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Assessing Plant Tolerance to Acute Heat Stress
植物对急性热胁迫耐受性的评估   

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Abstract

It is well-established that plants are able to acclimate to temperatures above or below the optimal temperature for their growth. Here, we provide protocols for assays that can be used quantitatively or qualitatively to assess the relative ability of plants to acquire tolerance to high temperature stress. The hypocotyl elongation assay described was developed to screen for mutants defective in the acquisition of tolerance to extreme temperature stress, and other assays were developed to further characterize mutant and transgenic plants for heat tolerance of other processes or at other growth stages. Although the protocols provide details for application to Arabidopsis thaliana, the same basic methods can be adopted to assay heat tolerance in other plant species.

Keywords: Thermotolerance(耐热性), Greening(绿化), Arabidopsis(拟南芥), Heat shock proteins(热休克蛋白)

Background

It is well-established that plants are able to acclimate to temperatures above or below the optimal temperature for their growth, and many studies have identified genes necessary for, or associated with temperature acclimation. Typically, acclimation requires a period of exposure to a non-damaging temperature treatment, either above optimal to induce heat tolerance, or below optimal to induce cold or freezing tolerance. In Arabidopsis and other plants, freezing tolerance can be achieved within a period of 24 h of cold treatment (cold hardening), with maximum freezing tolerance occurring after several days of hardening (Gilmour et al., 1988; Thomashow, 1999). Tolerance to normally lethal or damaging high temperatures can develop more rapidly, within a few hours. This type of rapid heat acclimation was extensively documented by Yarwood (1967) and others in the 1960s using a variety of plant species. Research on high temperature acclimation accelerated when it was recognized in the early 1980s that all organisms, including plants, responded to heat stress with a ‘heat shock response’ that involves transcription and translation of a conserved set of ‘heat shock proteins’ (Lindquist, 1986; Vierling, 1991). Work with soybean seedlings, similar to the earlier work of Yarwood, defined a simple assay for hypocotyl elongation that allowed investigation of the relationship of heat acclimation to the heat shock response (Lin et al., 1984). This assay was then extended to Arabidopsis and used to identify mutants altered in heat acclimation, in both forward and reverse genetic screens (Hong and Vierling, 2001; Larkindale et al., 2005; Kim et al., 2012). Other assays for heat acclimation were also developed and used for mutant screening in Arabidopsis, including heat acclimation of greening of dark grown seedlings (Burke et al., 2000) and seedling viability (Wu et al., 2013).

Because plants can experience rapid daily temperature cycles, it is perhaps not surprising that heat tolerance can be acquired on a time scale consistent with diurnal changes in temperature, and that acclimation treatments afford maximal protection if they are administered 24 h or less before the imposition of damaging heat stress. It is also important to recognize that plant responses to temperature vary significantly with the length and severity, as well as the developmental timing of the temperature treatment. A review by Yeh et al. (2012) provides an excellent description of how variations in temperature treatments can affect phenotypic outcomes. Here we describe in detail basic methods that have been used to assess the ability of plants to acclimate to severe high temperature using Arabidopsis thaliana. Done precisely, these assays can provide quantitative information on the relative heat tolerance of different plant genotypes or of plants grown under different conditions. They can also readily be developed to use with other plant species.

Materials and Reagents

  1. 100 x 15 mm square Petri dish with grid (Simport, catalog number: D210-16 )
  2. Any brand sterile pipette tips that fit pipettors in 3 above
  3. Parafilm (Bemis, catalog number: PM996 )
  4. Aluminum foil (any brand)
  5. 100 x 15 mm round Petri dish (Fisher Scientific, catalog number: S33580A )
  6. Filter paper
  7. Arabidopsis thaliana Columbia-0 seeds, hot1-3 mutant seeds (ABRC, catalog number: CS16284 )
  8. Household Bleach (Clorox or any brand that contains 5.25% sodium hypochlorite, 6 month shelf-life when stored at room temperature)
  9. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  10. MS basal salt mixture (Sigma-Aldrich, catalog number: M5524 ), store at 4 °C
  11. 2-(N-Morpholino) ethanesulfonic acid (MES) hydrate (Sigma-Aldrich, catalog number: M8250 )
  12. Phyto agar (Sigma-Aldrich, catalog number: A1296 )
  13. Sucrose (Fisher Scientific, catalog number: S5-500 )
  14. Potassium hydroxide (KOH) (Fisher Scientific, catalog number: P250-500 )
  15. Seed sterilization solution (see Recipes)
  16. Half-strength MS agar media (see Recipes)

Equipment

  1. Any brand of pipettors to dispense 2 to 20 μl, 20 to 100 μl, and 0.1 to 1.0 ml (e.g., Eppendorf, catalog numbers: 4924000037 , 4924000061 , 3123000063 ; or comparable)
  2. Growth chamber (Percival Scientific, model: AR41L2 )
  3. Oven incubator (Precision, model: Thelco incubator 3DM , catalog number: 51221120)
  4. Sharp pen
  5. Scale loupe (CWJ, Peak, model: 1983 10x Scale Loupe )
  6. Dissecting scope (Leica Microsystems, model: Leica MZ6 or comparable)
  7. Leveling table (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: H18310-0000 )
  8. 1 L glass bottles (Fisher Scientific)
  9. Autoclave

