CN111802155A - Azalea high-temperature resistance evaluation method based on chlorophyll fluorescence OJIP curve - Google Patents
Azalea high-temperature resistance evaluation method based on chlorophyll fluorescence OJIP curve Download PDFInfo
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Abstract
The invention provides a rhododendron high-temperature resistance evaluation method based on chlorophyll fluorescence OJIP curve, which comprises the following steps: selecting excellent potted seedlings which grow vigorously and consistently for 2 years and planting the excellent potted seedlings in a polyethylene culture pot; the seedling revival period is 15 days, and water and nutrient solution are regularly irrigated to the rhododendron seedlings once every 4 days in the whole seedling revival period; respectively placing the tested varieties in an illumination incubator by adopting a manual simulated climate identification method, pretreating for 4 days, and performing high-temperature stress treatment on the 5 th day; selecting leaves at the 4 th to 6 th leaf positions below the top leaves of the young shoots of the plants to measure various parameters of photosynthesis; measuring and analyzing a rapid chlorophyll fluorescence induction kinetic curve of rhododendron leaves by using a multifunctional plant efficiency analyzer; test data was processed, examined and graphed. The invention researches the high temperature resistance of different rhododendron varieties under high temperature stress by using a rapid chlorophyll fluorescence induction kinetic curve analysis technology, and determines the influence of the high temperature stress on two photosynthetic systems and the mechanism of the high temperature resistance.
Description
Technical Field
The invention relates to the field of general botany, in particular to a rhododendron high-temperature resistance evaluation method based on chlorophyll fluorescence OJIP curve.
Background
Rhododendron L belongs to evergreen shrubs or small trees of Rhododendron in Ericaceae, which is one of ten traditional flowers in China, has extremely high ornamental value and economic value, and is also recognized as one of famous and precious ornamental potted flowers and representative garden plants which have great influence on the gardens in the world. At present, the problems of few varieties, simple flower types, single flower colors, poor comprehensive ornamental value and the like mainly exist in the garden greening application of China, and the main reason is that the plants have strict requirements on the growth environment, like humid and cool environments, and along with the continuous warming of global climate in recent years, especially in summer high-temperature weather, the leaves of the rhododendron plants are often wilted to different degrees, so that the ornamental value in the garden application is greatly reduced. However, the evaluation of the high-temperature resistance of the rhododendron is mainly determined based on the growth condition of plants at present, and the evaluation method is unscientific due to the long period and the inaccurate method, so that the rhododendron on green land can die every summer.
Over the past 20 years, the analysis of the rapid chlorophyll fluorescence kinetics OJIP and JIP-test based on the "biomembrane energy flux theory" has the advantages of rapid, sensitive and nondestructive measurement, and is widely used as a research tool for plant photosynthesis. A typical rapid chlorophyll fluorescence induction kinetics shows a series of phases from initial (Fo) to maximum (Fm) fluorescence values, labeled as O phase (20 μ s, all PSII reflecting center open), J (2 ms), I (30 ms) and P (Fm, all PSII reflecting center closed). In addition to the basic OJIP phase, there may be other phases present in some cases, such as the L phase (reflecting the energy connectivity of the PSII unit), the K phase (inactivation of the associated OEC), etc. The morphological changes of the rapid fluorescence-induced kinetic profile (ojis) under different environmental conditions are closely related to their physiological conditions, and the quantitative analysis of the ojis transient is called "jis-test", which converts the shape changes of the ojis transient into quantitative changes of a set of structural and functional parameters that quantify the behavior of the photosynthetic organisms. The JIP-test can be used for accurately analyzing the PSII Reflection Center (RCs) energy capture and the electron transfer changes of the PSII donor side and the PSII acceptor side under high-temperature stress.
At present, the rapid chlorophyll fluorescence induction kinetic technology is widely applied to the research of the photosynthetic function of various plants, but the rapid chlorophyll fluorescence induction kinetic technology is not reported to be used for the evaluation of the high-temperature resistance of the plants, and only some basic parameters (Fv/Fm) are simply analyzed in the research aspect of the rhododendron photosynthetic function, and the deep knowledge of chlorophyll fluorescence information is lacked. The test uses 4 rhododendron varieties commonly used in gardens as materials, carries out comparative analysis on a rapid chlorophyll fluorescence induction kinetics OJIP curve and important parameters of leaves of the rhododendron varieties under high-temperature treatment, and aims to disclose the relationship between high-temperature stress and chlorophyll fluorescence characteristics and related indexes, thereby carrying out high-temperature resistance evaluation and classification on the 4 rhododendrons, providing reference for popularization and application range of the 4 rhododendrons in gardens and providing reference for stress resistance evaluation of other plants.
Disclosure of Invention
The invention aims to provide a rhododendron high-temperature resistance evaluation method based on a chlorophyll fluorescence OJIP curve.
