Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon

Abstract

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.

Figure 1.

Regenerating shoots and roots in transgenic and wild-type plants. A, Regenerating shoots of the transgenic plants. Arrows indicate adventitious buds. Bar = 3 mm. B, Growth of regenerated transgenic shoots. The shoots had pinnate leaflets during elongation. Bar = 2.0 cm. C, Regenerating transgenic roots. The plantlets producing roots had pinnate leaves. Bar = 1 cm. D, Regenerated plantlets of wild-type plants. Bar = 1 cm.

Figure 2.

Analysis of PaPopCel1 expression in the main vein with petiolule pulvinus. A, Reverse transcription-PCR Southern-blot analysis of PaPopCel1 mRNA. The relative amounts of mRNAs by reverse transcription-PCR analysis at 15 cycles are shown. B, Western-blot analysis of cell wall proteins. Five micrograms of protein was used for each. C, Level of cellulase activity. D, Level of cello-oligosaccharides. Three separate main veins for each plant were used for the determination.

Figure 3.

Effects of PaPopCel1 transgenes on stem growth. A, Increase in stem length. B, Increase in stem diameter. Black circles, trg1; white circles, trg2; black squares, trg3; white squares, trg4; white triangles, wild type.

Figure 4.

Wild-type and transgenic (trg1) plants. The wild-type and transgenic plants are shown on the left and right, respectively, at 390 d after adventitious shoot formation. A, Whole plants. Bar = 10 cm. B, Leaves. Bar = 1.5 cm.

Figure 5.

Leaf movements of upper, middle, and lower parts of petioles in the wild-type and transgenic plants. A, Opening. All of the leaves are open (left). Bar = 4 cm. B, Closing. Closing leaves are distinguishable as yellow and white leaves (left). Bar = 4 cm. The lower part of the petiole was defined as the second petiole from the bottom, the middle part as the fifth or sixth petiole from the bottom, and the upper part as the ninth or tenth (or newest) petiole. All of the leaves in the petiole were observed to determine the opening and closing times of leaf pairs from start to finish. se values were calculated from four lines of trg1, trg2, trg3, and trg4.

Figure 6.

Closing movements of leaves whose main vein was excised. Leaves in the vein at the upper, middle, and lower parts of petioles are shown from left to right. se values were calculated from four lines of trg1, trg2, trg3, and trg4. Bar = 2 cm.

Figure 7.

In situ xyloglucan endotransglucosylase activity incorporating green fluorescent whole xyloglucan for 15 min on the transverse section (200 μm) of the petiolule pulvinus attached to the main vein of trg1. The tissues of the pulvinus and vein are shown in the upper and lower areas, respectively, in the images of the wild-type (A) and transgenic (B) plants. The arrows indicate the incorporated whole xyloglucan in the parenchyma motor cells. The red color is due to the autofluorescence of chloroplasts. Bars = 0.5 mm.