Cell wall modification in tobacco by differential targeting of recombinant endoglucanase from Trichoderma reesei

Abstract

Background: The development of transgenic plants as a production platform for biomass-degrading enzymes is a promising tool for an economically feasible allocation of enzymes processing lignocellulose. Previous research has already identified a major limitation of in planta production such as interference with the structure and integrity of the plant cell wall resulting in a negative influence on plant growth and development.

Results: Here, we describe the in planta expression of endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei with differential intracellular targeting and evaluate its impact on the tobacco cell wall composition. Targeting of the enzyme to the apoplast leads to distinct changes in cell polysaccharides such as glucose level in the matrix polysaccharides (MPS). These effects are combined with severe changes in plant development. Retention of TrCel5A in the endoplasmic reticulum (ER) could avoid visible effects on plant growth under the chosen conditions, but exhibits changes in the composition of the MPS.

Conclusions: These results give new insights into the complex interaction of heterologous cellulase expression with cell wall development and it outlines novel promising strategies to engineer plant cell walls for improved biomass processing.

Figure 1

Generation of transgenic tobacco expressing TrCel5A. Genomic PCR of DNA prepared from wt and transgenic plants with primers binding in the CaMV 35SS expression cassette and below genomic PCR with primers binding in a highly conserved region in the chloroplast genome [38] (A); M, GeneRuler™ 1 kb wt: SR1 wild type, nc: H₂O, pc: control plasmid DNA pTRAkc-TrCel5A-ER. Western Blot for recombinant TrCel5A from three different TrCel5A_AP and TrCel5A_ER lines (B). Lanes contain 10 μg of TSP from transgenic plants; Antibody system used in is MαHis5 /GAM_Fc ^AP; wt: SR1 wild type, pc: purified His6 tagged Phenylammonium lyase from Zea mays (ZmPAL-His6). In all transgenic lines the degraded TrCel5A is detectable. Additionally in TrCel5A_ER lines also small amounts of the full enzyme are detectable. Expression level of TrCel5A in transgenic tobacco lines was determined by conversion of 4MUC (C). Three independent lines with five plants each were tested (coloured bars). White bars represent the average expression level in TrCel5A_AP and TrCel5A_ER lines respectively.

Figure 2

Localization of TrCel5A in transgenic tobacco leaf tissue. Plant tissue expressing TrCel5A targeted to the ER (A) and wt (B) were immunostained with the MαKDEL primary antibody and GAM_FC ^FITC. Apoplast targeting was monitored by staining transgenic plant tissue and wt with RαCell primary antibody and GAR_FC ^FITC (C-D). Electron microscopy with immunolabeling (RαCell/GAR_FC ^Gold) indicated localization for TrCel5A_AP inside the apoplastic space or at the cell walls (E, G) and for TrCel5A_ER inside the ER (F , H). Arrows mark accumulation of recombinant enzyme.

Figure 3

Transgenic tobacco plants expressing TrCel5A. Eight-week-old N. tabacum SR1 wt plants were grown in soil under standard conditions. Twenty plants of each line and wt were monitored on their stem size TrCel5A_AP plants showed a significant reduction in size and growth rate, whereas TrCel5A_ER plants were not significantly different from wt (D). The leaf shape of several transgenic plants was altered compared to wt (C). TrCel5A_ER (A) and TrCel5A_AP plants (B) displayed a curly leaf phenotype which in the case of TrCel5A_AP plants included additional necrotic lesions.

Figure 4

Cellulose content and cell wall convertibility in transgenic tobacco. The crystalline cellulose content and stem length was analyzed in six-week-old wt and TrCel5A transgenic tobacco plants grown in soil. Asterisks indicate a significant decrease (P ≤ 0.01) in the crystalline cellulose content for TrCel5A_AP compared to wt plants whereas TrCel5A_ER plants show no significant difference (A). Leaf tissue from transgenic tobacco lines TrCel5A_AP and TrCel5A_ER (B). Samples from both lines expressing ~30 mU/mg TSP (based on 4MUC conversion) were hydrolyzed with the appropriate amount of the commercial cellulase preparation [24]. Digestion was carried out at 55°C with constant shaking at 1000 rpm. The TrCel5A_AP leaves showed higher hydrolysis levels than the TrCel5A_ER and wt leaves after 8 h.