Software

  1. Microsoft Excel
  2. ImageJ (optional)

Procedure

  1. Hypocotyl elongation assay for testing acclimation to high temperature
    The assay described here can provide quantitative data on the ability of seedlings to develop tolerance to a normally lethal/severely damaging high temperature stress.
    1. Preparing plates with sterile seeds
      1. Surface-sterilize Arabidopsis thaliana seeds of the genotype of interest with 50% Bleach solution containing 0.1% Triton X-100 (seed sterilization solution [see Recipes]) by soaking in the solution for 10 min. Using relatively fresh seeds (less than one year of harvest) is recommended because there is less variability in germination rate.
      2. Rinse seeds with sterile water five times to remove any residual Bleach in a sterile environment.
      3. In a sterile environment, place the seeds on half-strength MS agar media (see Recipes) in square plates with grids using sterile pipette tips that are cut appropriately (a couple of millimeters) at the tip to allow passage of the seeds, ideally one by one. We usually put 12-13 seeds on each line with even spacing between seeds (Figure 1). Stagger the seed placement from one line to the next in order to avoid contact when the hypocotyls elongate. It is important to put appropriate control samples (wild-type and a heat sensitive mutant) on every plate.
      4. Wrap the plates with Parafilm and put the plates in 4 °C for 2-3 days, which helps synchronize germination.


        Figure 1. Diagram of a plate with seeds placed evenly on each line

    2. Seedling growth and heat treatments
      1. Growth prior to heat stress
        Wrap the plates carefully with aluminum foil so that the seedlings are kept in the dark; hypocotyls elongate more in the dark. Mark the foil to indicate the bottom edge of the plate relative to expected upward growth of the seedlings. Let the seeds germinate and grow along the surface of the media by supporting the plates in a vertical orientation in a growth chamber for about 2.5 days at 22 °C. Plates could also be kept in any dark place at the appropriate temperature. The length of the hypocotyl should be roughly about 6 to 9 mm (for the wild-type Columbia accession) when ready for heat stress treatment.
      2. Heat acclimation treatment
        Unwrap the plates from aluminum foil and put them in an oven incubator in the dark set at 38 °C for the acclimation treatment for 1.5 h. Place plates horizontally for even heat transfer from the shelf to the media (Figure 2). Another replicate set of plates needs to be kept at 22 °C (or whatever the normal growth temperature is) as a control. Minimize the time the incubator is open in order to reduce temperature fluctuation and exposure of the seedlings to light. Many heat shock proteins are induced during this period (Lin et al., 1984; Hong and Vierling, 2000).


        Figure 2. Plates are placed horizontally in an oven incubator

      3. Acclimation recovery period
        Take the plates out of the incubator and keep at 22 °C for two hours in a dark place, again placing them upright. The plants recover from the mild heat treatment during this period and continue synthesizing heat shock proteins.
      4. Severe, acute heat stress
        Put the plates in a 45 °C incubator horizontally for a desired length of time. Two hours are sufficient to completely inhibit the growth of hot1-3 (HSP101 knock-out Arabidopsis line that has a T-DNA insertion in AT1G74310) plants, while allowing recovery of wild-type plants (Hong and Vierling, 2001). Depending on the heat sensitivity of the plants being tested, the length of heat stress at 45 °C can be varied, e.g., 1.5 h, 2.0 h, 2.5 h, 3.0 h. You should also have as a control replicate plates that have received the acclimation treatment, but that have not been treated at 45 °C. Make sure you quickly open and close the incubator when taking out plates in order to minimize temperature drop of remaining plants inside.
      5. Recovery after severe heat stress
        After the heat stress treatment, mark on the plate the position of the tip of each hypocotyl with a sharp pen, and cover the plates again with foil, being sure to note the growth direction of the seedlings. Place the plates again in a vertical position and let the plants recover and grow at 22 °C for about 2.5 days. Minimize light exposure when the marking is done, as during the rest of the procedure. If the seedlings are exposed to light for a relatively long time, you will end up with cotyledon expansion instead of hypocotyl elongation.
      6. Hypocotyl length measurement
        After the period of recovery, remove the foil and mark the tip of the hypocotyls again. At this point the plates can be placed at 4 °C until measured. A typical result for wild-type Columbia is shown in Figure 3. The length of hypocotyl elongation can be measured by eye using a scale loupe. Alternatively, photograph the plates and use the measuring tool of ImageJ to perform the measurements (Schneider et al., 2012).


        Figure 3. Heat stress phenotype of wild-type A. thaliana (Columbia accession) seedlings. After the treatments indicated, seedlings were transferred to a new plate and photographed (Modified from Hong and Vierling, 2000). AC: Acclimation treatment: 38 °C/1.5 h followed by 22 °C/2 h.

  2. Heat stress assay for light-grown 7-10 day old seedlings
    1. Surface-sterilize seeds and plate them on round Petri dishes with MS agar media as described in section A1. Divide plates into equal sectors. If you divide 100 x 15 mm Petri dishes into six sectors, place 18-20 seeds on each sector. It is a good practice to put the same genotype in sectors on opposite sides of the plate for duplication.
    2. Wrap the plates with Parafilm and put them at 4 °C for 2-3 days for stratification.
    3. Move the plates to a growth chamber and let the seedlings grow for 7-10 days under long day conditions (22 °C/18 °C, 16-h-day/8-h-night cycle) with a light intensity at ~80 μmol m-2 sec-1. Short days can also be used if they provide better growth for a particular genotype.
    4. Perform heat treatments in an oven incubator as described above for the hypocotyl elongation assay, varying the length of time as necessary in order to distinguish between genotypes. Acclimation treatment is the same as for the hypocotyl elongation assay. Again, try to minimize temperature drop by acting quickly when opening and closing the incubator. Make sure you have control plates with no acclimation treatment, as well as plates maintained continuously at optimal growth temperatures for comparison.
    5. After the heat treatment, put the plates back in a growth chamber and let them recover for 5-8 days. You can take a picture when the phenotypic differences are optimal.

