RNAi-mediated silencing of the Arabidopsis thaliana ULCS1 gene, encoding a WDR protein, results in cell wall modification impairment and plant infertility

Plant Science 245 (2016) 71–83

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RNAi-mediated silencing of the Arabidopsis thaliana ULCS1 gene, encoding a WDR protein, results in cell wall modification impairment and plant infertility Despoina Beris a , Georgios Kapolas a , Pantelis Livanos a , Andreas Roussis a , Dimitra Milioni b , Kosmas Haralampidis a,∗ a b

University of Athens, Faculty of Biology, Department of Botany, 15784 Athens, Greece Agricultural University of Athens, Department of Agricultural Biotechnology, Iera Odos 75, 11855 Athens, Greece

a r t i c l e

i n f o

Article history: Received 25 September 2015 Received in revised form 19 January 2016 Accepted 23 January 2016 Keywords: Arabidopsis Cell wall CRL complex Infertility Lignification Ubiquitination WD40 motif WDR protein

a b s t r a c t Ubiquitin mediated protein degradation constitutes one of the most complex post translational gene regulation mechanisms in eukaryotes. This fine-tuned proteolytic machinery is based on a vast number of E3 ubiquitin ligase complexes that mark target proteins with ubiquitin. The specificity is accomplished by a number of adaptor proteins that contain functional binding domains, including the WD40 repeat motif (WDRs). To date, only few of these proteins have been identified in plants. An RNAi mediated silencing approach was used here to functionally characterize the Arabidopsis thaliana ULCS1 gene, which encodes for a small molecular weight WDR protein. AtULCS1 interacts with the E3Cullin Ring Ligase subunit DDB1a, regulating most likely the degradation of specific proteins involved in the manifestation of diverse developmental events. Silencing of AtULCS1 results in sterile plants with pleiotropic phenotypes. Detailed analysis revealed that infertility is the outcome of anther indehiscence, which in turn is due to the impairment of the plants to accomplish secondary wall modifications. Furthermore, IREGULAR XYLEM gene expression and lignification is diminished in anther endothecium and the stem vascular tissue of the silenced plants. These data underline the importance of AtULCS1 in plant development and reproduction. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The WD40 protein domain, or beta-transducin repeat (WDR), was initially identified in the G␤ subunit of the heterotrimeric G receptor. It was characterized as a repetitive, short structural motif of about 44–60 amino acids, which includes a conserved glycine-histidine (GH) dipeptide in the N-terminus and a signature tryptophan-aspartic (WD) dipeptide in the C-terminus of the domain [1–3]. The GH and WD dipeptides stabilize the WD40 fold

Abbreviations: COP1, constitutive photomorphogenic 1; TRIP1, TGF-beta receptor interacting protein 1; FY, flowering time control protein FY; PRL1, protein pleiotropic regulatory locus 1; DCAF1, DDB1-CUL4 associated factor 1; MSI4, multicopy suppressor of IRA1 4; ASK1, Arabidopsis SKP1 homolog; NST1 and NST2, NAC secondary wall thickening promoting factor 1 and 2. ∗ Corresponding author at: University of Athens, Faculty of Biology, Department of Botany, Molecular Plant Development Laboratory, 15784 Athens, Greece. Fax: +30 210 7274702. E-mail addresses: [email protected] (D. Beris), [email protected] (G. Kapolas), [email protected] (P. Livanos), [email protected] (A. Roussis), [email protected] (D. Milioni), [email protected] (K. Haralampidis). http://dx.doi.org/10.1016/j.plantsci.2016.01.008 0168-9452/© 2016 Elsevier Ireland Ltd. All rights reserved.

through a hydrogen bond network [4,5]. Today, it is considered one of the most abundant domains in eukaryotic proteins, while it is rarely found in bacteria [6]. WDR proteins constitute a large family of eukaryotic proteins with diverge functions. They contain 4–16 tandem WD40 repeats and fold into a tertiary ␤-propeller architecture [7,8], making them excellent scaffolds for protein–protein and protein–DNA interactions [9]. WDR proteins can dock a variety of substrates, with similar or distinct modes, utilizing the entire surface of the ␤-propeller architecture [6]. The diversity within the WD40 repeat sequence and the specificity of the protein, which is determined by the sequences outside the repeats, enables them to coordinate the assembly of various multi-protein complexes. Thus, WDR proteins are considered as key regulators in numerous cellular and developmental processes. These comprise mRNA synthesis, signal transduction, protein ubiquitination, histone methylation and cell cycle control [3,5,9–11]. In plants, an extended comparative study as well as a genome wide analysis revealed that Arabidopsis thaliana has about 269 low and high molecular weight proteins, categorized in 143 distinct families, which contain at least one WD40 repeat [10,12]. Several of these proteins are members of the ubiquitin-proteasome

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system (UPS) and have been previously identified as regulators and signaling hubs in many plant-specific processes [13]. The UPS marks substrate proteins with one or more ubiquitin units, that either be degraded by the 26S proteasome or transported to the vacuole/lysosome for breakdown [14]. This ubiquitination of target proteins is accomplished through the sequential action of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligase complexes (E3s). The latter constitute key elements to the specificity of ubiquitination and the arrangement of the bound ubiquitin units [15]. Amongst the identified E3s are the cullin-RING ligases (CRLs), which contain an isoform of a CULLIN protein, a RING/Ubox motif protein (RBX) and a protein responsible for substrate recognition [16–18]. In plants, three CRL complexes have been recognized (SCF, BTB, DWD) and are implicated in various processes, such as embryo development [19,20], light signaling [21], pollen development [22], cell cycle [23–25] and flowering time control [26]. Moreover, impaired CRL function has been related to pleiotropic phenotypes and developmental defects [27,28]. Many of the above developmental processes recruit E3 DWD CRL complexes. These contain an adaptor segment, consisting of the DNA damage-binding protein 1 (DDB-1a or DDB-1b) and a variety of DDB1-binding/WD40 repeat-containing (DWD) proteins as a linker for substrate recognition [29]. More than 80 WDR proteins have been identified in the A. thaliana proteome as putative E3 DWD CRLs subunits [28] including COP1, TRIP-1, FY, PRL1, DCAF1 and MSI4 [21,26,30–34]. However, the existence of many uncharacterized proteins containing solely WD40 repeats indicates, that both the complexity of the DWD CRLs and the range of processes affected by them may even be greater than anticipated. The aim of this study was the functional characterization of the A. thaliana ULCS1 (Ubiquitin Ligase Complex Subunit 1) gene. AtULCS1 encodes for a novel low molecular weight WDR protein, which interacts with the E3 DWD ligase complex subunit DDB1a. Downregulation of AtULCS1 causes pleiotropic phenotypes during plant development. Transgenic ulcs1i silencing lines display a sterile phenotype due to anther indehiscence, reduced lignification and altered seed size. The putative function of AtULCS1 as an E3 DWD CRL subunit and its role in plant development is discussed.

