A method for observing and identifying the light-collecting and light-avoiding movements of corn chloroplasts

Abstract

The present application belongs to the field of corn genetic engineering technology, and specifically relates to a method for observing and identifying the light-concentrating and light-avoiding movements of corn chloroplasts. The light movement includes two aspects: light-concentrating movement and light-avoiding movement. The method includes the steps of seed germination, cultivation into seedlings, pretreatment, sampling and processing, and observation. This application takes corn as the research object. Based on an in-depth study of the characteristics of chloroplast light movement in corn leaves, the observation and identification system of corn chloroplast light movement is optimized, and key observation and identification conditions such as the optimal growth days of leaves in slice materials are clarified, which can lay a certain technical foundation for the establishment of a standardized identification system for corn chloroplast light movement. At the same time, it also provides a good reference and reference for the establishment of microscopic movement identification systems for other organisms. Based on these works, it has very important basic technical significance for in-depth analysis of the mechanism of corn light movement, as well as related parent screening and new variety breeding.

Description

Technical Field

[0001] The present application belongs to the field of corn genetic engineering technology, and specifically relates to a method for observing and identifying the photokinesis of corn chloroplasts. Background Art

[0002] Maize (Zea mays L.) is an annual, monoecious, cross-pollinated herbaceous plant of the genus Zea in the Poaceae family. Native to Central and South America, it is widely cultivated in tropical and temperate regions around the world and is used in a wide range of applications, including food, feed, and bioenergy. To meet the growing global demand for maize production, breeders have been continuously selecting maize varieties that can be planted at higher densities to increase maize yield per unit area of ​​land. However, high-density planting can lead to insufficient light in the middle and lower leaves of the crop due to shading from adjacent plants, resulting in low-light stress. Effectively adjusting the repositioning of chloroplasts in maize leaves to optimize light capture in response to changes in the light environment is a new approach to improving maize productivity.

[0003] Chloroplasts are plastids that contain green pigments (including chlorophyll a and b) and are the site of photosynthesis in green plants. Chloroplasts exhibit aggregation and dispersion movements in response to changes in the light environment, primarily regulated by blue light receptors called phototropins (phot1 and phot2), which enhance the plant's ability to capture light.

[0004] In Arabidopsis, studies have shown that when light is limited, weak blue light (0.01–20 µmol m –2 s –1 ) will induce chloroplasts to aggregate on the mesophyll cell wall perpendicular to the direction of incident light (photoconcentration movement), maximizing light capture. In contrast, blue light intensity > 20 µmol m –2 s –1 When the light is in the direction of the incident light, it induces the chloroplasts to gather near the mesophyll cell wall parallel to the direction of the incident light (photophobic movement), protecting the chloroplasts from light damage and facilitating the distribution of light to other cell layers. In general, the specific physiological processes of different photokinesis are somewhat different. The mechanism of phototropism is as follows: the phototropism is redundantly regulated by the photoreceptors PHOT1 and PHOT2 (the phototropism is mainly regulated by PHOT2); the light signal is transmitted to actin through the signal network composed of genes such as CHUP1, PMI2, PMI1, and PMI15, and the actin (CP-actin) pulls the chloroplast to move to the side of the cell parallel to the direction of light; and the mechanism of light-gathering movement is as follows: the light-gathering movement is redundantly regulated by the photoreceptors PHOT1 and PHOT2 (mainly regulated by PHOT1); the light signal is transmitted to actin through the signal network composed of genes such as NCH1, RPT2, JAC1, KAC1, and KAC2, and the actin (CP-actin) pulls the chloroplast to move to the side of the cell perpendicular to the direction of light.

[0005] However, in actual research, the mechanisms of light-concentrating and light-avoiding movement in maize chloroplasts are unclear. In particular, maize chloroplast movement is a microscopic phenomenon that is difficult to observe directly, let alone quantify. This makes it difficult to assess chloroplast movement. Indirect leaf transmittance measurements or the FV / Fm ratio of photosynthesis are often used to assess chloroplast movement. However, these assessment methods do not provide a direct understanding of chloroplast movement within the cell.

