Abstract
Kobresia pygmaea Clarke is mainly distributed in the alpine region from 3800 to 4500 m elevation on the Qinghai-Tibet Plateau. It is excellent in low-temperature tolerance and trampling resistance and plays an important role in maintaining the stability of regional ecological environments. Mainly based on the chlorophyll fluorescence imaging technique, using grass sods blocks with a size of 20 × 20 cm2 collected from K. pygmaea forb meadow, simulated a low-temperature treatment in cultural room with 8 °C/4 °C (day/night) at diurnal photoperiod, we analyzed the effects of low temperature on PSII photochemical efficiency and non-photochemical quenching, as well as the interaction between low temperature and light intensity. The results showed that low temperature could significantly decrease the maximum quantum efficiency of PSII photochemistry (Fv/Fm) and inhibit the leaf growth of K. pygmaea. The light response curve of PSII relative linear electron transport rate was reduced by low temperature, while it recovered to almost normal level after two days of removing adversity. Further experiments under steady-state actinic light confirmed that light intensity was the main factor affecting the PSII photochemical efficiency, and its reduction caused by the low temperature showed a certain degree of reversibility. The non-photochemical quenching (NPQ) was significantly affected by light intensity, and its fast and slow components (NPQf and NPQs) in NPQ accounted for 36% and 64% of NPQ, respectively. The decrease in NPQf and corresponding increase in NPQs slowly recovered after relieving from low-temperature condition. The relative inhibition of photosynthesis (Pinh) was significantly enhanced by low temperature and reduced after removal of adversity. Strong light intensity could intensify the adverse effect of low temperature on PSII operating efficiency Fq′/Fm′ and efficiency factor Fq′/Fv′, as well as NPQ. In conclusion, low temperature was one of the main reasons limiting the photosynthetic performance of K. pygmaea on the Qinghai-Tibet Plateau and could aggravate photosynthetic photoinhibition. The adversity of low temperature frequently occurring in the plant growing season on the Qinghai-Tibet Plateau was constituted of the main factor of the low productivity level in alpine meadow.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
1 Introduction
The Qinghai-Tibet Plateau is the highest plateau in the world with an average altitude of more than 4000 m and is known as "the roof of the world" and "the third pole" (Chen et al. 2019). The region of plateau frequently presents a harsh and extreme climate with low temperature, strong winds and high solar radiation during the plant growing season. For this reason, alpine plants are often exposed to abiotic stress (Miehe et al. 2019). Kobresia pygmaea C. B. Clarke, an herbaceous perennial of Cyperaceae with dense rhizomes and strong vitality, is a constructive species in the alpine meadow belt of the Qinghai-Tibet Plateau and Himalayas (Zhou 2001). As an important dominant species in the alpine meadow, the alpine species of K. pygmaea has high nutritional value and occupies a large proportion in the available grassland in the region of the Qinghai-Tibet Plateau (Li et al. 2010). Due to its excellent characteristics of resistance to low temperature, drought, trampling and soil erosion (Wang et al. 2008), it is of great importance to maintain regional ecological environment stability.
Alpine K. pygmaea meadow is mainly distributed at an altitude of 3800 to 4500 m, and its distribution ranges from a low slope of 3000 m to an upland of 5960 m, indicating its successful adaptation to alpine habitats (Zhou 2001). Low temperature is a typical abiotic stress on the Qinghai-Tibet Plateau, especially in early spring and late autumn. Cold stress has influenced plant growth and geographical distribution, which can not only limit the growth and development of plants but also affect their yield and quality of forage (Tesso 2013; Valizadeh-Kamran et al. 2017). Therefore, the productivity level or primary productivity of meadows is very low, restricting the sustainable development of animal husbandry on the plateau. Moreover, natural disasters such as strong winds and snowfall are frequent happened. Because of extreme weather and overgrazing, the degradation of grassland is serious and the secondary bare land (named the black soil beach by local people) is often formed. However, due to the limitations of the unique habitat of the plateau and the configuration of dwarf plants, early much of the work is only focused on its distribution pattern, breeding strategies, phenological characteristics, and so on (Li et al. 2003; Sun et al. 2007), there is little work on the photosynthetic physiological processes and their adaptability to harsh climates.
Stronger solar radiation reaches the Earth’s surface due to the lower air pressure and higher atmospheric transparency in the region of the Qinghai-Tibet Plateau (Peng et al. 2015). Generally, except for driving photosynthesis, most of the solar light energy absorbed by the photosynthetic apparatus is in surplus (Xu 2002); in this case, the extra light energy must be dissipated safely through an effective approach (Tikkanen et al. 2013; Yamori et al. 2014), avoiding the potential damage of excess excitation energy to the photosynthetic apparatus. Savitch and his coworker (2009) researched the effects of low-temperature stress on excitation energy partitioning and photoprotection using C4 plant maize sensitized to cold stress and found that the fraction utilized by PSII photochemistry was twofold lower, whereas the proportion of thermally dissipated energy was only 30% higher in cold-stressed plants than in control plants. However, it is not clear whether the photoinhibition degree of the photosynthetic apparatus of alpine plants will be enhanced (synergistic effects), or whether resistance to another stress will be increased (cross-tolerance) after a period of exposure to low temperature. The hypotheses of this paper are as follows: (1) low temperature can inhibit the growth of K. pygmaea and reduce the PSII photochemical quantum efficiency in leaves; (2) after relieving low-temperature condition, plant growth can recover to its normal status; and (3) low-temperature adversity can accelerate the degree of photoinhibition induced by strong solar light.
The Qinghai-Tibet Plateau, as a sensitive and driving region of global change (Shrestha et al. 2012), is extremely prominent in its ecosystem degradation, species extinction and deterioration of natural environments (Yu and Lu 2011). Therefore, it is particularly important to reveal the physiological and ecological adaptation mechanisms of native species of K. pygmaea to extreme adversity and the potential degradation crisis of the alpine meadow ecosystem. Based on a chlorophyll fluorescence image analysis system (CF imager), we studied the photosystem II (PSII) photochemical efficiency and non-photochemical energy distribution characteristics impacted by simulating low-temperature adversity and discussed the interaction effect between low temperature and light intensity on alpine species of K. pygmaea.