In order to achieve the purpose, the invention adopts the following technical scheme:
a rhododendron high-temperature resistance evaluation method based on chlorophyll fluorescence OJIP curve comprises the following steps:
step 1): selecting 4 varieties of rhododendrons of cockscomb, phantom environment, royal generation and red yang, and planting excellent potted seedlings which are robust in growth vigor and consistent in growth for 2 years in a polyethylene culture pot;
step 2): the seedling revival period is 13-17 days, and water and nutrient solution are regularly irrigated to the rhododendron seedling once every 3-5 days in the whole seedling revival period;
step 3): respectively placing the tested varieties in a lighting incubator by adopting a manual simulated climate identification method, pretreating for 4 days at 25 ℃ in the day and 17 ℃ at night, and carrying out high-temperature stress treatment on the 5 th day, wherein the stress temperature is as follows: mild high temperature: day 30 ℃ night 20 ℃, moderate high temperature: 35 ℃ day, 25 ℃ night, severe high temperature: the temperature of 40 ℃ in the day and the temperature of 17 ℃ in the night are taken as a reference, 4 groups of treatments are carried out, 3 times of treatment are carried out, 3 plants are carried out in each time of treatment, and the illumination and the moisture conditions of the test group and the reference group are kept consistent except that the temperature is different;
step 4): selecting leaves at the 4 th to 6 th leaf positions below the top leaves of the young shoots of the plants to measure various parameters of photosynthesis, performing dark adaptation for 15 to 25 min before measurement of all treated leaves, measuring various fluorescence parameters at the normal temperature of 25 ℃ as initial values, measuring various indexes after high-temperature stress treatment is started, treating 4 groups, repeating treatment for 3 times in each group, and taking the average value as an observed value after 3 plants are repeated;
step 5): measuring a chlorophyll fluorescence OJIP curve of rhododendron leaf by using a multifunctional plant efficiency analyzer, and carrying out JIP-test analysis on the obtained chlorophyll fluorescence OJIP curve;
step 6): processing test data, performing single-factor analysis of variance and Duncan method difference significance test, and drawing a chart.
Furthermore, the culture medium is a mixture of peat, vermiculite and perlite.
Furthermore, the mass ratio of the peat to the vermiculite to the perlite is 2.8-3.2:0.8-1.2: 0.8-1.2.
Furthermore, the mass ratio of the peat to the vermiculite to the perlite is 3:1: 1.
Further, in the step 2), the seedling revival period is 15 days, and water and nutrient solution are regularly irrigated to the rhododendron seedlings once every 4 days in the whole seedling revival period.
Further, in the step 3), the illumination intensity of the climate box is 1900-.
Adopt above-mentioned technical scheme to have following beneficial effect:
the rhododendron varieties with the characteristics of 'Fengguan (FG) ",' Yudairong (YDZR)", 'Red Yang (HY) ", and' Phantom (HJ)" as materials are researched for high-temperature resistance of different rhododendron varieties under high-temperature stress by utilizing a rapid chlorophyll fluorescence induction kinetic curve analysis technology (JIP-test), and the influence of the high-temperature stress on two photosynthetic systems and a mechanism of the high-temperature resistance are determined. The results show that: it was found from the JIP-test analysis that the inhibition of the Oxygen Evolution Complex (OEC) and the inactivation of the PSII Reaction Centers (RCs) were mainly caused by high temperature stress. Under the stress of high temperature of 30 ℃, the damage of OEC in a chlorophyll fluorescence induction curve is light, and no obvious K phase is caused; under the high temperature stress of 35 ℃ and 40 ℃, obvious K phase occurs due to irreversible severe damage to OEC. In addition, there is deactivation of PSII RCs, reduced energy connectivity, destruction of antenna structure and loss of overall photosynthetic activity, and increased PSI activity. The photosynthetic capacity of 'YDZR' and 'HY' is less impaired than that of 'FG' and 'HJ'. At the high temperature of above 35 ℃, the energy flux parameters (ABS/CSm, TRo/CSm and ETo/CSm, RC/CSm) of unit area, the energy distribution ratio (phi Po, psi o, phi Eo) and the photosynthetic performance index (PI ABS) of the varieties 'FG', 'HJ' are obviously reduced compared with the varieties 'YDZR' and 'HY', and the energy flux parameters (PI ABS) and the photosynthetic performance index (PI ABS) are obviously increased; indicating that the light energy absorbed by the 'YDZR' and 'HY' leaves is more captured by the reaction center and enters an electron transfer chain; the varieties 'FG' and 'HJ' have more energy used for heat dissipation, so that more energy can be utilized for photochemical reactions in 'YDZR' and 'HY' than in 'FG' and 'HJ'. Meanwhile, the damage to the PSI of 'YDZR' and 'HY' is smaller than that of the PSII, and more PSI participates in ring-type electron transfer to dissipate redundant energy, so that the generation of Reactive Oxygen Species (ROS) is reduced.
