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Rheophytic Osmunda lancea (Osmundaceae) exhibits large flexibility in the petiole – Scientific Reports

Plant materials

In this study, wild populations of O. lancea and O. japonica were sampled from riparian (O. lancea: N35° 40′ 44.234″, E139° 04′ 25.068″) and inland (O. japonica: N35° 42′ 13.963″, E139° 00′ 53.744″) locations along the Tsuru River in Uenohara City, Yamanashi Prefecture, Japan (Fig. 1). Iwatsuki29 describes in Flora of Japan that O. lancea (Fig. 1a-I) and O. japonica (Fig. 1b-I) are distinguished by pinnules cuneate to acute (Fig. 1a-III) or subtruncate (Fig. 1b-III) at base, respectively, and the widest part of pinnules is less than 10 mm (Fig. 1a-III) for the former and 10–25 mm (Fig. 1b-III) for the latter. Therefore, we identified the samples collected according to this description (Fig. 1). This field research and sampling were carried out in areas where there were no collection restrictions under Japanese law. All methods were carried out in accordance with Japanese law. Voucher specimens are deposited in National Museum of Nature and Science (TNS). Following removal from the petiole base, the specimens were brought back to the laboratory on moistened newspapers to prevent water evaporation. Mechanical analyses were performed less than 12 h after of sample collection.

Morphological analyses

We measured Angle at base of pinnules and Length at widest part of pinnules of O. lancea and O. japonica base on Iwatsuki29.

In the morphological analysis, lamina area was analyzed using the graphic software Fiji (ImageJ Version 1.53e). The shape of the petiole cross-section was assessed by measuring the long and short diameters using an electronic caliper. While this method is a simplified approach, it allowed for the calculation of the long to short diameter ratio, which was found to be approximately 1.1 for both species. Based on this observation, the transverse shape of the petiole was assumed to be elliptical. The petiole transverse area A was calculated using the following formula:

$$A = frac{pi ab}{4}$$

where a and b are the long and short diameters of the petiole, respectively. The relationship between the petiole transverse area A calculated by the above formula, and the lamina area supported by the petiole transverse area A was analyzed.

Mechanical analyses

Due to the testing machine used in this study, a fixed span length of 50 mm was maintained for all conducted tests. Therefore, to apply the general bending theory, the petiole was selected to have a short diameter ranging from 3–4 mm. The samples were divided into sections approximately 60 mm in length, and the long and short diameters of the central portion of each sample were measured using calipers (CD-15CXR; Mitutoyo, Japan). These measurements were conducted to determine the transverse area of the petiole, following the same method employed in the morphological analyses. The span-depth ratio ranged from 13 to 16. To conduct the mechanical measurements, the same samples used in the anatomical measurements were subjected to a three-point bending test. This test was performed using a tabletop tensile and compression testing machine (MCT-1150, A&D, Japan) and an R5 bending test fixture (JM-B1-500N, A&D, Japan) with the test speed set to 10 mm/min.

Values of bending stress σ and strain ε were calculated using the following standard elliptical transverse formulas for the elastic loading of beams:

$$sigma = frac{M}{Z} = frac{PL}{{4Z}}$$

and

$$varepsilon = frac{48delta sigma I}{{PL^{3} }} times 100$$

where M is the bending moment of simply supported beams and Z is section modulus. Where P is the load, L is the span length, δ is the displacement, and I is the moment of inertia. In this study, strain ε is calculated as %.

The following formulae were used to calculate the section modulus Z and the area moment of inertia I of elliptical area:

$$Z = frac{{pi ab^{2} }}{32}$$

and

$$I = frac{{pi ab^{3} }}{64}.$$

In this study, the flexural stress and strain at the breaking point of the specimen were defined as the bending strength σmax and the breaking strain εmax, respectively. The bending modulus of elasticity E was calculated as follows:

$$E = frac{{sigma_{0.25} – sigma_{0.05} }}{{epsilon_{0.25} – epsilon_{0.05} }} times 100.$$

Strain values from 0.05 to 0.25% and bending stresses were used to determine the bending modulus of elasticity. A sampling frequency of 50 Hz was used in this study. The testing machine used in this study was not sufficiently powerful to accurately measure the bending modulus of elasticity owing to the limited resolution of the strain measuring instrument. Nevertheless, the measurement of bending modulus of elasticity was conducted to determine potential alterations in the mechanical properties of supporting organs in plants along the river, which allow them to evade mechanical stresses caused by water currents.