[0006] On the other hand, when studying the light-concentrating and light-avoiding movement of corn chloroplasts, since corn belongs to C4 plants, compared with C3 plants whose chloroplasts are located in mesophyll cells, the chloroplasts of C4 plant corn are distributed in both mesophyll cells and bundle sheath cells. The problem caused by this distribution difference is that the chloroplasts in mesophyll cells can make more obvious movements when the light environment changes, while the chloroplasts in bundle sheath cells are often distributed in the center of the cell and will not make more obvious movements when the light environment changes. At the same time, because corn leaves have a large number of vascular bundles arranged in close proximity, and most of the chloroplasts are distributed in the bundle sheath cells, this makes it easy for large human errors to occur even when using a microscope to visually observe the light-avoiding or light-concentrating movement of corn chloroplasts.

[0007] In summary, establishing a relatively standard and accurate method for identifying and evaluating chloroplast light-avoiding or light-focusing movement for different plants, especially various crops, is of great technical significance for accurately reflecting the effects of photosynthesis and for the cultivation and identification of new varieties. Summary of the Invention

[0008] Based on living corn seedlings, the purpose of the present invention is to provide a method for observing and identifying the light movement (light-avoiding movement and light-focusing movement) of corn chloroplasts, thereby laying a certain technical foundation for technical improvements in the study of corn photosynthesis, the cultivation of new corn varieties, and the improvement of corn yield.

[0009] The technical solutions adopted in this application are briefly described as follows.

[0010] A method for observing and identifying the light movement of corn chloroplasts, wherein the light movement includes two aspects: light-collecting movement and light-avoiding movement. The method comprises the following steps:

[0011] (1) Seed germination

[0012] Take corn seeds, soak them in water, and culture them in the dark to induce germination and form yellow flower seedlings. The specific operation is as follows:

[0013] Soak corn seeds in pure water for 12 hours, then place them on absorbent paper (reference: 4-5 seeds per row, about 4-6 rows in total). Roll up the absorbent paper along one side to form a paper tube around the seeds, and wrap the tube with plastic wrap.

[0014] The paper tube was then placed vertically in an incubator (with the seed endosperm on the upper side and the embryo on the lower side). Taking the wild type maize B73 as an example, it was cultured in the dark at 28°C and a relative humidity of 65-75 RH for 3 days to form etiolated maize seedlings.

[0015] (2) Cultivation of seedlings

[0016] Transplant the etiolated corn seedlings that have germinated in step (1) into a mixture of soil (nutrient soil and coconut soil in a ratio of 1:2) in an artificial climate incubator at 28°C, 65-75RH, 80% (full light) intensity, and a photoperiod of 16 h white / 8 h dark for 15-25 days (20-25 days is the optimal growth range), applying distilled water or tap water during this period;

[0017] (3) Pretreatment

[0018] Dark-treat the corn seedlings in step (2) for 1 day at a temperature and humidity of 22°C and 65-75 RH.

[0019] (IV) Sampling, processing and observation

[0020] Take the corn leaves from step (3), cut the middle part of the third or fourth leaf from the bottom to the top (about 5 cm in length), place it on a 1-1.5% single agar medium, and cover the leaf surface with a layer of tin foil with a slit (about 5 mm in width) that allows light to pass through; then, place it in an incubator at 22°C and 65-75 RH.

[0021] For light-avoidance exercise, the inventors designed a 50-300 µmol m -2 s -1 The subjects were uniformly irradiated under gradient blue light for 8 h, and the most significant blue light intensity for light avoidance was determined to be 200 µmol m -2 s -1 ; then at 200 µmol m -2 s -1 A time gradient of 1 to 8 hours of blue light irradiation was designed to confirm that light-avoidance movement had significantly occurred within 4 hours;

[0022] For the focusing motion, the inventors designed a 5~50µmol m -2 s -1 The gradient blue light was uniformly irradiated for 12 h, and the most significant blue light intensity for the focusing movement was determined to be 10 µmol m -2 s-1 ; then at 10 µmol m -2 s -1 A 6-12 h time gradient irradiation was designed under blue light to confirm that the converging movement occurred significantly after 12 h.

[0023] (It should be explained that preliminary experiments have found that when the light intensity is greater than 50µmol m -2 s -1 When the chloroplasts begin to move slowly to avoid light, therefore, 50µmol m -2 s -1 Critical light intensity for maize chloroplasts to gather light and avoid light)

[0024] After the blue light irradiation treatment, the treated leaves were placed on a light board and photographed. The images were converted to black and white using Photoshop. The grayscale values ​​of the strips at the slit and the blocked parts of the leaves were measured using ImageJ software (the measurements were repeated three times in different areas, and the average value was taken as the measurement result). The relative brightness of the leaves at the slit was calculated using a formula, and the relevant results were further statistically analyzed.

