Introduction

Isodon rubescens (Hemsley) H. Hara belongs to the genus Isodon of the Lamiaceae family1. The entire I. rubescens plant is designated as the traditional Chinese medicine (TCM) Rabdosia rubescentis herba2,3. Rabdosia rubescentis herba is used for the treatment of sore throats, throat inflammation and oesophageal cancer in traditional Chinese medicine2.

Isodon rubescens is a deciduous perennial subshrub widely distributed on mountain with abundant sunshine in China1. The roots and rhizomes of I. rubescens are woody and perennial. The upper herbaceous parts of I. rubescens stems wither in winter. The middle and lower woody parts of I. rubescens stems do not wither in winter and will sprout during the next spring. The majority of the branches are located on the lower part of the I. rubescens stem (2–6 branches in general). There is few branch on the upper part of I. rubescens stem. The leaves of I. rubescens are paired1. There are 5–15 nodes (leaf positions) on I. rubescens stems. The two leaves of each node are the same shape. The leaves on the upper part of the I. rubescens stem are light green. The leaves on the middle upper part of the I. rubescens stem are dark green. The leaves on the lower upper part of the I. rubescens stem are yellowish green.

The orders of leaf maturity and senescence on the stem were generally from top to bottom4,5. The degree of leaf maturity differs among the different leaf positions because the extent of leaf development and formation is not simultaneous. Concomitantly, the photosynthetic capacities of leaves on the plant are diverse6,7,8. The photosynthetic capacities of the lush leaves on the upper and middle parts of general plant stems are high, and those of wilted leaves at the bottom of the plant stems are low7. The apical buds of I. rubescens stems continuously generate young leaves in spring and summer. These apical buds develop into flowers in summer or autumn. The young leaves on I. rubescens stems gradually develop into mature leaves and wither after they fall from the stems. The differences in the photosynthetic capacity of I. rubescens leaves at different leaf positions have not been studied. As a result, the appropriate pruning strategy during the cultivation and management of I. rubescens is currently unclear. The photosynthetic rates, light response curves and chlorophyll fluorescence characteristics of I. rubescens leaves at different leaf positions were determined in this study to clarify the differences in photosynthesis capacity among these leaves. This study revealed differences in the photosynthetic capacity of leaves at different leaf positions on I. rubescens stems. This study provides a theoretical basis for pruning or topping in the cultivation and management of I. rubescens.

Results

The photosynthetic rates of leaves with the same leaf position on different plants were varied because of the different growth patterns of I. rubescens plants (Fig. 1). However, there were obvious distinctions among the photosynthetic rates of I. rubescens leaves with different leaf positions on the same stem and under the same light intensity (Fig. 1). These differences were detected for all 6 I. rubescens plants. The No. 1 leaf possessed a high photosynthetic capacity relative to that of the low leaf position, although the blades were incompletely opened. The photosynthetic rate of the No. 1 leaf was slightly lower than that of the No. 2 or No. 3 leaf. The photosynthetic rates of the No. 4, No. 5 and No. 6 leaves were generally the highest among the leaves on a given stem. The photosynthetic capacity of I. rubescens leaves gradually decreased with decreasing leaf position from the No. 5 or No. 6 leaves. The photosynthetic capacity of I. rubescens leaves at the lowest leaf positions was very poor. The leaves at lower leaf positions still possessed photosynthetic capacity, although more than half of the leaf was withered (Fig. 1).

Fig. 1
figure 1

The photosynthetic rates of Isodon rubescens leaves at different positions (one leaf on each position from top to bottom) on 6 stems under the same light intensity.

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The light response curves of I. rubescens leaves at different leaf positions all initially rose, stabilized, and then decreased with increasing light intensity (Fig. 2). There were distinctions among the light response curves of leaves at different leaf positions on the same I. rubescens stem (Fig. 2; Table 1). The light saturation points of the leaves on the upper and middle parts of the stem varied from 800 to 1000 µmol m− 2 s− 1. The light saturation points and the maximum photosynthetic rates of the leaves on the upper part of the stem were greater than those of the leaves on the middle and lower parts of the stem. The leaves on the lower I. rubescens stem exhibited low light saturation points and the maximum photosynthetic rates. The I. rubescens leaves at all leaf positions presented photosynthesis inhibition under high light intensity. The photosynthetic rates of the leaves at all leaf positions decreased with increasing light intensity from approximately 2000 µmol m− 2 s− 1. The light compensation points, light saturation points, dark respiration rates and maximum photosynthetic rates of the upper leaves were all greater than those of the lower leaves.

Fig. 2
figure 2

The light response curves of Isodon rubescens leaves at different leaf positions (one leaf on each position from top to bottom).