2. Materials and methods 2.1. Plant material and growth conditions A. thaliana (L.) Heynh. (ecotype Col-0) and Nicotiana benthamiana plants were used in this study. The AtULCS1 T-DNA transgenic lines, SALK 109060.29.20n and GABI 215H08, were obtained from ABRC Arabidopsis stock center. A. thaliana and N. benthamiana wild-type (WT) and transgenic plants were grown in a growth chamber under long-day conditions, 16 h light/8 h dark, at 22 ◦ C with 60–70% humidity and illumination of 110 ␮E m−2 s−1 PAR supplied by cool-white fluorescent tungsten tubes (Osram, Berlin, Germany). Arabidopsis seeds from individual T1, T2 and T3 generation lines were germinated under sterile conditions on selective half-strength Murashige and Skoog (MS) medium containing Cefotaxime (200 mg L−1 ), Hygromycin (20 mg L−1 ) or Kanamycin (40 mg L−1 ) and transferred to soil for further development.

2.2. Bioinformatic and phylogenetic analysis The DNA sequence of the AtULCS1 gene (At5g66240) was obtained from TAIR (http://www.arabidopsis.org). The protein sequences used for the phylogenetic analysis were obtained from NCBI (http://www.ncbi.nlm.nih.gov/pmc/) using the BLASTp software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were

fully aligned with CLUSTALW2 or CLUSTAL OMEGA at EBI (http:// www.ebi.ac.uk/). The ExPaSy software suite was used for all routine bioinformatics analysis (http://www.expasy.org/). The phylogenetic tree was constructed by using the neighborjoining method of the PHYLIP package, version 3.65 (Department of Genome Science, University of Washington, Seattle, WA, USA). Amino acid distances were calculated according to the Dayhoff PAM matrix or the Protein Parsimony method (PROTDIST and PROTPARS programs of PHYLIP package, respectively). Both methods produced trees with essentially identical topologies. The statistical significance was tested by using 1000 bootstrap replicates and the final tree was visualized with TreeView1.6.6 (http://taxonomy.zoology. gla.ac.uk/rod/treeview.html). Accession numbers and gene model names are given in Supplementary Table A1. 2.3. Nucleic acid extraction, cDNA synthesis and gene expression analysis DNA and total RNA was extracted from plant tissues using the NucleoSpin® Plant II and NucleoSpin® RNA Plant kits, respectively, according to the manufacturer’s instructions (Macherey Nagel, Düren, Germany). First-strand cDNA synthesis was performed using 1 ␮g of total RNA as template and the PrimeScript Reverse Transcriptase (Takara-Clontech, Kyoto, Japan). PCR products for cloning were amplified with Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Beverly, MA, USA). For semiquantitative (sq-) and quantitative (q) RT-PCR, Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) and KAPA SYBR® FAST qPCR Kits (Kapa Biosystems, Woburn, MA, USA) was used, respectively, according to manufacturer’s instructions. The primers used in this study are listed in Supplementary Table A2. All PCR products were separated by electrophoresis on 1% agarose gels and visualized under UV light after staining with ethidium bromide (100 ␮g L−1 ). 2.4. Construction of vectors for plant transformation For the RNAi silencing construct, the region between the 608th and 1087th nucleotide (including the 3 UTR) of the AtULCS1 full length cDNA was amplified by PCR using primer pairs XbaIULCS1-Fi/ClaIULCS1-Ri and EcoRIULCS1-Fi/KpnIULCS1Ri. The purified bands were then cloned as a KpnI/EcoRI (sense) and XbaI/ClaI (antisense) fragment into the respective cloning sites of the pHannibal vector. The hairpin construct [CaMV35Sp::608 AtULCS11087 ::INTRON:1087 AtULCS1608 ::ter] was subsequently introduced into the pCambia2200 (Cambia Canberra, Australia) binary vector as a PstI/SacI fragment, generating plasmid pCAtULCS1i. Transgenic lines overexpressing the AtULCS1 gene were made with plasmid pC35S::ULCS1. The full length coding sequence was amplified by PCR using the SalI35S::ULCS1-F and BstEII35S::ULCS1R primers and subsequently cloned into pCambia2200 as a SalI/BstEII fragment downstream of the CaMV35S promoter. For the AtULCS1promoter::GUS construct, the 2092 bp fragment upstream of the start codon, representing the 5 regulatory sequence of the AtULCS1 gene, was amplified by PCR from genomic DNA using primer pair ULCS1pGUS-F/ULCS1pGUS-R. The purified band was subsequently cloned as a BamHI/NcoI fragment into the pCambia1201 binary vector upstream of the ␤-glucuronidase (GUS) reporter gene, generating plasmid pCAtULCS1p::GUS. Subcellular localization experiments of the AtULCS1 protein were carried out by using an eYFP::AtULCS1 fusion construct. In brief, the enhanced Yellow Fluorescent Protein (eYFP) coding sequence from plasmid pGII0029 was PCR amplified and cloned as an XhoI/BamHI fragment into the pYES2 vector. The eYFP was then excised from pYES2 as an XbaI/SacI fragment and cloned

