA insertions. Pf and Pr are the positions of primers used for RT-PCR analysis. kob1-3 is a point mutation in C371T. (b) Relative ABI8 expression in the wild type and mutants. Data are mean ± SD of triplicate samples. (c) Photomorphogenic phenotypes of seedlings grown in the dark and under various light conditions. Bars indicate 2 mm. (d) Hypocotyl length of seedlings grown as shown in c. (e) Cotyledon angle of seedlings grown as shown in c. For c–e, seedlings were grown in the indicated light conditions for 5 d. Data are mean ± SD of more than 20 plants in (d and e).

Compared with the wild-type plants, the abi8-2 mutant seedlings accumulated more anthocyanin when grown in various light conditions (Supporting Information Fig. S3a). Furthermore, the expression levels of three genes encoding the light harvesting proteins of photosystem II, that is, LHCB1.3, LHCB2.1 and LHCB2.2, and also of CHS (encoding chalcone synthase in the anthocyanin biosynthesis pathway) were increased in abi8-2 relative to the wild

We examined the gene expression pattern of ABI8 in Col wild-type seedlings grown under various light conditions using reverse transcription followed by quantitative real-time PCR (RT-PCR). We found that the ABI8 transcript level in seedlings grown under white light was about 32% of that in dark-grown seedlings (Fig. 2a). When 5-day-old etiolated seedlings were irradiated with light for up to 12 h, ABI8 mRNA levels gradually decreased. By contrast, levels increased when light-grown seedlings were transferred to the dark (Fig. 2a). Furthermore, ABI8 expression was slightly down-regulated by red and blue light treatment, and was drastically reduced by far-red light relative to darkness (Fig. 2b). These data suggest that light negatively regulates ABI8 at the transcript level, with far-red light playing the predominant role under the conditions investigated. We then tested whether light regulation of ABI8 expression is mediated by photoreceptors. phyA, phyB, and cry1 mutants and Col wild-type plants were grown under far-red, red and blue light conditions, respectively. As shown in Fig. 2c, compared with Col, the ABI8 mRNA level was greatly increased in the phyA mutant. However, no difference in ABI8 expression was found between phyB or cry1 and the wild type. To explore the possibility that ABI8 is regulated in a tissuespecific manner in response to light, we generated transgenic plants expressing ABI8p::GUS (a glucuronidase reporter gene driven by a 2.0-kb promoter fragment of ABI8). The GUS reporter was expressed in the roots regardless of the light treatment (Fig. 2d). Interestingly, GUS was strongly expressed in the uppermost regions of hypocotyls in darkness, but was barely stained in light-grown hypocotyls (Fig. 2d), further confirming the tissue-specific mode of light regulation of ABI8.

Light promotes 26S proteasome-mediated protein degradation of ABI8 We next investigate whether ABI8 is also regulated at the post-translational level by light. To this end, we expressed 35S::ABI8-GFP in the abi8-2 mutant background. The ABI8GFP fusion protein was expressed and the transgene fully complemented the short-hypocotyl and dwarf phenotypes of abi8 (Supporting Information Fig. S4, two representative lines are shown), suggesting that ABI8-GFP is functional in the transgenic plants. Five-day-old dark-grown 35S::ABI8-GFP seedlings were exposed to white light for up to 24 h, and the ABI8 protein level was examined by immunoblotting using the GFP antibody. We found that the ABI8-GFP fusion protein level was gradually decreased after light irradiation, indicating that ABI8 protein is unstable in the presence of light (Fig. 3a). To test whether ABI8 turnover involves the 26S proteasome © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

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Figure 2. ABI8 transcript level is down-regulated by light. (a–c) Quantitative RT-PCR assay showing ABI8 expression under conditions of darkness, light and their transitions (a), different wavelengths of light (b) and in different photoreceptor mutants (c). In c, the phyB, cry1 and phyA mutants and the wild type were grown in continuous red, blue or far-red light, respectively, for 5 d. Relative ABI8 expression was normalized to the level of UBQ1. For a–c, data are mean ± SD of triplicate samples. (d) GUS staining of ABI8p::GUS transgenic plants grown under white light and in darkness. Insets show enlargements of the boxed regions. Bars denote 1 mm.