  3. Heat tolerance of germination
    Before germination seeds are significantly more heat tolerant and variation in heat tolerance of seeds between Arabidopsis accessions has been documented (Clerkx et al., 2004).
    1. It is not necessary to sterilize seeds for this test. However, because germination is sensitive to maternal effects, as well as seed storage conditions, only seeds grown and harvested at the same time and stored under the same conditions should be used in comparisons.
    2. Sow seeds in Petri dishes on filter paper saturated with the same volume of water for all plates. Volume should be sufficient to completely wet the filter paper without leaving excess water. 30 to 50 seeds of each genotype to be tested are spread evenly on the filter paper, using a separate plate for each genotype. Seeds are allowed to imbibe for 18 h at room temperature. To retain moisture, Petri dishes should be enclosed in plastic boxes lined on the bottom with water saturated filter paper throughout the assay.
    3. Perform heat treatment by direct transfer of the plates to 50 °C for 8 h, then return them to room temperature to score germination. Use identical plates maintained at room temperature as a control for germination potential of the seed lot.
    4. Determine germination percentage at an end point after 7 days, or record germination daily for estimates of differences in germination rate. Score small seeds, such as Arabidopsis, under a dissecting scope, or alternatively by photographing and scoring enlarged pictures viewed on a computer screen. Score seeds as germinated at the first sign of radical emergence.

  4. Heat tolerance of seedling greening
    The ability of dark grown seedlings to accumulate chlorophyll can be tested for heat tolerance either with or without an acclimation treatment. The assay below does not employ acclimation.
    1. Prepare plates with seedlings as for the hypocotyl elongation assay and grow seedlings for 2.5 to 3 days, vertically, in the dark.
    2. Treat plates with seedlings at 43 °C for 120 min in the dark. Leave a duplicate set of plates untreated. Different lengths of time at 43 °C can also be tested to provide resolution between genotypes if necessary.
    3. Place the plates in the light, horizontally or vertically, either at room temperature in room light or low light (50 to 80 µE m-2 sec-1) in a growth chamber at 22 °C. Any day length or continuous light can be used. Record the light intensity at the plate level for reference in future experiments.
    4. Record cotyledon greening daily, judged by eye as number of seedlings with green, light green, yellow green, or yellow cotyledons. Score again at an end point after three days in the light. Cotyledons can also be counted daily for expansion/opening.

  5. Other heat stress treatments
    The heat stress treatments described above have been used extensively in studies of Arabidopsis. However, the exact times and temperatures can also be varied to assay different phenotypes. For example, acclimation pretreatments can be performed by raising the temperature gradually from 20 to 22 °C to the stress temperature of 45 °C over the course of 3 to 4 h (Larkindale and Vierling, 2008). This requires an appropriate chamber with temperature ramping capabilities, which is a limiting resource for most laboratories. However, this gradual acclimation treatment was documented to provide better seedling viability after recovery from stress than the acclimation treatment described above. Another parameter than can be varied is the time between the acclimation treatment and the heat stress treatment, which can assess differences in the ability of different genotypes to maintain acclimation. Yeh et al. (2012) describe other parameters that can be modified in assays for heat tolerance.

  6. Adapting assays to other plant species
    As noted in the background section, the original assay for hypocotyl elongation was adapted from experiments with soybean (Lin et al., 1984), and other plant species can be similarly assayed for acclimation to high temperatures. For this purpose, it is simply necessary to perform careful preliminary experiments to establish the rate of seedling germination and extent of hypocotyl growth or other parameters, as well as the time and temperature that results in heat killing or seedling growth arrest for each species at different growth stages.

Data analysis

  1. Hypocotyl elongation assay
    Using Microsoft Excel, plot genotypes on the x-axis and corresponding hypocotyl lengths on the y-axis. When there are mutants that are defective in growth, normalize the length relative to the control (no heat treatment) growth for each genotype. An example is shown in Figure 4. Typical standard deviation from 45 °C/2 h treatment after acclimation is about 10% for wild-type Columbia, when the lengths are normalized to the control growth. Statistical significance can be tested by performing a Student’s t-test. At least three independent experiments must be performed with similar results for reproducibility.


    Figure 4. An example plot showing hypocotyl elongation of various Arabidopsis genotypes after heat treatment (45 °C/80min followed by heat acclimation, modified from Kim et al., 2012). The shot1-2 mutant grows more slowly than the wild-type and hot1-3, so the hypocotyl growth was normalized by the growth during the same time period for seedlings that had not been heat treated. Error bars indicate SE; n = 12.

  2. Heat stress assay for light-grown 7-10 day old seedlings
    Take a picture when the phenotypic differences are optimal, which happens usually 5 to 8 days after heat treatment. An example is shown in Figure 5. To make the assay more quantitative, count seedlings that have survived and graph the percentage of survival as in Larkindale et al. (2005). As detailed in the procedure, it is also good practice to plant sectors of the same genotype on opposite sides of the plate, to account for differences in heat distribution. Even small temperature differences can result in different outcomes. It is also critical to have genotypes to be compared on the same plate. At least three independent experiments must be performed with similar results for reproducibility.