[0025] The calculation formula is:

[0026]

[0027] in:

[0028] VGS is the relative gray value,

[0029] AC is the integrated gray value of the leaf at the occlusion point,

[0030] AL is the integrated gray value of the blade at the slit;

[0031] After taking the photo, cut the illuminated part of the leaf (about 1 cm x 1 cm), transfer it to a new 50 ml EP tube, add fixation buffer, evacuate the tube, and fix it at 4°C overnight.

[0032] The components of the Fixation buffer are:

[0033] 50 mM PIPES-NaOH, pH 6.9,

[0034] 4 mM MgSO4,

[0035] 10 mM EGTA,

[0036] 0.1% (w / v) TritonX-100,

[0037] 200 μM phenylmethylsulfonyl fluoride (PMSF),

[0038] 5% (v / v) formaldehyde and 1% (v / v) glutaraldehyde;

[0039] After fixation, the fixed leaves were embedded in 5% agarose gel and sliced ​​into 70-80 μm thickness using an oscillating microtome. The slices were observed and photographed using an optical microscope at 20X.

[0040] In actual research on chloroplast movement, the type of plant species and the physiological state of the leaves have a certain impact on chloroplast movement research. For example, combined with relevant preliminary experimental results, for corn, young leaves (5-15 days after germination) are dark green, but the leaves are thin, the mesophyll cells and chloroplasts are not fully developed, the size is small, and they have less cellulose and lignin, making it difficult to make slices for observation. On the other hand, old leaves (more than 25 days after germination) are light green, the mesophyll cells and chloroplasts are fully developed, but the cellulose and lignin are high, making it difficult to make slices for observation. Therefore, in actual research, when studying the problem of light movement, the physiological state of the leaves needs to be paid special attention and clarified.

[0041] Overall, this application uses corn as the research object. Based on an in-depth study of the characteristics of chloroplast photomovement in corn leaves, the observation and identification system of corn chloroplast photomovement is optimized. Key observation and identification conditions such as the optimal growth days of leaves in sliced ​​materials are clarified, which can lay a certain technical foundation for the establishment of a standardized identification system for corn chloroplast photomovement. At the same time, it also provides a good reference and reference for the establishment of microscopic movement identification systems for other organisms. Based on this work, it has very important basic technical significance for the in-depth analysis of the corn photomovement mechanism, as well as the screening of related parents and the breeding of new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the light-avoiding and light-collecting movements of corn chloroplasts;

[0043] Figure 2 The blue-white pattern observation of maize chloroplasts avoiding light under different light intensities and time treatments;

[0044] A is 50-300 µmol m -2 s -1 The results of light-proof white spots on chloroplast slits after 8 hours of irradiation under gradient light intensity;

[0045] B is 200 µmol m -2 s-1 The results of light-proof white spots on chloroplast slits after 1-8h time gradient irradiation under light intensity;

[0046] C and D are the grayscale value statistics of Figures A and B respectively. The larger the grayscale value, the stronger the light transmittance and the more obvious the light avoidance.

[0047] Figure 3 This is the phenotypic observation of maize chloroplasts avoiding light induced by high-intensity blue light. In the figure, B represents vascular cells and M represents mesophyll cells.

[0048] Figure 4 To observe the phenotypic pattern of maize chloroplast light-concentrating movement induced by blue light of different intensities;

[0049] Figure 5 Phenotypic observation of the light-concentrating movement of maize chloroplasts induced by low-intensity blue light. DETAILED DESCRIPTION

[0050] The present application will be further explained below with reference to the following examples. Before introducing the specific examples, a brief description of the experimental background of some of the following examples is given below.

[0051] Biomaterials:

[0052] Maize wild-type inbred line B73, a commonly used and common germplasm material in existing maize research, is available through public channels;

[0053] Experimental reagents and main instruments and equipment:

[0054] Vibrating microtome VT1200S, Leica Microsystems;

[0055] The relevant test reagents are all chemically pure products and can be purchased from public channels.

[0056] Example 1

[0057] The anatomy of maize physiological structures shows that due to the unique structure of maize leaves, the phenotype of chloroplast photokinesis is difficult to observe. Therefore, it is necessary to establish a targeted, rapid and direct method and system for observing the photokinesis state of chloroplasts. The method for observing and identifying maize chloroplast photokinesis provided in this application specifically includes the following steps.