2 Materials and methods
Plant material and sample plots
– Plant material was taken from a typical K. pygmaea forb meadow near the Alpine Grassland Ecosystem Research Station of the Source Region of Three Rivers, Qinghai University and Tsinghua University. The station is located in Zhenqin Town, Chengduo County, Yushu Prefecture, Qinghai Province, China. Its geographical coordinates are 33°N and 97°E, with an altitude of 4270 m. The average annual temperature is − 5.6 to 3.8 °C, and the average annual precipitation is 562.2 mm. Precipitation is mainly distributed from June to September during the plant growing season, accounting for approximately 75% of the annual precipitation. In July and August 2019, the average temperature at station is 7.4 °C; the average maximum and minimum temperatures are 14 °C and 1.8 °C, respectively. There is no absolute frost-free period during plant growing season. The climate is a typical plateau continental climate with a longer cold season and shorter warm season, and there are almost no frost-free periods. The soil is alpine meadow soil (Mat Crygelic Cambisols) with rich humus content but poor decomposition, which has low effective fertility. The soil pH was 6.92, organic matter content was 2.36%, available nitrogen was 14.0 mg L−1, ammonia nitrogen was 5.1 mg L−1, nitrate nitrogen was 8.9 mg L−1, available phosphorus was 7.0 mg L−1, and available potassium was 76.5 mg L−1.
K. pygmaea C. B. Clarke is the dominant herbage in the K. pygmaea forb meadow (Fig. 1); the subdominant species are: Kobresia humilis (C. A. Mey.) Serg., Stipa aliena Keng, Poa poiphagorum Bor, Polygonum viviparum L., Potentilla. nivea L., Anaphalis lactea Maxim., Thalictrum alpinum L., etc.; associated plants include Festuca ovina L., Elymus nutans Griseb., Aster tsarungensis (Griers.) Ling, Saussurea superba Anthony, Taraxacum mongolicum Hand. –Mazz; miscellaneous and poisonous weeds include Oxytropis kansuensis Bunge, Gentiana sino-ornata Balf., Stellera chamaejasme L., Euphorbia sieboldiana Morr. et Decne., Pedicularis kansuensis Maxim., Lamiophlomis rotata (Benth.) Kudo, etc. There was no grazing utilization by sheep and yaks in the alpine K. pygmaea forb meadow near the site of the station during the period of field experiments in the warm season.
Experimental design
– The experiments were conducted from June 6 to 15, 2019 (forage green up period), and August 5 to 15, 2019 (exuberance period). This paper mainly analyzed the results from the experiment conducted in the exuberance period. We chose 3 flat fields in K. pygmaea forb meadow and dug up 2 blocks of grass sods with a size of 20 × 20 cm2 and depth of approximately 15 cm in each field. After sealing 4 edges of grass block with plastic bandages, all turfs were set outdoors in natural light for 1 day; during this period, we carefully removed other plant species, withered grass and dopants from the grass cortex and kept the K. pygmaea in its natural growth state. On August 7, the aboveground biomass was mowed near above the yellow leaf sheath to ensure that the stubble height was consistent; a piece of turf block was picked from each field site and placed in an artificial low-temperature climate chamber, setting a low-temperature group (Low Temp). The control group (Control) was placed under an artificial light source in the cultivation rack of a semi-automatic illumination culture room, adjusting to a light intensity similar to that in an artificial climate chamber (Table 1). The light intensity in the climate chamber was 600 µmol photons m−2 s−1, the air temperature was 8 °C during the day and 4 °C at night with a 12-h photoperiod. The intensity of the artificial light source in the culture room was 600 ± 20 µmol photons m−2 s−1, and the air temperature was 23 ± 1 °C during the day and 18 ± 2 °C at night with a 12-h photoperiod.
Sufficient water was sprayed in the morning and evening to keep the meadow moist in the control group; a small amount of water was sprayed on the turf block in the low-temperature group every day to maintain a moisture level similar to that of the control group. Chlorophyll fluorescence parameters and leaf growth status were measured during the low-temperature treatment and the recovery phase. On the 7th day, the low-temperature group was transferred to a culture room irradiated with artificial light for 2 days of restoration. After completing all tests on August 15, the soil water contents were measured, and soil samples were taken from the inside of turf blocks using a cutting ring. The relative water contents of the soil in the low-temperature group and control group were 25.20 ± 1.44% and 24.75 ± 1.25%, respectively, showing no significant difference.
Determination of chlorophyll fluorescence parameters. Chlorophyll fluorescence images were determined nearly every two days from the 3rd day. The chlorophyll fluorescence parameters were measured by using a chlorophyll fluorescence image analyzer (chlorophyll fluorescence imager, CF imager: Technological Ltd, UK) with a light-emitting diode (LED) red and blue light source. The turf block was put in the sample chamber, and the focal length of the charge-coupled device camera (CCD camera) was adjusted. The indoor light was turned off, the built-in protocol was run for experimental purposes, and fluorescence images of plants were measured chlorophylafter 15 min of dark adaptation. During measurement, the indoor temperature was maintained at 20 ± 1.5℃, and the air humidity was kept constant.
Measurement protocol.
Rapid light response curve: The minimum chlorophyll fluorescence yield (Fo) in the open state of the PSII reaction center and the maximum chlorophyll fluorescence yield (Fm) in the closed state were determined after accurate dark adaptation for 15 min. The steady-state actinic light was turned on, and fluorescence parameters were measured with 25, 50, 100, 150, 200, 300, 500, 800, 1000, 1200, 1500, 1800 µmol photons m−2 s−1 successively. To determine every fluorescence parameter at light intensities of 25, 50, 100, 150 µmol photons m−2 s−1, stable illumination lasted for 2 min. After light intensities higher than 200 µmol photons m−2 s−1, each light intensity was stabilized for 1 min. The maximum fluorescence yield (Fm′) and the steady-state fluorescence yield (Fs) were measured for each point of light intensity. The saturation pulse light in the determination of Fm and Fm′ was 6840 µmol photons m−2 s−1 and lasted for 60 ms.
