Study of plants affected in the expression of the GDP‐mannose 3,5‐epimerase (GME) gene

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3.8.3 Study of plants affected in the expression of the GDP-mannose 3,5-

insertion was detected about 1.286 bp downstream of the ATG in exon 4 (see Figure 54).

Figure 55: Amplification of a DNA fragment consisting of a GME‐specific and a T‐DNA left border specific  sequence for confirmation of presence of T‐DNA by sequencing.  

The DNA fragment was amplified with primers GMEmutscreenrev and Lba1 and sequenced with primer LBb1. Lane  1 –Fermentas 100 bp DNA Ladder Plus. Col‐0 WT – Columbia wildtype was used as a control. 

The line gme#2 has been allowed to self-fertilisation and the next generation was screened in order to detect homozygous knockout plants. The PCR screen using flanking as well as left border specific primers was performed on 67 plants in the offspring generation, but no homozygous candidate, indicated by absence of a signal in contrast to the wildtype control (Figure 56 A, lane 4), could be detected (Figure 56 A, lane 2 and 3). But including T-DNA specific primer LBa1 in another screen resulted in signals (Figure 56 B, lane 2 and 3) which indicated that heterozygous plants were present.

Figure 56: PCR screening of two GME knockout plants gme#2/1 and gme#2/2 in the offspring generation.  

A) Detection of homozygous knockout plants with T‐DNA insertion flanking primers GMEmutscreenfor and  GMEmutscreenrev which gave a signal of 1.200 bp in the Col‐0 wildtype control (lane 4). The same signal was  detected in the two putative knockout candidates (lane 1 + 2), indicating that they are either wildtype plants or  heterozygous plants. B) Screening of gme#2/1 and gme#2/2for presence of T‐DNA left border with primers  GMEmutscreenrev and Lba1 (lane 2 + 3). Lane 1 –Fermentas 100 bp DNA Ladder Plus. Col‐0 WT – Columbia  wildtype was used as a control. 

In theory, according to the Mendelian laws, one could have expected in the first generation one quarter of wildtype plants and three quarter of heterozygous plants. As

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one plant could be identified in the first generation as a heterozygous plant and was allowed to self-fertilise, the second generation could have been expected to produce one quarter of wildtype plants, two quarter of heterozygous plants and one quarter of homozygous mutant plants. But in this case just wildtype or heterozygous plants could be identified. Our hypothesis is that a complete knockout of the GME gene is lethal due to a defect in RG-II biosynthesis. In addition to this process, GME is involved in ascorbic acid biosynthesis in plants but the complementation of the growth medium of the heterozygous plants and their offspring with this compound did not lead to any rescue.

To overcome this situation in order to study these effects, it was decided to down regulate the GME gene by RNAi silencing instead of knocking this gene out.

3.8.3.2 Silencing of GME by RNAi

As no homozygous GME plant knockouts could be identified, it was decided to silence GME gene by RNAi technique. We interpreted these results as being the potential consequence that complete absence of GME activity, and in consequence L- galactose, cause lethality by leading to incomplete RG-II structure. In the same way, it has already been described that even silencing of an UDP-D-apiose/UDP-D-xylose synthase acting on RG-II in N. benthamiana cause lethality (Ahn et al., 2006).

Silencing was achieved by using pSAT- and pRCS binary expression vector system described by Tzfira et al. (2005) and Dafny-Yelin et al. (2007). The RNAi construct that we generated contained the full coding sequence of GME.

3.8.3.3 Analysis of sugar nucleotides from GME RNAi plants

At first, to confirm that GME was down regulated, the analysis of the levels of the sugar nucleotide GDP-L-galactose was estimated by LC-ESI-TOF-MS from A. thaliana Col-0 wildtype and GME RNAi plants. These results have been published in (Pabst et al., 2010).

A strong reduction of the level of GDP-L-galactose caused by RNAi silencing of the GME gene was observed (Figure 57 B). The amount of GDP-L-galactose per milligram of fresh plant weight in the GME-RNAi plant was drastically reduced in comparison to what was observed in wild type (Figure 57 A). Endogenous GDP-D-Mannose was used as internal reference. No phenotype could be observed, but this result confirmed the knockdown status of GME so that these plants were used for further investigations of RG II.

Figure 57: Analysis of sugar nucleotides of A. thaliana Col‐0 wildtype and the GME‐knockdown plant GME RNAi  by LC‐ESI‐TOF‐MS.  

A strong reduction of the peak corresponding to GDP‐L‐galactose was observed in the knockdown plant (red  arrow) compared to the wildtype. Endogenous GDP‐D‐Mannose was included as an internal reference.  

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3.8.3.4 Analysis of RG-II from GME RNAi plants

Side chains A of RG-II from Col-0 wildtype and GME RNAi plants were analysed in order to investigate the influence of suppressed GDP-L-galactose biosynthesis of GME RNAi plants on the structure this side chain.

Analysis of side chain A showed a clear difference of the mass profile of GME plants silenced by RNAi (Figure 58 B). Masses corresponding to a side chain A-structure without L-galactose represented more than one third of the chain A. A small fraction of chain A had a mass suggesting a replacement of L-galactose by a deoxyhexose, probably L-fucose. The number of peaks without L-galactose is identical to the number of peaks initially present in wild type plants, separated by 14 Da.

Figure 58: Analysis of RG‐II side chain A from Col‐0 wildtype and GME RNAi plants.  

Side chain A was present as four main chains which differed by a mass of 14 Da in the wildtype. The same was  found in the GME RNAi plant which showed in addition structures corresponding to structures without L‐galactose.