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into the respective sites of the linearized pBI121 binary vector, generating construct pBI35SYFPc. Subsequently, the full length coding sequence of AtULCS1 was amplified by PCR using primer pair ULCS1YFP-F/ULCS1YFP-R and cloned, as a AscI/SacI fragment into the pBI35SYFPc vector, in frame with the C-terminus of the eYFP protein, producing construct pBI35Sp::eYFP::AtULCS1. A second subcellular localization vector was also generated by using the endogenous AtULCS1 promoter sequence. The 2092 bp upstream of the ATG start codon was amplified using primer pair ULCS1pYFP-F/ULCS1pYFP-R and cloned as a HindIII/XbaI fragment into the pBIeYFP::AtULCS1 vector, generating plasmid pBIAtULCS1p::eYFP::AtULCS1. Multicolour Bimolecular Fluorescence Complementation (BiFC) was carried out with the pSAT vector system [35,36]. AtULCS1 was amplified with primers ULCS1cCFP-F/ULCS1cCFP-R and cloned into pSAT1 cCFP-N as an EcoRI/XmaI fragment, in frame with the C-terminus of CFP (Cyan Fluorescent Protein) generating construct pSAT1 ULCS1cCFP-N. DDB1a and DDB1b cDNAs were amplified with primers DDB1anCER-F/DDB1anCER-R and DDB1bnCER-F/DDB1bnCER-R and cloned as EcoRI/XmaI and EcoRI/BamHI fragments into pSAT4 nCeruleanN, generating the pSAT4 DDB1anCeruleanN and pSAT4 DDB1bnCeruleanN constructs, respectively. All constructs were checked for integrity and cloning correctness by restriction enzyme analysis and DNA sequencing. A schematic presentation of the constructs used in this study is shown in Supplementary Fig. A2.

2.5. Plant transformation Agrobacterium tumefaciens strain GV3101 competent cells were transformed with the aforementioned vectors by using the general freeze-thaw method, as described by An et al. [37]. The transformed bacteria were then used, either for stable transformation of Arabidopsis (Col-0) plants via the floral dip method [38] or for transient expression experiments, using N. benthamiana leaves. Transient transformation of tobacco leaf tissues was carried out according to Voinnet et al. [39] with minor modifications. The Agrobacterium strains harboring the translation fusion constructs pBI35Sp::eYFP::AtULCS1 and pBIAtULCS1p::eYFP::AtULCS1, were co-infiltrated with a strain containing the p19 suppressor from Tomato Bushy Stunt Virus (TBSV), in a OD600 ratio of 0.7:0.9, into the abaxial leaves of 4-6-week-old plants. Epidermal cell layers of tobacco leaves were evaluated by fluorescence microscopy 48–72 h after infiltration. For A. thaliana protoplast isolation and transfection, the TapeArabidopsis-Sandwich method was used [40]. Protoplasts were isolated from 5-week-old Arabidopsis Col-0 rosette leaves, grown under long day conditions. For each pSAT co-transformation experiment 30 ␮g of plasmid DNA was added to the protoplasts.

2.6. Histochemical GUS assay Histochemical assays for GUS activity were performed on T2 and T3 offspring of 8–12 individual transgenic lines using 5-bromo-4-chloro-3-indolyl-␤-d-glucuronide (X-Gluc) as a substrate. Seedlings or dissected tissues were incubated for 2 h at 37 ◦ C in X-Gluc reaction buffer (50 mM sodium phosphate buffer, pH 7.2, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 2 mM X-Gluc), dehydrated by a series of ethanol washes, and kept in a solution containing 3.7% (w/v) formaldehyde, 50% (w/v) ethanol, and 5% (w/v) acetic acid at 4 ◦ C [41]. Samples were treated with the clearing agent chloral hydrate (5 g chloral hydrate dissolved in 1 mL 30% glycerol) before microscopy.

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2.7. Semi thin sections of anthers Dissected A. thaliana flowers, at stage 12 from WT and transgenic plants, were double-fixed with 3% (v/v) glutaraldehyde and 1% (w/v) OsO4 in sodium cacodylate buffer pH 7.0. The samples were subsequently dehydrated in an acetone series and infiltrated in a graded series of Spurr’s resin (Serva, Heidelberg, Germany) in propylene oxide. Finally, they were embedded in small plastic dishes or capsules filled with fresh pure resin. Semi-thin sections of the flowers were prepared using an LKB ultratome III (LKB Produkter AB, Stockholm, Sweden) and stained with 1% (w/v) toluidine blue in 1% (w/v) borax solution. 2.8. Staining and microscopy DPBA (diphenyl-boryloxy-ethylamine) staining was performed in order to determine the seed coat total flavonoid content. A. thaliana seeds were incubated in 0.1% (w/v) DPBA in 0.1 M potassium acetate and 1% (w/v) NaCl for 10 min [42] and visualized under UV light. Pollen grain viability was tested using the differential Alexander staining technique [43]. To evaluate the lignification of endothecium cells, a modified ethidium bromide staining was used [44]. In brief, dissected flowers were incubated in PBS containing 2% (v/v) Tween-20 for 10 min and then 0.00005% (w/v) ethidium bromide in PBS for 1 h at room temperature. Samples were washed twice with PBS for 10 min and observed with an epifluorescence microscope [45]. Before microscopy and when appropriate, fresh stained tissues were fixed with acetic acid:ethanol (1:8) buffer for 1 h, washed with 100% and 90% ethanol, 30 min each and incubated overnight at room temperature in chloral hydrate buffer. The samples were visualized with a Zeiss Axioscope epifluorescence microscope (Zeiss, Oberkochen, Germany), equipped with a Zeiss Axiocam MRc5 digital camera, a differential interference contrast (DIC) system and proper filters. In particular, a set filter with exciter BP450-490 and barrier BP515-595, a set with exciter G-365 and barrier LP420 and a set with exciter BP510-560 and barrier LP590 were used. Stereoscopic photographs were obtained with a Zeiss Stemi 2000-C stereomicroscope, equipped with Jenoptic ProGres3 digital camera (Jenoptik, Jena, Germany). All images were processed by using Adobe Photoshop CS5 Extended software (Adobe Systems Inc., San Jose, CA, USA). 3. Results 3.1. AtULCS1 encodes for a highly conserved WD40-repeat-containing protein In order to investigate the functional involvement of the low molecular weight WDR proteins in plant development, we focused on the Arabidopsis gene locus 66240, located on the fifth chromosome. At5g66240, named hereafter AtULCS1, is organized in 11 exons and 10 introns (Fig. 1A). The gene locus encodes for a protein of 331 amino acids, annotated as a subunit of the Cul4-RING E3 ubiquitin ligase complex. AtULCS1 contains four WD40 repeats, which constitute a single WD40 repeat-containing domain that covers almost the entire protein sequence (Fig. 1B). It is a conserved protein that exhibits a 40–83% amino acid identity across the plant and animal kingdom. Yeast contains an orthologous sequence with an amino acid identity and similarity of 29% and 47%, respectively (Supplementary Table A3). The phylogenetic analysis showed that AtULCS1 is clustered with other WD40 plant proteins in a distinct clade, from that formed by the yeast and animal orthologs. However, in contrast to animals, all plant genomes contain a second closely related sequence, which form a separate clade in the tree (Fig. 1D). Phylogenetic clustering of the Arabidopsis WD40 proteins