Measurement of cell wall area, cell area and cell length

After the mechanics test, petioles were air-dried in a draft chamber without heating and used to observe the petiole transverse tissue. The subepidermal cortex just below the epidermal layer on the abaxial side of the petiole cross-section was recorded using a tabletop electron microscope (TM3000, HITACHI, Japan), and analyzed using the ImageJ software. To determine the cell wall per unit area of the petiole, the cell wall area was measured within a 50 µm per side square section in the subepidermal cortex. As the cytoplasm, intracellular organelles, and intercellular spaces were compressed, in the dried petiole samples used, it was possible to measure only the cell wall. The cell wall fraction per unit area was calculated using the following formula:

$${text{cell}};{text{wall}};{text{fraction}};{text{per}};{text{unit}};{text{area}} = frac{{left( {{text{cell}};{text{wall}};{text{area}};{text{within}};2500;{upmu}text{m}^{2} } right)}}{{2500;{upmu}text{m}^{2} }}.$$

Cell area was measured starting from cells in the subepidermal cortex adjacent to the epidermis. After measuring 10 cell area in the subepidermal cortex, the mean value was calculated and compared between the two species. The cell length along the vertical direction of petiole was measured using the maceration protocol of Gärtner and Schweingruber30. To macerate the tissue, a 0.1 mm thick razor (FA-10, Feather Safety Razor CO.,LTD, Japan) was used to cut the petiole longitudinally through the epidermis. Two types of cells were observed using the light microscopy (CX43, Olympus); one was the epidermis based on morphology, and the other was the sclerenchyma cell based on morphology. We measured the latter. The cell length was averaged by measuring 30 cells from one individual in the subepidermal cortex. Also, cells were stained with safranin to see if they were lignified. Base on Kijima31, a 5% safranin staining solution in 50% ethanol was prepared and maceration samples were stained on slide glass for 5 min, followed by thorough rinsing with water and observation of cells remaining on slide glass.

Cell wall mass per unit volume

The various types of tissues were involved in the petiole and cell composition at the same position in petiole may be different among closely related species. Therefore, it was very difficult to compare the precise value of ‘cell wall mass per unit volume’. However, ‘cell wall mass per unit volume’ played an important role to contribute the plant strength28,32. In this study, we selected ‘cell wall mass per unit volume’ to explain anatomical and mechanical results. The cell wall mass per unit volume is one of the factors that determine the mechanical properties of plants and is used as an indicator of the mechanical stability of stems. The volume Vfresh of a fresh petiole was calculated using the following formula:

$$V_{fresh} = frac{pi abl}{4}$$

where l represents the length of petiole. After volume measurements, the samples were dried in an incubator (DRM420DD, ADVANTEC, Japan) at 100 °C for 72 h, and the dry weight [g] was measured using an electronic balance (AG204, Mettler Toledo). The cell wall mass per unit volume in each petiole was calculated using the following formula:

$${text{cell}};{text{wall}};{text{mass}};{text{per}};{text{unit}};{text{volume}} = frac{Dry;weight}{{V_{fresh} }} .$$

Statistical analysis

Microsoft Excel was used for statistical analysis. After obtaining equal variances for the comparison of each measured item (F-test), a t-test was performed under the assumption of “equal variances” or “variances not equal”. In this study, the F-test results indicated unequal variances in the width at widest part of pinnules, bending strength, bending modulus of elasticity, cell wall fraction per unit area, transverse cell area and cell length along the vertical direction of petiole. Equal variances were observed in the angle at base of pinnules and breaking strains.

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