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Table 1 The fitted results of the light response curve of Isodon rubescens leaves at different leaf positions (from top to bottom).
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There was obvious diversity among the rapid light curves of chlorophyll fluorescence in I. rubescens leaves at different leaf positions. The maximum electron transport rates in chlorophyll fluorescence in leaves at the middle leaf positions were slightly higher than those in the leaves at the upper leaf positions. Leaves at the lower leaf positions had the lowest maximum electron transport rate in chlorophyll fluorescence of all I. rubescens leaves (Fig. 3).

Fig. 3
figure 3

The maximum electron transport rates in photosynthetic system II in Isodon rubescens leaves (one leaf on each position).

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Discussion

Different plant species have different phyllotaxies (leaf arrangements on stems). There are long internodes between the nodes in plants with alternate, decussate or verticillate phyllotaxis. The leaf positions with these types of phyllotaxies are clearly separated. The development time, formation time and maturity of leaves at different leaf positions are diverse. Therefore, the photosynthetic capacity of leaves at different positions is also varied. There are distinctions among leaf photosynthesis at different leaf positions in many plant species9. The variance in leaf photosynthesis with leaf position differs among species.

Isodon rubescens is a deciduous subshrub. The leaves on the I. rubescens stem are continuously renewed from the apical bud. There are long internodes on the I. rubescens stem. Therefore, the leaves at different positions are greatly separated. There are few reports regarding differences in photosynthesis among I. rubescens plants relative to leaf position. In this study, it was found that not only the degree of maturity but also the light intensity contributed to variation in the photosynthetic capacity of I. rubescens leaves at different leaf positions. Although the I. rubescens leaves at the No. 1 and No. 2 positions were only barely developed and the blades were incompletely opened, the photosynthetic capacities of these leaves was similar to those of the mature leaves below them in this study and the results of published report10. The cells in the tender leaves had been approaching maturity and possessed intact chloroplasts. Therefore, the tender leaves possessed similar photosynthetic capacities with that of mature leaves.

The rapid light curves of chlorophyll fluorescence in I. rubescens leaves showed the same pattern as the photosynthetic rates in these leaves. This pattern has been observed in several other plants11. The chlorophyll fluorescence characteristics of leaves can indicate the photosynthetic potential of the leaf. The photosynthetic potential of the leaf was related to the leaf longevity with active photosynthesis also12.

The leaves of the middle part of the I. rubescens stem were the main site of photosynthesis because of their large area and strong photosynthetic capacity. But there was not definite boundary between the tender leaf and the developed leaf. The photosynthetic capacity of leaves on the lower part of the stem was very weak because the leaves were partially withered. There were some visible withered spots on the lower leaves. The withered portion of each of the lowest pair of leaves represented approximately half of the entire leaf area. There were 1–3 withered areas on the leaves above the lowest pair of leaves. Many of the cells in the lower leaves had aged. The content of chlorophyll in decreased in the aged leaves13. Therefore, the photochemical potential of these leaves reduced. The majority of chloroplasts in these aged cells have disintegrated to provide the recycled nutrients for plant reproduction13,14. The changes of leaf senescence degree and photosynthetic capacity were gradual.

The tender leaves on the upper part of the I. rubescens stem not only possessed strong photosynthetic capacity but could also be used as a traditional Chinese medicine to increase economic performance. The aged leaves on the lower part of the stem possessed both weak photosynthetic capacity and inferior pharmacodynamics15. Pruning wilting leaves could reduce the losses of carbon and water12. Targeted pruning during the cultivation and management of I. rubescens could prompt the formation of tender leaves. Thus, the photosynthetic efficiency of the plant would be increased and the productivity of rabdosia rubescentis herba would also be enhanced.

Conclusion

The I. rubescens leaves on the upper part of the stem possessed a similar photosynthetic capacity with that of the mature leaves. The leaves on the middle part of the stem were the main site of photosynthesis. The photosynthetic capacity of the leaves decreased with decreasing leaf position on the lower part of the stem. The leaves at the lower leaf positions still possessed photosynthetic capacity, although more than half of the leaves were withered.

Materials and methods

Instruments

A Li-6400 Photosynthesis system (made in LI-6400 Inc., Lincoln, NE, USA) and a PAM-2500 portable chlorophyll fluorescence apparatus (made in Walz, Würzburg, Germany) were used in this study.