Drawings
The invention will be further described with reference to the accompanying drawings in which:
FIG. 1 is a graph showing the effect of high temperature stress on rapid fluorescence induction curves of rhododendron leaves;
FIG. 2 is the effect of high temperature stress on the relative variable fluorescence Δ Vt of the leaves of the rhododendron;
FIG. 3 is the effect of high temperature stress on the L-band of the rapid fluorescence induction curve of rhododendron leaves;
FIG. 4 is a graph showing the effect of high temperature stress on the K band on the rapid fluorescence induction curve of rhododendron leaves;
FIG. 5 is a graph of the effect of high temperature stress on some of the parameters of the JIP-test selection of rhododendron leaves;
FIG. 6 is a graph showing the effect of high temperature stress on the energy distribution per unit cross-sectional area (leaf model) of rhododendron leaves and the reflection of the center density;
FIG. 7 is a graph of the effect of high temperature stress on the energy distribution ratio (quantum yield) of rhododendron leaves;
FIG. 8 is a graph showing the effect of high temperature stress on PSI of rhododendron leaves participating in circulating electron transfer;
FIG. 9 shows the effect of high temperature on the photosynthesis index (PI abs) and the overall performance index (PI total) of rhododendron leaves.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and specific examples.
The invention provides a rhododendron high-temperature resistance evaluation method based on chlorophyll fluorescence OJIP curve, which comprises the following steps:
step 1): selecting 4 varieties of cuckoo tree (FG), Yudai Rong (YDZR), Hongyang tree (HY) and Phantom tree (HJ), wherein the varieties are all good varieties with good growth, high ornamental value and high application value. Excellent potted seedlings which grow vigorously and consistently for 2 years are selected and planted in a polyethylene culture pot (the upper caliber is 21.5 cm, the height is 19.5 cm, and the lower caliber is 14.5 cm).
In the embodiment, the culture medium is a mixture of peat, vermiculite and perlite, and the mass ratio of the peat, the vermiculite and the perlite is 3:1: 1.
Step 2): the seedling revival period is 13-17 days, and water and nutrient solution are regularly irrigated to the rhododendron seedling once every 3-5 days in the whole seedling revival period;
step 3): respectively placing the tested varieties in a lighting incubator by adopting a manual simulated climate identification method, pretreating for 4 days at 25 ℃ in the day and 17 ℃ at night, and carrying out high-temperature stress treatment on the 5 th day, wherein the stress temperature is as follows: mild high temperature: day 30 ℃ night 20 ℃, moderate high temperature: 35 ℃ day, 25 ℃ night, severe high temperature: the temperature of 40 ℃ in the day and the temperature of 30 ℃ in the night are controlled by using 25 ℃ in the day and the temperature of 17 ℃ in the night as a control, 4 groups of treatments are carried out, 3 treatment times are set, 3 plants are repeated, and the illumination and the water content of the test group and the control group are kept consistent except that the temperature is different. The illumination intensity of the climate box is 1900-.
Step 4): selecting leaves at the 4 th to 6 th leaf positions below the top leaves of the young shoots of the plants to measure various parameters of photosynthesis, performing dark adaptation for 15 to 25 min before measurement of all treated leaves, measuring various fluorescence parameters at the normal temperature of 25 ℃ as initial values, measuring various indexes after high-temperature stress treatment is started, treating 4 groups, repeating treatment for 3 times in each group, and taking the average value as an observed value after 3 plants are repeated;
step 5): measuring a chlorophyll fluorescence OJIP curve of rhododendron leaf by using a multifunctional plant efficiency analyzer, and carrying out JIP-test analysis on the obtained chlorophyll fluorescence OJIP curve;
the fast fluorescence OJIP curve was induced by a 2s red pulse (650 nm, 3500 μmol m-2 s-1). O (50 mus) is the initial fluorescence level, K (300 mus), J (2 ms), I (30 ms) are intermediate levels, and P (500 ms-1 s) is the peak level. The OJ phase is largely photochemically driven primarily, where only a single turn of QA reduction is carried out; the JI phase mainly reflects that the reduced Plastoquinone (PQ) library is completely reduced in the electron transfer process; the IP phase reflects the PSI receptor side reduction part and is considered as one step of the final speed limit of the PSII receptor side reduction, and the amplitude of the step can be used as a rough index of PSI content.
The fluorescence ojis transients can be analyzed by using a jis-test which defines the maximum (subscript "m") energy flux in the energy cascade for Absorption (ABS), capture (TRo), electron transfer (ETo), dissipation (DIo), excitation leaf section (CS). The raw data used herein are as follows: fo: initial fluorescence when all RCs are open; fk: fluorescence intensity at point K (300 μ s); fj: fluorescence intensity at J-spot (2 ms); fi: spot I fluorescence intensity (30 ms); fm: maximum fluorescence intensity, all RCs were off. Table 1 summarizes the formulation and interpretation of the fluorescence data for the OJIP curve, as well as the JIP-test parameters to be used in this study.
TABLE 1 chlorophyll fluorescence (OJIP) parameters List
Step 6): processing test data, performing single-factor analysis of variance and Duncan method difference significance test, and drawing a chart.