    Figure 5. 10 day old seedlings were heat-stressed as shown above the picture. Picture was taken 7 days after the treatment (modified from Kim et al., 2012).

  3. Heat tolerance of germination
    Express results of seed germination as a percentage of untreated seeds of the same seed lot. If data are collected daily, results can be plotted as a line graph. If data are collected only at an end point, use a table to display the data.
  4. Heat tolerance of seedling greening
    Express results of cotyledon greening as a bar graph showing for each day the percentage of cotyledons at different stages of greening compared to seedlings that were not heat treated. Similarly, percent of open cotyledons can also be graphed as a percentage of untreated seedlings. Statistical significance can be tested by performing a Student’s t-test. At least three independent experiments should be performed with 20 to 30 seedlings per genotype and treatment.

Notes

  1. Be careful not to damage the aluminum foil covering for dark grown seedlings. Also, minimize light exposure of seedlings during handling. Light leakage makes plants undergo photomorphogenesis with cotyledons opening and expanding, while preventing hypocotyl elongation.
  2. It is important to not overload the incubator and to be aware of any dramatic differences in temperature in different parts of your incubator. It is best to use a thermometer to check the incubator temperature in case the calibration of the incubator is not accurate, and also to compare different incubators. Seedlings are very sensitive to the exact temperature, and differences of as little as ± 1 °C can alter the outcome significantly.
  3. We use an oven incubator for our dark heat treatment. However, other researchers put tightly sealed plates in a water bath, which may help even heat transfer and prevent temperature fluctuation (Charng et al., 2006).
  4. Marking seedling growth is important, as it allows you to separate growth before heat stress from growth after heat stress. This can normalize for not only differences in hypocotyl length between genotypes, but also differences in germination rates.
  5. When making plates with MS agar media, pour the same amount of media (e.g., 10 ml for the 100 x 15 mm square Petri dish) in each plate on a leveling table. This will reduce temperature variability between plates and also within a plate.
  6. When measuring heat tolerance of germination, all genotypes should be tested in three replicates for statistical analysis. Results should be expressed relative to germination of the unheated controls. The temperature or time of heat stress can be varied to increase resolution of differences between genotypes as necessary. If seeds have germinated after the 18 h imbibition period, shorten the time of imbibition prior to heat treatment.
  7. For all assays described here, comparisons between genotypes are made by comparing percentages calculated based on untreated seeds or seedlings of the same genotype. In addition, whenever possible, it is preferable to use seeds from plants that were grown at the same time and under the same conditions, and to store the seeds under the same conditions. This is important for determining if there are significant differences in heat tolerance between genotypes, especially if differences are small. Repeating with more than one batch of seeds is critical for confirming small differences between genotypes.
  8. For assays of heat stressed light grown seedlings or for heat tolerance of cotyledon greening, quantitative results can also be obtained by extracting chlorophyll from equal numbers of seedlings from control and heat stressed samples as developed by Burke et al. (2000).

Recipes

  1. Seed sterilization solution (for 100 ml)
    50 ml Bleach
    50 ml distilled water
    500 µl 20% Triton X-100 (0.1% final concentration)
    Note: The solution can be kept in a dark place for up to 1 month at room temperature.
  2. Half-strength MS agar media (for 1,000 ml)
    2.15 g MS basal salt mixture (stored at 4 °C)
    0.5 g 2-(N-Morpholino) ethanesulfonic acid (MES) hydrate
    5 g sucrose
    Add 900 ml of distilled water and stir to dissolve
    Adjust pH to 5.7 using 1 N KOH
    Add distilled water to the final volume of 1,000 ml
    Divide the media into two 1 L glass bottles. Add 4 g of phyto agar into each bottle
    Autoclave for 30 min at 121 °C, 15 psi
    Note: After the solution is autoclaved, let it cool to approximately 60 °C and pour a fixed amount of media (10-30 ml) in each plate on a leveling table in a sterile environment. The amount of agar influences the rate at which the temperature increases in the interior of the plate, and it is therefore important to add the exact same amount of media per plate. After pouring the plates, let them solidify and dry for 20 min in a sterile environment. Unused media plates can be stored upside down at 4 °C for months in a sealed container.

Acknowledgments

Assays reported here have been developed with support of grants from the US Department of Energy’s Basic Biosciences program, the US Department of Agriculture’s National Research Initiative Competitive Grants program, and the National Science Foundation Molecular Biosciences Division in the Directorate for Biological Sciences to EV. Seed germination assays were developed with support of a Guggenheim Fellowship to EV. These protocols were adapted and modified from Hong and Vierling, 2000 and Larkindale et al., 2005.

