CN110199708B

CN110199708B - Method for detecting distribution condition of chloroplasts in rice leaves after light treatment

Info

Publication number: CN110199708B
Application number: CN201910568289.XA
Authority: CN
Inventors: 于彦春; 张燕莉; 武丽敏; 陈飞; 熊二辉; 徐俊锋
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2021-09-28
Anticipated expiration: 2039-06-27
Also published as: CN110199708A

Abstract

The invention discloses a method for detecting distribution condition of chloroplasts in rice leaves after light treatment, and belongs to the technical field of plant light signal research. The method comprises the following steps: (1) taking rice seeds to be detected, and obtaining rice seedlings by a water culture method; (2) putting the rice seedlings in a dark environment for dark treatment for 12-36 h; (3) culturing the rice seedlings subjected to dark treatment under different illumination conditions; (4) cutting the middle area of flag leaves of the rice seedlings after the illumination treatment, placing the flag leaves in a fixing solution, and fixing the leaves; (5) and taking out the fixed leaves from the fixing solution, flaking, and observing and recording the distribution condition of the rice chloroplast in the cells by using a laser confocal microscope. The method provided by the invention is simple in operation process, clear, real and intuitive in observation result, can be used for researching the relation between the rice chloroplast and the optical signal, and provides help for further analyzing the rice chlorophyll optical signal regulation and control mechanism.

Description

Method for detecting distribution condition of chloroplasts in rice leaves after light treatment

Technical Field

The invention relates to the technical field of plant optical signal research, in particular to a method for detecting distribution condition of chloroplasts in rice leaves after optical treatment.

Background

The chloroplast is used as the place of plant photosynthesis to absorb carbon dioxide, release oxygen and synthesize organic matters, and along with the gradual deepening of light signal research in various plants, the chloroplast has important significance in research and identification of different light intensities and different light quality sensitivity degrees.

Rice (Oryza Sativa) is one of important food crops, and the problem of ensuring the high yield of the rice is related to national civilization. Rice is a monocotyledon of the family Gramineae, is fond of warm and humid, and an indoor water culture method of rice seedlings is widely applied to various researches of the rice due to the rapidness and convenience of the method. The key point of ensuring the high yield of the rice seedlings is to improve the photosynthetic efficiency of the rice seedlings, and the method of observing the distribution condition of chloroplast of rice leaves of different rice varieties under different illumination conditions is utilized, so that convenience is provided for researching rice optical signal paths, the planting illumination conditions suitable for different rice varieties can be quickly screened out, the rice breeding work is facilitated, the possibility is provided for solving the food problems of China and many other countries in the world, and the method has incomparable effect on agricultural production of the whole human society.

At present, along with the discovery of more and more genes in plant optical signals, a set of precise regulation and control system exists in the reaction of plants to light, chloroplasts can make different reactions to different light intensities and light qualities, the position distribution and the form change of the chloroplasts in cells are adjusted to provide light energy for photosynthesis to the maximum extent, the most organic matters are accumulated, and the damage of over-strong light to the chloroplasts is avoided at the same time, so that the chloroplasts in plant mesophyll cells are flatly paved on the upper and lower surfaces of the cells to the maximum extent under the weak illumination condition, the effective area vertical to incident light is maximized, and the photosynthetic efficiency is maximized; on the contrary, under the strong illumination condition, the chloroplast is parallel to the peripheral side wall of the vertical cell and is parallel to the incident light, so that the direct projection area is reduced, and the light damage caused by direct irradiation of strong light is reduced. Chloroplasts make sensitive and rapid changes in position to light, and as a self-protection response of plants, rice is not exceptional, but has been found in a plurality of species.

However, rice leaves have the following characteristics: firstly, the rice leaves have dense vascular bundles and are different in size, and the rice leaves are composed of rich xylem and phloem, so that the leaves are uneven, when observed by a common fluorescence microscope, the number of cells in the same plane is small, and the area without the vascular bundles is extremely narrow and is difficult to observe; secondly, the surface of the rice leaf has a waxy layer which is a first barrier for resisting external stimulation, so that the fixation treatment of the leaf is challenged; thirdly, the rice leaf cells have irregular shapes, the epidermis also has epidermal hair, and the mesophyll cells are thin and are not well observed. Therefore, clear chloroplast distribution is extremely difficult to observe by the conventional method, so that the method is not suitable for rice light-related research.