degradation pathway, we treated seedlings with MG132, a proteasome-specific inhibitor, following transfer from darkness to light. Immunoblot experiments showed that the ABI8-GFP fusion protein was more stable in seedlings treated with MG132 for 12 and 24 h in light conditions than in the DMSO mock-treated seedlings (Fig. 3b). Furthermore, we observed the GFP fluorescence in the hypocotyls by confocal microscopy, and found that ABI8-GFP was likely distributed in the plasma membrane, cell wall and nucleus, and in a punctate pattern within the cytoplasm (Pagant et al. 2002; Lertpiriyapong & Sung 2003; Brocard-Gifford et al. 2004). Nevertheless, the intensity of ABI8-GFP fluorescence was greatly reduced after 1 d of light treatment (blank and DMSO panels), whereas it was not altered when seedlings were treated with MG132 inhibitor (Fig. 3c). These data confirm that ABI8 degradation is mediated by the 26S proteasome pathway.

ABI8 promotes hypocotyl cell elongation Since the hypocotyl length is greatly inhibited in the abi8 alleles (Fig. 1), we decided to explore the underlying molecular mechanism by which ABI8 regulates hypocotyl growth. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

We found that ABI8 predominantly regulates hypocotyl cell length, but not cell number (Supporting Information Fig. S5), consistent with a previous report of the eld1 mutant allele (Cheng et al. 2000). Next, we compared the expression of several cell elongation-related genes, including INDOLE-3ACETIC ACID INDUCIBLE 19 (IAA19), EXPANSIN 2 (EXP2) and an uncharacterized gene At2g43050, between Col and abi8-2 plants (Oh et al. 2012; Jing et al. 2013). Quantitative RT-PCR experiments showed that the transcript levels of these genes were greatly decreased in abi8-2 compared with the wild type (Fig. 4), suggesting that ABI8 contributes to the activation of these genes, in agreement with its role in regulating hypocotyl cell elongation.

Cellulose content and cellulose synthetic gene expression are impaired by ABI8 mutation Prompted by the observation that cell wall and cellulose biosynthesis are defective in kob1 mutants (Pagant et al. 2002), we grew Col and abi8-1 and abi8-2 mutant seedlings both in the dark and light conditions for 5 d, and determined their cellulose content. As shown in Fig. 5a, light repressed cellulose levels both in the wild-type and abi8 plants. The

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Figure 4. ABI8 activates cell elongation-related gene expression. Relative expression of cell elongation-related genes in the wild type and the abi8 mutant. Plants were grown in darkness for 5 d. Expression levels were normalized to the level of UBQ1. Data are mean ± SD of triplicate samples.

Figure 3. Light regulates ABI8 protein stability. (a) The abi8/35S:ABI8-GFP transgenic seedlings were grown in darkness for 5 d and were then transferred to white light for up to 24 h. Dark-grown Col seedlings were set as a negative control. (b) Dark-grown abi8/35S:ABI8-GFP seedlings were treated without (blank) or with dimethyl sulfoxide (DMSO) or 50 μm MG132 under white light conditions and grown for a further 12 or 24 h. ABI8-GFP fusion protein was immunoblotted with GFP antibody. Immunoblotting with tubulin antibody served as an equal loading control. (c) Confocal imaging of green fluorescence protein (GFP) fluorescence of abi8/35S:ABI8-GFP seedlings. The seedlings were treated without (blank) or with DMSO or 50 μm MG132 under white light conditions for 1 d. Bars indicate 50 μm.

