Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis

Abstract

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO2 uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.

Figure 1.

Identification of autotetraploid willow genotypes by chromosome counting (using 4′,6-diamidino-2-phenylindole [DAPI] stain) and flow cytometric analysis of relative DNA content (using propidium iodide). Shoots and plantlets that emerged from colchicine-treated axillary buds were rooted and sampled as described in “Materials and Methods.” Representative data of at least three repetitions are shown. Bars = 5 µm.

Figure 2.

Variation in characteristics of shoot development in diploid and tetraploid genotypes of willow plants. A, Comparison of green pixel-based shoot surface area monitored by digital photography to record aboveground biomass growth of willow plants from different genotypes in the greenhouse. The graph extension at top right shows interquartile ranges (25th and 75th percentiles) at week 7 for corresponding data points. Seventh week data points having the lowest (PP-E13) and highest (PP-E2) mean values are connected to the corresponding interquartile ranges with dashed lines. B, Box-plot presentation of average shoot lengths after 7 weeks of growth shows reductions in primary growth of autotetraploid plants in comparison with diploid plants. C, Box-plot presentation of average stem diameter after 7 weeks of growth shows enhanced secondary growth of autotetraploid plants in comparison with diploid plants. Based on Welch’s t test, statistically significant events compared with diploids are indicated below the sample labels as ***, P < 0.01 and **, P < 0.05. Underlined asterisks indicate the level of significance based on posthoc comparisons made with Tukey’s honestly significant difference (HSD) test. Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Alternate boxes are shaded to differentiate neighboring boxes. n = 15, 15, 10, 11, 5, and 15 sample points.

Figure 3.

Altered plant architecture and growth characteristics of autotetraploid willow plants grown under greenhouse conditions. Stem cuttings were planted into cultivation pots, and the outgrowing shoots with characteristic phenotypic traits are presented. Note the development of larger, densely packed leaves of autotetraploid (PP-E7 and PP-E13) plants. Insets show thresholded binary images corresponding to the plants for a given view. Bar = 6.5 cm.

Figure 4.

Wider stems with enlarged wood regions in stem sections of tetraploid willow plants. The chart shows the quantification of section areas representing bark, wood, and pith regions (n = 4). Statistically significant events (based on both Welch’s t test and Tukey’s HSD posthoc test) compared with diploids are indicated for wood and bark regions as ***, P < 0.01. Dissection microscope images of a set of cross sections are shown below the chart. Samples were collected at 120 cm from the shoot tip of plants. Bar = 0.5 cm.

Figure 5.

The autotetraploid genomic constitution of energy willow increases the foliage capacity of plants. A, Leaf morphology variations of willow plants grown in the field. B, Differences in leaf width between diploid and tetraploid willow plants grown in the greenhouse (n > 52). C, Differences in lamina length between diploid and tetraploid willow plants grown in the greenhouse (n > 37). D, Tetraploid willow plants produce more leaf biomass in comparison with diploid ones under greenhouse conditions (n > 10). Based on Welch’s t tests, statistically significant events compared with diploids are indicated below the sample labels as ***, P < 0.01, **, P < 0.05, and *, P < 0.1. Underlined asterisks indicate the level of significance based on posthoc comparisons made with Tukey’s HSD test. Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots.

Figure 6.

Enhanced midrib-xylem development in leaves from tetraploid plants compared with a diploid plant. Midrib cross-sectional areas were measured by manually tracing white-colored midrib regions sampled from the midpoint of each leaf. Representative images of hand-sectioned material are shown. Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles (n = 9). Statistically significant events (based on both Welch’s t test and Tukey’s HSD posthoc test) compared with diploids are indicated below the sample labels as ***, P < 0.01. Bar = 0.5 mm for all images.

Figure 7.

Tetraploid willow plants have enlarged palisade parenchyma cells. A, Comparison of leaf cross-sections from diploid and tetraploid willow plants. Calcofluor White-stained cell wall fluorescence (blue) was merged with transmission images. Arrows indicate the palisade parenchyma layer of leaves. Bar = 20 µm for all images. B, Quantification of average palisade parenchyma cell size as cross-sectional area (n = 200). C, Quantification of average number of parenchyma cells per 100-µm-long distance (n = 40). Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Statistically significant events (based on both Welch’s t test and Tukey’s HSD posthoc test) compared with diploids are indicated below the sample labels as ***, P < 0.01.

Figure 8.