Disclosure of Invention

The invention aims to provide a method for detecting the distribution condition of chloroplasts in rice leaves after light treatment, so as to solve the problem that the rice leaves are difficult to observe. The method can be used for researching the sensitivity of rice seedlings to different illumination conditions or the reaction of different varieties of rice to the same illumination conditions so as to promote the deep research of rice optical signals.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting distribution condition of chloroplasts in rice leaves after light treatment comprises the following steps:

(1) taking rice seeds to be detected, and obtaining rice seedlings by a water culture method;

(2) putting the rice seedlings in a dark environment for dark treatment for 12-36 h;

(3) culturing the rice seedlings subjected to dark treatment under different illumination conditions;

(4) cutting the middle area of flag leaves of the rice seedlings after the illumination treatment, placing the flag leaves in a fixing solution, and fixing the leaves;

(5) and taking out the fixed leaves from the fixing solution, flaking, and observing and recording the distribution condition of the rice chloroplast in the cells by using a laser confocal microscope.

In the step (1), the rice seedlings can be quickly and conveniently obtained by using a water culture method.

Preferably, the hydroponic method comprises: soaking rice seeds in water for accelerating germination, sowing after germination, carrying out water culture for 2-4 days, replacing with rice culture solution for culture for 2 weeks under the conditions of 30 ℃, carrying out illumination culture for 12-14 hours every day and carrying out dark culture for 10-12 hours.

In the step (2), before the rice seedlings are treated under different illumination conditions, strict dark treatment is carried out, so that the distributed induction effect of chloroplasts under the influence of light can be effectively removed, all chloroplasts sink to the bottom of cells due to gravity, and a uniform starting point is provided for the subsequent illumination treatment.

Preferably, the rice seedlings are placed in a dark incubator in a dark room for dark treatment for 24 hours.

In the step (3), different illumination conditions (such as different illumination intensities and different light qualities) are selected according to different research requirements to carry out illumination treatment on the dark-treated rice seedlings, and at the moment, chloroplasts in the leaves can make different responses to the different illumination conditions, so that the position distribution and the shape of the chloroplasts in the cells are changed.

Preferably, the time for the light culture is 2 hours. Studies have shown that chloroplasts have responded adequately after 2 hours of culture.

And (4) after the illumination treatment is finished, fixing the rice leaves to quickly fix the state of cells in the leaves.

Preferably, 2-3mm segments are cut in the middle area of the flag leaf of the rice seedling and placed in a fixing solution, and 3-4 segments are cut for each sample.

Preferably, the fixative solution contains 20mM PIPES and 5mM MgCl₂5mM EGTA, 0.5mM phenylmethylsulfonyl fluoride, 1% dimethyl sulfoxide, pH 7.0.

The fixing liquid is fully filled in the cells, and the effect of fully fixing all organelles in the cells is achieved. Preferably, the leaves are placed in a stationary liquid, treated for 5-10min under a vacuum degree of-0.1 MPa, and then placed for 5-7 days at 4 ℃. The rice leaves cut into small sections are subjected to vacuum treatment, and the fixing liquid fully enters the rice leaves, so that a better fixing effect is achieved.

And (5) observing the distribution condition of chloroplasts in mesophyll cells of the rice leaves by using a laser confocal microscope. Chloroplasts are located in mesophyll cells of plants, and mainly contain chlorophyll a, chlorophyll b, carotene and lutein, the chlorophyll a and b absorb blue-violet light and red light more, the other two absorb blue-violet light mainly, and part of light which is not used for photosynthesis after the blue-violet light is absorbed by the pigments forms fluorescence to be radiated out again, but the radiated fluorescence is red light with less energy than the blue-violet light because part of energy is lost as heat energy in the radiation process. The invention utilizes the principle of autofluorescence and adopts a laser confocal microscope to observe the distribution condition of chloroplasts in mesophyll cells of rice leaves to obtain a clear and strong signal result. Meanwhile, the laser confocal microscope shooting technology can solve the problem that the number of cell layers is too large and the cells cannot be separated during the observation of a common fluorescence microscope, and clearly observe the distribution condition of chloroplasts in mesophyll cells of single-layer rice leaves.

