Polyploidy Affects Plant Growth and Alters Cell Wall Composition

Abstract

Polyploidization has played a key role in plant breeding and crop improvement. Although its potential to increase biomass yield is well described, the effect of polyploidization on biomass composition has largely remained unexplored. Here, we generated a series of Arabidopsis (Arabidopsis thaliana) plants with different somatic ploidy levels (2n, 4n, 6n, and 8n) and performed rigorous phenotypic characterization. Kinematic analysis showed that polyploids developed slower compared to diploids; however, tetra- and hexaploids, but not octaploids, generated larger rosettes due to delayed flowering. In addition, morphometric analysis of leaves showed that polyploidy affected epidermal pavement cells, with increased cell size and reduced cell number per leaf blade with incrementing ploidy. However, the inflorescence stem dry weight was highest in tetraploids. Cell wall characterization revealed that the basic somatic ploidy level negatively correlated with lignin and cellulose content, and positively correlated with matrix polysaccharide content (i.e. hemicellulose and pectin) in the stem tissue. In addition, higher ploidy plants displayed altered sugar composition. Such effects were linked to the delayed development of polyploids. Moreover, the changes in polyploid cell wall composition promoted saccharification yield. The results of this study indicate that induction of polyploidy is a promising breeding strategy to further tailor crops for biomass production.

Figure 1.

Phenotype of polyploid Arabidopsis. A, Bolting time of polyploid Arabidopsis (2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid). B, The average projected rosette area at time of bolting. C, Rosettes of representative Arabidopsis plants at the start of bolting. Images were digitally extracted for comparison. Error bars represent SD; different lowercase letters indicate statistically significant difference; n = 3 biological replicates of 20 plants; Statistical analysis was done with univariate analysis and Scheffe post-hoc testing in SPSS; α = 0.05.

Figure 2.

Growth parameters of polyploid Arabidopsis. A, Rosette growth rate over time of polyploid Arabidopsis (2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid). Blue diamonds indicate the measured values and a curve was fitted using LEAF-E. Leaf elongation rate (LER), Maximal LER (LERmax), the time at which LER reaches a maximum (tm), the end-point of growth (te). B, The values for all parameters determined using the LEAF-E tool. C, The average growth rate of the main inflorescence stem of polyploid Arabidopsis. Error bars represent SD. Different lowercase letters indicate statistically significant difference; n = 20 plants. Statistical analysis was done with univariate analysis and Scheffe posthoc testing in SPSS; α = 0.05.

Figure 3.

Cellular analysis of polyploid Arabidopsis. A, Leaf area of the first two leaves (L1/2) over time in polyploid Arabidopsis (2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid). The arrow indicates the time point where L1/2 were harvested for further analysis. B, Leaf area of L1/2 on day 31, which was chosen as the time point to make cellular drawings of abaxial epidermal cells. C, Representative cellular drawings of abaxial epidermal cells of polyploid plants. D, Average size of abaxial epidermis cells of L1/2 leaves of polyploid plants. E, Average size of stomatal guard cells of polyploid plants. F, Average circularity of abaxial epidermis cells of L1/2 leaves of polyloid plants. G, Average number of cells per L1/2 of polyploid plants. Error bars represent SD; Different lowercase letters indicate statistically significant difference; n = 8 plants per time point; Statistical analysis was done with univariate analysis and Scheffe posthoc testing in SPSS; α = 0.05.

Figure 4.

Polyploid Arabidopsis stem biomass. A, Fully-grown representative polyploid Arabidopsis plants (2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid). Scale bars represent 5 cm. B, The average length of the primary inflorescence stem of polyploid Arabidopsis plants. C, The average dry weight of the primary inflorescence stem of polyploid Arabidopsis plants. Error bars represent SD; Different lowercase letters indicate statistically significant differences; n = 3 biological replicates of 20 plants; Statistical analysis was done with univariate analysis and Scheffe posthoc testing in SPSS; α = 0.05.

Figure 5.

Morphological analysis of polyploid Arabidopsis stems. A and B, Transverse sections stained with toluidine blue of representative polyploid Arabidopsis stems (2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid). A, Detail of the vascular bundle. The arrow indicates the interfascicular fibers. Scale bars represent 100 µm. B, Detail of pith region. Scale bars represent 200 µm. C to F, Transmission Electron Microscopy (TEM) of different tissues including (C) cortex, (D) phloem, (E) xylem, and (F) pith. The arrowheads indicate collapsed cells or the remainder of crushed cells. Scale bars represent either 10 or 2 µm, as indicated. G, The average diameter of the primary inflorescence stem taken 1 to 2 cm above the rosette. H, The average amount of collapsed vessels per total amount of vessels. I, The average thickness of the cell wall of the interfascicular fibers. The thickness of primary cell wall is depicted in gray; the amount of secondary cell wall is shown in white. Error bars represent SD; Different lowercase letters indicate statistically significant difference; n = 4 biological replicates; statistical analysis was done with univariate analysis and Scheffe posthoc testing in SPSS; α = 0.05.

Figure 6.

Apical dominance of polyploid Arabidopsis. A, The ratio of the main inflorescence stem dry weight (without side branches and secondary inflorescences) to the total dry weight (with side branches) in polyploid Arabidopsis (2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid). B, The average number of additional stems emerging from the rosette, besides the main stem. Error bars represent SD; different lowercase letters indicate statistically significant difference; n = 3 replicates of 20 plants; statistical analysis was done with univariate analysis and Scheffe posthoc testing in SPSS; α = 0.05.

Figure 7.

Cell wall composition of polyploid Arabidopsis stems. A, The cell wall residue (CWR) of Arabidopsis polyploids (2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid) is given as percentage of dry weight. B, The amount of CW polymers is given as percentage of CWR. Error bars represent SD; different lowercase letters indicate statistically significant difference; n = 8 pools of 2 or more biological replicates; statistical analysis was done with univariate analysis and Scheffe posthoc testing in SPSS; α=0.05.

Figure 8.

Carbohydrate microarray on different fractions of cell wall extracts. All spot intensity values are relative to the highest fluorescence value. Pectins are extracted with 1,2-cyclohexylenedinitrilotetraacetic acid (CDTA), hemicelluloses with NaOH. 2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid. HG = homogalacturonan, AGP = arabinogalactan protein; intensity of green coloration is indicative of higher values. n = 8 pools of 2 or more biological replicates.

Figure 9.

Cell wall composition of polyploid Arabidopsis compared to different developmental stages of diploid main stems. A, The amount of cell wall residue (CWR) and CW components of fully senesced polyploids (4n, tetraploid; 6n, hexaploid; 8n, octaploid) are given relative to fully senesced diploid stems (2n), a series of diploid stems at different stages of development (5 cm, 10 cm, 15 cm, 27 cm), and fully grown diploid stems that had not yet started to senesce (Green). B, The amount of monosaccharides in the MPS fraction relative to that in fully senesced diploid stems (2n). Error bars represent SD; n = 8 pools of 2 or more biological replicates.

Figure 10.

Saccharification yield of polyploid Arabidopsis. The amount of Glc released per Arabidopsis polyploid (2n, diploid; 4n, tetraploid; 6n, hexaploid; 8n, octaploid) cell wall residue (CWR) is given for saccharification without pretreatment (None), after acid pretreatment (Acid), and after alkaline pretreatment (Alkaline). Error bars represent SD; different lowercase letters indicate statistically significant difference; n = 8 pools of 2 or more biological replicates; statistical analysis was done with univariate analysis and Scheffe posthoc testing in SPSS; α = 0.05.