cellulose contents of the two abi8 alleles were greatly decreased, to approximately 50% of wild-type levels, both in the dark and light conditions. Cellulose biosynthesis is catalysed by the cellulose synthase complex, which is encoded by the cellulose synthase (CESA) gene family (Doblin et al. 2002). The Arabidopsis genome contains CESAs that are involved in cellulose synthesis in primary (primary CESAs) and in secondary (secondary CESAs) cell walls. We found that the transcript levels of several primary CESAs, that is, CESA1 and CESA6, and secondary CESAs, that is, CESA4, CESA7 and CESA8, were reduced in abi8-2 compared with Col (Fig. 5b). In addition, CSI1 (CELLULOSE SYNTHASE INTERACTING 1), a gene encoding an interacting protein of CESA6 (Gu et al. 2010), was also down-regulated in abi8 (Fig. 5b). These results indicate that ABI8 promotes the expression of genes involved in cellulose biosynthesis.

Next, we tested whether these cellulose biosynthesisrelated genes are regulated by light. Similar to ABI8, the transcript levels of these genes were much lower in lightgrown seedlings than in dark-grown seedlings (Fig. 5c). We also observed that the expression of CESA1, CESA3, CESA6 and CSI1 was down-regulated following dark-to-light transition, but up-regulated following the light-to-dark transition (Fig. 5d,e).

Chemically reducing cellulose synthesis mimics the abi8 mutant phenotype DCB is an inhibitor of cellulose synthesis (Wells et al. 1994). We hypothesized that application of DCB might cause shorthypocotyl response, mimicking the effect of the ABI8 mutation. To test this hypothesis, Col wild-type seedlings were grown in MS medium with or without 1 μm of DCB under different light conditions and their phenotypes were characterized. Compared with the untreated controls (blank and DMSO), DCB treatment drastically inhibited hypocotyl elongation, while promoting cotyledon opening in red, farred and blue light conditions (Fig. 6a,b). In addition, after DCB treatment, the seedlings accumulated more anthocyanin than did the untreated control (Fig. 6c). Furthermore, when seedlings were grown in medium containing increasing concentrations of DCB under dark conditions, the hypocotyl length was gradually decreased, while the cotyledons were gradually opened (Fig. 6d,e). Accordingly, the cellulose levels declined as the DCB concentration increased (Fig. 6f). These results demonstrate that inhibition of cellulose synthesis by DCB results in reduced hypocotyl growth in a dosagedependent manner and mimics the abi8 defects. In addition, when abi8/kob1 mutants were treated with 1 μm DCB, their hypocotyl lengths were shorter than those of DMSO mocktreated seedlings. The cotyledon angles were wider in DCB© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

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Figure 5. Cellulose content and cellulose biosynthesis-related gene expression are modulated by ABI8 and light. (a) Cellulose contents in Col and abi8 mutants. Seedlings were grown in darkness or under white light for 5 d. The hypocotyls were used for determination of light-grown plants. (b) Relative expression of cellulose synthesis-related genes in 5-day-old dark-grown Col and abi8 seedlings. (c–e) Relative expression of cellulose synthesis-related genes in light and darkness (c), or following the dark-to-light transition (d) or light-to-dark transition (e). Expression levels were normalized to the level of UBQ1. Data in a to e are mean ± SD of three replicate samples.

treated mutants than in the mock control (Supporting Information Fig. S6), suggesting that the effects of DCB and ABI8 mutation are additive.

Photoreceptors and COP1 modulate cellulose synthesis Since cellulose synthesis is regulated by light, we asked whether this regulation is mediated by the phytochrome and cryptochrome photoreceptors. Therefore, phyA, phyB and cry1 mutants and wild-type seedlings were grown in far-red, red and blue light conditions, respectively, and their cellulose levels were determined. As shown in Fig. 7, the phyA, phyB and cry1 mutant plants contained higher amounts of cellulose than the wild-type plants under matching light conditions, indicating that these photoreceptors negatively regulate cellulose synthesis. Furthermore, we found that the cellulose level was drastically reduced by mutation of COP1 (Fig. 7), suggesting that COP1 is a positive regulator of cellulose synthesis.