References

  1. Burke, J. J., O’Mahony, P. J. and Oliver, M. J. (2000). Isolation of Arabidopsis mutants lacking components of acquired thermotolerance. Plant Physiol 123(2): 575-588.
  2. Charng, Y. Y., Liu, H. C., Liu, N. Y., Hsu, F. C. and Ko, S. S. (2006). Arabidopsis Hsa32, a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation. Plant Physiol 140(4): 1297-1305.
  3. Clerkx, E. J., El-Lithy, M. E., Vierling, E., Ruys, G. J., Blankestijn-De Vries, H., Groot, S. P., Vreugdenhil, D. and Koornneef, M. (2004). Analysis of natural allelic variation of Arabidopsis seed germination and seed longevity traits between the accessions Landsberg erecta and Shakdara, using a new recombinant inbred line population. Plant Physiol 135(1): 432-443.
  4. Gilmour, S. J., Hajela, R. K. and Thomashow, M. F. (1988). Cold acclimation in Arabidopsis thaliana. Plant Physiol 87(3): 745-750.
  5. Hong, S.W., and Vierling, E. (2000). Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proc Natl Acad Sci U S A 97: 4392-4397.
  6. Hong, S.W., and Vierling, E. (2001). Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress. Plant J 27(1): 25-35.
  7. Kim, M., Lee, U., Small, I., des Francs-Small, C. C. and Vierling, E. (2012). Mutations in an Arabidopsis mitochondrial transcription termination factor-related protein enhance thermotolerance in the absence of the major molecular chaperone HSP101. Plant Cell 24(8): 3349-3365.
  8. Larkindale, J., Hall, J. D., Knight, M. R. and Vierling, E. (2005). Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol 138(2): 882-897.
  9. Larkindale, J. and Vierling, E. (2008). Core genome responses involved in acclimation to high temperature. Plant Physiol 146(2): 748-761.
  10. Lin, C. Y., Roberts, J. K. and Key, J. L. (1984). Acquisition of thermotolerance in soybean seedlings: synthesis and accumulation of heat shock proteins and their cellular localization. Plant Physiol 74(1): 152-160.
  11. Lindquist, S. (1986). The heat-shock response. Annu Rev Biochem 55: 1151-1191.
  12. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  13. Thomashow, M. F. (1999). PLANT COLD ACCLIMATION: Freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50: 571-599.
  14. Vierling, E. (1991). The roles of heat shock proteins in plants. Annu Rev Plant Physiol Plant Mol Biolo 42: 579-620.
  15. Wu, T. Y., Juan, Y. T., Hsu, Y. H., Wu, S. H., Liao, H. T., Fung, R. W. and Charng, Y. Y. (2013). Interplay between heat shock proteins HSP101 and HSA32 prolongs heat acclimation memory posttranscriptionally in Arabidopsis. Plant Physiol 161(4): 2075-2084.
  16. Yarwood, C. E. (1967). Adaptation of plants and plant pathogens to heat. In: Prosser, C. L. (Ed.). Molecular mechanisms of temperature adaptation. Amer Ass Adv Sci pp: 75-89.
  17. Yeh, C. H., Kaplinsky, N. J., Hu, C. and Charng, Y. Y. (2012). Some like it hot, some like it warm: phenotyping to explore thermotolerance diversity. Plant Sci 195: 10-23.

简介

植物能够适应温度高于或低于最佳温度以达到其生长的目的是确定的。 在这里,我们提供可用于定量或定性评估植物获得耐高温胁迫耐受性的相对能力的测定方案。 开发了描述的下胚轴延伸测定以筛选获得对极端温度胁迫的耐受性的缺陷突变体,并且开发了其他测定以进一步表征突变体和转基因植物用于其它过程的耐热性或在其它生长阶段。 虽然方案提供了应用于拟南芥的细节,但是可以采用相同的基本方法来测定其他植物物种的耐热性。
【背景】植物能够适应温度高于或低于其最佳温度以达到其生长的目的已经确定,许多研究已经确定了与温度适应有关的基因。通常,适应需要暴露于非破坏性温度处理的时期,要么超过最佳以诱导耐热性,要么低于最佳诱导感冒或冷冻耐受性。在拟南芥和其他植物中,冷冻耐受性可以在冷处理(冷硬化)的24小时期内实现,在几天硬化后发生最大的冷冻耐受性(Gilmour等人,1988; Thomashow,1999)。在几个小时内,对正常致命或破坏性高温的耐受性可以更快地发展。这种类型的快速驯化被Yarwood(1967)和其他人在20世纪60年代使用各种植物物种进行了广泛的记录。高温适应性研究在20世纪80年代早期得到认可时加速,包括植物在内的所有生物体都受到热应激的反应,“热休克反应”涉及转录和翻译一组保守的“热休克蛋白”(Lindquist, 1986; Vierling,1991)。与早期的Yarwood的工作类似,与大豆幼苗合作,定义了一种用于下胚轴伸长的简单测定法,允许研究热适应与热休克反应的关系(Lin等人,1984)。然后将该测定扩展到拟南芥,并用于鉴定正向和反向遗传筛选中热驯化中改变的突变体(Hong和Vierling,2001; Larkindale等人, 2005; Kim等人,2012)。还开发了用于热驯化的其他测定法,并用于拟南芥中的突变筛选,包括黑化生长的幼苗的绿化驯化(Burke等,2000)和幼苗生存力(Wu等人,2013)。
因为植物可以经历每日温度周期的快速增长,所以可以在与昼夜温度变化相符的时间尺度上获得耐热性也许不足为奇,如果在适应条件下施用24小时以上施加有害的热应激。同样重要的是认识到植物对温度的反应随着长度和严重程度以及温度处理的发育时间而显着变化。 Yeh等人的综述。 (2012)提供了一个很好的描述温度处理的变化如何影响表型结果。在这里,我们详细描述已经用来评估植物适应严重高温的能力的基本方法,使用拟南芥(Arabidopsis thaliana)。准确地说,这些测定可以提供关于不同植物基因型或在不同条件下生长的植物的相对耐热性的定量信息。它们也可以容易地开发成与其他植物物种一起使用。