Steady state chlorophyll fluorescence parameterssec: After dark adaptation for 15 min, the Fo and the Fm were determined. Successively turning on 400 µmol photons m−2 s−1 low steady-state actinic light and 1500 µmol photons m−2 s−1 high steady-state actinic light, the Fm′ and the Fs were determined for 5, 10 and 15 min of continuous illumination with each light intensity. The actinic light was turned off, and the Fo and Fmr were measured at accurate dark recovery times of 5, 10 and 15 min. The saturation pulse light intensity in the determination of Fm, Fm′ and Fmr was 6840 µmol photons m−2 s−1 and lasted for 60 ms.
Leaf localization and image analysis. The paired measurement data of the 6th day and 8th day (2 days after moving to the culture room) were used for analysis in this paper. In the graphic edit frame of the fluorescent image, the linear small leaves of K. pygmaea were located and segmented and then resolved into data values. Chlorophyll fluorescence parameter data of 30 leaves were obtained as one group for each tested turf block. More details on the specific operational steps can be found in the CF imager manual (http://www.technologica.co.uk/).
PSII photochemical efficiency and non-photochemical dissipation: The PSII maximum photochemical quantum efficiency (Fv/Fm) of the dark-adapted leaves was calculated with the minimum chlorophyll fluorescence yield (Fo) and maximum chlorophyll fluorescence yield (Fm) after accurate dark adaptation for 15 min, where Fv = Fm − Fo. PSII maximum efficiency (Fv′/Fm′) and PSII operational efficiency (Fq′/Fm′) were calculated by the following equations (Genty et al. 1989): Fv′/Fm′ = (Fm′ − Fo′)/Fm′; Fq′/Fm′ = (Fm′ − Fs)/Fm′. The PSII efficiency factor (Fq′/Fv′) and the non-photochemical quenching (NPQ) were calculated according to the formulas of Bilger and Björkman (1990): Fq′/Fv′ = (Fm′ − Fs)/(Fm′ − Fo′); NPQ = Fm/Fm′ − 1. Fo′ in the above formulas was estimated by the empirical formulas of Oxborough and Baker (1997): Fo′ = Fo/(Fv/Fm + Fo/Fm′); among them, Fo and Fm were the chlorophyll fluorescence yield after accurate dark-adaption for 15 min.
Photosynthetic photoinhibition and components of non-photochemical quenching: The components of fast and slow relaxation of non-photochemical quenching (NPQf and NPQs) were calculated by referring to the formulas of Maxwell and Johnson (2000): NPQs = (Fm − Fmr)/Fmr; NPQf = (Fm/Fm′) − (Fm/Fmr). In the above formula, Fm is the fluorescence yield of accurate dark adaptation for 15 min; Fmr selected the fluorescence yield from accurate dark recovery for 5, 10 and 15 min. The relative photoinhibition of photosynthesis was estimated by the method of Dodd and coworkers (Dodd et al. 1998): Pinh = 100 × (1 − (Fvr/Fmr)/(Fv/Fm)), of which Fv = Fm − Fo, Fvr = Fmr − For.
Leaf growth and relative growth rate
On the 4th and 7th days after low-temperature treatment and the 9th day after normal temperature was restored for 2 days, the leaf length of the plants was measured by a steel ruler. The longest leaves were selected from each plant for determination, and 10 plants were measured from each sod block with a total of 30 samples. The average relative growth rate of leaf length was calculated as: R = (LnLm − LnLn)/(m − n), where Lm and Ln represent leaf length at day m and day n, respectively. Ln, natural logarithm.
Statistical analysis
SPSS 16.0 software was used for statistical analysis. The independent sample t test was used to compare the significance of differences between the low-temperature and control groups. The differences between different treatments and different recovery times were analyzed by using multivariate ANOVA; the minimum significant difference method (LSD) was used for multiple comparisons, and the significance level was set as α = 0.05. The interaction effects between light intensity and low temperature were judged by general linear model univariate variance analysis (GLM univariation). Using Microsoft Excel software to collate data and make graphs, the data in the graph were represented by the mean, and the vertical bar represents the standard deviation (SD).
3 Results
The inhibition of low-temperature adversity on the leaf growth of K. pygmaea
– After mowing, the leaf growth of the control group was significantly greater than that of the plants treated with low temperature in each corresponding period (p < 0.001), of which leaf length had distinct differences on day 4, day 7 and day 9 (p < 0.05) (Fig. 2). There was no significant difference between day 4 and day 7 (p > 0.05); however, after 2 days of normal temperature growth resumption (day 9), the leaf length was significantly higher than that on the day 4 and day 7 (p < 0.05).
Figure 3 shows the changes in the average relative growth rate (R) of leaf length in different time periods after mowing. The results indicated that compared with the control group, low temperature caused the R value to be significantly reduced during days 0 to 3 and days 3 to 6 (p < 0.001). As it grew, the R of leaves decreased significantly in turn (p < 0.05). After moving the low-temperature group to the normal temperature culture room, its R increased significantly, showing no significant difference from the R value on days 0 to 3 (p > 0.05).
Response of the PSII photochemical efficiency to low-temperature adversity
– The relative linear electron transport rate (rETR) of the PSII reaction center increased with increasing actinic light intensity (Fig. 4). The rETR increased rapidly when the light intensity was less than 500 µmol photons m−2 s−1 but only slightly increased when the light intensity was more than 800 µmol photons m−2 s−1. There were obvious differences in the light response curve of rETR between groups or treatments; in the middle to high light intensity range, Control 8 Days > Control 6 Days > Low Temp and Recovery 6 + 2 Days > Low Temp 6 Days.