[0058] (1) Seed germination

[0059] Soak corn seeds in pure water for 12 hours, then place them horizontally on absorbent paper (4-5 seeds per row, about 4-6 rows in total). Roll up the absorbent paper along one side to form a paper tube around the seeds, and wrap the tube with plastic wrap.

[0060] The paper tube was then placed vertically in an incubator (with the seed endosperm on the upper side and the embryo on the lower side of the paper tube) and cultured in the dark at 28°C and 70RH for 3 days to form yellowing maize seedlings (when cultivating materials, unified germination dark treatment was performed to ensure that the materials were exposed to light at the same time for photomorphogenesis, so as to eliminate interference caused by inconsistent growth and development of the materials).

[0061] (2) Cultivation of seedlings

[0062] Transplant the etiolated corn seedlings that have germinated in step (1) into the proportioned soil (nutrient soil and coconut soil ratio is 1:2) in an artificial climate incubator at 28°C, relative humidity 65-75RH, 80% (full light) intensity, and a photoperiod of 16 hours white / 8 hours dark for 15-25 days, applying distilled water during the period;

[0063] (3) Pretreatment

[0064] Dark-treat the corn seedlings in step (2) for 1 day at a temperature and humidity of 22°C and 65-75RH.

[0065] (IV) Sampling, processing and observation

[0066] Take the corn leaves from step (3), cut the middle part of the third or fourth leaf that is in good growth from bottom to top (about 5 cm in length), place it on a 1.5% agar plate, and cover the surface of the leaf with a layer of tin foil with a slit (about 5 mm in width) that allows light to pass through; then, place it in an incubator at 22°C and 65-75 RH.

[0067] For light-avoiding movement and light-focusing movement, different light intensities and different illumination durations are used for processing respectively.

[0068] After the blue light irradiation treatment, the treated leaves were placed on a light board and photographed. The images were converted to black and white using Photoshop. The grayscale values ​​of the strips at the slit and the blocked parts of the leaves were measured using ImageJ software (the measurements were repeated three times in different areas, and the average value was taken as the measurement result). The relative brightness of the leaves at the slit was calculated using a formula, and the relevant results were further statistically analyzed.

[0069] The calculation formula is:

[0070] ;

[0071] Where: VGS is the relative gray value, AC is the integrated gray value of the leaf at the occlusion, and AL is the integrated gray value of the leaf at the slit.

[0072] After taking the photo, cut the illuminated part of the leaf (about 1 cm x 1 cm), transfer it to a new 50 ml EP tube, add fixation buffer, evacuate the tube, and fix it at 4°C overnight.

[0073] The components of the Fixation buffer are:

[0074] 50 mM PIPES-NaOH, pH 6.9,

[0075] 4 mM MgSO4,

[0076] 10 mM EGTA,

[0077] 0.1% (w / v) TritonX-100,

[0078] 200 μM phenylmethylsulfonyl fluoride (PMSF),

[0079] 5% (v / v) formaldehyde and 1% (v / v) glutaraldehyde;

[0080] After fixation, the fixed leaves were embedded in 5% agarose and sliced ​​into 70 μm thickness using an oscillating microtome. The slices were observed and photographed using an optical microscope at 20X.

[0081] During the experiment, the inventors selected leaves of different growth periods for slice observation experiments based on the growth time of corn seedlings in step (2). The results showed that:

[0082] When the growth period is 5-15 days, the leaves have less cellulose and lignin, making them tender, which is not conducive to subsequent fixation. In addition, their chloroplasts and mesophyll cells are not fully developed, which is not conducive to fiber observation after sectioning.

[0083] After growing for more than 25 days, the leaves are green and the light-avoiding movement phenotype can be clearly observed. However, due to the high content of cellulose and lignin, the difficulty of slicing is greatly increased.

[0084] In general, when the growth time is 15-25 days, the chloroplast development of the leaves has been basically completed, the shape is full and the vitality is good, which is suitable for observation under a microscope. At this time, the cellulose and lignin content of the leaves is moderate, which is suitable for slicing operations.