关键字:耐热性, 绿化, 拟南芥, 热休克蛋白

材料和试剂

  1. 100 x 15毫米方格带网格的培养皿(Simport,目录号:D210-16)
  2. 任何品牌的无菌移液器提示,适合移液器在3以上
  3. 石蜡膜(Bemis,目录号:PM996)
  4. 铝箔(任何品牌)
  5. 100 x 15 mm圆形培养皿(Fisher Scientific,目录号:S33580A)
  6. 滤纸
  7. 哥伦比亚-000种子,拟南芥突变种子(ABRC,目录号:CS16284)
  8. 家用漂白剂(Clorox或含有5.25%次氯酸钠的任何品牌,室温下储存6个月保质期)
  9. Triton X-100(Sigma-Aldrich,目录号:T8787)
  10. MS基础盐混合物(Sigma-Aldrich,目录号:M5524),在4℃下储存
  11. 2-(N-吗啉代)乙磺酸(MES)水合物(Sigma-Aldrich,目录号:M8250)
  12. 植物琼脂(Sigma-Aldrich,目录号:A1296)
  13. 蔗糖(Fisher Scientific,目录号:S5-500)
  14. 氢氧化钾(KOH)(Fisher Scientific,目录号:P250-500)
  15. 种子灭菌方案(见食谱)
  16. 半强度MS琼脂培养基(见食谱)

设备

  1. 任何品牌的移液器分配2至20μl,20至100μl和0.1至1.0ml(例如,Eppendorf,目录号:4924000037,4924000061,33123000063或相当的)
  2. 生长室(Percival Scientific,型号:AR41L2)
  3. 烤箱孵化器(Precision,型号:Thelco孵化器3DM,目录号:51221120)
  4. 锋利笔
  5. 尺寸放大镜(CWJ,Peak,型号:1983 10x Scale Loupe)
  6. 解析范围(Leica Microsystems,型号:Leica MZ6或可比较)
  7. 调平台(SP Scienceware - Bel-Art产品 - H-B仪器,目录号:H18310-0000)
  8. 1升玻璃瓶(Fisher Scientific)
  9. 高压灭菌器

软件

  1. Microsoft Excel
  2. ImageJ(可选)

程序

  1. 用于测试适应高温的下胚轴延伸测定
    这里描述的测定可以提供关于幼苗对通常致命/严重破坏性高温胁迫耐受的能力的定量数据。
    1. 用无菌种子准备板
      1. 通过在溶液中浸泡10分钟,将含有0.1%Triton X-100(种子灭菌溶液[参见食谱])的50%漂白溶液表面灭菌拟南芥种子。推荐使用比较新鲜的种子(收获不到一年),因为发芽率的变异性较小
      2. 用无菌水冲洗种子五次,以在无菌环境中除去任何残留的漂白剂
      3. 在无菌环境中,将种子放在具有网格的正方形板上的半强度MS琼脂培养基(参见食谱)上,使用无菌移液器吸头,在尖端适当地切割(几毫米)以允许种子通过,理想的是一个一个我们通常在每条线上放置12-13个种子,种子间距甚至均匀(图1)。将种子放置从一条交错到另一条,以避免下胚轴伸长时接触。在每个板上放置适当的对照样品(野生型和热敏感突变体)很重要。
      4. 用Parafilm包裹板,并将板放在4℃2-3天,这有助于同步发芽。


        图1.具有均匀放置在每条线上的种子的平板图

    2. 幼苗生长和热处理
      1. 热应激前的生长
        用铝箔仔细包裹板,使幼苗保持在黑暗中;下胚轴在黑暗中延伸更多。标记箔片以指示板材的底部边缘相对于幼苗的预期向上生长。让种子通过在22℃下在生长室中以垂直取向支撑板约2.5天,沿着培养基表面发芽并生长。板也可以在适当的温度下保存在任何黑暗的地方。准备进行热应激治疗时,下胚轴的长度约为6〜9毫米(野生型哥伦比亚登陆号)。
      2. 热适应治疗
        将板从铝箔上打开,并将它们放置在38℃的黑暗中的烘箱培养箱中,以适应1.5小时。水平放置平板,以便从架子到介质均匀传热(图2)。需要在22℃(或任何正常的生长温度)作为对照保持另一组重复的板。尽量减少孵化器开放的时间,以减少幼苗的温度波动和暴露。在此期间诱导了许多热休克蛋白(Lin et al。,1984; Hong and Vierling,2000)。