COP1 positively regulates the expression of ABI8 and cellulose synthesis-related genes in darkness We then examined how light signalling regulates the expression of ABI8 and cellulose biosynthesis-related genes. Since HY5, PIFs and COP1 are key light signalling transducers in © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

the pathway, we compared the relative expression levels of ABI8 and cellulose synthesis genes in Col and mutants of HY5, PIFs and COP1. When seedlings were grown in the dark for 5 d, the transcript levels of ABI8, CESA1, CESA3, CESA6 and CSI1 in the hy5, pif1, pif3, pif1/pif3 and pifq (the quadruple loss of PIF1, PIF3, PIF4 and PIF5) mutants were similar as in the wild type (Supporting Information Fig. S7a). Most remarkably, the expression of these genes was drastically inhibited by the cop1 mutation (Supporting Information Fig. S7b), indicating that COP1 positively regulates their expression.

HY5 directly represses ABI8 and CESA gene expression in light conditions We next evaluated the expression of ABI8, CESAs and CSI1 in the hy5 and pifs mutant background under continuous white light conditions. The transcript levels of ABI8, CESA1, CESA6 and CSI1, and to a lesser extent of CESA3, were increased in hy5 (Fig. 8a). The expression levels of these genes were slightly reduced in the pif1, pif3, pif1/pif3 and pifq mutants (Fig. 8a). HY5 is a basic domain/leucine zipper transcription factor that directly regulates the expression of a wide range of genes through binding to their promoters (Lee et al. 2007). We further investigated whether HY5 could bind to ABI8, CESA1, CESA6 and CSI1 in vivo using a chromatin immunoprecipitation (ChIP) assay. After precipitation with HY5 antibody or an IgG control, the DNA was quantified by

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Figure 6. 2,6-dichlorobenzonitrile (DCB) treatment mimics the abi8 mutant phenotype. (a–c) Hypocotyl length (a), cotyledon angle (b) and anthocyanin content (c) of Col wild-type seedlings grown in medium containing dimethyl sulfoxide (DMSO), DCB or neither DMSO nor DCB (blank) in the indicated light conditions for 5 d. Data are mean ± SD of more than 20 plants for a and b, and of triplicate experiments for c. The relative amount of anthocyanin is expressed as A530-0.25A657 per 100 seedlings. (d) Representative phenotype of 5-day-old dark-grown Col seedlings in medium containing a series of concentrations of DCB. Bar indicates 2 mm. (e, f) Hypocotyl length (e) and cellulose content (f) of seedlings grown as shown in d. Data are mean ± SD of more than 20 plants for (e and f).

real-time PCR with primer sets spanning the upstream promoter and the coding region. The ChIP results showed that the promoter fragments of ABI8 (fragments 1 and 3), CESA1 (fragment 4), CESA6 (fragments 3 and 4) and CSI1 (fragment 1) containing an E-box (CANNTG) or G-box (CACGTG) were significantly enriched in samples pulled down by the HY5 antibodies, but not in samples precipitated by the IgG control (Fig. 8b–e). These data confirm that HY5 is directly associated with the promoter regions of these

Figure 7. Cellulose contents are affected by mutations in phyA, phyB, cry1 and COP1. phyA, phyB, cry1 and cop1 mutants and wild-type seedlings were grown in the indicated conditions for 5 d, and the hypocotyls were used for determination. Data are mean ± SD of five replicate samples.

cellulose biosynthesis-related genes. Consistently, the cellulose content was increased in hy5 relative to the wild type (Fig. 8f).