Experimental data processing single-factor analysis of variance (one-way ANOVA) and Duncan method significance test (a =0.05) were performed using Microsoft Excel 2010 with SPSS 22.0 software and plotted using Origin 2017 software.
Results and analysis:
(1) influence of high-temperature stress on rapid chlorophyll fluorescence induction kinetic curve of rhododendron leaf
As shown in FIG. 1, the rhododendron leaves are treated for 4 days under mild high temperature stress (30 ℃), moderate high temperature stress (35 ℃) and severe high temperature stress (40 ℃), and the high temperature stress obviously causes a plurality of changes of rapid fluorescence induction kinetics.
From the figure we observe that under different high temperature treatments, the fast fluorescence kinetics curves of 4 rhododendron varieties, normalized, exhibit a typical multiphase rising ojis phase. With increasing temperature, the OJIP curves of the 4 varieties are similar in shape, and the time reaching the P point is consistent. At the high temperature of 35-40 ℃, the OJIP curve of the fluorescence rising kinetics of 4 varieties is gradually transformed into an OKJIP fluorescence kinetic curve, and a new intermediate 'K' phase appears around 300 mus. Compared with the control, after the high temperature treatment at 30 ℃ for 4d, the OJIP curves of 4 varieties have no obvious change, while the OJIP curves of the varieties 'FG' and 'HJ' start to change at 35 ℃ respectively, and the change of the OJIP curves is more obvious as the treatment temperature is higher; the same treatment temperature has no obvious difference on varieties 'HY' and 'YDZR', and the OJIP curve changes obviously when the temperature rises to 40 ℃. And the degree of deviation of the OJIP curves of the 4 varieties from the control increases with the increase of the temperature, and it can be seen that there is a correlation between the change of the OJIP curve of the fluorescence rising kinetics and the change of the fluorescence intensity with the temperature.
In order to accurately understand the response of electron transfer in PSII to high temperature stress, the study plots the relative variable fluorescence difference (Δ Vt), which can more intuitively reflect the changes of the OEC and PSII unit complexes of rhododendron leaves.
As shown in FIG. 2, under the high temperature stress of 35 ℃, the Δ K and the Δ J of 4 kinds of rhododendrons are both <0, wherein the Δ K and the Δ J of 'FG' and 'HJ' are the most remarkably reduced than 'HY' and 'YDZR', which indicates that the OEC of the leaves of the rhododendron seedlings is damaged and inactivated. When the seedlings are treated at the high temperature of 40 ℃, the chlorophyll fluorescence yield of 4 varieties at the characteristic site of about 300 microseconds is obviously increased, namely the positive increase of the K site (K & gt 0) occurs, wherein the Δ K value of the FG seedling is increased most obviously than that of the other 3 varieties, which shows that the donor side of the PSII of the FG seedling is more seriously injured, the capability of supplying electrons to the downstream is weakened, and the injuries of HJ ', HY ' and YDZR ' are obviously lower than that of the FG seedling. The J value is more than 0, which indicates that QA-accumulation exists, the high-temperature rib forces the QA to inhibit the electron transfer to the QB, at the high temperature of 40 ℃, the varieties ' FG ' and ' HJ ' are more remarkable than the ' HY ' and the ' YDZR ' in rising, and the inhibition degree is shown as ' FG ', HJ ', and ' HY ' and ' YDZR '.
To compare the response of rhododendron seedling leaves in the OK, OJ phases under high temperature stress, the present study also performed additional normalization of the rapid fluorescence-induced kinetics ojis curve (kinetic curve differences). FIG. 3 is a fast chlorophyll fluorescence induction curve normalized between O (50 μ s) -K (300 μ s) phases for 4 rhododendron varieties, i.e., WOK = (Ft-Fo)/(Fk-Fo), and the kinetic difference between hyperthermia treatment and CK is plotted (Δ VOK = VOK treatment-VOK control). We can observe that an additional L band is hidden at about 150 mus between O-K phases by the difference of WOK, and the L band is used as an indicator of energy connection (combination) of PSII units, and the higher the connectivity is, the larger the damage is. Thus, as can be seen in fig. 3-a, the variety 'FG' leaves, even under mild high temperature stress (30 ℃), resulted in a significant decrease in energy connectivity based on the positive L-band with temperature changes. Under the same high-temperature treatment, the energy connectivity of the variety 'FG' is obviously higher than that of the other 3 varieties, and the damage degree is sequentially as follows: ' FG ' > TsHJ ' > TsHY ' & TsYDZR '.
