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RNAi silencing of the Arabidopsis thaliana ULCS1 gene results in pleiotropic phenotypes during plant growth and development.

WD40-repeat-containing proteins (WDRs) are highly abundant in all eukaryotes. Several of them have been implicated as subunits of multi-protein CRL E3 ligase complexes that regulate ubiquitination mediated protein degradation and thus various cellular and developmental processes. Impairment of the WDR protein ULCS1 from Arabidopsis causes pleiotropic phenotypes during plant development, including reduced lignification, anther indehiscence, and sterility. Here we show that RNAi-mediated downregulation of ULCS1 results in a fast-growing phenotype during vegetative development. Due to accelerated growth, ulcs1i mutants reach their vegetative to reproductive transition point earlier than WT plants. However, their comparable germination rate and their similar number of secondary branches and rosette leaves at bolting indicate that ulcs1i is not an early flowering time mutant. GUS staining of progeny, obtained from crosses between ulcs1i and CYCB1::GUS plants, revealed an increased number of mitotic cell divisions in the root meristems of ulcs1i compared to WT. Immunolabeling of homogalacturonans (HGAs) epitopes showed significant fluorescent signal differences at the cell walls and the mucilage of the seeds between ulcs1i and WT. Furthermore, we demonstrate that ULCS1 interacts with the UBA-like protein in a yeast two-hybrid assay, suggesting a direct or indirect physical coupling of these proteins in Arabidopsis.


Introduction
WDR proteins (WD40 protein domain, or beta-transducin repeat-WDRs) constitute a large prokaryotic and eukaryotic family, that act as scaffold subunits for the assembly of We have previously shown that the ULCS1 gene from A. thaliana encodes for a small molecular weight WDR protein and interacts with the E3 Cullin Ring Ligase subunit DDB1a, probably regulating the degradation of proteins involved in secondary wall modifications (Beris et al., 2016). RNAi-mediated silencing mutants (ulcs1) exhibit a reduced deposition of secondary cell wall components and lignin, both in the vascular system of stems and in anthers, resulting in anther indehiscence and sterility. However, ulcs1 mutants display several other pleiotropic phenotypes, which are most likely related to the characteristics of the protein to act as a scaffold subunit. As previously suggested, WDR proteins are crucial and indispensable in the assembly of multiple functional complexes that regulate various aspects of plant growth and development. In this report, we present several growth phenotypes associated with the impairment of the AtULCS1 gene and discuss their nature and putative origin in the context of plant development. Furthermore, we show that ULCS1 physically interacts with the UBA-like protein from Arabidopsis thaliana. Seed germination assays were carried out in petri dishes with two layers of water-saturated filter paper (Whatman). Germination was scored in 6-h time intervals for up to 44 h and the germination frequency (%) was calculated. For seedling root growth measurements, seeds were germinated vertically on half-strength MS plates without sucrose for 1-8 days. Before sowing, all seeds were imbibed at 4 0 C for 48 h in darkness. Each experiment was repeated three times using biological replicates.

Bioinformatic and phylogenetic analysis
The DNA sequence of the AtULCS1 gene (At5g66240) was obtained from TAIR (http://www.arabidopsis.org). The full-length amino acid 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). Alignment of the protein sequences was performed with the CLUSTAL OMEGA server at EBI (http://www.ebi.ac.uk/). The ExPaSy software suite (http://www.expasy.org/) was used for all routine bioinformatics analyses.
The phylogenetic tree was constructed by using the neighbor-joining method as implemented in the NEIGHBOR program of the PHYLIP package, version 3.65 (Department of Genome Science, University of Washington, Seatle, 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). The statistical significance was tested by bootstrap analysis for 1000 replicates. The final tree was visualized using TreeView 1.6.6.
(http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). The numbers indicate the bootstrap replications (of 1000) in which the given branching was observed. The protein parsimony method produced trees with essentially identical topologies. Accession numbers and gene model names are given in Supplementary Table S1.

Nucleic acid extraction, cDNA synthesis and gene expression analysis
DNA and total RNA were 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 μg of total RNA as template and the PrimeScript Reverse Transcriptase All experiments were performed in triplicate. All PCR products were separated by electrophoresis on 0,9% agarose gels and visualized under UV light after staining with 80 μg L -1 ethidium bromide. The primers used in this study are listed in Supplementary   Table S2.

