Increased lodging resistance in long-culm, low-lignin gh2 rice for improved feed and bioenergy production

Abstract

Lignin modification has been a breeding target for the improvements of forage digestibility and energy yields in forage and bioenergy crops, but decreased lignin levels are often accompanied by reduced lodging resistance. The rice mutant gold hull and internode2 (gh2) has been identified to be lignin deficient. GH2 has been mapped to the short arm of chromosome 2 and encodes cinnamyl-alcohol dehydrogenase (CAD). We developed a long-culm variety, 'Leaf Star', with superior lodging resistance and a gh phenotype similar to one of its parents, 'Chugoku 117'. The gh loci in Leaf Star and Chugoku 117 were localized to the same region of chromosome 2 as the gh2 mutant. Leaf Star had culms with low lignin concentrations due to a natural mutation in OsCAD2 that was not present in Chugoku 117. However, this variety had high culm strength due to its strong, thick culms. Additionally, this variety had a thick layer of cortical fiber tissue with well-developed secondary cell walls. Our results suggest that rice can be improved for forage and bioenergy production by combining superior lodging resistance, which can be obtained by introducing thick and stiff culm traits, with low lignin concentrations, which can be obtained using the gh2 variety.

Figure 1. The natural gh2 mutation in Chugoku 117 and Leaf Star.

(a) Plant appearance. Koshihikari (left); Leaf Star (center); Chugoku 117 (right). (b) Lodging during grain ripening in a paddy field immediately after a strong typhoon hit. Koshihikari (left); Leaf Star (right). (c) Gold hull phenotype. Kinmaze-gh2 mutant (left); Koshihikari (lower center); Chugoku 117 (upper center); Leaf Star (right). (d) Gold internode phenotype in Leaf Star. (e) Lignin concentrations of fifth internodes 20 days after heading. Blue bar: Koshihikari; yellow bar: Chugoku 117; green bar: Leaf Star. Each column represents the mean ± s.d. (three replicates). The means followed by different letters are significantly different (5% confidence level) according to Tukey's test. The values in parentheses are the percentages relative to Koshihikari. (f, g) Coniferyl alcohol dehydrogenase activity (f) and sinapyl alcohol dehydrogenase activity (g) in fifth internodes from 30 days before heading to 10 days after heading. Blue circles: Koshihikari; yellow circles: Chugoku 117; green triangles: Leaf Star. The means followed by different letters are significantly different (5% confidence level) according to Tukey's test. (h) Location of gh2 estimated by RILs and introgression lines between Chugoku 117 and Koshihikari. The location of gh2 was predicted to be between markers RM3703 and RM6375 on chromosome 2. The putative gh2 region was narrowed down to the 3.8 Mb region between RM3703 and RM3390 by mapping. (i) Graphical genotype of NIL-gh2 on rice chromosomes. The red bar indicates the chromosome segment of gh2 including OsCAD2. Photographs (a)–(d) were taken by Taiichiro Ookawa.

Figure 2. Properties of OsCAD2 in the gh2 varieties Leaf Star and Chugoku 117.

(a) Expression of OsCAD2 relative to UBQ5 in the fifth internode from 30 days before heading to 10 days after heading. Blue circles: Koshihikari; yellow circles: Chugoku 117; green triangles: Leaf Star. The means followed by different letters are significantly different (5% confidence level) according to Tukey's test. (b) OsCAD2 gene structure and mutation sites in Leaf Star. There were no differences in the exon sequences of Leaf Star and Koshihikari, but a 506-bp region of a retrotransposon is inserted in the first intron in Leaf Star.

Figure 3. Lignin properties and culm strength in a gh2 mutant.

(a) Lignin concentration in the fifth internode 20 days after heading. (b) Coniferyl alcohol dehydrogenase activity (C) and sinapyl alcohol dehydrogenase activity (S) in the fifth internode 20 days before heading. (c) M, SM and MS of the fifth internode 20 days after heading. Open bars: Kinmaze-WT; red bars: Kinmaze-gh2. Each column represents the mean ± s.d. (three replicates). The means followed by different letters are significantly different (5% confidence level) according to Tukey's test. The values in parentheses are the percentages relative to Kinmaze-WT.

Figure 4. Accumulation of cell wall components and thickness of cortical fiber tissue in culms.

(a) The densities of cellulose (shaded) and hemicellulose (unshaded). (b) Thickness of cortical fiber tissue. Blue bars: Koshihikari; yellow bars: Chugoku 117; green bars: Leaf Star. Each column represents the mean ± s.d. (three replicates). The means followed by different letters are significantly different (5% confidence level) according to Tukey's test. The values in parentheses are the percentages relative to Koshihikari.

Figure 5. Morphological characteristics of transverse sections of cortical fiber tissues in culms.

(a, d) Koshihikari, (b, e) Chugoku 117, (c, f) Leaf Star. Scale bar: 100 μm. Transverse sections of fifth internodes 20 days after heading were stained with crystal violet. CF: cortical fiber tissue; SVB: small vascular bundle; P: parenchyma; A: aerenchyma.

Figure 6. Accumulation of cell wall components in basal culms of Leaf Star and both parents.

Transverse sections of fifth internodes 20 days after heading were stained with phloroglucinol (left) or calcofluor (right). Phloroglucinol staining (red color) was observed in cortical fiber tissues and in small vascular bundles. Calcofluor staining was stronger in the parenchyma cells of Leaf Star than in the parenchyma cells of both parents. (a,d) Koshihikari, (b,e) Chugoku 117, (c,f) Leaf Star. Scale bar: 100 μm.

Figure 7. Development of cortical fiber cells in culms.

(a) Koshihikari, (b) Chugoku 117, (c) Leaf Star. Scale bar: 50 μm. Transverse sections of fifth internodes 20 days after heading were observed using a scanning electron microscope.