FIG. 4 double normalizes fluorescence data Fo (50 μ s) and FJ (2 ms) of rhododendron leaves, respectively, VOJ = (Ft-Fo)/(FJ-Fo) and Δ VOJ = VOJ treatment-VOJ control (inset). As can be seen, under moderate (35 ℃) or severe (40 ℃) high temperature stress, the K phase can be excited in the rapid fluorescence induction kinetic curve of 4 varieties VOJ, compared with CK, the K phase below 35 ℃ is not obvious, and at high temperature of 40 ℃, the variety 'FG' is obviously higher than that of the other 3 varieties. As can be seen from the inset, there is an obvious positive K band in the different Δ VOJ, which is the same as the trend in FIG. 3. The K-point (peak) strength of the 'FG' and 'HJ' leaves was significantly higher than those of the other two species at moderate high temperature (35 ℃), and the increase in K-point for VOJ indicated that the OEC center inactivation of the 'FG' and 'HJ' leaves was more severe than that of 'HY' and 'YDZR'.
(2) Effect of high temperature stress on relevant fluorescence parameters of Azalea leaves OJIP-test
As can be seen from FIG. 5-A, when the temperature was increased to 35 ℃, the relative variable fluorescence of the leaves of cultivars ' FG ' and ' HJ ' was significantly increased at a value of 300 μ s, i.e., Vk versus cultivars ' HY ' and ' YDZR, and further significantly increased with an increase in stress temperature. Vj reflects the degree of closure of the active PSII reaction center when illuminated for 2 ms, and also reflects the degree of reduction of QA. As can be seen in FIG. 5-B, the relative variable fluorescence at J-point at 2 ms increases at 40 ℃, whereas the Vj values for 4 varieties decrease slightly from CK at 35 ℃, indicating that K and J-points are two independent points of rapid chlorophyll fluorescence kinetics.
As can be seen from FIG. 5-C, the high temperature stress temperature was increased from 30 ℃ to 40 ℃, the Wk and Δ Wk values depending on temperature change started to increase significantly at moderate high temperature (35 ℃), where the K levels of the 'FG' and 'HJ' leaves increased faster relative to the varieties 'YDZR' and 'HY'. The Vi values of the 4 rhododendron leaves showed a tendency to increase continuously with increasing temperature (FIG. 5-D), wherein the varieties 'FG' and 'HJ' increase significantly at 35 ℃, indicating that the high temperature causes the accumulation of QA-in the photosynthetic electron transport chain in large quantities and the PSII electron transport capacity is reduced; and the Vi value increases greatly with the increase of the high temperature degree (40 ℃), which shows that the RCs of 'FG' and 'HJ' are completely destroyed by the high temperature, QA-in the photosynthetic electron transport chain is continuously accumulated, and the PSII electron transport capacity is reduced to the minimum. Whereas 'YDZR' and 'HY' change significantly at 40 ℃, indicating that the PSII receptor-side electron transfer is significantly inhibited only when the temperature rises to a certain extent.
(3) Influence of high temperature stress on rhododendron leaf energy distribution per unit cross-sectional area (leaf model) and reaction center density
As shown in FIG. 6, under the high temperature treatment of 35 ℃, the energy of ABS/CSm, TRo/CSm and ETO/CSm of the varieties 'FG' and 'HJ' blades is significantly reduced compared with CK, while DIO/CSm on the unit area is sharply increased, and the rising amplitude is significantly higher than that of the other 3 varieties, which shows that the energy share of the blades for electron transfer is reduced, the electron transfer is blocked, and the energy share of the heat dissipation is increased. When the variety 'HY' and 'YDZR' leaves are exposed to the high temperature of 35 ℃, the influence of the high temperature on the chlorophyll concentration of the antenna, the energy flux captured by each excited leaf section and the electron transfer energy of the leaves is small; as the temperature increased to 40 ℃, all 3 variables were gradually decreased. In addition, ABS/CSm, TRo/CSm and ETO/CSm of the variety ' FG ' are more reduced than those of the varieties ' YDZR ', ' HY ' under the high-temperature stress of 40 ℃, and the damage degree is ' FG ' > HJ ' > TsHY ' > YDZR ' in sequence.
(4) Influence of high temperature stress on rhododendron leaf energy distribution ratio (quantum yield)
In order to better compare the differences of absorption, capture, transfer and dissipation of energy of 4 varieties, the energy distribution ratios of rhododendron leaves are compared in the research, and phi Po represents the maximum photochemical efficiency of PSII; φ Do represents the PSII heat dissipation quantum ratio; phi Eo is the quantum yield for electron transfer; Ψ o is the ratio at which excitons trapped at the PSII reaction center transfer electrons to other electron acceptors in the electron transfer chain beyond QA.
As shown in FIG. 7, in the high temperature treatment above 35 ℃, φ Po is reduced along with the temperature rise, and the variety 'YDZR' is always higher than the other three varieties; phi Do increases with increasing temperature, and 'YDZR' is consistently less distinct than varieties 'FG' and 'HJ', and 'HY'; phi Eo and psi o of variety 'YDZR' begin to decrease at 35 ℃ high temperature treatment, while phi Eo and psi o of 'FG' begin to decrease at 30 ℃; under high temperature stress above 35 ℃, phi Eo and psi o of 'YDZR' are both significantly higher than 'FG'. The above results show that under high temperature stress, more light energy absorbed in 'YDZR' is captured by the reaction center and enters the electron transport chain, and more light energy absorbed in 'FG' is used for heat dissipation.