In view of the short balance time (1 and 2 min) at each light intensity in measuring the light response curve, the PSII photochemical efficiency of K. pygmaea leaves at steady-state light intensities of 400 and 1500 µmol photons m−2 s−1 was further determined. Figure 5 shows that under continuous irradiation with 400 µmol photons m−2 s−1 low light intensity for 10 min, the PSII operating efficiency (Fq′/Fm′), the maximum efficiency (Fv′/Fm′) and the efficiency factor (Fq′/Fv′) were all significantly higher than those under high light intensity (p < 0.01 and p < 0.001). Except for Fq′/Fv′, the values of Fq′/Fm′ and Fv′/Fm′ under different treatments showed a similar variation trend; that is, the Control 8 Days group was higher, while the Low Temp 6 Days group was lower; meanwhile, the variation trend from the two steady-state light intensities was relatively consistent.
PSII non-photochemical quenching and component analysis
– The light response curve of PSII non-photochemical quenching (NPQ) also showed an increasing process of rapid to slow (Fig. 6). In the control group (Control 6 Days and Control 8 Days), the NPQ increased rapidly when the light intensity was less than 500 µmol photons m−2 s−1, but only slightly increased when the light intensity was more than 800 µmol photons m−2 s−1. The low-temperature group increased rapidly before 200 µmol photons m−2 s−1 and then increased slowly, while it was the demarcation point of the light response curve for recuperated plants (Low Temp and Recovery 6 + 2 Days) near approximately 300 µmol photons m−2 s−1. Within the range of medium to high light intensity, the difference of NPQ between different treatments was more obvious, that was: Control 8 Days > Control 6 Days > Low Temp and Recovery 6 + 2 Days > Low Temp 6 Days.
The determination of NPQ under steady-state light intensity showed that there was an extremely significant difference between low and high light intensities (p < 0.001). At 1500 µmol photons m−2 s−1 high light intensity, the relative changes of each treated group were consistent with the order of the light response curves and showed significant differences (p < 0.05) except for Control 6 Days and Control 8 Days. At two actinic lights, the recuperated plants (Low Temp and Recovery 6 + 2 Days) were significantly higher than those of the low-temperature group (p < 0.05) (Fig. 7a).
After continuous low and high steady-state irradiation for a total of 30 min and then accurate dark relaxation restoration for 15 min, the fast component (NPQf) and the slow component (NPQs) of PSII non-photochemical quenching were further analyzed. The results showed that NPQf decreased slightly and NPQs increased slightly with increasing dark relaxation time; however, there was no significant difference among the three time points of dark relaxation for low-temperature-treated and recovered plants (Fig. 7b, c). On average, NPQf and NPQs accounted for 36% and 64% of PSII non-photochemical quenching, respectively; among them, NPQf and NPQs accounted for 30% and 70% of NPQ in the low-temperature group, 39% and 61% for recuperated plants; 36% and 64% for control group on 6th day and 38% and 62% on 8th day.
Influence of low-temperature adversity on photoinhibition of photosynthesis
– Figure 8a shows that the maximum quantum efficiency of PSII photochemistry Fv/Fm was higher in the control group (Control 6 Days and Control 8 Days) and significantly lower in the low-temperature group; however, after the plants were restored in the normal temperature culture room for 2 days, the Fv/Fm value (Low Temp and Recovery 6 + 2 Days) was significantly higher than that of the low-temperature group and significantly lower in the control group (p < 0.05). The relative inhibition of photosynthesis (Pinh) was higher in the low-temperature group; with removal of low-temperature adversity, the Pinh decreased and was significantly lower than that of the control at 6 days and in the low-temperature group (p < 0.05) (Fig. 8b).
4 Discussion
K. pygmaea is a kind of hardy plant in alpine meadows on the Qinghai-Tibet Plateau
– When environmental temperature is below the lower limit of the optimal growth temperature of plants, it can cause an adversity of low temperature, which varies with plant species and growth period (Larcher 1980; Kuss 1986). Low-temperature stress can result in a series of changes from molecules to cells, tissues to phenotypes in plants (Moellering et al. 2010; Liu et al. 2019), which are often expressed as acute or chronic abiotic environmental stresses. Physiological responses mainly include changes in cell membrane structure (Lu et al. 2019), enzyme function (Pearce 2001; Theocharis et al. 2012), soluble sugars and proteins, osmotic regulatory substances such as proline (Meena et al. 2019), stomatal conductance and photosynthesis (Foyer et al. 2002; Shi et al. 2016). It is generally recognized that the adversity of low temperature is one of the important environmental limiting factors affecting plant growth, development and geographical distribution; the effects on leaves are usually expressed as a decrease in growth rate and photosynthetic rate.
The alpine species of K. pygmaea is a typical cold mesophyte with a dense rhizome tufted. As an herbaceous perennial, the aboveground is lower, and its height is only 1 to 3 cm; the leaf shape is in linear, approximately 1 mm wide and 3 cm long. Extremely developed roots often form a dense interlayer with soil, and its thickness can be more than 10 cm (Li et al. 2010). Owing to the cold climate in the Qinghai-Tibet Plateau region, K. pygmaea starts to turn green in mid-early May and becomes yellow wilt in early October. The growth and expansion of leaves are slow, especially from May to early July, and the productivity of alpine meadows is maintained at a very low level. The dwarf shape of plants and linear leaves can effectively reduce the wind area; in addition, they also have the advantages of heat preservation, water retention and transpiration reduction, avoiding damage from physiological drought and day and night temperature differences (Jiang and Jia 1999). At the same time, the leaf stomata distributed on the abaxial surface with higher density and more sunken also reflected the adaptability to the low pressure and cold climate (Zhang et al. 2006; Zhao et al. 2007). The morphology and life characteristics of K. pygmaea exhibit adaptation to extreme adversity, and it is also the product shaped by alpine harsh habitats (Yamori et al. 2010). During the field experiments, we always found that the blade tip of K. pygmaea gradually emerges yellow and there are signs of drying up and dying; it has been considered mainly induced by extremely cold adversity such as frost at night (Li et al. 2003; Sun et al. 2007). Low temperature seems to constitute the main adversity restricting plant growth and development in the Qinghai-Tibet Plateau region, and frost frequently appears in the plant growing season and is the key factor accelerating leaf apoptosis.