Tetraploid willow plants transpire more water, as shown by the elevated stomatal conductance in leaves. Leaf stomatal conductance (g_s) was measured on the fifth/sixth fully developed younger leaves (from top) of willow plants. The measurements were recorded in an air CO₂ concentration of 400 μg mL⁻¹, leaf temperature of 22°C, and photosynthetic active radiation of 400 to 430 µmol photons m⁻² s⁻¹ (n = 5). Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Based on Welch’s t test, statistically significant events compared with diploids are indicated below the sample labels as ***, P < 0.01 and **, P < 0.05. Underlined asterisks indicate the level of significance based on posthoc comparisons made with Tukey’s HSD test.

Figure 9.

Autotetraploid willow plants absorb CO₂ more efficiently from the atmosphere. The rate of net CO₂ fixation was measured on the fifth/sixth fully developed young leaves (from top) of willow plants at 400 to 430 µmol photons m⁻² s⁻¹ light intensity, 22°C temperature, and 400 μg mL⁻¹ ambient CO₂ level (n = 5). Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Statistically significant events (based on both Welch’s t test and Tukey’s HSD posthoc test) compared with diploids are indicated below the sample labels as ***, P < 0.01.

Figure 10.

Improved photosynthetic capacity of tetraploid willow plants as indicated by ETRs of PSI and PSII measured on leaf samples. Simultaneous light response curves of ETR(I) and ETR(II) were measured in the dark-adapted fifth/sixth fully developed young leaves (from top) for both field and greenhouse genotypes using Dual PAM as described in “Materials and Methods.” A, ETR(I) under field conditions. B, ETR(II) under field conditions. Leaves of field-grown plants were collected in wet tissue and kept in an ice box, and ETR measurements were carried out within 2 h of sample collection. C, ETR(I) under greenhouse conditions. D, ETR(II) under greenhouse conditions. Tetraploid willow genotypes are indicated as black symbols and the diploid genotype by white symbols. Data are means ± se of six independent plants per genotype. Based on Welch’s t test, statistically significant events (for the highest photosynthetic photon flux density [PPFD] measurement) compared with diploids are indicated next to corresponding data points as ***, P < 0.01, **, P < 0.05, and *, P < 0.1. Underlined asterisks indicate the level of significance based on posthoc comparisons made with Tukey’s HSD test.

Figure 11.

Spider plot of chlorophyll fluorescence parameters deduced from OJIP fast kinetics measurements. Shown are the values of initial (F_o) and maximal (F_m) fluorescence levels, the F_v/F_m and F_v/F_o (maximal PSII quantum yield) ratios, the (1 − V_j)/V_j parameter, where V_j = (F_2ms – F_o)/F_v, the performance index (PI), the area parameter, as well as the dissipated energy flux per active reaction center (RC/ABS) measured on fifth/sixth young fully developed leaves. The data are shown for the tetraploid lines (white symbols) after normalization to respective values obtained in the diploid line (black symbols). Data are means ± se of six to seven independent greenhouse-grown plants per genotype.

Figure 12.

Significant stimulation of root development after duplication of the genome size of energy willow. A, Side and bottom views of roots from the diploid and tetraploid (PP-E12) plants grown in soil in transparent wall plexiglass columns. Digital images were taken at week 3 of cultivation. Bar = 2 cm for all images. B, Total surface area (in mm²) occupied by white pixels was used to monitor root biomass growth to compare diploid and tetraploid willow plants during early development. Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Based on Welch’s t test, statistically significant events compared with diploids are indicated as **, P < 0.05 and *, P < 0.1. Underlined asterisks indicate the level of significance based on posthoc comparisons made with Tukey’s HSD test. n = 10 sample points.

Figure 13.

Autotetraploidization resulted in energy willow genotypes with increased root biomass. A, Experiment 1. Plants from tetraploid genotypes developed significantly more root than the control diploid ones based on fresh weight measurements (g plant⁻¹; n = 10). B, Experiment 2. Increased root biomass of tetraploid willow genotypes as compared with diploid plants based on dry weight measurements (g plant⁻¹; n = 10). Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Based on Welch’s t test, statistically significant events compared with diploids are indicated below the sample labels as ***, P < 0.01, **, P < 0.05, and *, P < 0.12. Underlined asterisks indicate the level of significance based on posthoc comparisons made with Tukey’s HSD test.

Figure 14.

Differences in root anatomy detected between diploid and tetraploid willow plants. A, Calcofluor White-stained, hand-sectioned roots (from the maturation zone) of diploid and tetraploid plants were imaged using confocal laser scanning microscopy. Note the larger cortical cells of the tetraploid samples. Bar = 50 µm for all images. B, Cortical cells of diploid and tetraploid roots were manually traced on hand-sectioned material using Olympus Fluoview software, and average cross-sectional areas of cortical cells were calculated and plotted for diploid and tetraploid samples (n > 362). Box-plot center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Statistically significant events (based on both Welch’s t test and Tukey’s HSD posthoc test) compared with diploids are indicated below the sample labels as ***, P < 0.01.