Construction of vectors for plant transformation and yeast two-hybrid assays
For generating the RNAi silencing construct, the DNA fragment spanning the region between the 608 th and 1087 th 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/KpnIULCS1-Ri. 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 was subsequently introduced into the binary vector pCambia2200 (Cambia Canberra, Australia) as a PstI/SacI fragment (plasmid pCAtULCS1i) and used to transform Arabidopsis thaliana plants.
For the yeast two-hybrid vectors, the first-strand cDNA, synthesized as described above, was used to amplify by PCR the ULCS1 (At5g66240) and UBA-like (Ubiquitin All constructs were checked for integrity and cloning correctness by restriction enzyme analysis and DNA sequencing.

Plant transformation and histochemical GUS assays
The pCAtULCS1i vector was transformed into Agrobacterium tumefaciens strain GV3101 competent cells by using the general freeze-thaw method, as described by An Histochemical assays for GUS activity were performed using 5-bromo-4-chloro-3indolyl-β-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. Samples were treated with the clearing agent chloral hydrate (5 g chloral hydrate dissolved in 1 ml 30% glycerol) before microscopy.

Ruthenium Red Staining and Calcofluor White Staining
Mature dry seeds were hydrated in distilled water for 24 h and stained with 0.01% (w/v) Ruthenium Red (Sigma-Aldrich) for 30 to 60 min. Subsequently, seeds were rinsed several times with distilled water and imaged using a Zeiss Axioplan microscope (Zeiss, Berlin, Germany). For cellulose staining, seeds were stained for 10 min with Calcofluor White solution (Sigma-Aldrich) and observed with an epifluorescent microscope.
For investigating the functional involvement of the ULCS1 protein in plant development, we generated a hairpin construct to obtain A. thaliana RNAi-mediated posttranscriptional silencing mutants that were impaired in AtULCS1 function (Fig. 1B). RT PCR analysis performed in individual transgenic T1 and T2 progeny lines showed that AtULCS1 transcripts were significantly downregulated in several of them. Fig. 1C shows four representative lines, displaying moderate and low levels of AtULCS1 mRNA. Seeds from lines 1 and 4 were selected for further phenotypic analysis.

AtULCS1 impaired plants display pleiotropic phenotypes during plant growth and development.
We have previously shown that ulcs1 silencing lines exhibited a predominant phenotype of infertility and that the degree of sterility is correlated with the AtULCS1 transcript levels. The analysis revealed that infertility resulted from the inability of the  (Fig. 2C). Furthermore, and as the number of primary inflorescence internodes is concerned, WT plants usually acquire, under long-day light conditions, two to three inflorescence internodes. As shown in Fig 2D, ulcs1i plants display the same number of internodes during bolting, suggesting that under long-day photoperiod conditions, soil-grown ulcs1i mutants exhibited a similar flowering time phenotype to WT plants. Taken together, the above data clearly indicate that ulcs1i plants may reach their reproductive transition point 2-3 days earlier than WT plants, but this can obviously be attributed to their accelerated growth and, thus, cannot be characterized as early flowering time mutants. However, due to their increased growth rate, ulcs1i plants are at full maturity significantly higher than WT plants and contain an increased number of secondary and lateral branches (Fig. 2E).
To further investigate the overall faster growth phenotype of the ulcs1i plants, we evaluated seed morphology, the germination rate, as well as the primary root length of WT and mutant plants during seedling development at 1-8 days after sowing. As shown in Fig. 3A and B, while ulcs1 seeds displayed a greater amplitude of size, compared to the relatively uniform size of WT seeds, they both displayed similar germination curves.
Although ulcs1i seeds showed a slightly faster germination rate (by ca. 3-4 hours), compared to WT, their germination curves were almost identical (Fig. 3B). However, after germination, ulcs1i seedlings displayed a constant accelerating growth rate, compared to WT plants, which was statistically significant and evident 7 days after sowing ( Fig. 3C and D).