        图2.将平板水平放置在烤箱培养箱中

      3. 适应恢复期
        将板从培养箱中取出,并在黑暗的地方保持在22°C两个小时,再次将它们放置在直立位置。在此期间,植物从轻度热处理中恢复,并继续合成热休克蛋白
      4. 严重急性热应激
        将板放在45°C的孵化器中水平放置一段所需的时间。两个小时足以完全抑制在AT1G74310植物中具有T-DNA插入的热缺陷拟南芥线(HSP101淘汰拟南芥线)的生长,同时允许野生型植物(Hong and Vierling,2001)。根据所测试的植物的热敏感性,45℃下的热应力的长度可以变化,例如1.5小时,2.0小时,2.5小时,3.0小时。您还应该拥有接受适应治疗的控制复制板,但在45°C时尚未处理过。确保您在取出板材时快速打开和关闭培养箱,以尽量减少其余植物的温度下降。
      5. 严重热应激后恢复
        热应激处理后,用锋利的笔在板上标记每个下胚轴的尖端位置,再用箔盖住板,确保注意幼苗的生长方向。将板再次放置在垂直位置,让植物在22℃恢复并生长约2.5天。当标记完成时,尽可能减少曝光,如在其余的过程中。如果幼苗长时间暴露在光照下,最终会出现子叶扩张而不是下胚轴伸长。
      6. 下胚轴长度测量
        恢复期后,取出箔片,再次标记下胚轴的尖端。此时,板可以放置在4℃直到测量。野生型哥伦比亚的典型结果如图3所示。下胚轴伸长的长度可以通过使用鳞片放大镜的眼睛来测量。或者,拍摄板并使用ImageJ的测量工具进行测量(Schneider等人,2012)。


        图3.野生型的热应激表型A。 th a)。。。。。。。。 indicated,,,,,,,,,and and and and and and and and from from from from from,,,。。。 AC:驯化处理:38℃/ 1.5小时,随后22℃/ 2小时。

  2. 轻度生长的7-10日龄幼苗的热胁迫测定
    1. 用种子琼脂培养基对圆形培养皿进行表面消毒并将其放置在A1部分中。将板块分成相等的部分。如果将100 x 15毫米的培养皿分成六个部门,每个部门放置18-20粒种子。将相同的基因型放在板对面的扇区中是一个很好的做法。
    2. 用Parafilm包裹板,并在4℃下放置2-3天进行分层。
    3. 将板移动到生长室,让幼苗在长的天气条件(22℃/ 18℃,16小时/ 8小时夜间循环)下生长7-10天,光强度为〜80 μmolm -2 sec -1 。如果对特定基因型提供更好的生长,也可以使用短时间。
    4. 在如上所述的下胚轴延伸测定的烘箱培养箱中进行热处理,根据需要改变时间长度以便区分基因型。驯化处理与下胚轴延伸测定相同。再次,尝试通过在打开和关闭培养箱时快速作用来最小化温度下降。确保您没有驯化处理的控制板,以及连续保持最佳生长温度的板材进行比较。
    5. 热处理后,将板放回生长室,使其恢复5-8天。当表型差异最佳时,您可以拍照。

  3. 发芽耐热性
    在萌发之前,种子具有显着更高的耐热性,并且已经记录了拟南芥种质之间种子耐热性的变化(Clerkx等人,2004)。
    1. 为了进行这种测试,不需要对种子进行灭菌。然而,因为萌发对母体效应以及种子储存条件敏感,所以只能在相同条件下种植和收获种子并储存的种子进行比较。
    2. 在用相同体积的水饱和的滤纸上将培养皿中的种子播种到所有板上。体积应足以使滤纸完全润湿而不会留下多余的水分。将待测试的每种基因型的30至50粒种子均匀地分布在滤纸上,使用每种基因型的单独的板。允许种子在室温下吸收18小时。为了保持水分,培养皿应在整个测定过程中用水饱和的滤纸封闭在底部衬里的塑料盒中。
    3. 通过将板直接转移到50℃8小时进行热处理,然后将其返回到室温以评定发芽。使用在室温下保持的相同的板作为种子批发芽势的对照
    4. 在7天后确定终点处的发芽率,或每天记录发芽率以估计发芽率差异。在解剖范围内评分小种子,例如拟南芥,或者通过拍摄和评分在计算机屏幕上观看的放大图片。将种子评为在激进出现的第一个迹象上发芽。

  4. 幼苗绿化耐热性
    黑色生长的幼苗积累叶绿素的能力可以在有或没有驯化处理的情况下测试耐热性。下面的测定不适用驯化。
    1. 用下胚轴延伸测定法准备具有幼苗的板材,并在黑暗中垂直生长幼苗2.5至3天。
    2. 在阴凉处处理43℃,120分钟的幼苗板。留下重复的一组板未经处理。也可以测试43℃的不同长度的时间,以便在需要时提供基因型之间的分辨率
    3. 将板在水平或垂直方向放置在室温或室内光照或低光(50至80μEm -2至 sec -1 )的生长中室在22°C。可以使用任何日期长度或连续光线。记录板级光强度,供日后的实验参考。
    4. 每天记录子叶绿化,用眼睛判断为绿色,浅绿色,黄绿色或黄色子叶的幼苗数量。在光明三天后,在终点再次得分。子午线也可以每天进行扩展/打开。

  5. 其他热应激治疗
    上述的热应激处理已广泛用于拟南芥研究中。然而,确切的时间和温度也可以变化以测定不同的表型。例如,驯化预处理可以通过在3〜4小时内将温度从20℃逐渐升高到22℃至45℃的应力温度来进行(Larkindale和Vierling,2008)。这需要具有温度升高能力的适当的室,这是大多数实验室的限制性资源。然而,这种逐渐适应的处理被证明可以在恢复应力之后提供比上述驯化处理更好的幼苗生存力。另一个可以改变的参数是驯化处理和热应激处理之间的时间,可以评估不同基因型保持适应能力的差异。 Yeh等人。 (2012)描述了可在耐热测定中修改的其他参数。

