Overexpression of vesicle-associated membrane protein PttVAP27-17 as a tool to improve biomass production and the overall saccharification yields in Populus trees

Abstract

Background: Bioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth.

Results: In this study, we report on transgenic hybrid aspen (Populus tremula × tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula × tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 that was selected from a gene-mining program for novel regulators of wood formation. Analytical-scale enzymatic saccharification without any pretreatment revealed for all greenhouse-grown transgenic lines, compared to the wild type, a 20-44% increase in the glucose yield per dry weight after enzymatic saccharification, even though it was statistically significant only for one line. The glucose yield after enzymatic saccharification with a prior hydrothermal pretreatment step with sulfuric acid was not increased in the greenhouse-grown transgenic trees on a dry-weight basis, but increased by 26-50% when calculated on a whole biomass basis in comparison to the wild-type control. Tendencies to increased glucose yields by up to 24% were present on a whole tree biomass basis after acidic pretreatment and enzymatic saccharification also in the transgenic trees grown for 5 years on the field when compared to the wild-type control.

Conclusions: The results demonstrate the usefulness of gene-mining programs to identify novel genes with the potential to improve biofuel production in tree biotechnology programs. Furthermore, multi-omic analyses, including transcriptomic, proteomic and metabolomic analyses, performed here provide a toolbox for future studies on the function of VAP27 proteins in plants.

Keywords: Bioprocessing; Growth; Metabolomics; Populus; Proteomics; Transcriptomics; VAMP; VAMP-associated protein; VAP27; Vesicle-associated membrane protein.

Fig. 1

Phylogeny and expression profile of VAP27 proteins. a Phylogenetic analysis and protein domain characterization of VAP27 proteins in A. thaliana and P. trichocarpa. b The expression profile of the PttVAP27-12, -13, -14, -15, -16 and -17 genes in the Populus stem. Relative expression values throughout the different stages of wood development were obtained from the RNA sequencing data in the AspWood database [10]. Scaled VST expression indicates scaled variance-stabilizing transformation. P/Ca phloem/cambium, Ex expanding xylem, SCW maturing xylem, CD xylem cell death

Fig. 2

Overexpression of PttVAP27-17 in greenhouse-grown hybrid aspen trees. a The expression of PttVAP27-12, -13, -14, -15, -16 and -17 genes in stem tissues of three transgenic P. tremula × tremuloides lines (1–3) carrying the 35S::PttVAP27-17 construct. The expression data were derived from the transcriptomic analysis of secondary xylem tissues collected from the basal part of 2-months-old greenhouse-grown trees. The log2 fold-change indicates log ratio of PttVAP27-17 expression in each transgenic line compared to WT. The figure also shows relative expression of PttVAP27-17 by qPCR (b), stem height (c), stem diameter (d), wood density (e), and total stem biomass (f) in the three transgenic P. tremula × tremuloides lines carrying the 35S::PttVAP27-17 construct and the WT grown for two months in the greenhouse. Stem volume was estimated using the formula: volume = π × radius² × height/3. n = 5 for transgenic line 1 and 2, n = 3 for transgenic line 3 and n = 7 for the WT where “n” indicates number of biological replicates. Vertical bars (in a, b) indicate ± SD. The box plots show the median (horizontal line) with the outer limits at the 25th and 75th percentiles, the 1.5 inter-quartile ranges (whiskers) and the outliers (gray spots). Asterisks indicate statistically significant differences compared to WT: p ≤ 5% (*), p ≤ 0.1% (**), using Student’s t-test (b, c, f) and differential expression analysis DESeq2 (a). Percentages indicate increase (+) or decrease (−) of corresponding data values for lines overexpressing PttVAP27-17 compared to WT

Fig. 3

Overexpression of PttVAP27-17 did not cause major changes in wood chemistry of greenhouse-grown trees. a–f Pyrolysis–gas chromatography/mass spectrometry analysis of cell wall components in wild type (WT) and three PttVAP27-17 lines (1–3). The values are relative, and depict the combined area of GC peaks assigned to carbohydrates (C), lignin (L), syringyl lignin (S-lignin), guaiacyl lignin (G-lignin) and p-hydroxyphenyl lignin (H-lignin) as a percentage of total GC peak area. S/G, ratio between syringyl and guaiacyl-type lignin. g–l Total carbohydrate content in WT and PttVAP27-17 lines measured by high-performance anion-exchange chromatography. The fractions of arabinose, galactose, glucose, xylose, mannose, and total monosaccharides (arabinose + galactose + glucose + xylose + mannose) after acid hydrolysis are indicated as percentages (g of monosaccharide sugar in anhydrous form per 100 g of dry weight of wood). Wood chemical analyses were performed using mature wood from the basal part of the stem of two-months-old trees grown in the greenhouse. For both type of analyses, n = 5 for transgenic line 1 and 2, n = 3 for transgenic line 3 where “n” indicates number of biological replicates. For the WT, 33 biological replicates were used without pooling for pyrolysis–gas chromatography/mass spectrometry analysis and with pooling to five replicates for total carbohydrate content analysis. The box plots show the median (horizontal line) with the outer limits at the 25th and 75th percentiles, the 1.5 inter-quartile ranges (whiskers) and the outliers (gray spots). Asterisks indicate significant differences from the WT at p ≤ 5% (*) and p ≤ 0.1% (**) according to Student’s t-test. Percentages indicate increase (+) or decrease (−) of values of lines overexpressing PttVAP27-17 in comparison to WT