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Fig. 1. Bioinformatic and phylogenetic analysis of AtULCS1 (At5g66240). (A) Schematic presentation of the AtULCS1 gene structure. (B) AtULCS1 amino acid residues and distribution of the four WD40 repeats along the sequence. (C) Phylogenetic relationship of the AtULCS1 protein with other homologs from Arabidopsis. (D) Phylogenetic analysis of AtULCS1 orthologous proteins from selected species. Trees were constructed by using the neighbor-joining method of PHYLIP after ClustalW alignment of the full-length protein sequences. Numbers indicate bootstrap support values from 1000 replicates. Abbreviations and the corresponding accession numbers are given in Supplementary Table A1.

is shown in Fig. 1C. AtULCS1 exhibits a 26–50% amino acid identity with these homologs and is more closely related to proteins that are annotated as putative components of the Cul4-RING E3 ubiquitin ligase complexes. To further analyze the WD40-repeat-containing domain and to inspect the potential differences between the two closely related homologs that are present in all plant genomes, the amino acid sequence of AtULCS1 was aligned with the homologous At1 and other orthologs from yeast, plants and animals (a representative alignment is shown in Supplementary Fig. A1). The aligned proteins share a number of identical and similar amino acids, mainly in the region of the WD40 domain, along with distinctive amino acids at various positions. The characteristic WDxR ubiquitin-ligase binding motif is located in AtULCS1 at position 146–149 and is present in all these orthologs. Despite the expected variability, a unique cysteine instead of glycine (at position 34) was found to be present only in the AtULCS1 protein, while a methionine instead of leucine (at position 48) was present only in the AtULCS1 and Zm2 proteins. A third amino acid residue (isoleucine instead of valine at position

174) was found to be identical only between AtULCS1 and the WD repeat domain 82 orthologs of animals (Supplementary Fig. A1 and A4). Both methionine and cysteine play critical roles in protein folding. As one of the most hydrophobic amino acids, methionine may participate in the formation of interior hydrophobic protein cores, whereas oxidized methionine residues within proteins may lead to alterations in protein structure and catalysis. Cysteine on the other hand plays a vital role in the tertiary and quaternary conformation of proteins, due to its ability to form inter- and intrachain disulfide bonds with other cysteines. 3.2. AtULCS1 is expressed in seedlings and flowering tissues The tissue specific expression pattern of AtULCS1 was studied in planta by analyzing stable transgenic Arabidopsis plants harboring a promoter-GUS transcriptional fusion construct. The results revealed a temporal and spatial expression pattern of AtULCS1 in the course of development. During germination strong GUS activity was detected in almost all tissues, including the root and shoot

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Fig. 2. Histochemical localization of AtULCS1 promoter-GUS expression during development. Images showing the representative GUS staining pattern in progeny from transgenic line pCAtULCS1p::GUS T2L6. Similar staining was observed in the majority of independent GUS transgenic lines. (A–F) Images showing pAtULCS1::GUS activity in the root tip of 2-d-old seedlings (A), part of the hypocotyl and the cotyledons of 3-d-old-seedlings (B), the aerial part of 4-d-old seedlings (C), the leaf primordia of 12-d-old seedlings (D), the stomata (E) and the vascular tissue of cotyledons (F). (G and H) Activity of pAtULCS1::GUS in reproductive tissues. Images showing staining in anthers (G–J), ovules (K) and during seed development (L–N). Bars: 100 ␮m for A–C and G–I; 50 ␮m for D and E and J–N; 10 ␮m for F.

apical meristem, except the hypocotyl area (Fig. 2A and B). However, AtULCS1 displayed a more specialized expression pattern in subsequent developmental stages. In 5-day-old seedlings GUS staining was restricted to the aerial part of the plants and the root cap cells (Fig. 2C). In 12-day-old plants, expression was evident in hydathode cells and the vascular system of cotyledons as well as in the incipient leaf primordia (Fig. 2D–F). During the reproductive developmental phase, AtULCS1 was expressed in the two fertile whorls of the flower. GUS activity was observed in early stages of microgametogenesis and throughout the maturation of pollen grains (Fig. 2G–J). After fertilization, AtULCS1 was expressed in the developing embryonic and endospermic tissues (Fig. 2K–N). In general, the above GUS staining pattern is consistent with the data from the publicly available Arabidopsis Microarray Databases (GENEVESTIGATOR and eFP Browser). The minor deviations observed between the microarray data and the GUS expression profile are probably attributed to tissue or developmental stage differences.