(5) Influence of high-temperature stress on participation of rhododendron leaves in circulating electron transfer PSI
Ro represents a decrease in PSI acceptor side terminal electron acceptor quantum yield. As shown in fig. 8, Ro increased with increasing temperature at high temperature treatment of 35 ℃ and above, and Ro for varieties 'YDZR', 'HY' was significantly lower than both varieties 'FG' and 'HJ'. Under high temperature stress, compared with 'FG', the variety 'YDZR' shows that more PSI participates in ring-type electron transfer, provides pH difference for ATP generation, dissipates excess excitation energy, and reduces the generation of Reactive Oxygen Species (ROS).
(6) Influence of high temperature stress on rhododendron leaf photosynthetic performance index (PI abs) and comprehensive performance index (PI total)
The photosynthetic performance index PI abs is a performance index based on absorbed light energy and is also the OJIP test parameter which reflects the most sensitive overall photosynthetic activity of PSII. As shown in FIG. 9-A, PI abs decreased sharply when leaves of varieties 'FG' and 'HJ' were exposed above 35 ℃; however, significant reduction in PI abs occurs only at temperatures of 40 ℃ or higher for 'YDZR' and 'HY' blades; at 30 ℃, the damage to the leaves of 4 varieties was not obvious, and there was no significant difference in PI abs between them. The comprehensive performance parameter PI total is mainly used for researching the electron transfer activity between optical systems and can further reflect the influence of temperature stress on the PS I. FIG. 9-B shows that PI total values for 4 varieties continued to increase with increasing stress temperature. When the temperature is stressed to 35 ℃, the varieties 'YDZR' and 'HY' are obviously increased compared with CK, and the 'FG' has no obvious change.
In conclusion, photosynthesis is one of the most sensitive physiological processes to high temperature stress, and maintaining high photosynthetic activity is critical to the high temperature stress resistance of plants. Numerous studies have shown that high temperature stress leads to deactivation of OEC, hindered electron transfer and reduced photochemical efficiency of PSII. PSII in photosynthesis is located on the inner side of a thylakoid membrane and is very sensitive to changes, and the K point in a chlorophyll fluorescence rapid induction curve is mainly caused by that the cleavage process of water in electron transfer is inhibited and the QA part is inhibited, which marks that OEC is damaged, so PSII is often considered as an original site and a main action site of photoinhibition.
(7) Influence of high-temperature stress on rapid chlorophyll fluorescence induction kinetic curve of rhododendron leaf
As can be seen from FIGS. 1-2, our experiments show that the appearance of the K phase is the main change in the rapid fluorescence induction kinetics of rhododendron leaves under high temperature stress, and this phenomenon occurs particularly due to the destruction of OEC by the release of manganese clusters. According to the model of De Rode et al, it was found that when the high temperature stress causes an imbalance in OEC to RCs and electron flow to the PSII receptor side after dissociation of OEC, another internal electron donor such as proline can donate electrons to PSII instead of H2O, which would result in a transient increase in the concentration of Pheo- (QA-) producing a K peak at about 300 μ s. The K peak (phase) is followed by a drop, the possible explanation being that PSII RCs are reopened by electron transfer from QA-to QB, and subsequent accumulation of low fluorescence yield P680+ centers; when the high temperature treatment is carried out at 40 ℃, the Δ K value of the ' FG ' blade is increased most obviously compared with that of other 3 varieties, which shows that the donor side of the PSII of the ' FG ' seedling blade is more seriously injured, QA-accumulation exists, the electron transfer from QA to QB is inhibited, the comprehensive comparison shows that the Δ K and the Δ J values after the high temperature stress are found, the varieties ' HY ' and ' YDZR ' are more resistant to high temperature obviously than the varieties ' FG ' & HJ ', which shows that the ' YDZR ', ' HY ' can better maintain the stability of the PSII OEC under high temperature stress, and the photosynthetic mechanisms of the ' FG ', ' J ' blade are more easily injured by high temperature.
As can be seen from FIG. 3, PSII is interconnected in structure and function, and under high temperature stress, the energy synergy among PSII units is greatly influenced by the destruction of the photosynthesis structure. This assumption is supported by positive L-band theory when high temperature stress causes the sample energy connectivity to drop, and lower connectivity results in inefficient use of excitation energy and lower stability of the PSII units.