The alpine K. pygmaea meadow is a unique vegetation type in Northwest China, and it has the widest distribution and largest area on the Qinghai-Tibet Plateau; its distribution is co-limited by low rainfall, a short growing season and livestock grazing (Li et al. 2010; Miehe et al. 2019). Early studies have revealed that in the alpine region where the average annual temperature is − 0.1 to − 1.6 °C and the accumulated temperature greater than and/or equal to 5 °C is only 543.1~886.9 °C, K. pygmaea can still complete its growth and development well, demonstrating its strong ability of cold tolerance (Sun et al. 2007). We simulated 8℃/4℃ (day/night) of the low-temperature treatment lasting for 5 days, compared with normal control growing condition of 23 °C/18 °C, and determined the influence of low-temperature adversity on the growth of leaves length. Similar to a typical finite growth process of plant organs, plants, or even populations (Hunt 1982), the leaf length of K. pygmaea increased, and its average relative growth rate decreased gradually after mowing. The results indicated that the adversity of low temperature restrained leaf length growth and reduced the relative growth rate. However, this suppression of growth could partially or even fully recover after relief from low-temperature stress, meaning that the PSII photosynthetic function of K. pygmaea still maintained activity and that the potential growth trend was preserved. Therefore, the photosynthetic and physiological processes of this typical native species had strong adaptability and/or tolerance to low-temperature adversity.
The adversity of low temperature restricts the activity performance of the photosynthetic apparatus of K. pygmaea leaves
– The PSII photochemical quantum efficiency of the photosynthetic apparatus is influenced by many biotic and abiotic factors and is very sensitive to variations in external environmental factors (Baker 2008). Fv′/Fm′ is the PSII maximum efficiency, which represents the maximum quantum efficiency of the PSII opening trap in light-adapting leaves. A decrease in Fv′/Fm′ means the deficiency of the energy capture traps. The PSII operating efficiency Fq′/Fm′ is the actual primary light energy capture efficiency when the PSII reaction center is partially closed under actinic light, which is directly related to the linear electron transfer flux through the PSII reaction center; furthermore, Fq′/Fm′, also known as the PSII actual photochemical quantum efficiency (ΦPSII), can reflect the actual operating efficiency of the PSII absorption light energy used in the reduction of the primary quinone electron acceptor QA. Fq′/Fm′ is a fast reflection of the efficiency of PSII reaction centers under varied light intensities or other environmental conditions, and its reduction will not only limit the efficiency of light energy conversion in plants but also affect the demand for the accumulation of homogenization forces (ATP and NADPH) in the carbon assimilation process of the dark reaction (Xu 2002). In addition to driving the photochemical reactions of PSII reaction centers, excessive energy absorbed by the photosynthetic apparatus needs to be dissipated in safe ways. Non-radiative energy dissipation in the PSII reaction center is the main way to eliminate excessive excitation energy; it can effectively dissipate excited energy accumulation in the PSII core and reduce the probability of photoinhibition or even photodamage (Bilger and Björkman 1990; Govindjee 2002; Niyogi and Truong 2013). The non-photochemical quenching, NPQ, is thought to protect the photosynthetic apparatus under excessive light conditions via controlled dissipation of absorbed light energy as heat (Ruban 2016). Studies have shown that NPQ is susceptible to a variety of environmental factors; under the condition of adversity, the closure of leaf stomata can block the electron flow from CO2 to O2, resulting in a decrease in the energy conversion efficiency of the PSII reaction center and the accumulation of ATP and NADPH in the carbon assimilation process of the dark reaction (Sáez et al. 2013), which indicates that the change in energy status in chloroplasts is the key factor in determining the non-photochemical quenching process. In our studies, Fv′/Fm′, Fq′/Fm′ and NPQ all decreased under low-temperature adversity; obviously, low temperature inhibited the PSII photochemical efficiency and the non-radiative heat dissipation capacity. It was clear that the light intensity played an important role in determining the photochemical efficiency of the PSII reaction center; however, the adversity of low temperature also led to a reduction in the PSII photochemical efficiency, and it could be improved to some extent after restoration to normal temperature growing conditions. As a result, the photosynthetic apparatus would accumulate a large amount of excessive light energy in the low-temperature state due to the reduction of Fv′/Fm′, Fq′/Fm′ and NPQ, which easily induced photoinhibition and even photodamage in K. pygmaea when exposed to high light intensity. The relative electron transfer rate, rETR, was also sensitive to growing temperature. The light response curves showed that the photosynthetic electron transport capacity was limited by low temperature, while two days of recovery after removal of adversity could increase the rETR.