3.3. AtULCS1 is localized in both nucleus and cytoplasm, and interacts with DDB1a To obtain insights into the subcellular localization of the AtULCS1 protein, two translational fusion constructs of AtULCS1 with YFP were generated. The first one was driven by the constitutive CaMV35S promoter and the second by the native AtULCS1 promoter. The analysis of transiently expressed N. benthamiana epidermal cells confirmed that, regardless of the promoter used, AtULCS1 was localized in both the nucleus and cytoplasm (Fig 3A). In view of the annotated function of the protein, tests were carried out to examine whether AtULCS1 is a putative E3CRL subunit. By using the multicolor BiFC vector system, the appropriate con-

structs were co-transformed into isolated Arabidopsis protoplasts. As shown in Fig. 3B, the strong cytoplasmic fluorescence signal indicates an in planta interaction of AtULCS1 with DDB1a (DNA damage-binding protein 1a), the main subunit of the DWD E3CRL complexes. The specificity of this interaction is further supported by the negative result obtained, when AtULCS1 and DDB1b were used as interacting partners (Fig. 3B).

3.4. Analysis of AtULCS1-impaired plants To decipher the functional role of AtULCS1 protein in plant development, the two available T-DNA transgenic lines (heterozygous SALK 109060.29.20n line and homozygous GABI 215H08 line) were obtained and analyzed. Based on the information provided by TAIR, the T-DNA insertions were mapped approximately 150 bp and 125 bp upstream of the ATG start codon, respectively. Plants of both lines were grown on half strength MS plates in order to verify the T-DNA insertion loci and for segregation analysis. The homozygous GABI plants displayed a normal WT phenotype, whereas RT-PCR analysis showed that AtULCS1 transcript accumulation was comparable to that of WT plants (Fig. 4A and B). As far as the SALK line is concerned, no homozygous offspring could be identified from any of the self-pollinated independent heterozygous lines. A closer examination of the siliques revealed that they contained approximately 25% of aborted seeds (data not shown). In view of the viable homozygous GABI plants and the relative position of the insertion in the two lines (the SALK insertion is located upstream of the GABI insertion), the absence of homozygous plants could be due to a secondary lethal mutation, which is co-inherited along with the AtULCS1 insertion. The heterozygous SALK progeny showed also normal AtULCS1 transcript levels and WT phenotypes (Fig. 4A and B).

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Fig. 3. Subcellular localization of AtULCS1 and in planta interaction with DDB1a. (A) Images showing the localization of the chimeric AtULCS1::GFP fusion protein, expressed under the CaMV35S promoter (upper panel) and under the native AtULCS1 promoter (lower panel) in transiently transformed N. benthamiana epidermal cells. AtULCS1 was localized in the nucleus and the cytoplasm. (B) Images showing the specific in planta interaction of AtULC1 with DDB1a in transformed Arabidopsis protoplasts by using the multicolor BiFC vector system (upper and middle panel). No interaction signal was detected when AtULC1 and DDB1b were used as interacting partners (lower panel). Cells were visualized under an epifluorescence microscope equipped with DIC optics and GFP filter sets.

Given the lack of T-DNA knockout mutants, an RNAi-mediated post-transcriptional silencing approach was used to generate AtULCS1-impaired plants (ulcs1i). The pCambia-based vector construct contained two head-to-head copies of the AtULCS1 3 cDNA region, cloned downstream of the CaMV 35S promoter sequence (Fig. 4C). More than twelve independent T1 transgenic lines were obtained and RT-PCR analysis verified that AtULCS1 transcripts were downregulated in both T1 plants and their T2 progeny (Fig. 4D). Transcripts of the At1 homolog were not affected in the ulcs1 RNAi lines (data not shown). All RNAi lines exhibited a predominant phenotype of infertility, while the mRNA levels of AtULCS1 were shown to correlate with the degree of sterility (Fig. 4E and Fig. 5). Depending on the severity of

impairment, ulcs1 mutants exhibited also some other phenotypic abnormalities, such as increased growth, deformed leaves (data not shown) and variable seed size. As demonstrated in Fig. 6D, while WT seeds had a relatively uniform size, ulcs1 seeds displayed a greater amplitude of size. However, seed germination was unaffected, as designated by the similar curves obtained with WT and ulcs1 seeds (Fig. 6D and E). Moreover, seed coat flavonoid content, as indicated by the fluorescence signal emitted after DPBA staining, was higher than in CHALCONE SYNTHASE (CHS) ttg4-1 mutants and comparable to WT (Fig. 6C). In order to investigate in depth the cause of infertility, two strong (ulcs1-1 and ulcs1-3) and one weak (ulcs1-6) mutants were chosen and their T2 and T3 offspring were used for further analysis.

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Fig. 4. Phenotypic and molecular analysis of AtULCS1 T-DNA and RNAi mutant lines. (A) Schematic map of the AtULCS1 gene. Exons (black boxes), introns (lines) and untranslated regions (white boxes) are indicated. The SALK and GABI T-DNA insertions as well as the predicted transcription start site (TSS) are indicated by black arrows. RT-PCR analysis showed normal AtULCS1 transcript levels both in homozygous (HoZ) GABI and heterozygous (HeZ) SALK lines. The GAPDH gene was used as an internal control. (B) Images of the homozygous GABI and heterozygous SALK mutants, showing their WT phenotype. (C) Schematic representation of the hairpin RNAi construct used to generate the AtULCS1 impaired plants. (D) RT-PCR analysis showing down-regulation of AtULCS1 expression in six independent transgenic RNAi lines. Lines ulcs1-1, ulcs1-3 and ulcs1-6 displayed the lowest transcript levels compared to WT plants. Expression of the GAPDH gene was monitored as a control. (E) Phenotypes of 4-week-old WT and infertile Atulcs1-3 plants.

