Plant lines used in this study

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Electroporation of A. tumefaciens UIA143: A frozen aliquot of -80 °C competent cells was thawed up on ice and mixed with the ligation mix. The cell mix was transferred into a cooled electroporation cuvette (Peqlab Electroporation Cuvette, 25 x 1mm electrode gap). Electroporation was performed on a BioRad MicroPulser with 1 pulse at 1,8 kV.

Subsequently, 900 µL of SOC medium prewarmed at 29 °C was transferred into the electroporation cuvette and carefully mixed by pipetting. The cell solution was transferred into a 1.5 mL Eppendorf tube and incubated at 29 °C for 2 – 3 hours. After incubation, 10 µL of cell mix were plated on LB supplemented with 50 mg/mL kanamycin diluted 1:1000 (final concentration 50 µg/mL) and 50 mg/mL gentamycin diluted 1:2000 (final concentration 25 µg/mL). Incubation followed at 29 °C over night for 1 – 2 days.

2.1.1.6 Cryostock preparation

In order to allow long-term storage of bacterial cells of both E. coli and A.

tumefaciens, cryostocks were prepared and stored at – 80 °C. A volume of 900 µL of a dense over night culture was mixed with 900 µL of sterile 87 % glycerol (Merck) and frozen immediately.

gene name locus stock name line predicted T-DNA insertion point AtFuc95A At4g34260 GK-440B01-018208 440B01 Exon

AtFXG1 At1g67830 SALK_016739 N516739 Exon

AtFuT1 At2g03220 SALK_101729 N601729 Exon

AtFuT2 At2g03210 SALK_027979 N527979 Exon

AtFuT3 At1g74420 SALK_073920.47.05.n N573929 Promoter AtFuT4 At2g15390 SALK_125310 N625310 Promoter

AtFuT5 At1g15370 GK_872B07 N317969 Promoter

AtFuT6 At1g14080 SALK_078348.41.20.x N578348 Exon

AtFuT7 At1g14070 SALK_049570 N859993 Exon

AtFuT8 At1g14100 SALK_010965 N510965 Exon

AtFuT10 At2g15350 SALK_020408 N859950 Exon

GMD1 At5g66280 SALK_136531.36.80.x N636531 Promoter GMD2 At3g51160 SALK_027379.55.75.x N527379 Exon GME At5g28840 SALK_150208.40.70.n N650208 Exon Table 4: Overview about all plant lines used in this work. 

Information about available lines, number and predicted T‐DNA insertion points were collected from TAIR  including sequence viewer, and NASC website. 

General information about the investigated genes was obtained from TAIR (The Arabidopsis Information Resource, www.Arabidopsis.org), which were: DNA sequences (gDNA, cDNA, coding sequence CDS), availability of T-DNA insertion lines and their position within the sequence (exon, intron, promoter or UTR). Tools like sequence viewer and eFP-browser were used to check predicted T-DNA insertion point and expression profile (based on microarray data) which influenced experiments at the beginning, like knockout screening or isolation of plant RNA from different plant organs for cloning.

2.1.2.1 Surface sterilization of A. thaliana seeds, sowing on soil, and stratification

Seed sterilisation was performed with 1 mL of water/Tween-20 (75 µL Tween-20 (Merck) in 50 mL of HQ-water) and 300 µL of 12 % NaOCl (sodium hypochlorite solution in water; Roth). Sterilisation followed for 10 minutes on a shaker. The seeds were immediately washed with 1 mL of water during 3-4 washing steps and then sowed on soil using a pipette. All pots were covered with plastic foil and stratified. For that reason the seeds were protected from light by covering in aluminium foil and incubation

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at 4 °C for 2 days. After stratification, the aluminium foil was removed and pots were incubated in a plant climate room or in a plant incubator.

2.1.2.2 Cultivation of plants on soil

Plants were grown in soil in common plastic pots and cultivated in plant growth chambers with 22 °C constant temperature, 50 % humidity and 16 h light period (long day). The used soil composed of around 1 part filling material (perlite) and 2 parts common potting soil was sterilized by 2 freezing (at - 30 °C) and thawing cycles (at room temperature).