NPQ is linearly related to heat dissipation, and it is known as an important mechanism by which plants protect their photosynthetic apparatus in a rapidly varying light environment (Lima Neto et al. 2014). Recent studies suggest that most NPQ occurs in the light-harvesting antenna complex of PSII (LHCII), but the core antenna complexes CP43 and/or CP47 are also likely to be additional PsbS protein-dependent quenching sites in the PSII core (Nicol et al. 2019). The reduction in NPQ caused by cold stress is related to LHCII damage and the destruction of CP43 and/or CP47 proteins (Niyogi and Truong 2013; Tikkanen et al. 2013). When NPQ is increased under high light irradiation, most of the PSII reaction center on the thylakoid membrane of the matrix is in an inactive status; inactivated PSII can participate in the dissipation of excessive excitation energy together with light-harvesting antenna pigments, protecting the functional reaction center from injury (Murchie and Niyogi 2011). Usually, dark relaxation within a few minutes is considered to be a photoprotective process, which mainly depends on the presence of a low pH in the lumen of the thylakoid (ΔpH) and partly depends on the change in the light absorption cross-section and state transition of excited energy overflowing (Quick and Stitt 1989). For most cases, the relaxation over a time scale of a few minutes following the cessation of irradiation can be regarded as photoprotective processes (Maxwell and Johnson 2000); however, a long-term dark relaxation (lasting up to a few hours) is often seen as photoinhibition, which is associated with the presence of zeaxanthin and thought to occur in the LHCII complex (Lima Neto et al. 2014). It has also been confirmed that photoinhibition itself is involved in both the protection and injury mechanisms of the PSII reaction center (Xu 2002). The NPQ was obviously affected by light intensity; however, the low-temperature growing conditions also influenced the value of NPQ. Six days of low-temperature adversity caused a relative decrease in NPQ, combined with a reduction in the PSII maximum photochemical quantum efficiency Fv/Fm and an enhancement in the relative photoinhibition of photosynthesis Pinh, which indicated an inhibition of the activity performance of the photosynthetic apparatus in the leaves of K. pygmaea. The values of NPQ and Fv/Fm showed some recovery or even more after 2 days of resuming in a normal temperature culture room, and Pinh also showed lower values than those after treatment in low-temperature adversity for 6 days. The injury to LHCII and destruction to CP43 and/or CP47 proteins caused by low-temperature adversity still needed a slow recovery process to reach the control level.
Furthermore, we divided the NPQ into two components, fast and slow relaxing quenching (NPQf and NPQs), by referring to Maxwell and Johnson (2000). The results were slightly different from those of alpine plants Anisodus tanguticus and Rheum tanguticum (Shi et al. 2007). Here, the NPQs occupied a main component, accounting for nearly 64% of NPQ on average, which probably resulted from the relatively lower growth light intensity in the culture room and artificial climate chamber. It has been proven that shaded plants of the same species exhibit reduced light use, and an increased likelihood of photoinhibition (Demmig-Adams et al. 1995). Our results demonstrated that the photosynthetic apparatus of K. pygmaea did not dissipate the accumulated excessive excitation energy quickly and effectively, and there was an increased chance of photoinhibition. During 15 min of dark relaxation, the NPQf of the control plants continued to decrease, while NPQs increased, and the variation tendency was relatively steady; it was slightly different from early soil drought trials conducted under the natural field sunlight conditions (Shi et al. 2015), and its recovery of photosynthetic function from the photoinhibition state was slower due to the unperfected development of the photosynthetic apparatus (Demmig-Adams et al. 1995). It should be kept in mind that the proportion of NPQs and NPQf only represented the value determined after 15 min of dark relaxation, providing a relative inter-comparison among the control group and the low-temperature group and its resumption treatment. It cannot be compared with the fully relaxed absolute state, which usually needs 30 min to one hour of dark relaxation (Maxwell and Johnson 2000). The NPQ and its two components, NPQf and NPQs, were relatively lower in the low-temperature group, but NPQf was slightly reduced; after the 2 days of resuming treatment, NPQ recovered slightly and mainly belonged to the increase in NPQf. The fast component NPQf in NPQ was more sensitive to environmental variation (Shi et al. 2015); the decrease in photosynthetic function caused by low temperature was not permanent, but its recovery still required a longer time. Our results demonstrated that low-temperature adversity could aggravate the degree of photoinhibition or photodamage of photosynthesis, being a main environmental factor restricting plant growth and development; however, it did not constitute the completely irreversible damage to the photosynthesis apparatus of K. pygmaea, showing a strong tolerance to low-temperature stress.
Analysis of the interaction effect between low temperature and high light intensity
– Two or more environmental factors often exhibit inter-effectiveness due to the synergistic effects of their ecological factors and/or the cross-tolerance or cross-adaptation of biological organisms to adversity during the period of acclimation (Shi et al. 2017). Does low-temperature stress exacerbate the effect of strong light intensity on PSII photochemical and non-photochemical quenching abilities in plants? Based on the univariate variance analysis, we found that although both low temperature and strong light intensity could cause a significant reduction in the PSII maximum efficiency Fv′/Fm′, there was no inter-effectiveness on Fv′/Fm′ between light intensity and temperature; that is, the reduction in the maximum quantum efficiency of the PSII opening trap caused by low-temperature stress did not tend to be aggravated by the presence of strong light intensity. PSII operating efficiency Fq′Fm′ and efficiency factor Fq′/Fv′ displayed inter-effectiveness, even though low temperature had no remarkable effect on Fq′/Fv′. The NPQ showed the interaction effect of two stress factors, but its contribution to the total variation (η2) was small (Table 2). The inter-effectiveness was not remarkable when irradiated with light for only 5 and 10 min (the results were not listed), indicating that only long enough of high light illumination could aggravate the adverse impact caused by low-temperature adversity.
Table 2 shows that the contribution of high light intensity to total variation η2 was much greater than that of low-temperature adversity, which further confirmed that light intensity was the main factor influencing the PSII photochemical efficiency and non-photochemical quenching ability in plant leaves. Except for the PSII efficiency factor Fq′/Fv′, the values of η2 in other chlorophyll fluorescence parameters were as follows: high light intensity > low temperature > high light intensity × low temperature, which was completely consistent with early studies on the inter-effectiveness of strong light intensity and soil drought stress in K. pygmaea (Shi et al. 2017). Except for the PSII operating efficiency Fq′/Fm′, the η2 was relatively small, which was also similar to early studies in soil drought trials. The results demonstrated that low-temperature adversity could aggravate the photosynthetic photoinhibition or that the photoinhibition caused by strong light intensity was more obvious in a low-temperature environment. Although the relative contributions of the interaction effect to total variation were all lower, the adverse impacts on physiological processes caused by low temperature and strong light intensity could be exacerbated. Combined with early studies on soil drought (Shi et al. 2015, 2017), we should think if other adverse factors, such as low air pressure, strong wind, hail attack coupling, or disastrous and extreme weather occurring frequently due to the global changes, can the activity performance of the photosynthetic apparatus of K. pygmaea still withstand harsh environmental adversity? remaining a matter of great concern.