Fig. 5. Phenotypes of ulcs1 RNAi transgenic lines. (A) Images showing the primary inflorescence stem of WT, ulcs1-3, ulcs1-1 and ulcs1-6 plants. The AtULCS1 impaired plants were infertile and produced an increased number of tiny siliques. The weak RNAi line ulcs1-6 produced occasionally fully filled normal-sized siliques (white arrow). (B) Comparative depiction of the size and number of siliques produced by WT and Atulcs1 plants. (C) Fresh or cleared stereo- and microscopic images of the 3rd and 12th siliques from WT and ulcs1 plants. In WT plants the fertilized ovules developed normally into mature seeds during silique ripening. ulcs1 siliques contained only desiccated whitish unfertilized ovules. Bars = 500 ␮m for B and 100 ␮m for C.

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Fig. 6. ulcs1 seed development and morphology. (A) Dissected fully-grown siliques of WT and ulcs1 plants. In WT all ovules developed into seeds after successful fertilization, leading to the formation of enlarged siliques. ulcs1 plants were infertile with tiny siliques containing desiccated unfertilized ovules (white arrows). In the severe lines ulcs1-1 and ulcs1-3 developed seed were rarely observed. The semi-sterile ulcs1-6 plants developed nearly normal-sized siliques that contained an increased number of viable seeds. (B) Images of cleared seeds at different developmental stages from WT siliques (upper row) showing normal embryo development and from Atulcs1 siliques (lower row) containing unfertilized ovules. Black arrowheads designate the central cell nuclei. (C) DPBA stained WT and Atulcs1 seeds, showing no significant differences in flavonoid content compared to the CHALCONE SYNTHASE (CHS) ttg4-1 mutant seeds. (D and E) Images and charts showing the amplitude of size and the germination rate of WT and ulcs1 seeds.

3.5. Silencing of AtULCS1 results in plant infertility due to anther indehiscence The AtULCS1 impaired plants displayed a sterile phenotype and produced tiny siliques that contained small white bulges instead of fully developed seeds (Fig. 5). Lines ulcs1-1 and ulcs1-3, which displayed the lowest AtULCS1 mRNA level exhibited also the most severe infertility phenotype. In both lines, some siliques contained sporadically a few fully developed and viable seeds (Fig. 6A). In a similar manner, the silencing lines that showed slightly reduced AtULCS1 transcripts (e.g. ulcs1-6) displayed a semi-sterile phenotype. These plants developed both small and normal-sized siliques in random numbers and arrangement (Fig. 5A). Infertility may be the result of a mechanistic fertilization impairment or be due to abnormal development of either the reproductive cells and/or the embryo. To further unravel the cause of sterility, newly formed and fully-grown siliques from WT, ulcs1-1 and ulcs1-3 plants were dissected and inspected under the stereoscope. In young WT siliques, successful fertilization leads to the

enlargement of the ovules as a result of simultaneous development of the embryo and the endospermic tissue. In later developmental stages, the fully grown embryo is surrounded by a green colored seed coat, while siliques reach gradually their full size. As shown in Fig. 5, ulcs1 siliques remained stunted, almost in the size of the gynoecium throughout development (Fig. 5A and B). They contained small-sized white structures that correspond to desiccated unfertilized ovules (Fig. 5C). When ulcs1 mutants were pollinated with WT pollen, seed set and silique development was restored. Furthermore, the semi-sterile ulcs1-6 plants were able to develop occasionally a few normal seeds in slightly elongated siliques (Fig. 6A), as well as fully filled normal-sized siliques (Fig. 5A). The above data indicate that sterility is not caused by abnormal gametogenesis, but is rather the result of a mechanistic impairment of fertilization upon AtULCS1 downregulation. In order to verify that sterility of the ulcs1 mutants was not the result of abnormal embryo differentiation, developing seeds were dissected, cleared and microscopically observed. The results showed that, in contrast to WT, ulcs1-1 and ulcs1-3 ovules remained unfertilized at all

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Fig. 7. Infertility of ulcs1 RNAi mutants due to anther indehiscence. (A) Alexander staining of WT and ulcs1 anthers (flower stage 15a), showing the viable magentared stained

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stages of flower development and finally desiccated during silique ripening (Fig. 6B). In view of the semi-sterile ulcs1-6 phenotype and to further exclude a possible gametophytic lethality, a closer examination of the male reproductive tissues was also carried out. Alexander staining of whole anthers revealed that ulcs1 plants produced viable pollen, whereas randomly released pollen grains from the weak ulcs1-6 mutant were able to germinate in planta on the stigma of the gynoecium. However, in the strong ulcs1 mutants, the majority of pollen grains were not released from the pollen sacs, suggesting an anther dehiscence abnormality (Fig. 7 A). Considering that anther dehiscence is directly connected with the secondary wall thickening of the endothecium cells [45], ethidium bromide stained anthers from WT and ulcs1 plants were examined under the fluorescence microscope. In WT endothecium cells, secondary wall deposition of cellulose and lignin occur as bands of striated spring-like thickenings. These thickenings were significantly reduced in mild ulcs1 plants and almost absent in the strong ulcs1 phenotypes (Fig. 7B). Observations of toluidine blue stained thin sections of flowers, at stage 13, were consistent with the above data. Although anthers of ulcs1 plants were fully expanded and morphologically similar to WT, the lack of secondary wall modifications was evident in the ulcs1 endothecium cells (Fig. 7C). In agreement with the above, the connective tissue contained an increased number of cells with dense cytoplasm. In WT plants and at this stage of development, the connective tissue consists of vacuolated cells that undergo secondary wall thickening and programmed cell death (Fig. 7C) [46]. 3.6. AtULCS1 mRNA alterations influences IRREGULAR XYLEM (IRX) gene expression and lignification To further investigate the involvement of AtULCS1 in secondary wall modifications, transverse stems sections (last internode) from fully-grown WT and ulcs1 plants were examined under the fluorescence microscope. In WT Arabidopsis stems, the basal internode contains vascular bundles with several layers of lignified xylem cells and differentiated extraxylary fibers that possess a characteristic autofluorescence pattern. As shown in Fig. 8A, ulcs1 plants exhibited a reduced number of lignified layers in both the xylem and the interfascicular region compared to WT. These data indicate that AtULCS1 plays a role in cell wall modifications not only in anthers but probably throughout the plant. To strengthen the above hypothesis, transgenic lines harboring the AtULCS1 gene under the control of the CaMV35S promoter (35S::ULCS1 lines) were generated. As anticipated, the overexpressing plants exhibited a stronger autofluorescence signal compared to WT, whereas the lignified cell layers appeared to cover a more extended area in the stem. More importantly, in all overexpressors an additional ectopic lignification of the cortex cells adjacent to the phloem was observed, indicating their differentiation into fiber cells (Fig. 8A). Considering the altered lignification phenotype of the mutant lines, the expression level of several genes involved in the biosynthesis and modification of the cell wall were examined. In particular, qRT-PCR was performed to evaluate the transcript levels of the catalytic subunits of cellulose synthase complexes IRX1 and IRX5, the glycosyl transferase IRX8, which is involved in xylan biosynthesis [47,48], the lignin biosynthetic gene IRX12 [49] and the MYB26 transcription factor, previously found to regulate secondary wall modifications in anthers [45]. In agreement with the phenotypic observations, the analysis showed that the expression