2.1.2.3 Surface sterilisation of A. thaliana seeds and cultivation of plants on solid MS medium

A. thaliana Col-0 wildtype, overexpression or knockout plants were cultivated on solid MS medium (MSS with 3 % sugar, Table 5). Seeds were surface sterilised with 1 mL of water/Tween-20 (75 µL Tween-20 in 50 mL of HQ-water) and 300 µL of 12 % NaOCl (sodium hypochlorite solution in water; Roth). Sterilisation followed for 15 minutes on a shaker. The seeds were immediately washed with 1 mL of water for 3-4 washing steps in a laminar flow bench before plating on MS(S) plates. After last washing with water, seeds were mixed with 0.2 % (w/v) low-melting-point agar (Table 5) and spotted on the plate. The plates were sealed with parafilm or a textile tape and stratified as described above.

For growing of the knockout plant mur1 which has a dwarfed phenotype, the medium was supplemented with L-fucose (Jennerwein) at final concentration of 40 mM. In case of screening of overexpression plants, MSS plates supplemented with 50 mg/mL of kanamycin (final concentration 50 µg/mL) were used. Screening of T1-generation was made on large squared MSS-Kan plates to allow screening of a big number of seeds.

For screening of T2 and T3 generation, 20 seeds were plated on MSS-Kan plates in round Petri dishes. Transformed plants were transferred on soil after 2 – 3 weeks and cultivated as described above.

2.1.2.4 Cultivation of plants as in vitro liquid culture

Plant liquid cultures were used to produce a big amount of plant material used for enzymatic activity assays or analytical analysis. A. thaliana seeds were surface- sterilised and transferred into 100 mL (for 50 mL medium) or 250 mL (for 100 mL medium) sterile Erlenmeyer flasks containing liquid MS medium (Table 5). Cultivation was performed in a plant growth chamber on a shaker at 100 rpm agitation for a few days up to 3 weeks before harvesting.

media compounds

MS liquid medium (MS), 1 L 4,3 g/L Murashige and Skoog basal medium (M5519, Sigma), pH 5.7-5.8 with 1N KOH (Roth)

MS liquid medium with sucrose (MSS), 1 L

4,3 g/L Murashige and Skoog basal medium (M5519, Sigma), 30 g sucrose (Wiener Zucker), pH 5.7-5.8 with 1N KOH

MS solid medium with sucrose (MSS), 1 L

4,4 g/L Murashige and Skoog basal medium (M5519, Sigma), 30 g sucrose (Wiener Zucker), 8 g agar-agar (Fluka), pH 5.7-5.8 with 1N KOH 0.2 % agar 0.2 g agar-agar (Fluka),in 100 mL of water, autoclaved

(low-melting agar for sowing of seeds on MSS plates)

Table 5: Overview about the composition of media used for cultivation of plant, either on solid plates or as  liquid culture.  

MS (Murashige and Skoog) was the common medium for growth of A. thaliana wildtype, overexpression and  knockout lines. MS supplemented with 3 % sugar (to give MSS) was used in most of the cases for both plates and  liquid cultures. Medium for plates was poured either into squared plates or into round petri dishes. To cultivate on  plates, seeds were spotted onto the plate with 0.2 % agar to avoid swimming of seeds on agar surface. 

2.1.2.5 Transformation of plants: transient expression of proteins by leaf infiltration of N. benthamiana

The following protocol describes transient production of proteins in Nicotiana benthaminana leaves and is based on the publication of (Batoko et al., 2000).

