Study on the cause of peeling and withering in Isodon rubescens stems in winter

1 Introduction

Isodon rubescens (Hemsley) H. Hara belongs to the Lamiaceae family (Flora of China Committee, 1979). I. rubescens is widely distributed in China (Flora of China Committee, 1979). The dry leaves and stems of Isodon rubescens are called rabdosiae rubescentis herba used in traditional Chinese medicine for the treatment of sore throat, inflammation, gastrointestinal problems, oesophageal cancer and so on (Chinese Pharmacopoeia Committee, 2020). I. rubescens is a kind of perennial subshrub. The root and rhizome of I. rubescens are woody and can grow for several years. The shoot tip of the I. rubescens stem is herbaceous and withers in winter. The middle and lower parts of I. rubescens stems are woody and able to sprout in spring, although their leaves fall in winter.

The upper part of the living stem of I. rubescens peeled and withered when the air temperature was below 0 °C in winter (Wang et al., 2008; Lu et al., 2007). The living stem of I. rubescens peeled and withered piece by piece if the air temperature fell below 0 °C several times. This was very unfavorable for I. rubescens growth. A field investigation and paraffin sectioning were performed to study the causes of peeling and withering in the living stem of I. rubescens in winter. This study can contribute to I. rubescens cultivation and resources protection.

2 Materials and methods

Instruments used were a camera (Nikon D5200, Nikon Group), a microtome (RM2125, Leica Microsystems Trading Co., Ltd) and a microscope (BA310, MoticMedical Diagnosis System Co., Ltd). All solvents, reagents and other chemicals (ethanol, dimethylbenzene, formalin, acetic acid, glycerol, paraffin, safranin and fast green) used were analytical grade.

The peeled I. rubescens stem and healthy (intact) one were collected from Jiulingshan Mountain in Henan province of China in January 2019.

The surface features and changes of I. rubescens stems were investigated at air temperatures below 0 °C on the Taihang Mountain in January 2019. The I. rubescens stems were photographed.

The slide specimens of peeled and intact I. rubescens stems were prepared using the modified paraffin section method detailed below (Liu et al., 2016).

The specimens were placed in the fixation solution (90 ml of 70% (v/v) ethanol, 5 ml of acetic acid and 5 ml of formalin) for 24 h at first. Then the fixation solution was rinsed away, and the specimens were softened by soaking in solution containing 10 ml of glycerol and 10 ml of 95 % (v/v) ethanol for 2 h. The specimens were dehydrated in gradient solutions of 30 % (v/v), 50 %, 70 %, 85 %, 95 %, and 100 % ethanol for 2 h each time. The dehydrated specimens were successively soaked in the mixture of ethanol and dimethylbenzene (1:1, v/v) followed by dimethylbenzene (2 times) for 1 h each time to gradually clear. The cleared specimens were successively soaked in the mixture of dimethylbenzene and melted paraffin (1:1, v/v) followed by melted paraffin (2 times) for 1 h each time. Then the specimens were embedded in paraffin before sectioning. The embedded materials were sectioned (8–10 μm in thickness) on microtome. The sections were attached to a glass slide with gelatin and then baked in an oven at 40 °C for 5–10 min. The paraffin sections were successively soaked in dimethylbenzene (2 times), mixture of dimethylbenzene and ethanol (1:1, v/v) and then in ethanol (2 times) for 10 min each time. The dried paraffin sections were dyed with 0.3 % (w/v) fast green solution (dissolved in ethanol) for 1 min and then dyed with 0.5 % (w/v) safranin solution (dissolved in water) for 5 min. The dyed paraffin sections were sealed with gelatin and cover glasses.

The prepared paraffin sections were photographed under the microscope with a micro camera.

3 Results and discussion

Based on the field investigation, it was found that there were thin feathery ice ribbons on the ridge of living I. rubescens stems at air temperature below 0 °C. The ice ribbons burst the bark (Fig. 1). The detached barks appeared as longitudinal strips. The peeled I. rubescens stem and the upper section of stem died soon. There no ice ribbons on I. rubescen stems and the barks of stems were intact at air temperature above °C. The I. rubescens stems with intact barks did not die.

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Fig. 1

The burst bark on I. rubescens stem due to ice ribbons.

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The paraffin section of intact I. rubescens stem was shown as Fig. 2. There was phloem around the xylem in the intact I. rubescens stem. The phloem was dyed green and the xylem was dyed red in the paraffin section of intact I. rubescens stem. There was imperfect epidermis on the surface of I. rubescens stem.

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Fig. 2

The paraffin section of living intact I. rubescens stem.

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The paraffin section of peeled I. rubescens stem was shown as Fig. 3. The ice ribbons were formed between xylem and cambium. The inside cells of cambium were torn by ice ribbons. There was no phloem around the xylem on the ridge of peeled I. rubescens stem. The xylem was exposed on the surface of peeled I. rubescens stem. There was residual phloem on the ravine of peeled I. rubescens stem. The phloem in the detached bark withered.

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Fig. 3

The paraffin section of I. rubescens stem peeled due to ice ribbons.

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Some plants have detached bark, such as Lonicera japonica Thunb., Ulmus lamellosa Wang et S. L. Chang ex L. K. Fu, Platanus occidentalis Linn., Pinus bungeana Zucc. ex Endl., and Lagerstroemia indica Linn. at normal temperature. The detached bark of these plants is entirely made of phellem. There are very few species of plant that peel their barks as the result of ice ribbons at air temperatures below 0 °C. Studies have reported that Verbesina virginica and Helianthemum canadense develop ice ribbons at air temperatures below 0 °C (Carter 2009; Carter, 2013; Hofmann et al., 2015). These studies did not report whether the stems of these plants died after the ice ribbons formation. I. rubescens is a species that peels and withers when ice ribbons arise on its stems at air temperatures below 0 °C in winter. The upper parts of the I. rubescens stems would not wither (only the tender tip of stem withered in autumn) in winter if the air temperature was above 0 °C. I. rubescens stems peeled and withered because the stems extruded ice ribbons at air temperatures below 0 °C. The ice ribbons separated the phloem from the xylem. The phloem and even the whole bark of the I. rubescens stems were torn by the enlarged ice ribbons. The broken phloem could not transport organic compounds. The water in the xylem in the damaged stem could not be transported to the upper stem (Venturas et al., 2017). Therefore, the upper stems of I. rubescens died because it receives no water. I. rubescens should be cultivated in regions where the air temperature generally stayed above 0 °C to prevent freezing injury. The I. rubescens variety that resists ice ribbons should also be bred to expand its cultivation area to cold regions (Li et al., 2011).

4 Conclusions

The phloem was separated from the xylem by the enlarged ice ribbons. The phloem and the whole bark of the I. rubescens stems were torn when ice ribbons appeared. The damaged stem could not transport water and nutrition to the upper section. The upper stem above the ice ribbons withered because it could not receives water and nutrition.

5 Interests declaration

The authors of this article declare that they have no conflicts of interest.

6 Abbreviations

Not applicable.

7 Ethics approval and consent to participate

Not applicable.

8 Consent for publication

Not applicable.

9 Availability of data and materials

Data has been permanently archived: https://doi.org/10.5281/zenodo.3830395.

10 Competing interests

The authors declare that they have no competing interests.

Funding

Funding information is not applicable.