DISCUSSION ABI8 is involved in several plant growth and developmental processes, including skotomorphogenesis, the ABA- and sugar-mediated growth response, plasmodesmatal permeability and stomatal patterning (Cheng et al. 2000; Pagant et al. 2002; Brocard-Gifford et al. 2004; Kong et al. 2012). Our collected evidence further demonstrates a negative role for ABI8 in regulating light-inhibition of hypocotyl growth. First, loss-of-function or point mutation of abi8/kob1 seedlings resulted in reduced hypocotyl elongation, increased cotyledon opening, accumulation of anthocyanin and increased light-responsive gene expression (Fig. 1, Supporting Information Fig. S3). Second, the transcript levels of cell elongationrelated and cellulose biosynthetic genes were decreased by ABI8 mutations relative to the wild type (Fig. 4 & 5). Consistently, cellulose synthesis was reduced in the abi8 mutant seedlings compared with wild-type plants (Fig. 5). Third, a reduction in cellulose synthesis by the DCB inhibitor mimicked abi8 mutant defects under light conditions and in darkness (Fig. 6). Brocard-Gifford et al. (2004) reported that ABI8 is involved in ABA signalling during germination, stomatal regulation and the regulation of gene expression. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

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Figure 8. HY5 binds to the promoters of ABI8 and cellulose biosynthesis-related genes and represses their expression in the light. (a) Expression of ABI8 and cellulose biosynthesis-related genes in hy5 and pifs mutants. Seedlings were grown in continuous white light for 5 d. Relative gene expression levels were normalized to the level of UBQ1. Data are mean ± SD of three replicate samples. (b–e) ChIP assay showing the relative enrichment of various genomic fragments of ABI8 (b), CESA1 (c), CESA6 (d) and CSI1 (e) upon precipitation with HY5 antibody or the IgG control. Diagram of promoter structure and the positions used for the ChIP-PCR assay were shown. CR, coding region. Data are mean ± SD of triplicates. (f) Cellulose contents in the hypocotyls of Col and hy5 mutant. Seedlings were grown in white light for 5 d. Data are mean ± SD of five replicate samples.

Thus, ABI8 might represent an integrative point of the ABA and light signalling pathways in the regulation of plant growth and development. Similarly, ABI5 regulates both ABA and light responses in Arabidopsis (Brocard et al. 2002; Chen et al. 2008). In agreement with its role in seedling de-etiolation, the expression of ABI8 is modulated by light both at the transcriptional and post-translational levels. At the mRNA level, ABI8 was down-regulated by white light and, markedly, by far-red light but not by red or blue light (Fig. 2a,b). Consist© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

ently, this far-red light-inhibited ABI8 expression is dependent on the far-red light photoreceptor phyA (Fig. 2). Most strikingly, we found that ABI8 is largely degraded by light through the 26S proteasome degradation system (Fig. 3). COP1 is a well-documented E3 ubiquitin ligase that destabilizes of a number of positive signalling components, such as HY5, HFR1 and LAF1 (Lau & Deng 2012). However, it is unlikely that COP1 interacts with ABI8 and regulates its stability because both of these proteins are negative regulators of the light signalling pathway.The question of