Agrobacteria containing the cloned gene in a plant expression vector were inoculated in 3 mL LB liquid medium supplemented with antibiotics kanamycin and gentamycin (Table 3) and cultivated at 29°C for 1 – 2 days. 300 µL of a dense over night culture were centrifuged at 5 000 rpm for 5 minutes. The pellet was washed with 1 mL of infiltration medium (500 mM MES (2-(N-Morpholino)ethanesulfonic acid sodium salt) from Sigma, 20 mM Na3PO4*12H2O from Sigma, 1 M acetosyringone (Fluka) in DMSO (Sigma-Aldrich), 50 mg D-glucose from Merck) and centrifuged. The pellet was resuspended in 1 mL of infiltration medium and transferred into plastic cuvettes to measure optical density at 600 nm. The cells were then diluted with infiltration medium.

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For assessment of enzymatic activity assays, cells were diluted to OD600 0.3. For subcellular localisation of GFP-fusion proteins, cells were diluted to OD600 0.01, 0.03, 0.1 and 0.3 in order to verify ideal expression level for detection by confocal laser scanning microscopy (CLSM). Infiltration volume ranged between 1 mL (for enzymatic activity) and 2 mL (for localisation studies). Plants used were 5 – 6 weeks old, and younger leaves were used preferentially. Infiltration was performed into the lower epidermis using sterile 1 mL syringes (Injekt-F 1 mL syringes, Braun/Roth). A tiny hole was made with a needle into the leaf epidermis in order to facilitate infiltration. The syringe was gently pressed on the hole and the bacterial solution was carefully injected.

The infiltrated area was labelled and cautiously dried with towel paper. After infiltration, plants were watered and cultivated in a plant climate chamber. Leaves were harvested 48 hours later (2 days post infiltration, dpi) and again after 72 hours in the case of subcellular localisation studies.

2.1.2.6 Transformation of plants: stable transformation of A. thaliana by floral dipping

The method follows the floral dipping protocol published by Clough and Bent (1998) and was slightly modified.

Agrobacteria clones were inoculated in 10 mL LB liquid medium (preculture) supplemented with antibiotics, in most of the cases kanamycin and gentamycin (see Table 3), and cultivated at 29 °C for 1 – 2 days. The next day, 500 mL prewarmed LB liquid medium in a 2 L baffled shaker flask were inoculated with the preculture and cultivated at 29 °C up to an optical density of 0.8 (maximally 1.2). The agrobacteria cells were harvested by centrifugation at 5 500 g for 20 minutes at 20 °C on a Sorvall RC-6 PLUS 46910 centrifuge. Subsequently, the cell pellet was resuspended in 150 mL of 5

% fresh sugar solution (Wiener Zucker) and the optical density at 600 nm was taken.

The cells were diluted to OD600 0.8 and supplemented with 300 µL/L of the detergent Silwet L-77 (product code 510010; Van Meeuwen Chemicals B.V.) to reduce surface tension of the bacterial solution. The bacterial solution was transferred into a plastic bag, as it was found that a flexible plastic bag ease dipping and increase control of plants, especially potential washing out of soil into the liquid. Used plants were approximately 3 – 6 weeks old and had a lot of closed buds. Already opened flowers and ripening siliques were removed. The plants were not watered the day before and the soil was gently compressed in order to avoid washing out of soil. The plants were

carefully put over into the liquid, approximately to one quarter of the pot, and incubated for 2 minutes. The bag was slightly compressed with one hand to allow agitation and mixing. After incubation, the pots were placed on their side into a tray and covered with plastic- and aluminium foil. The dipped plants were incubated for 24 hours in a cool room and then watered and cultivated in a plant cultivation chamber. Seeds were harvested and screened on MSS-plates supplemented with kanamycin (Table 3).

2.1.2.7 Screening of A. thaliana overexpression lines

After floral dipping, seeds were collected and screened on a selection medium, MSS supplemented with 50 µg/mL kanamycin. Nomenclature of dipped plants and their successor plants followed the guide presented at www.gabi-kat.de (Universität Bielefeld). According to this guide, the dipped plant was named T0 (transformed). This plant produces T1-seeds. A high number of these seeds was screened by cultivation on MSS plates with kanamycin in order to identify transformed T1 plants, which are hemizygous (one copy of the transgene is present). Kanamycin causes death of untransformed plants while transformed T1 plants survive. These surviving plants were transferred on soil and cultivated until T2 seeds could be harvested. 20 T2 seeds were sowed on MSS medium supplemented with 50 µg/mL kanamycin to give T2 plants.