ulcs1i seeds release larger "halos" upon hydration in water and contain more methylesterified pectins
In comparison to WT, hydrated ulcs1i seeds appear to release larger mucilage "halos" (Fig. 5A and B). The "halos" formed by the adherent layer of the mucilage was around 10-15% wider in ulcs1i seeds than the ones formed around the WT seeds (Fig. 5C).
Both "halos" displayed a darker disc at the edge of the mucilage "halo", as observed by Ruthinium Red staining. . Immunolabeling revealed that ulcs1i seeds displayed an intense LM20 fluorescent signal at the mucilage released in rays associated with the tops of the columella of the seed coat epidermal cells (Fig. 5D and F) in comparison to that observed at the cells of the WT seeds ( Fig. 5E and I). Labeling with LM19 revealed a signal at the radial cell wall of the WT epidermal cells (Fig. 5H), while in ulcs1i, the signal was observed at the mucilage present at the top of the columella of the seed coat epidermal cells (Fig. 5G). When labeling with 2F4 antibody, WT seeds displayed a fluorescent signal at the outer tangential and the radial cell walls. An intense fluorescent signal was also observed at the tops of the columella of the seed coat epidermal cells ( Fig. 5K and N). On the contrary, most of the ulcs1i seeds did not display 2F4 fluorescent signaling nor the cell walls of the seeds or at the mucilage present at the top of the columella cells ( Fig. 5J and M). After calcofluor white staining, no significant differences were observed between WT and ulcs1i seeds ( Fig. 5L and O). (fly1) mutation, which displayed primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence, probably due to the lower degree of methylesterification in the mucilage. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. However, neither the target of FLY1 nor its exact mode of action has been determined. The fact that the DM of HGAs appears to be regulated by protein degradation through two distinct mechanisms (ARA12/SBT1.7 and FLY1) and by at least one PMEI, indicates that mucilage production and extrusion is highly dependent on the tight regulation of the degree of pectin methylesterification (Sola et al., 2019). It is rather intriguing to further elucidate the involvement of a putative E3 ULCS1 CRL Ligase complex in the regulation of pectins demethylesterification processes during seed coat development, given that they seem to play a crucial role in mucilage formation and seed germination.

AtULCS1 interacts physically with the UBA-like protein
ULCS1 interacts with DDB1a in planta (Beris et al., 2016), suggesting a direct or indirect physical coupling of these proteins and a potential existence of a CRL DDB1a/ULCS1 E3 ligase complex in plants (Beris et al., 2016). Here we show that ULCS1 also interacts physically with the UBA-like protein from Arabidopsis. UBA-like is a basic salivary proline-rich-like protein that, when impaired, results in plants that display similar phenotypes to those observed in ulsc1i mutants (unpublished data). Both ULCS1 and UBA cDNAs were cloned into the appropriate yeast two-hybrid vectors and assayed for their putative association. Fig. 6 shows two representative independent clones, which showed a positive interaction, as indicated by the ability of the yeast cells to grow on selective media (Fig. 6A). To further verify the interaction, series dilution (1/1 to 1/500) of the two independent clones were spotted on selection plates and allowed to grow for 3-4 days. As shown in Fig. 6B colonies from both clones were visible up to dilution 1/500. As negative controls, yeast cells of strain SG335 were transformed with plasmid pGBKT7/UBA or pGBKT7/ULCS1 along with plasmid pGADT7/T and monitored for growth on SD medium selection plates without Trp and Leu. Even at low dilution (1/1), no colonies were visible ( Fig. 6B).

Conflict of interest statement
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.    Data are means ±SE of three biological replicates (n=20). P value < 0.05 (Student's t-test).  Heterodimerization of AtULCS1 and two independent clones of AtUBA-like in yeast cells.

Figure legends
(B) Series dilution (1/1 to 1/500) of the two positive clones on selection plates. Double transformed SG335 yeast strain colonies were selected on synthetic dropout (SD) medium without Trp and Leu (SD, trp-, leu-) and restreaked on SD/−Trp/−Leu medium containing 1% raffinose and 2% galactose. As a positive control (+), yeast cells SG335 harboring plasmids pGBKT7/p53 and pGADT7/T were used. As negative controls (-) yeast cells of strain SG335 were transformed with plasmid pGBKT7/UBA (A) or pGBKT7/ULCS1 (B) along with plasmid pGADT7/T and monitored for growth on SD selection plates without Trp and Leu. Yeast growth was evaluated after 3-4 d at 30 0 C.