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how ABI8 is modified at the post-translational level remains to be answered in future studies. Nevertheless, repression of ABI8 at different levels might ensure that plants maintain relatively low ABI8 levels in response to light, since this protein negatively regulates light responses. COP1 is a central repressor in the light-mediated seedling de-etiolation pathway (Lau & Deng 2010). The cop1 mutant displays typical constitutive and enhanced photomorphogenic phenotypes in both dark and light conditions. The abi8 mutant seedlings show strong short-hypocotyl phenotypes similar as cop1. In addition, mutation in ABI8 leads to other defects, including impaired root elongation and reproductive growth, thus resulting in severe growth retardation or even death at the adult stage (Cheng et al. 2000; Pagant et al. 2002). However, at the molecular level, COP1 positively regulates ABI8 expression. Hence, COP1 and ABI8 play both overlapping and distinct roles. HY5 and PIFs are master transcription factors in the light signalling pathway and regulate the expression of a large set of downstream genes (Lee et al. 2007; Leivar et al. 2009). We failed to detect expression changes of ABI8 in the mutants of HY5 or PIFs, compared with the wild type in darkness (Supporting Information Fig. S7). Intriguingly, we found that HY5 is associated with the promoter regions of ABI8, CESA1, CESA6 and CSI1 and inhibits their gene expression in light conditions (Fig. 8), indicating that HY5 is an important regulator of the genes that modulate cellulose synthesis. However, PIF proteins are not involved in this process. This finding also suggests a direct role of light signalling in modulating cellulose biosynthesis. Cellulose is thought to be synthesized by cellulose synthase at the plasma membrane, and its biosynthesis is tightly regulated (Li et al. 2014). CESAs belong to family 2 glycosyltransferases (Somerville 2006), and ABI8 encodes a putative glycosyltransferase-like protein (Kong et al. 2012). A previous study demonstrated that ABI8 is required for cellulose biosynthesis during cell elongation (Pagant et al. 2002). In this work, we provide multiple lines of evidence that light negatively regulates cellulose synthesis. First, the cellulose content is lower in light-grown seedlings than in darkgrown plants (Fig. 5a). Importantly, phytochrome and cryptochrome photoreceptors repress cellulose synthesis, whereas COP1 has the opposite effect (Fig. 7). Furthermore, the expression of cellulose synthase and its related genes is down-regulated by light treatment and is activated by COP1 (Fig. 5, Supporting Information Fig. S7b). Similar to abi8, loss-of-function mutants of CESA6, CESA1 and CSI1 showed reduced hypocotyl elongation in darkness (Fagard et al. 2000; Bringmann et al. 2012). Previous studies suggest that the expression of CESAs is regulated by ethylene and brassinosteroid signalling (Hamann et al. 2004; Xie et al. 2011). Our findings further reveal that cellulose biosynthesis and CESA gene expression are modulated by the light environment. Light affects cellulose synthesis, at least in part, through regulating the transcription level of CESAs and ABI8 and the post-translational modification of ABI8. A previous study documented that movement of the cellulose synthase complex is inhibited in darkness through an inter-

action with microtubules, and that activation of phyB overrules this inhibition (Bischoff et al. 2011). It will be interesting to test whether additional regulatory layers are involved in regulating CESAs by light. The cell wall is the fundamental determinant of cell shape and form. As a major structural component of the cell wall, cellulose plays an important role in plant growth and development. During etiolated growth, cells of the hypocotyl undergo rapid expansion at right angles to the predominant orientation of cellulose microfibrils (Arsovski et al. 2012). As photoautotrophs, plants undergo photomorphogenesis, a critical developmental switch triggered by light. Therefore, light-regulated cellulose synthesis and cell wall modification are likely the main events underlying photomorphogenesis.

ACKNOWLEDGMENTS We thank the Arabidopsis Biological Resource Center for providing the T-DNA mutants, and Dr. Elena Shpak (University of Tennessee) for the kob1-3 mutant seeds. No conflict of interest is declared. This work was supported by grants from the National Natural Science Foundation of China (31170221, 31325002) to R.L. and 31125019 to Y.Z., and the Ministry of Agriculture of China (2011ZX08009-003) to R.L.

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Received 27 February 2014; received in revised form 16 June 2014; accepted for publication 23 June 2014

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. HLS1 expression in Col and abi8. Figure S2. Characterization of ABI8 overexpression transgenic plants. Figure S3. Anthocyanin contents and light-responsive gene expression in Col and abi8.

Figure S4. Complementation of abi8 by 35S::ABI8-GFP. Figure S5. ABI8 regulates cell elongation. Figure S6. Phenotype of abi8/kob1 mutants treated with DCB. Figure S7. Relative transcript levels of ABI8 and cellulose synthesis genes in hy5, pif and cop1mutants. Table S1. List of primers used in this study.

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 411–422

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KOB1 promotes Arabidopsis hypocotyl elongation through regulating cellulose biosynthesis.

Seedling de-etiolation (photomorphogenesis) is an important light-regulated developmental process in plants. Here, we showed that disruption of the ge...
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