of all IRX genes was significantly reduced (2.5–3-fold) in the RNAi plants. In contrast, MYB26, IRX3 and NST1 mRNA levels were not affected in these lines, even though AtULCS1 transcripts were 5 to 6 fold lower than in WT plants (Fig. 8B and Supplementary Fig. A3). On the other hand, while overexpression of AtULCS1 resulted in a 5–6-fold increase of ULCS1 transcripts and a significant elevation of IRX1 and IRX8 gene expression, no significant differences were observed in the mRNA levels of IRX5, IRX12 and MYB26 (Fig. 8B). 4. Discussion WD40-repeat-containing proteins are highly abundant in all eukaryotes. Numerous of these proteins have been implicated as potential subunits of multi-protein E3 ligase complexes that regulate various cellular and developmental aspects [50]. Here, it was shown that downregulation of AtULCS1 caused anther indehiscence and thus infertility, probably due to alterations in the expression of genes involved in secondary wall modifications. AtULCS1 encodes for a 331 amino acid WDR protein that contains four WD40 repeats. Phylogenetic clustering of AtULCS1 from Arabidopsis and other plant species depicted the conserved WD40 motif within this protein. Unlike other eukaryotes, plants seem to contain two closely related homologs in their genome, which implies the occurrence of a gene-duplication event before the divergence of the plant lineage [51]. The unique amino acids, identified at specific positions within the AtULCS1 protein sequence may contribute to the alternative tertiary folding of the protein and thus could determine its ability to bind specific target proteins [52]. The interaction of AtULCS1 with DDB1a in planta, suggested a direct or indirect physical coupling of these two proteins and a potential existence of a DDB1aULCS1 complex in plants. This assumption was strengthened by the results, showing that AtULCS1 interacted specifically with DDB1a but not with DDB1b. Previously, the small WD40 repeat containing protein WDR55 has also been shown to interact with DDB1a [53]. The authors suggested that the characteristic E3 DWD CRL signature motif WDxR within the WDR55 protein might be sufficient for this interaction [28,29,33]. Similarly, the DCAF1 protein was shown to interact with DDB1 via the WDxR motif, whereas mutation of WDxR in MSI4 diminished DDB1 binding and negatively affected its function in flowering time control [28,33,34]. The presence of the conserved amino acid WDxR docking site for DDB1, in the second WD40 repeat of AtULCS1, further supports its role as a putative subunit of an E3 DWD CRL complex. Several reports have shown that partial or complete lossof-function mutations in genes encoding for WDR proteins in plants caused pleiotropic phenotypes. For example, underexpression of the Solanum chacoense NOTCHLESS (ScNLE) gene, encoding for a WDR protein, affected organ number and size, stomatal index, flowering and seed development. ScNLE is localized in both cytoplasm and nucleus and is essential for cellular growth and proliferation [54]. Similarly, DCAF1 has also been shown to form a distinct Arabidopsis CUL4-DDB1-DCAF1 E3 ubiquitin ligase complex that is involved in multiple developmental processes. Homozygous dcaf1 T-DNA insertion mutants were embryonic lethal, while co-suppression lines exhibited several developmental defects including smaller seedlings with abnormal phyllotaxy, multiple primary shoots and irregular branching [33]. One of the most well characterized small WDR proteins is VERNALIZATION INDEPENDENT 3 (VIP3), a negative regulator of flowering time in

pollen grains. Despite the advanced developmental stage of the flower, ulcs1 pollen grains remained inside the indehiscent pollen sacs. In the semi-sterile plants, the sporadic dehiscence of some anthers resulted in the release of pollen grains that were able to germinate normally on the stigma. (B) Ethidium bromide staining of anthers at different developmental stages, showing the absence of lignification in ulcs1 (bottom row) compared to the WT (upper row). Arrows show the regions of the magnified images (inserts). (C) Images of toluidine blue-stained semi-thin sections of flowers, showing the presence (arrows) and absence of lignin deposition in WT (upper row) and ulcs1 (bottom row) endothecium cells, respectively. In contrast to WT, the connective tissues of ulcs1 anthers contained an increased number of cells with dense cytoplasm (right column).

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Fig. 8. Microscopic phenotype of stems and quantitative expression analysis of secondary wall modification-related genes. (A) Images showing alternate vascular bundles (DIC) and lignin autofluorescence in stem sections of WT, ulcs1 and overexpression plants (35S::ULCS1). The number of lignified cells were significantly reduced in the RNAi mutants compared to WT plants, whereas an ectopic lignification of cortex cells was observed in the overexpression lines. Bar = 50 ␮m for all images. (B) Quantitative RT-PCR analysis showing the reduction of IRX1, IRX5, IRX8 and IRX12 gene expression in ulcs1 compared to WT plants. Expression of IRX1 and IRX8 were increased in the overexpression lines, while MYB26 transcripts were not significantly affected in any of the transgenic lines. Values ± SD were normalized to GAPDH and represent the mean of three biological samples analysed in triplicates.