In conclusion, the alpine plant K. pygmaea is a typical cold mesophyte, but low-temperature stress could still reduce the photosynthetic performance of leaves. Low temperature led to a decrease in PSII photosynthetic efficiency and caused an increase in the degree of photosynthetic photoinhibition or even photodamage, playing a main environmental factor restricting plant growth and development. The low temperature at 8 °C day/4 °C night did not constitute a complete irreversible damage to the photosynthetic apparatus, and the photosynthetic function could be partially recuperated after two days of resumption under normal temperature, reflecting the strong tolerance to low-temperature adversity. There was an interaction effect between low temperature and strong light intensity, and the adverse impact of low-temperature adversity could be aggravated by strong light intensity. Therefore, the frequent adversity of low temperature in the plant growing season on the Qinghai-Tibet Plateau is an important factor that affects the effective operation of the photosynthetic function of K. pygmaea and limits the productivity of alpine meadows.
Data Availability
The data presented in this study are available in the article.
References
Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu Rev Plant Biol 59:89–113
Bilger W, Björkman O (1990) Role of the xanthophyll cycle protoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth Res 25:173–185
Chen FH, Fu BJ, Xia J, Wu D, Wu SH, Zhang YL, Sun H, Liu Y, Fang XM, Qin BQ, Li X, Zhang TJ, Liu BY, Dong ZB, Hou SG, Tian LD, Xu BQ, Dong GH, Zheng JY, Yang W, Wang X, Li ZJ, Wang F, Hu ZB, Wang J, Liu JB, Chen JH, Huang W, Hou JC, Cai QF, Long H, Jiang M, Hu YX, Feng XM, Mo XG, Yang XY, Zhang DJ, Wang XH, Yin YH, Liu XC (2019) Major advances in studies of the physical geography and living environment of China during the past 70 years and future prospects. Sci China Earth Sci 62:1665–1701
Demmig-Adams B, Adams WW III, Logan BA, Verhoeven AS (1995) Xanthophyll-cycle-dependent energy dissipation and flexible photosystem II efficiency in plants acclimated to light stress. Aust J Plant Physiol 22:249–260
Dodd IC, Critchley C, Woodall GS, Stewart GR (1998) Photoinhibition in differently coloured juvenile leaves of Syzygium species. J Exp Bot 49:1437–1445
Foyer CH, Vanacker H, Gomez LD, Harbinson J (2002) Regulation of photosynthesis and antioxidant metabolism in maize leaves at optimal and chilling temperatures: review. Plant Physiol Bioch 40:659–668
Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92
Govindjee (2002) A role for a light-harvesting antenna complex of photosystem II in photoprotection. Plant Cell 14:1663–1668
Hunt R (1982) Plant growth analysis: second derivatives and compounded send derivatives of splined plant growth curves. Ann Bot 50:317–328
Jiang GC, Jia XH (1999) Comparison of the microstructures of vegetative organs of three species of Kobresia growing in different altitude. J Henan Univ (nat Sci) (chinese Version) 29:63–68
Kuss FR (1986) A review of major factors influencing plant responses to recreation impacts. Environ Manag 10:637–650
Larcher W (1980) Physiological plant ecology, 2nd edn. Springer, Berlin, pp 18–50
Li XL, Yang YW, Zhang J, Nuzhou YJ (2003) Growth characteristics of Kobresia pygmaea clones in the “black soil beach” with different degradation. Acta Prataculturae Sinca (chinese Version) 12:51–56
Li YK, Lin L, Zhang FW, Liang DY, Wang X, Cao GM (2010) Kobresia pygmaea community—disclimax of alpine meadow zonal vegetation in the pressure of grazing. J Mt Sci (chinese Version) 28:257–265
Lima Neto MC, Lobo AKM, Martins MO, Fontenele AV, Silveira JAG (2014) Dissipation of excess photosynthetic energy contributes to salinity tolerance: A comparative study of salt-tolerant Ricinus communis and salt-sensitive Jatropha curcas. J Plant Physiol 171:23–30
Liu LL, Ji HT, An JP, Shi KJ, Ma JF, Liu B, Tang L, Cao WX, Zhu Y (2019) Response of biomass accumulation in wheat to low-temperature stress at jointing and booting stages. Environ Exp Bot 157:46–57
Lu YZ, Hu YG, Snyderb RL, Kentc ER (2019) Tea leaf’s microstructure and ultrastructure response to low temperature in indicating critical damage temperature. Inf Process Agric 6:247–254
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51:659–668
Meena M, Divyanshu K, Kumar S, Swapnil P, Zehra A, Shukla V, Yadav M, Upadhyay RS (2019) Regulation of L-proline biosynthesis, signal transduction, transport, accumulation and its vital role in plants during variable environmental conditions. Heliyon 5:1–20
Miehe G, Schleuss PM, Seeber E, Babel W, Biermann T, Braendle M, Chen FH, Coners H, Foken T, Gerken T, Graf HF, Guggenberger G, Hafner S, Holzapfel M, Ingrisch J, Kuzyakov Y, Lai ZP, Lehnert L, Wesche K (2019) The Kobresia pygmaea ecosystem of the Tibetan highlands - Origin, functioning and degradation of the world’s largest pastoral alpine ecosystem: Kobresia pastures of Tibet. Sci Total Environ 648:754–771
Moellering ER, Muthan B, Benning C (2010) Freezing tolerance in plants requires lipid remodeling at the outer chloroplast membrane. Science 330:226–228
Murchie EH, Niyogi KK (2011) Manipulation of photoprotection to improve plant photosynthesis. Plant Physiol 155:86–92
Nicol L, Nawrocki WJ, Croce R (2019) Disentangling the sites of non-photochemical quenching in vascular plants. Nat Plants 5: 1177–1183
Niyogi KK, Truong TB (2013) Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenic photosynthesis. Curr Opin Plant Biol 16:307–314