Preferably, in tableting, the leaf is taken out of the fixative solution, the solution is blotted, the long arm end of the leaf is cut off with the proximal face facing upwards, placed in 50% glycerol, and mounted.

Through the long arm end of cutting off the blade, reduce the unevenness nature of rice blade, do benefit to the observation of one deck cell, add 50% glycerine during the film-making, be favorable to reducing because the cavity that the blade unevenness arouses causes the problem of observing unclearly, guarantee simultaneously that the blade can not be because the desiccation is flat.

When the compound is observed by a laser confocal microscope, exciting light with the wavelength of 568nm is adopted.

Preferably, mesophyll cells at the extreme edge of the short arm end of the rice leaf are selected for observation. The mesophyll cell layer at the edge of the short arm end is selected uniformly, and the mesophyll cell layer under the epidermal cells is observed uniformly, so that the error among samples can be reduced, and the result has great comparability and reliability.

The invention has the following beneficial effects:

according to the invention, rice seedlings are quickly and conveniently cultured, a complete system for different illumination treatment of the rice seedlings is established, a proper sampling, fixing, sheet making and observing method is found out, the principle that chloroplasts can perform autofluorescence at 568nm wavelength is utilized, the distribution condition of rice leaf chloroplasts can be efficiently and clearly observed by adopting a laser confocal microscope shooting technology, the operation process is simple, the observation result is clear, real and intuitive, and the problem that the rice leaves are difficult to observe in the prior art is effectively solved.

The method provided by the invention can be used for researching the relation between the rice chloroplast and the optical signal, provides help for further analyzing the rice chlorophyll optical signal regulation mechanism, can effectively screen out the optimal light intensity required by the cultivation of different rice varieties, is beneficial to performing proper light supplement operation on the rice when the rice is cultivated in different areas, can also be used for genetic improvement of crops, and has important significance for creating high-light-efficiency rice varieties.

Drawings

FIG. 1 is a drawing of a rice seed germination process, wherein A is a selected rice seed; b, accelerating germination of rice seeds; c is rice seed budding.

FIG. 2 shows the dark treatment of seedlings, wherein A is a rice seedling cultured for 2 weeks; and B, culturing in a non-light incubator.

FIG. 3 shows the treatment of rice seedlings under different light intensity and light quality conditions for 2 hours, wherein A is the illumination intensity of 50. mu. mol/m²White light in,/s; b is the illumination intensity of 300 mu mol/m²White light in,/s; c is illumination intensity of 50 mu mol/m²Blue light in/s; d is the illumination intensity of 250 mu mol/m²Blue light in/s.

FIG. 4 is a schematic diagram of a sampling, wherein A is a selected sampling location; b is a sampling blade.

Fig. 5 shows the blade fixed in the fixing liquid.

Fig. 6 shows temporary mounting.

FIG. 7 is a diagram illustrating selected portions of a map, where the boxes are labeled as regions.

FIG. 8 shows rice leaf epidermal cells.

FIG. 9 is a graph showing the results observed in the laser confocal measurement. A illumination intensity is 0 mu mol/m²S; the illumination intensity of B is 50 mu mol/m²White light in,/s; the illumination intensity of C is 300 mu mol/m²White light in,/s; d the illumination intensity is 50 mu mol/m²Blue light in/s;e illumination intensity is 250 mu mol/m²Blue light in/s.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

The compositions of the solutions used in the examples are as follows:

rice culture solution: 9.14% ammonium nitrate; 8.86% calcium chloride; 32.4% magnesium sulfate heptahydrate; 7.14% potassium sulfate; 4.03% of sodium dihydrogen phosphate dihydrate; 0.9315% disodium edetate dihydrate; 0.6965% iron sulfate heptahydrate; 0.15% manganese chloride tetrahydrate; 0.0934% boric acid; 0.0074% ammonium molybdate; 0.0035% zinc sulfate heptahydrate; 0.0031% copper sulfate pentahydrate.