A. thaliana. vip3 mutants exhibited also pleiotropic phenotypes such as stunted growth, small rosette leaves and flower abnormalities. The association VIP3 with DDB1 and their assemblage in a putative E3 DWD CRL complex was thought to regulate these developmental processes through ubiquitin-mediated degradation of specific target proteins [26,28]. Likewise, WDR55 was shown to be required for apical patterning in the Arabidopsis embryo and for vegetative development. wdr55 seedlings displayed multiple and/or asymmetric cotyledons, while mature plants exhibited phenotypes similar to those related to auxin misregulation [53,55]. The existence of many uncharacterized proteins in the Arabidopsis proteome, containing solely WD40 repeats, implies that the scaffold of DWD CRLs may be characterized by a great diversity. Depending on the target proteins, additional factors could facilitate the binding of different WDRs to these complexes [16]. However, E3 DWD CRLs that may regulate secondary wall modifications have not yet been recognized. To the best of our knowledge, SECONDARY WALL THICKENING ASSOCIATED F-BOX 1 (SAF1) is the only WDR so far that has been implicated in endothecial secondary wall thickening, while the overexpression mutants were shown to be sterile [56]. Nevertheless, SAF1 is an A. thaliana WDR F-box containing protein and interacts with the SKP1/ASK1 subunit of an E3 SCF CRL complex [16]. Consistent with the role of AtULCS1 as a subunit of a potential DDB1aULCS1 (DWD CRL) complex, the respective RNAi silencing lines exhibited also multifaceted developmental abnormalities, including a ULCS1 transcript-dependent infertility phenotype. Detailed analysis showed that all ulcs1 plants produced perfectly normal and viable pollen grains capable of germinating on the

stigma. The completely sterile plants exhibited tiny siliques at full maturity, filled with undeveloped white bulges that represented desiccated unfertilized ovules. However, in some siliques a few normal and viable seeds were occasionally observed, the number of which was significantly higher in the weak semisterile plants. Microscopic examination confirmed that embryo and endosperm development in those seeds were not inhibited. Furthermore, regardless of the severity of the phenotype, aborted seeds were never observed, whereas pollination of ulcs1 with WT pollen restored seed set and normal silique development. Taken together, the above data indicated that the AtULCS1 impaired plants were neither male nor female gametophytic mutants. The infertility of ulcs1 was the consequence of the inability of the anthers to dehisce. Anther dehiscence is one of the most synchronized processes during reproductive plant development. It facilitates the release of mature pollen grains and their attachment to the stigma of mature flowers. Successful double fertilization will lead thereafter to embryo and endosperm development and finally to seed and fruit maturation [57]. The dehiscence of anthers involves a sequence of events including cellular differentiation, expansion and thickening of the endothecial cells, tapetum degeneration and septum degradation [58,59]. An important phase before dehiscence is the deposition of secondary wall elements, mainly cellulose and lignin, in the endothecium tissue. The main genes involved in this step include the IRREGULAR XYLEM (IRX) family and the genes encoding the NST (NAC secondary wall thickening promoting factors) and MYB transcriptional regulators of IRXs [60,61]. IRX1 and IRX5 are the catalytic subunits of cellulose-synthase complexes CesA8 and CesA4 respectively. These proteins are required

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for the deposition of cellulose during secondary-wall formation, while the respective impaired plants exhibit collapsed xylem cells and reduced growth [47,62]. IRX8 is involved in xylan biosynthesis and the respective mutants display more severe phenotypes than the cellulose deficient mutants, including low levels of lignin, dwarfism and sterility [48,63,64]. Finally, IRX12 belongs to the lignin pathway and gene mutations lead to a 50% reduction in lignin content [49]. IRX gene expression depends on a number of transcription factors that constitute a complex regulatory network controlling cell wall thickening and lignification [60,61]. The A. thaliana MYB26/MALE STERILE35 (MS35) transcription factor has been shown to regulate NST1 and NST2, which in turn control the expression of downstream genes involved in cellulose and lignin biosynthesis. In ms35 mutants the endothecium cells lack secondary wall elements and fail to expand, leading to anther indehiscence and thus male sterility. This impairment is anther-specific since ms35 plants do not exhibit any lignin deficient phenotype in tissues other than the endothecium [45]. On the contrary, ulcs1i plants display secondary wall modification defects both in the anthers and the stem, as shown by ethidium bromide and toluidine blue staining. In accordance with the observed phenotypes, the levels of IRX gene expression were significantly reduced in AtULCS1 impaired plants. Interestingly, no alterations in the expression of MYB26 was detected. In view of the putative function of AtULCS1, the post-translational regulation of MYB26 by a potential DDB1aULCS1 cannot be excluded. DDB1aULCS1 may also affect any other downstream factor involved in the complex regulatory network of cell wall modifications. Alternatively, ULCS1 could participate in the assembly of diverse DWD CRL complexes that target multiple regulators of IRX gene expression. Whatever the mode of action, the significant role of AtULCS1 in secondary wall thickening is further supported by the ectopic lignification of cortex cells in the overexpression lines and moreover by the increased expression of IRX1 and IRX8. In conclusion, the above data suggest a possible link between AtULCS1 and a DWD E3CRL complex, and provide evidence regarding its involvement in secondary wall thickening. Impairment of AtULCS1 expression results in anther indehiscence and ultimately in plant infertility. Clearly, further research is necessary to elucidate the participation of AtULCS1 in integral CRL complexes, which may function in diverse developmental processes, as dictated by the pleiotropic phenotypes of mutants. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgements This work was supported by the European Social Fund and National Resources (EPEAK II) PYTHAGORAS and the UoA Special Account For Research Grants (S.A.R.G.). We thank the Salk Institute and the NASC for providing the sequence-indexed Arabidopsis TDNA insertion lines. We also thank the Benaki Phytopathological Institute (Athens, Greece) for technical assistance. The authors also wish to apologize to those authors whose excellent work could not be cited due to space restrictions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2016.01. 008.

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