Transgenic rice plants expressing the α-L-arabinofuranosidase of Coprinopsis cinerea exhibit strong dwarfism and markedly enhanced tillering

Abstract

Lignocellulosic materials are potential renewable sources of fermentable sugars for bioethanol production. In this study, we used the CcAbf62A gene encoding CcAbf62A, a putative extracellular α-L-arabinofuranosidase, cloned from the mycotrophic basidiomycete Coprinopsis cinerea. CcAbf62A acts on arabinoxylan, the major hemicellulose of grasses, releasing arabinose. CcAbf62A was introduced into rice with the aim of enhancing delignification efficiency and the availability of lignocellulosic materials without reducing lignin content. Among the 32 lines of regenerated transgenic rice, 13 exhibited markedly disrupted elongation growth and excessive tillering (dwarf), seven showed delayed elongation growth (retarded-growth), and 12 showed phenotypes similar to those of control plants (normal). Additionally, the dwarf lines showed reduced acclimation. RT-PCR analysis revealed that dwarf lines had higher levels of CcAbf62A expression than retarded-growth and normal lines. Although the lignin content of transgenic rice plants expressing CcAbf62A did not differ significantly from that of control rice plants, dwarf lines were characterized by delayed deposition of lignin in the culms compared with the controls. The reduced acclimation ability of dwarf lines is believed to be associated with increased water loss and reduced water conductivity concomitant with delayed lignin deposition. Contrary to expectations, the alkaline delignification rates of dwarf and retarded-growth Abf lines were slightly lower than those of control rice plants. Our findings indicate that CcAbf62A reduces ferulate-lignin cross-links by detaching arabinose side chains from arabinoxylan and increases the relative abundance of alkaline-resistant benzyl ether cross-links. CcAbf62A is anticipated to provide new approaches for breeding plants containing altered lignocellulosic materials or lodging-resistant crops.

Keywords: Coprinopsis cinerea; arabinofuranosidase; delignification; dwarfism; lignocellulosic material.

Figure 1. Phenotypes of pIG121-GUS(−)-Abf lines. (A–F) Phenotypes of the T₀ generation. (A) Dwarf lines showed abnormal elongation growth and excessive tillering on the hormone-free medium, similar to turf. (B) Dwarf lines were also characterized by short roots and showed repressed rooting. (C and D) A comparison of growth between the retarded-growth and dwarf lines (C) and between the normal and dwarf lines (D) grown in hormone-free medium. (E) The retarded-growth lines showed delayed elongation growth, whereas the heights of normal lines plants were comparable to those of the control plants. (F) Only a single dwarf lines could acclimate and form ears following the thinning of excess tillers. (G, H) Phenotypes of T₁ generation plants. (G) The dwarf trait was inherited in the T₁ generation, although the extent of the dwarf trait was less pronounced than in the T0 generation. (H) The leaves of the dwarf line were characterized by slightly yellowish patches. As controls, we used pActnos (T₃) and pIGnos (T₁) line empty vector control plants.

Figure 2. Infrared spectra of the surface of leaves cut from transformed rice plants (T₀). All absorbance values were normalized to that of the band at 1,040 cm⁻¹, which is associated with the C-O stretching vibration of cellulose. Each spectrum is the average of triplicate measurements.

Figure 3. RT-PCR analyses of gene expression among three lines showing different phenotypes (T₁). Abf-2, Abf-20, and Abf-26 are typical lines selected from among the normal, retarded-growth and dwarf lines, respectively. Two specimens were analyzed for each line, and the Actin gene was used as an internal standard.

Figure 4. Histochemical analysis of lignin deposition for T₁ plants based on phloroglucinol staining. Cross sections were prepared by cutting culms 2 cm above the ground at the vegetative stage, the middle of the second internode at the heading stage, and the middle of the third internode at the ripening stage. Bar=100 µm.

Figure 5. Lignin contents determined using the acetyl bromide method (A), delignification rates (B), and mass recovery rates (C) determined in alkaline-treated rice straw samples (T₁). Values are the means±standard deviations from quadruplicate (A) and triplicate (B and C) analyses. Statistical analyses were performed by Tukey’s test. Different letters indicate significant differences at p<0.05.