Fixing liquid: 20mM PIPES, 5mM MgCl₂5mM EGTA, 0.5mM phenylmethylisolfonyl fluoride, 1% dimethyl sulfoxide, pH 7.0, and fresh preparation is preferred.

And (3) temporary mounting and manufacturing: 50% of glycerol.

The rice variety used in the examples was a light-sensitive normal wild-type rice variety NIP. Example 1

1. Cultivation of rice seedlings

1) The filled seeds are selected from the harvested seeds and placed in a plate, preferably purified water, to submerge the seeds, as shown in FIGS. 1A-B.

2) The plate is put in an environment of 30 ℃ for germination for 2-3 days. Purified water is replaced every morning and evening, and germination is noticed, as shown in fig. 1C.

3) When the seeds are sowed in the PCR plate after budding, the bottom end of each hole is cut off by the PCR plate before sowing, the sowing density can not be too large, and 3 holes are sowed with one seed to prevent the influence on growth due to excessive close planting.

4) And (3) finishing sowing, firstly culturing with water for 3 days, then preventing the green algae caused by excessive nutrition from flooding, then culturing with the rice culture solution, and replacing once a week, ensuring the culture solution to be sufficient every day, continuously culturing for 2 weeks under the culture conditions of 30 ℃, 14-hour illumination and 10-hour darkness.

Aiming at different rice varieties, fine adjustment is carried out according to the germination efficiency and the germination time, the experimental material can be quickly and conveniently obtained by the culture method, the complexity of field sampling is reduced, and various researches on the rice seedling stage are facilitated.

2. Dark treatment of rice seedlings

1) After 2 weeks of cultivation, the young rice plants (as shown in FIG. 2A) were divided into several portions (depending on how many light intensity treatments were applied at the later stage), cultured in a dark incubator for 24 hours, and dark-treated (as shown in FIG. 2B).

The dark treatment process is strict, and in view of the sensitivity of rice to light, if light leakage occurs, the initial state is inconsistent, and the final observation result is greatly influenced, so that during dark treatment, the lightless incubator is placed in a dark room, and the next seedling taking process is ensured not to influence 0 mu mol/m²The materials treated by the treatment of/s and the materials treated by different light intensities do not influence each other.

3. Treatment of rice seedlings under different illumination conditions

1) Taking out the dark-treated rice seedlings from the incubator in a dark room, and respectively placing the rice seedlings under different illumination conditions (the illumination intensity is 0 mu mol/m 2/s; the illumination intensity is 50 mu mol/m²White light in,/s; the illumination intensity is 300 mu mol/m²White light in,/s; the illumination intensity is 50 mu mol/m²Blue light in/s; the illumination intensity is 250 mu mol/m²Blue light/s) for 2 hours to prevent the interaction of the lights, or one by one (as shown in FIG. 3).

The operation can select various illumination conditions, is favorable for more finely researching the reaction of the rice seedlings to different illumination intensities and different light qualities, and can also be used for the reaction of different rice varieties to the same illumination conditions.

4. Method for sampling rice seedlings after treatment under different illumination conditions

1) Before finishing the treatment under different illumination conditions, preparing a 1.5 ml centrifuge tube added with 1 ml of stationary liquid, determining the leaves and the position (the middle area of the flag leaf of the rice seedling) to be sampled (as shown in figure 4A), rapidly cutting off the leaves when the treatment time is up, cutting the selected area into small sections of 2mm (as shown in figure 4B), rapidly placing the small sections into the stationary liquid, and carrying out vacuum treatment in a vacuum device for 5 minutes at a vacuum degree of-0.1 MPa (as shown in figure 5).

2) Placing the sample subjected to vacuum treatment in a refrigerator at 4 deg.C for 5-7 days, and supplementing in time if the fixing solution is volatilized.

The operation of this step is fast and requires 0. mu. mol/m for light intensity²For the treatment group/s, sampling was performed in a dark room. 3-4 pieces of each sample are cut, so that excessive leaves do not influence the vacuum fixing effect.