Therefore, the high temperature response mechanism of 4 rhododendron seedlings can be summarized as follows: the 4 varieties partially inhibit OEC and PSII RCs under mild high-temperature stress (less than or equal to 30 ℃), but do not cause obvious K phase; therefore, we believe that inhibition of OEC and PSII RCs is the initial cause of high temperature damage. Subsequently, with the appearance of the K-phase and the massive deactivation of PSII RCs, the energy connectivity of the PSII units of 'YDZR' and 'HY' and the overall photosynthetic activity of PSII decreased at moderate high temperature rib (35 ℃), but had no significant effect on the structure, the primary photochemical reaction, the energy dissipation and the electron transfer activity of the antenna complex; the varieties 'FG' and 'HJ' cause irreversible damage to OEC, which further degrades the energy connectivity and overall photosynthetic activity of PSII, and additionally reduces antenna size, while significant K-phase will occur with complete destruction of OEC under severe high temperature stress (40 ℃).
(8) Effect of high temperature stress on relevant fluorescence parameters of Azalea leaves OJIP-test
When the donor side of PSII (OEC) was injured, the chlorophyll fluorescence yield increased before the J-point (at about 300. mu.s), so the increase in the relative fluorescence value WK at this point was taken as an indication of the injury on the donor side of PSII, and the increase in WK represents the degree of destruction on the donor side of PSII. When Vj and Vi rise, it indicates that the electron transport chain is blocked, mainly due to the accumulation of QA-and the limitation of the oxidation-reduction reaction of the PQ library. The experimental result shows that Vj and Vi of different varieties of rhododendrons are in a continuously increasing trend under high-temperature stress, and the increasing trend shows an increasing trend along with the weakening of the high-temperature resistance of the rhododendron seedlings. An increase in Vj indicates an increased rate of QA reduction, reflecting a specific sign of the obstruction of QA to QB electron transfer on the PSII receptor side. Vi increase indicates that high temperature stress significantly reduces the ability of PQ to accept electrons, which is also a significant reason for its reduced QA to QB transport, so Vi reflects the heterogeneity of the PQ pool during QA to QB transport.
(9) Influence of high temperature stress on rhododendron leaf energy distribution per unit cross-sectional area (leaf model) and reaction center density
The reduction of ABS/CSm, TRo/CSm, ETo/CSm and RC/CSm under adversity stress is probably caused by the degradation or inactivation of RCs, the change of RCs can also be regarded as a self-defense mechanism of plants, and the increase of DIo/CSm indicates that RCs start a corresponding self-protection mechanism, so that the excess excitation energy can be dissipated in time to reduce the damage of excess light energy to the plants, which is consistent with the result of the experiment.
The reduction of ABS/CSm is due to the degradation or inactivation of the reflection center part caused by temperature stress on one hand, and the damage of the pigment structure of the antenna on the other hand, which causes the reduction of the captured light energy, thus reducing the RCs excitation energy (TRo/CSm) and the reduction energy (ETO/CSm) and influencing the electron transfer. Meanwhile, the heat dissipation of the unit leaf area is increased due to high-temperature stress, which shows that the defense mechanism of RCs of rhododendron is started after the rhododendron is subjected to the high-temperature stress, and redundant excitation energy in the leaves is dissipated in the form of heat dissipation and fluorescence.
(10) Influence of high temperature stress on rhododendron leaf energy distribution ratio (quantum yield)
Under the combined influence of high temperature and drought stress, phi Po, phi Eo and psi o of quercus acutissima plants show a significant decrease. In the research, the values of varieties 'FG' and 'HJ' are reduced more obviously than 'HY' and 'YDZR', and the higher phi Po, phi Eo and phi o of 'YDZR' and 'HY' under high-temperature stress indicate that more energy is absorbed by reaction centers and is used for electron transfer; whereas a higher φ Do of 'FG' and 'HJ' indicates that more energy is used for heat dissipation, less energy is utilized by photochemical reactions. Under high temperature stress, the photochemical efficiencies of 'YDZR', 'HY' are higher than that of 'FG' and 'HJ' and are weaker than the light inhibition degrees of 'FG' and 'HJ', so that more absorbed energy in 'YDZR' and 'HY' is distributed to electron transfer chains to be beneficial to maintaining higher photosynthetic capacity, and the excessive heat dissipation of 'FG' and 'HJ' ensures that the energy available for photochemical reactions is less than that of 'YDZR' and 'HY'.
(11) Influence of high-temperature stress on participation of rhododendron leaves in circulating electron transfer PSI
The change in the balance between cyclic and linear electron transport is considered to be an important mechanism for plants to adapt to environmental stress. A low Ro indicates a decrease in the number of PSI involved in linear electron transfer and an increase in the number of PSI involved in cyclic electron transfer, possibly because PSI or cytb6/f is physically distant from PSII, thereby compromising linear electron transfer. Under high temperature stress, the transmission of ring type electrons around PSI is enhanced, and the damage of high temperature to a photosynthetic system is reduced. A low Ro in 'YDZR' indicates that more PSI is involved in cyclic electron transport, thereby assisting it in dissipating excess energy, reducing ROS production due to excess excitation energy, and generating more ATP for protein repair and other defense mechanisms.