Materials

The applied plant materials were identified as I. rubescens by JIAN Zaiyou, who was an expert in botany. The voucher specimens (02111443) were deposited in the publicly available herbarium at the Henan Institute of Science and Technology. I. rubescens is not an endangered or protected species in China. It was permitted by the local government to collect and study the I. rubescens plants as experimental materials. The methods for the collection of plant materials and the performance of experimental research on I. rubescens plants complied with the national guidelines of China. More than 1000 seeds of I. rubescens were collected from the cultivated plants at the experimental site in Xinxiang, Henan Province, China, in October 2021. These seeds of I. rubescens were sown at the experimental site in Xinxiang, Henan Province, China, in March 2022. These I. rubescens seeds germinated and developed into plants in the same year. Approximately 100 I. rubescens plants were transplanted to a sunny experimental site in March 2023. These I. rubescens plants were 40–80 cm in height in May 2024. There were 2–8 branches on the lower I. rubescens stems. There were 5–12 pairs of leaves on the I. rubescens stems.

Methods

A total of 6 I. rubescens plants were randomly selected for sampling on May 16, 2024. A stem was randomly selected from each plant to study the photosynthetic characteristics of I. rubescens leaves. The first pair of leaves with recently opened blades on the upper part of each I. rubescens stem were designated as the No. 1 leaf position (of which the area was larger than 0.5 cm2). The area of the No. 1 leaf was approximately half the area of a mature leaf on the middle part of the I. rubescens stem. There were young leaves above the No. 1 leaf in which the blades presented at 30–60° angles. The area of these young leaves was less than 1 cm2. The withered portion of each of the lowest pair of leaves represented approximately half of the entire leaf area. There were 1–3 withered areas on the leaves above the lowest pair of leaves.

All of the data were measured on the middle parts of leaf blades.

The photosynthetic rate of a leaf on each leaf position was determined with a Li-6400 photosynthesis system. The light intensity, CO2 density, temperature and flow rate of gas in the leaf chamber were set at 1000 µmol m− 2 s− 1, 450 µmol mol− 1, 30 °C and 500 µmol s− 1, respectively, during the measurement of photosynthetic rates.

The rapid light curve of chlorophyll fluorescence of a leaf on each leaf position was determined with a PAM-2500 portable chlorophyll fluorescence apparatus. The leaves were subjected to dark adaptation for 30 min before the determination of chlorophyll fluorescence light curves.

The light response curves of the No. 1, No. 3, No. 5 and No. 8 leaves on a I. rubescens stem were also determined with a Li-6400 photosynthesis system (continued from May 16 to 17 at the same conditions). The temperature, CO2 density and flow rate of gas in the leaf chamber were set at 30 °C, 450 µmol mol− 1, and 500 µmol/s, respectively, when the light response curves of the leaves were determined. The light intensities in the leaf chamber at each stage of the light response curve were set at 2500, 2200, 2000, 800, 1600, 1400, 1200, 1000, 800, 600, 400, 200, 150, 50, 20 and 10 µmol m− 2 s− 1.

Data analysis

These light response curves were fitted with a modified rectangular hyperbola model16,17. The modified rectangular hyperbola model is shown as below.

$${\rm PSR}={\rm E}^{*}(1-{\rm M}^{*}{\rm LI})^{*}({\rm LI}-{\rm LCP})/(1+{\rm N}^{*}{\rm LI})$$

The PSR is the net photosynthetic rate, LI is the value of light intensity in the leaf chamber, LCP is the light compensation point, E is the apparent quantum yield, and M and N are parameters in the model. The dark respiration rate (DRR) was calculated as E*LCP. The light saturation point (LSP) was calculated according to the following formula:

$${\rm LSP}=((({\rm M}+{\rm N})^{*}(1+{\rm N}^{*}{\rm LCP})/{\rm M})^{1/2})-1)/{\rm N}$$

The maximum photosynthetic rate (MPSR, the net photosynthetic rate at the light saturation point) was calculated according to the following formula:

$${\rm MPSR}={\rm E}^{*}(1-{\rm M}^{*} {\rm LSP})^{*}({\rm LSP} -{\rm LCP})/(1+N^{*}{\rm LSP})$$

The rapid light curve of chlorophyll fluorescence was automatically fitted by the apparatus according to the model of Eilers and Peeters18. The model of Eilers and Peeters is shown below.

$${\rm ETR}={\rm FI}/({\rm a}^{*}{\rm FI}^{2}+{\rm b}^{*}{\rm FI}+{\rm c}).$$

The ETR is the electron transport rate in photosynthetic system II, FI is the fluorescence intensity of the leaf under illumination, and a, b and c are parameters in the model.

The minimum saturation of the light intensity (Ik) was calculated according to the following formula:

$${\rm Ik}=({\rm c/a})^{1/2}.$$

The maximum electron transport rate (ETRmax) was calculated according to the following formula:

$${\rm ETRmax}={\rm Ik}/({\rm a}^{*}{\rm Ik}^{2}+{\rm b}^{*}{\rm Ik}+{\rm c})$$

All data were analysed with Statistical Product and Service Solutions (SPSS, International Business Machines Corporation, USA).