Fig. 4

The impact of the overexpression of PttVAP27-17 on the enzymatic saccharification of greenhouse-grown trees. a, b Glucose production rate (GPR) after 2 h of enzymatic saccharification (gL⁻¹ h⁻¹) for samples without (untreated) and with acid pretreatment (pretreated) in wild type (WT) and three transgenic lines (1–3). c, d Glucose yields after 72 h of enzymatic saccharification [g glucose per g of wood (dry weight) in samples without (untreated) and after acid pretreatment (pretreated)]. e Total wood glucose yield (TWG) after pretreatment and 72 h of enzymatic saccharification per whole tree biomass. TWG was calculated by using the formula 1/3 × π × stem radius² × stem height × wood density × glucose yield after pretreatment and 72 h of enzymatic saccharification. n = 5 for transgenic line 1 and 2, n = 3 for transgenic line 3 and n = 33 for the WT where “n” indicates number of biological replicates. Samples from the 33 WT trees were pooled to five replicate samples in all these experiments. The box plots show the median (horizontal line) with the outer limits at the 25th and 75th percentiles, the 1.5 inter-quartile ranges (whiskers) and the outliers (gray spots). Asterisks indicate significant differences from the WT at p ≤ 5% (*) according to Student’s t-test. Percentages indicate increase (+) or decrease (−) of values for lines overexpressing PttVAP27-17 compared to the WT

Fig. 5

Overexpression of PttVAP27-17 can increase stem biomass in field-grown trees. Stem height (a), stem diameter (b), wood density (c) at the base of the stem and total stem biomass (d) in field-grown trees of the wild type and four transgenic lines (1–4) during four seasons of growth (a, b) or at the end of the 5-year field trial (c, d). n = 3 for transgenic line 1, n = 5 for transgenic line 2, 3, 4 and 19 for the WT where “n” indicates number of biological replicates. The box plots show the median (horizontal line) with the outer limits at the 25th and 75th percentiles, the 1.5 inter-quartile ranges (whiskers) and the outliers (gray spots). Asterisks indicate significant differences compared to the WT at p ≤ 5% (*), according to Student’s t-test. The volumes of the stems were estimated using the formula: volume = π × radius² × height/3. Total stem biomass was defined on the basis of the stem volume and density. Percentages indicate increase (+) or decrease (−) for lines overexpressing PttVAP27-17 in comparison to the WT

Fig. 6

Overexpression of PttVAP27-17 affected only slightly the wood chemistry of field-grown trees. a–f Py–GC/MS analysis of cell wall components in WT and four PttVAP27-17 lines (1–4). The values are relative, and depict the combined area of GC peaks assigned to carbohydrates (C), lignin (L), syringyl lignin (S-lignin), guaiacyl lignin (G-lignin) and p-hydroxyphenyl lignin (H-lignin) as a percentage of total GC peak area. S/G, ratio between syringyl and guaiacyl-type lignin. g–l Total carbohydrate content for WT and PttVAP27-17 lines 1–4 determined by high-performance anion-exchange chromatography. The fractions of arabinose, galactose, glucose, xylose, mannose, and total monosaccharides (arabinose + galactose + glucose + xylose + mannose) released after acid hydrolysis are indicated as percentages (g of monosaccharide sugar in anhydrous form per 100 g of dry weight of wood). For all analyses, mature wood was collected from the base of the stem of 5-year-old trees grown in the field. n = 3 for transgenic line 1, n = 5 for transgenic line 2, 3, 4 and n = 8 for the WT where “n” indicates number of biological replicates. The box plots show the median (horizontal line) with the outer limits at the 25th and 75th percentiles, the 1.5 inter-quartile ranges (whiskers) and the outliers (gray spots). Asterisks indicate significant differences from the WT at p ≤ 5% (*) according to Student’s t-test. Percentages indicate increase (+) or decrease (−) in values for lines overexpressing PttVAP27-17 compared to the WT

Fig. 7

The impact of the overexpression of PttVAP27-17 on the enzymatic saccharification in woody tissues of field-grown trees. a, b Glucose production rate (GPR) after 2 h of enzymatic saccharification (gL⁻¹ h⁻¹), for samples without (untreated) and with acid pretreatment (pretreated) in wild type and four transgenic lines (1–4). c, d Glucose yields after 72 h of enzymatic saccharification [g glucose per g of wood (dry weight) without (untreated) and with acid pretreatment (pretreated)]. e Total wood glucose yield (TWG) after pretreatment and 72 h of enzymatic saccharification per whole tree stem biomass. TWG was calculated by using the formula 1/3 × π × radius² × height × wood density × glucose yield after pretreatment and 72 h of enzymatic saccharification. n = 3 for transgenic line 1, n = 5 for transgenic line 2, 3, 4 and n = 8 for the WT; where “n” indicates number of biological replicates. Mature wood was collected from the base of the stem of 5-year-old trees grown in the field. The box plots show the median (horizontal line) with the outer limits at the 25th and 75th percentiles, the 1.5 inter-quartile ranges (whiskers) and the outliers (gray spots). Asterisks indicate significant differences from the WT at p ≤ 5% (*), according to Student’s t-test. Percentages indicate increase (+) or decrease (−) in values for lines overexpressing PttVAP27-17 compared to the WT