5. Observing the distribution condition of chloroplast in rice leaves by using a laser confocal microscope

1) The sample was taken out from the 4 ℃ freezer, and the slide glass and the cover slip were prepared, first, a drop of 50% glycerin was dropped on the slide glass, the leaf to be observed was gently taken out with a small forceps, the fixative was sucked off with a water-absorbent paper, placed in glycerin with the front side up, the long-arm end of the rice leaf was cut off with scissors, covered with the cover slip, and mounted (as shown in fig. 6).

2) The temporary mount was placed upside down and the mesophyll cell layer with the observation site being the edge-most of the short arm end was selected (as shown in FIG. 7, indicated by a square).

3) After the position is selected, the shooting wavelength is set to be 568nm, and scanning is started from the surface layer of the blade. First, rectangular serrated epidermal cells are seen, with stomata distributed (as shown in FIG. 8); scanning downwards, the mesophyll cells can be seen, the chloroplast morphology and distribution (as shown in figure 9) appear, and pictures are taken.

As shown in FIG. 9, when the rice plants are continuously dark-treated, chloroplasts in the leaves sink to the bottom of the cells due to gravity, and when the chloroplast distribution of the cell epidermis is photographed by vertical leaves, there is basically no chloroplast autofluorescence signal, as shown in FIG. 9A; when the concentration is 50 mu mol/m²In white light or weak blue light treatment of/s, chloroplasts aggregate to the upper and lower surfaces of the cells due to aggregation reaction, and then the mesophyll cell surfaces of the leaves are covered with chloroplast autofluorescence signals, as shown in FIGS. 9B and 9D; when the concentration is 300 mu mol/m²White light of/s or 250. mu. mol/m²When leaves were treated with strong blue light at/s, chloroplasts moved to the periphery of the cells in the dark, allowing chloroplast autofluorescence to appear at the periphery of the cells, as shown in FIGS. 9C and 9E.

Therefore, according to the method provided by the invention, the sensitivity of different rice varieties to light can be effectively analyzed. According to different research purposes, the method can be used for observing the distribution of chloroplasts.

When the rice is cultivated in a greenhouse, reference and support are provided for selection of illumination intensity; providing a basis for light supplement operation of rice cultivation in different latitudes and different areas; provides an effective method for researching the relation between rice chloroplast and optical signals.

Claims

1. A method for detecting distribution condition of chloroplasts in rice leaves after light treatment is characterized by comprising the following steps:

(4) cutting the middle region of flag leaf of rice seedling, placing in fixing solution, treating at-0.1 MPa for 5-10min, standing at 4 deg.C for 5-7 days, and fixing the leaf;

(5) taking out the fixed leaves from the fixing solution, sucking the liquid, cutting off the long arm end of the leaves with the paraxial surface upward, placing in 50% glycerol, sealing, and observing and recording the distribution of rice chloroplast in cells under 568nm excitation light by using a laser confocal microscope.

2. The method for detecting the distribution of chloroplasts in rice leaves after light treatment according to claim 1, wherein in the step (1), the hydroponics method comprises the following steps: soaking rice seeds in water for accelerating germination, sowing after germination, carrying out water culture for 2-4 days, replacing with rice culture solution for culture for 2 weeks under the conditions of 30 ℃, carrying out illumination culture for 12-14 hours every day and carrying out dark culture for 10-12 hours.

3. The method for detecting the distribution of chloroplasts in rice leaves after light treatment according to claim 1, wherein in the step (2), the rice seedlings are placed in a dark incubator in a dark room for 24 hours.

4. The method for detecting the distribution of chloroplasts in rice leaves after light treatment according to claim 1, wherein the period of light culture in step (3) is 2 hours.

5. The method for detecting distribution of chloroplasts in rice leaf blades after light treatment as claimed in claim 1, wherein in step (4), 2-3mm small segments are cut from the middle area of flag leaves of rice seedlings and placed in a stationary liquid, and 3-4 segments are cut from each sample.

6. The method according to claim 1, wherein in step (4), the fixing solution contains 20mM PIPES and 5mM MgCl₂5mM EGTA, 0.5mM phenylmethylsulfonyl fluoride, 1% dimethyl sulfoxide, pH 7.0.

7. The method for detecting the distribution of chloroplast in rice leaves after light treatment as claimed in claim 1, wherein in step (5), mesophyll cells at the extreme edge of the short arm end of rice leaves are selected for observation.