(12) Influence of high temperature stress on rhododendron leaf photosynthetic performance index (PI abs) and comprehensive performance index (PI total)
The PI abs and the PI total can accurately reflect the transmission of electrons between PSII and PSI and the state of a photosynthetic system. The PIabs comprises three parameters of phi Po, phi Eo and psi o to comprehensively reflect the activity of a photosystem, so that the PIabs can be used as an effective fluorescence parameter index of different varieties of rhododendrons in response to high-temperature stress. In this study, both PI abs and φ Po were more sensitive to high temperature stress, but it can be seen from FIGS. 7-A and 9-A that PI abs are more sensitive. The transformation condition of PSIRCs cannot be reflected by PI abs, the chlorophyll fluorescence induction kinetics research scope is no longer limited to PSII due to the appearance of PI total, the transmission capability of electrons between the PSII and the PSI and the related performance of the PSI can be further reflected, and the increase of the PI total is considered as the expression that the PSI has stronger stress tolerance, and the damage to the PSI is smaller than that of the PSII.
In conclusion, the rhododendron varieties with strong high temperature stress adaptability can be effectively screened by utilizing the rapid chlorophyll fluorescence induction kinetics OJIP-test, the influence on the acceptor side, the donor side OEC and RCs of rhododendron leaves PSII after the high temperature stress (not less than 35 ℃) is carried out for 4 days, the rhododendron varieties 'FG' and 'HJ' are more sensitive to high temperature, the high temperature causes the damage of the PSII of the 'FG' and the 'HJ' to be more serious than that of the other two varieties, the whole electron transfer chain is seriously hindered, and the photosynthetic structure is protected from being damaged by increasing the heat dissipation; on the contrary, the varieties 'YDZR' and 'HY' can maintain higher photosynthetic activity and have better high-temperature resistance.
Other embodiments of the present invention than the preferred embodiments described above will be apparent to those skilled in the art from the present invention, and various changes and modifications can be made therein without departing from the spirit of the present invention as defined in the appended claims.
Claims (6)
1. The azalea high-temperature resistance evaluation method based on chlorophyll fluorescence OJIP curve is characterized by comprising the following steps:
step 1): selecting 4 varieties of rhododendrons of cockscomb, phantom environment, royal generation and red yang, and planting excellent potted seedlings which are robust in growth vigor and consistent in growth for 2 years in a polyethylene culture pot;
step 2): the seedling revival period is 13-17 days, and water and nutrient solution are regularly irrigated to the rhododendron seedling once every 3-5 days in the whole seedling revival period;
step 3): respectively placing the tested varieties in a lighting incubator by adopting a manual simulated climate identification method, pretreating for 4 days at 25 ℃ in the day and 17 ℃ at night, and carrying out high-temperature stress treatment on the 5 th day, wherein the stress temperature is as follows: mild high temperature: day 30 ℃ night 20 ℃, moderate high temperature: 35 ℃ day, 25 ℃ night, severe high temperature: the temperature of 40 ℃ in the day and the temperature of 17 ℃ in the night are taken as a reference, 4 groups of treatments are carried out, 3 times of treatment are carried out, 3 plants are carried out in each time of treatment, and the illumination and the moisture conditions of the test group and the reference group are kept consistent except that the temperature is different;
step 4): selecting leaves at the 4 th to 6 th leaf positions below the top leaves of the young shoots of the plants to measure various parameters of photosynthesis, performing dark adaptation for 15 to 25 min before measurement of all treated leaves, measuring various fluorescence parameters at the normal temperature of 25 ℃ as initial values, measuring various indexes after high-temperature stress treatment is started, treating 4 groups, repeating treatment for 3 times in each group, and taking the average value as an observed value after 3 plants are repeated;
step 5): measuring a chlorophyll fluorescence OJIP curve of rhododendron leaf by using a multifunctional plant efficiency analyzer, and carrying out JIP-test analysis on the obtained chlorophyll fluorescence OJIP curve;
step 6): processing test data, performing single-factor analysis of variance and Duncan method difference significance test, and drawing a chart.
2. The method for evaluating fire resistance of azalea based on chlorophyll fluorescence ojis p curve according to claim 1, wherein the culture medium is a mixture of peat, vermiculite and perlite.
3. The method for evaluating fire resistance of azalea based on chlorophyll fluorescence OJIP curve according to claim 2, wherein the mass ratio of peat, vermiculite and perlite is 2.8-3.2:0.8-1.2: 0.8-1.2.
4. The method for evaluating fire resistance of azalea based on chlorophyll fluorescence OJIP curve according to claim 3, wherein the mass ratio of peat, vermiculite and perlite is 3:1: 1.
5. The method for evaluating the high temperature resistance of azalea based on chlorophyll fluorescence OJIP curve according to claim 1, wherein in the step 2), the seedling revival period is 15 days, and the seedling is watered with water and nutrient solution periodically every 4 days during the whole seedling revival period.
6. The method for evaluating the high temperature resistance of azalea based on the chlorophyll fluorescence OJIP curve as claimed in claim 1, wherein in the step 3), the illumination intensity of the climate box is 1900-.
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