Brassinosteroid overproduction improves lignocellulose quantity and quality to maximize bioethanol yield under green-like biomass process in transgenic poplar

Abstract

Background: As a leading biomass feedstock, poplar plants provide enormous lignocellulose resource convertible for biofuels and bio-chemicals. However, lignocellulose recalcitrance particularly in wood plants, basically causes a costly bioethanol production unacceptable for commercial marketing with potential secondary pollution to the environment. Therefore, it becomes important to reduce lignocellulose recalcitrance by genetic modification of plant cell walls, and meanwhile to establish advanced biomass process technology in woody plants. Brassinosteroids, plant-specific steroid hormones, are considered to participate in plant growth and development for biomass production, but little has been reported about brassinosteroids roles in plant cell wall assembly and modification. In this study, we generated transgenic poplar plant that overexpressed DEETIOLATED2 gene for brassinosteroids overproduction. We then detected cell wall feature alteration and examined biomass enzymatic saccharification for bioethanol production under various chemical pretreatments.

Results: Compared with wild type, the PtoDET2 overexpressed transgenic plants contained much higher brassinosteroids levels. The transgenic poplar also exhibited significantly enhanced plant growth rate and biomass yield by increasing xylem development and cell wall polymer deposition. Meanwhile, the transgenic plants showed significantly improved lignocellulose features such as reduced cellulose crystalline index and degree of polymerization values and decreased hemicellulose xylose/arabinose ratio for raised biomass porosity and accessibility, which led to integrated enhancement on biomass enzymatic saccharification and bioethanol yield under various chemical pretreatments. In contrast, the CRISPR/Cas9-generated mutation of PtoDET2 showed significantly lower brassinosteroids level for reduced biomass saccharification and bioethanol yield, compared to the wild type. Notably, the optimal green-like pretreatment could even achieve the highest bioethanol yield by effective lignin extraction in the transgenic plant. Hence, this study proposed a mechanistic model elucidating how brassinosteroid regulates cell wall modification for reduced lignocellulose recalcitrance and increased biomass porosity and accessibility for high bioethanol production.

Conclusions: This study has demonstrated a powerful strategy to enhance cellulosic bioethanol production by regulating brassinosteroid biosynthesis for reducing lignocellulose recalcitrance in the transgenic poplar plants. It has also provided a green-like process for biomass pretreatment and enzymatic saccharification in poplar and beyond.

Keywords: Bioethanol; Brassinosteroid; Green-like pretreatment; Lignocellulose modification; Populus; Saccharification; Xylem differentiation.

Fig. 1

Collection of PtoDET2 transgenic poplars. a PtoDET2 expression in different developmental stages throughout the most periods of life cycle in poplar. b PtoDET2 expression profiling by Q-PCR analysis. c Endogenous BRs levels in transgenic and wild-type stems. d The expression of BRs biosynthesis relative genes. Data represent mean ± SD of three biological replicates. Statistical analyses were performed using Student’s t test as **P < 0.01

Fig. 2

Measurement of plant growth and biomass yield in transgenic poplar plant. a Images of 5-month-old transgenic poplar lines and wild type (WT); Scale bar as 10 cm. b, c Observation of plant growth in the transgenic lines and WT during time course of 6 months. d–f Plant height, stem diameter and dry weight (biomass yield) in the transgenic lines and WT of 6-month-old. Data represent mean ± SD of five biological replicates. Student’s t test was performed between the transgenic lines and WT as **P < 0.01

Fig. 3

Observations of plant cell walls in transgenic poplar plant. a Toluidine blue staining of the 6th internode stems of 5-month-old transgenic line and WT (Ph: phloem, C: cambium, Xy: xylem, Xf: xylem fiber cells, P: pith, Ep: epidermis. Scale bars as 50 μm). b Numbers of xylem cell layers and lumen area of individual xylem vessel cell and fiber cell. c Calcofluor staining specific for glucans (scale bars as 100 μm). d Immunohistochemical fluorescence (green) specific for xylan using LM10 antibody (scale bars as 100 μm). e Scanning electron microscopy (SEM) images (Xv: xylem vessel, scale bars as 5 μm). f Cell wall composition and cell wall thickness of SEM observation. All data as mean ± SD. Student’s t test was performed between the transgenic line and WT as **P < 0.01 (n = 3 for cell wall composition, n = 30 for cell wall thickness, technical replicates)

Fig. 4

Analyses of biomass enzymatic saccharification in the transgenic lines and WT. a Total sugar yields and hexose yields released from enzymatic hydrolysis after the pretreatment with 4% H₂SO₄, b 4% NaOH, c 10% CaO or d Na₂S + Na₂CO₃ pretreatments. Data represent mean ± SD of three technical replicates. All data as mean ± SD. Student’s t test between transgenic line and WT as **P < 0.01

Fig. 5

Detection of bioethanol yield and sugar–ethanol conversion rate in the transgenic lines and WT. a Bioethanol yields (% biomass) or b bioethanol yields (per plant) obtained from yeast fermentation using total hexose contents released from enzymatic hydrolysis after pretreatments. c Sugar–ethanol conversion rates under pretreatments. Data represent mean ± SD of three technical replicates Student’s t test was performed between the transgenic line and WT as **P < 0.01

Fig. 6

Comparison of lignocellulose features between the transgenic lines and WT. a Crystalline index (CrI) of crude cellulose. b Degree of polymerization (DP) of crude cellulose. c Glucose yield of the cellobiose released from time-course CBHI hydrolyzes using crude cellulose as substrate. d Xyl/Ara rate of total hemicelluloses. All data as mean ± SD of three technical replicates; increased percentage (%) obtained by subtracting transgenic line value with WT divided by WT. Student’s t test was performed between the transgenic line and WT as **P < 0.01

Fig. 7

Characterization of biomass porosity and cellulose accessibility in the transgenic lines and WT. a SEM images of raw material and biomass residue obtained from green liquor (Na₂S + Na₂CO₃) pretreatment. Scale bar is 10 μm. Allows as rough point. b Cellulose accessibility by measuring Congo red (CR) dye area. c, d Surface area and average pore diameter of biomass residues obtained from green liquor (Na₂S + Na₂CO₃) pretreatment. All data as mean ± SD of three technical replicates. Student’s t test was performed between the transgenic plants and WT as **P < 0.01