About 1/3 of these plants should by homozygous. To identify homozygous individuals, surviving T2 plants were transferred on soil and cultivated until T3 seeds could be harvested. 20 T3 seeds were sowed on MSS medium supplemented with 50 µg/mL kanamycin. In case of homozygoty of the T2 mother plant it was expected that all seeds germinated and grow on the selection medium.

2.1.2.8 Screening of A. thaliana knockout lines

Arabidopsis T-DNA insertion lines (Table 4) were screened on gDNA and cDNA level and confirmed by sequencing.

As a first step, genomic DNA of young plants cultivated on soil was extracted as described at point 2.1.4.2. All ordered knockout lines were described as segregating T3 lines, leading to a direct PCR screen using gene specific primers flanking the putative T- DNA insertion point (for primers see Table 8). The exact PCR settings are described at point 2.1.5.2. As presence of T-DNA prolongs the reading frame, no amplification was expected in the given elongation time during PCR. Col-0 wildtype was included as a

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control where a signal was expected. To confirm the presence of T-DNA, a PCR screen using gene specific and T-DNA left border specific primer LBa1 (see Table 11) has been performed on the candidates. The resulting DNA fragment was purified from the PCR reaction mixes (see point 2.1.4.4) and sequenced in order to verify the identity of T-DNA border (see point 2.1.7). The obtained sequencing result was analysed at first with SeqMan from DNASTAR and the mismatching sequence part - as here the insertion point was suggested - was further analysed by BLAST search in order to confirm the identity of the T-DNA left border. As the T-DNA insertion was not always located in an exon but was also present in an intron or in the promoter region, it was necessary to proof absence of gene product on protein level. RNA was isolated from knockout plant candidates and converted into cDNA as described at point 2.1.4.1 and 2.1.6. The cDNA of the knockout plants was then screened by PCR using cloning primers in order to try amplification of the full reading frame. No signal was expected for the knockout plants but for the control Col-0 wildtype.

In case of crossing of knockouts it was necessary to screen the obtained heterozygous double knockout plants for presence of T-DNA using a T-DNA left border specific primer and a gene specific primer. After selecting double knockout candidates, the next generation was screened for homozygous double knockout plants using gene specific primers as described above.

2.1.2.9 Crossing of A. thaliana

Crossing of Arabidopsis followed according to the guideline of NASC (“Crossing Arabidopsis” at http://Arabidopsis.info/). For crossing of A. thaliana knockout lines, pollen donor (male plant) and the pollen recipient (female plant) were treated differently.

Siliques, already opened flowers and very young buds or buds at disturbing positions were removed from the recipient in order to assure proper identification of the treated bud. Selected buds were carefully opened with very fine forceps in order to remove petals, sepals and anthers without hurting pistil and stigma. An open flower from the pollen donor was clipped off with forceps in a way to open the flower extensively and to present the anthers. The anthers of the flower of the pollen donor were then rubbed several times onto the stigma of the pollen recipient. To increase success, a second flower from the male plant was used to fertilize the same pistil. The fertilised bud was labelled and the plants were cultivated in a plant climate room. Successful fertilisation could be observed a few days later by formation of siliques. After ripening of the silique,

dried seeds were collected and sowed on soil. To detect double-knockout plants, young plants were analysed by PCR-screening on gDNA-level.

2.1.2.10 Harvesting of A. thaliana seeds

Seeds from A. thaliana were harvested after drying and opening of the siliques. The seeds were wiped off with clean fingers over clean paper and sieved to remove large particles. For fine purification, seeds were transferred to another sheet of paper so that small flower particles could glue onto the paper. Residual material was carefully blown away. The seeds were transferred into a 1.5 mL Eppendorf tube, covered with parafilm with tiny holes and dried over night. Storage of seeds was performed in closed 1.5 mL Eppendorf tubes at 4 °C.

2.5 Molecular biologic methods