Knockdown of PCBER1, a gene of neolignan biosynthesis, resulted in increased poplar growth

Brügmann, Tobias; Wetzel, Hendrik; Hettrich, Kay; Smeds, Annika; Willför, Stefan; Kersten, Birgit; Fladung, Matthias

doi:10.1007/s00425-018-3021-8

Cited by 14 publications

(8 citation statements)

References 36 publications

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“…To verify the transgenic status of the putative transgenic regenerants, PCR amplifications were carried out under standard conditions and separated by TBE-agarose gel electrophoresis [62]. Here, PCR primer pairs (each 5′–3′) were used which were suitable for all transformation vectors for both selection markers NptII (forward: TTG GGT GGA GAG GCT ATT CGG; reverse: GAA GGC GAT AGA AGG CGA TGC, or forward: TTG AAC AAG ATG GAT TGC ACG; reverse: AAG AAG GCG ATA GAA GGC GA) or HptII (forward: GAG AAG TTT GAT AGC GTG TCT G; reverse: TAG CGT CAC AGC GGC CTT G), respectively, and partly for Cas9 endonuclease (forward: GCT CCA GAC AAG AAG TAC AGC; reverse: TGT TCA CGC GAA GGA TGT CG) and part of the gRNA sequence (forward: TCA AAA GGC CCC TGG GAA TC; reverse: AAA AAA GCA CCG ACT CGG TG).…”

Section: Methodsmentioning

confidence: 99%

Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Brügmann¹,

Deecke²,

Fladung³

2019

IJMS

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CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

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Section: Methodsmentioning

confidence: 99%

Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Brügmann¹,

Deecke²,

Fladung³

2019

IJMS

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“…Dwarf poplar mutants have also been reported after transfer of the meristem identity gene LEAFY (LFY) [22,23], the TaLEA (Tamarix androssowii LATE EMBRYOGENESIS ABUNDANT GENE) [24], of two flowering time genes (SUPPRESSOR OF CONSTANS [SOC1] and FRUIT-FUL [FUL] [25], the ROLC gene from Agrobacterium rhizogenes [26][27][28][29], or by adopting an Ac/Ds-transposon activation tagging-based mutation system in poplar [30]. On the other hand, poplar with increased plant size have also been reported after e.g., transfer and overexpression of the GA20-OXIDASE gene [31][32][33] or knock-down of PHENYLCOUMARAN BENZYLIC ETHER REDUCTASE1 (PCBER1), a gene of the neolignan biosynthesis pathway [34].…”

Section: Introductionmentioning

confidence: 99%

Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth

Fladung

2021

Forests

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Pyramidal-, erect- or upright-growing plant forms are characterized by narrow branch angles of shoots and leaves. The putative advantage of upright-leaf and shoot habit could be a more efficient penetration of light into lower canopy layers. Pyramidal genotypes have already been reported for various tree genotypes including peach. The paralogous rice ortholog TILLER ANGLE CONTROL 1 (TAC1) has been proposed to be the responsible gene for upright growth. However, it has not really been demonstrated for any of the pyramidal tree genotypes that a knock-out mutation of the TAC1 gene is causing pyramidal plant growth. By in silico analyses, we have identified a putative rice TAC1 ortholog (Potri.014G102600, “TAC-14”) and its paralog (Potri.002G175300, “TAC-2”) in the genome of P. trichocarpa. Two putative PcTAC1 orthologs in the P. × canescens clone INRA 717-1B4 were successfully knocked-out by applying a transgenic CRISPR/Cas9-approach. The mutants were molecularly analyzed and phenotyped over a period of three years in a glasshouse. Our results indicate that the homozygous knock-out of “TAC-14” is sufficient to induce pyramidal plant growth in P. × canescens. If up to twice as many pyramidal individuals were planted on short rotation coppices (SRCs), this could lead to higher wood yield, without any breeding, simply by increasing the number of trees on a default field size.

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“…Unexpected effects concerning gene overexpression were reported for example for the biosynthesis of lignin [37,38]. It was reported that overexpression of PCBER1 (phenylcoumaran benzylic ether reductase 1), a well-known gene in lignin biosynthesis, in poplar did not lead to a significantly modified phenotype, wood composition or lignin, cellulose or hemicellulose level [38].…”

Section: Dl3βhsdsmentioning

confidence: 99%

Overexpression and RNAi-mediated Knockdown of Two 3β-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis

et al. 2023

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Abstract3β-hydroxy-Δ5-steroid dehydrogenases (3βHSDs) are supposed to be involved in 5β-cardenolide biosynthesis. Here, a novel 3βHSD (Dl3βHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3βHSD1 and Dl3βHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3βHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3βHSD1, Dl3βHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3βHSDs was realised by Agrobacterium-mediated transfer of Dl3βHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3βHSD1 and 35S:Dl3βHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3βHSD1 lines than in the controls. In the 35S:Dl3βHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3βHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3β-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3βHSD1 knockdown. These results can be taken as the first direct proof that Dl3βHSD1 is indeed involved in 5β-cardenolide biosynthesis.

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Knockdown of PCBER1, a gene of neolignan biosynthesis, resulted in increased poplar growth

Cited by 14 publications

References 36 publications

Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth

Overexpression and RNAi-mediated Knockdown of Two 3β-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis

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