[0085] During the experiment, the results of the phenotypic difference analysis of different light intensities and different irradiation durations (leaves with a growth time of 20 days) showed that ( Figure 2 (shown), blue light intensity 200µmol m-2 s -1 When the irradiation time was 4 h, the light-avoiding movement phenotype showed significant differences (the slice results are shown in Figure 3 As shown in the figure, chloroplasts are located on the left and right sides of the mesophyll cells, and no chloroplasts are found on the upper and lower sides of the cells. -2 s -1 When the leaves were irradiated with blue light for 12 h, they showed typical focusing motion (the results are shown in Figure 4 As shown, chloroplasts in mesophyll cells are mostly distributed on the upper and lower sides, and there are almost no chloroplasts on the left and right sides of the cells).

[0086] Furthermore, based on the above results, combined with the results of the focal motion phenotype and tissue observation analysis ( Figure 5 As shown), the inventors constructed Figure 1 The light-avoiding and light-focusing movement patterns are shown.

[0087] Overall, this application has developed an observation and identification system that can quickly and directly observe the phenomenon of maize chloroplast movement to avoid light and focus light, and has achieved significant experimental results. Using this method and system, we can not only quickly screen for mutants with abnormal chloroplast movement to avoid light in maize, but also use it to identify known mutants with abnormal chloroplast movement to avoid light and focus light.

Claims

1. A method for observing and identifying the light-collecting and light-avoiding movements of corn chloroplasts, characterized in that: The method comprises the following steps: (1) Seed germination Take corn seeds, soak them in water, and culture them in the dark to induce germination and form yellow flower seedlings; (2) Cultivation of seedlings Transplant the etiolated corn seedlings that have germinated in step (1) into soil and cultivate them for 15-25 days; (3) Pretreatment Dark-treat the corn seedlings prepared in step (2) for 1 day; (IV) Sampling, processing and observation Take the corn leaves from step (3), cut the middle part of the third or fourth leaf from the bottom up, place it on an agar plate, and cover the surface of the leaf with a layer of tin foil with a narrow slit to allow light to pass through; For light-avoiding exercise, blue light irradiation was performed for 1–8 h; the blue light intensity was 50–300 µmol m -2 s -1 ; For light-focusing motility, blue light irradiation was performed for 6–12 h; the blue light intensity was 5–50 µmol m -2 s -1 ; After the blue light irradiation treatment is completed, the treated leaves are placed on a light board and photographed. After black and white conversion, the grayscale values ​​of the strips at the slit of the leaves and the blocked part are measured respectively, and the relative brightness of the leaves at the slit is calculated; The calculation formula is: in: VGS is the relative gray value, AC is the integrated gray value of the leaf at the occlusion point, AL is the integrated gray value of the blade at the slit; After taking the photo, cut the illuminated part of the leaf, transfer it to an EP tube, and add Fixation buffer to fix it; After fixation, the fixed leaves were embedded, sliced ​​using an oscillating microtome, and observed under a light microscope.

2. The method for observing and identifying the light-collecting and light-avoiding movement of corn chloroplasts according to claim 1, wherein: In step (1), the corn seeds are B73.

3. The method for observing and identifying the light-gathering and light-avoiding movement of corn chloroplasts according to claim 1, wherein: In step (1), the specific operation is as follows: after soaking the corn seeds in pure water for 12 hours, placing them on absorbent paper, wrapping them into a paper tube, and wrapping the paper tube with plastic wrap, with the seed endosperm on the upper side and the embryo on the lower side of the paper tube; then placing the paper tube vertically in an incubator, and incubating them in the dark at 28°C and a relative humidity of 65-75RH for 3 days to form yellow corn seedlings.

4. The method for observing and identifying the light-gathering and light-avoiding movement of corn chloroplasts according to claim 1, wherein: In step (2), the culture conditions are: 28°C, relative humidity 65-75RH, 80% full light intensity, photoperiod 16 hours white / 8 hours dark, and cultivation for 20-25 days.

5. The method for observing and identifying the light-gathering and light-avoiding movement of corn chloroplasts according to claim 1, wherein: In step (3), the temperature and humidity requirements are: 22°C, 65-75RH.

6. The method for observing and identifying the light-gathering and light-avoiding movement of corn chloroplasts according to claim 1, wherein: In step (4), the width of the slit on the tin foil is 5 mm.

7. The method for observing and identifying the light-gathering and light-avoiding movement of corn chloroplasts according to claim 1, wherein: In step (4), for light-avoiding exercise, blue light irradiation was performed for 4 h, and the blue light intensity was: 200 µmol m -2 s -1 For focusing motion, the blue light was irradiated for 12 h with a blue light intensity of 10 µmol m -2 s -1 .

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