Oxborough K, Baker NR (1997) Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components: calculation of qP and Fv′/Fm′ without measuring Fo′. Photosynth Res 54:135–142
Pearce RS (2001) Plant freezing and damage. Ann Bot Lond 87:417–424
Peng SM, Du QY, Lin AW, Hu B, Xiao K, Xi YL (2015) Observation and estimation of photosynthetically active radiation in Lhasa (Tibetan Plateau). Adv Space Res 55:1604–1612
Quick WP, Stitt M (1989) An examination of factors contribution to non-photochemical quenching of chlorophyll fluorescence in barley leaves. Biochim Biophys Acta 977:287–296
Ruban AV (2016) Nonphotochemical chlorophyll fluorescence quenching: mechanism and effectiveness in protecting plants from photodamage. Plant Physiol 170:1903–1916
Sáez PL, Bravo LA, Latsague MI, Toneatti MJ, Sánchez-Olate M, Ríos DG (2013) Light energy management in micropropagated plants of Castanea sativa, effects of photoinhibition. Plant Sci 201:12–24
Savitch LV, Ivanov AG, Gudynaite-Savitch L, Huner NPA, Simmonds J (2009) Effects of low temperature stress on excitation energy partitioning and photoprotection in Zea mays. Funct Plant Biol 36:37–49
Shi SB, Li HP, Wang XY, Li HM, Han F (2007) Utilization and dissipation of strong solar radiation in two alpine plants, Anisodus tanguticus and Rheum tanguticum. J Plant Ecol (chinese Version) 31:129–137
Shi SB, Li TC, Li M, Liu SZ, Li AD, Kang CZ, Ma JP (2015) Interaction effects analysis of soil-drought and strong light on PSII non-photochemical quenching in Kobresia pygmea leaves. Plant Physiol J (chinese Version) 51:1678–1689
Shi DW, Wei XD, Chen GX (2016) Effects of low temperature on photosynthetic characteristics in the super-high-yield hybrid rice ‘liangyoupeijiu’ at the seedling stage. Genet Mol Res 15:1–10
Shi SB, Li TC, Wang W, De KJ, Xu XY, Wang Q, Ma JP, Li AD, Kang CZ (2017) Interaction effects of soil-drought and strong light on PSII performance of Kobresia pygmea in Qinghai-Tibetan Plateau. Acta Agrestia Sinica (chinese Version) 25:724–731
Shrestha UB, Gautam S, Bawa KS (2012) Widespread climate change in the Himalayas and associated changes in local ecosystems. PLoS ONE 7:1–10
Sun BG, Long RJ, Wang CT (2007) A study on the plant population phenology in Qinghai-Tibet plateau Kobrecia pygmaea meadow. Prataculturae Sci (chinese Version) 24:16–20
Tesso MT (2013) Cold temperature episode at seedling and flowering stages reduces growth and yield components in sorghum. Crop Sci 53:564–574
Theocharis A, Christophe C, Essaid AB (2012) Physiological and molecular changes in plants grown at low temperatures. Planta 235:1091–1105
Tikkanen M, Mekala NR, Aro EM (2013) Photosystem II photoinhibition-repair cycle protects Photosystem I from irreversible damage. Biochim Biophys Acta 1837:210–215
Valizadeh-Kamran R, Toorchi M, Mogadam M, Mohammadi H, Pessarakli M (2017) Effects of freeze and cold stress on certain physiological and biochemical traits in sensitive and tolerant barley (Hordeum vulgare) genotypes. J Plant Nutr 41:102–111
Wang QJ, Li SX, Wang WY, Jing ZC (2008) The despondences of carbon and nitrogen reserves in plants and soils to vegetation cover change on Kobresia pygmaea meadow of Yellow River and Yangtze River source region. Acta Ecol Sinica (chinese Version) 28:885–894
Xu DQ (2002) Photosynthetic efficiency. Shanghai Scientific and Technical Press, Shanghai
Yamori W, Noguchi K, Terashima HI (2010) Phenotypic plasticity in photosynthetic temperature acclimation among crop species with different cold tolerances. Plant Physiol 152:388–399
Yamori W, Hikosaka K, Way DA (2014) Temperature response of photosynthesis in C3, C4, and CAM plants: temperature acclimation and temperature adaptation. Photosynth Res 119:101–117
Yu BH, Lu CH (2011) Assessment of ecological vulnerability in the alpine region of the Qinghai-Tibet Plateau. Geogr Res 30(12):229–2295
Zhang XQ, Qiang KB, Guo M, Long RJ, Zhao LC (2006) Leaf epidermal micro-morphology of four species in the genus Kobresia from alpine grassland. J Gansu Agric University (chinese Version) 6:89–93
Zhao QF, Cui Y, Ma SR, Li QX, Wang G (2007) Ecological adaptation study on anatomical structure of Kobresia leaf from East Qinghai-Tibet Plateau. Guihaia (chinese Version) 27:821–825
Zhou XM (2001) Kobresia Meadow of China. Science Press, Beijing
Acknowledgements
We would like to thank Miao Li from the University of Pennsylvania for helpful comments on the early manuscript.
Funding
This work was financially supported by the Qinghai Province Science Foundation (2019-ZJ-7016), the Innovation Team Project of Basic Research Program of Qinghai Province (2022-ZJ-902), and the National Natural Science Foundation of China (31660237).
Author information
Authors and Affiliations
Contributions
SBS supervised the experiments and wrote, reviewed and edited the manuscript. RS and DWZ performed the statistical analysis of the data and prepared the draft of the manuscript. TCL and KJD performed the research design and participated in paper revision. XZG, JLM and FLW performed field data collection and laboratory experimental items. All authors contributed to data analysis, writing of the paper and discussion.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflicts of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Shi, S., Shi, R., Zhou, D. et al. Reduction of PSII photosynthetic performance by low temperature is the reason for the growing inhibition of Kobresia pygmaea Clarke. Braz. J. Bot 46, 527–539 (2023). https://doi.org/10.1007/s40415-023-00901-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40415-023-00901-z