  6. 适应其他植物物种的分析
    如背景技术部分所述,原始的胚轴伸长测定法适应于大豆实验(Lin等人,1984),其他植物物种可以类似地测定适应高温。为此,只需进行仔细的初步实验来确定幼苗萌发率和下胚轴生长程度或其他参数,以及导致不同种类的不同种类的热杀死或幼苗生长停滞的时间和温度成长阶段

数据分析

  1. 下胚轴延长分析
    使用Microsoft Excel绘制x轴上的基因型和y轴上相应的下胚轴长度。当有突变体生长缺陷时,相对于每个基因型的对照(无热处理)生长,标准化长度。一个例子如图4所示。适应驯化后45°C / 2 h处理的典型标准偏差约为野生型Columbia的10%,当长度与对照生长正规化时。统计学意义可以通过执行学生的测试来测试。必须进行至少三次独立实验,重复性相似。


    图4.显示热处理后各种拟南芥基因型(45℃/ 80分钟,然后热适应,Kim等人修改)的下胚轴延伸的实例图。 ,2012)。突变体的突变体生长比野生型和 更慢,因此下胚轴生长通过生长正常化未经热处理的幼苗的相同时间段。错误栏表示SE; n = 12.

  2. 轻度生长的7-10日龄幼苗的热胁迫测定
    当表型差异最佳时,拍照,通常在热处理后5〜8天发生。一个例子如图5所示。为了使测定更定量,计算已经存活的幼苗,并绘制生存百分比,如Larkindale等人所述。 (2005年)。如程序详述,也是良好做法,在板的对面植入相同基因型的部分,以解释热分布的差异。即使温差较小也可能导致不同的结果。将基因型与同一板进行比较也是至关重要的。必须进行至少三次独立实验,重复性相似。


    图5.如图所示,10天龄的幼苗受热应激。照片在治疗后7天(从Kim等人,2012修改)拍摄。

  3. 发芽耐热性
    将种子发芽的结果作为相同种子批次的未处理种子的百分比表示。如果每天收集数据,结果可以绘制为线图。如果仅在终点收集数据,请使用表格显示数据。
  4. 幼苗绿化耐热性
    将子叶绿化的结果作为条形图显示,与不进行热处理的幼苗相比,每天显示不同绿化阶段的子叶百分比。类似地,开放子叶的百分比也可以作为未处理幼苗的百分比绘制。统计学意义可以通过执行学生的测试来测试。应进行至少三次独立实验,每基因型和处理20至30个幼苗

笔记

  1. 小心不要损坏黑色生长的幼苗的铝箔覆盖层。另外,尽量减少处理过程中幼苗的光照。漏光使植物在子叶开放扩张的同时发生光形态发生,同时防止下胚轴伸长
  2. 重要的是不要使培养箱超载,并且要注意孵化器不同部位的温度差异。如果培养箱的校准不准确,还要比较不同的培养箱,最好使用温度计检查培养箱的温度。幼苗对精确的温度非常敏感,差异只有±1°C可以显着改变结果
  3. 我们使用烤箱保温箱进行黑热处理。然而,其他研究人员将紧密密封的板放在水浴中,这可能有助于传热并防止温度波动(Charng等人,2006)。
  4. 标记幼苗生长是重要的,因为它允许您在热应激之前将热分解后的生长与热应激后的生长分开。这可以使不同基因型之间的下胚轴长度不同而发芽率也有差异。
  5. 当用MS琼脂培养基制备板时,在校平台上的每个板中倒入相同量的培养基(例如,10×100mm×15mm方形陪替氏培养皿)。这将降低板之间以及板内的温度变化。
  6. 当测量发芽的耐热性时,所有基因型应在三次重复测试中进行统计分析。结果应相对于未加热对照的发芽表达。可以改变热应激的温度或时间,以增加基因型之间差异的分辨率。如果种子在18小时吸收期后发芽,可缩短热处理之前的吸收时间。
  7. 对于本文所述的所有测定,通过比较基于未处理的种子或相同基因型的幼苗计算的基因型进行基因型之间的比较。此外,只要有可能,优选使用在同一时间和相同条件下生长的植物中的种子,并在相同条件下储存种子。这对于确定基因型之间耐热性是否存在显着差异很重要,特别是如果差异很小。重复使用多批种子对于确认基因型之间的小差异至关重要。
  8. 对于热应激光生长的幼苗或子叶绿化的耐热性的测定,也可以通过从由Burke等人开发的对照和热应激样品的等量的幼苗中提取叶绿素来获得定量结果。 (2000)

食谱

  1. 种子灭菌溶液(100 ml)
    50ml漂白剂
    50ml蒸馏水
    500μl20%Triton X-100(0.1%终浓度)
    注意:解决方案可以在室温下保存在阴暗的地方长达一个月。
  2. 半强度MS琼脂培养基(1000毫升)
    2.15克MS基础盐混合物(4℃保存)
    0.5g 2-(N-吗啉代)乙磺酸(MES)水合物 5克蔗糖
    加入900毫升蒸馏水,搅拌溶解 使用1N KOH调节pH至5.7 加入蒸馏水至最终体积为1000 ml
    将介质分成两个1升玻璃瓶。在每个瓶子里加入4g植物琼脂 高压灭菌器在121℃,15psi下进行30分钟 注意:将溶液进行高压灭菌后,将其冷却至约60℃,并在无菌环境中的校平台上的每个板中倒入固定量的培养基(10-30ml)。琼脂的量影响板内部温度升高的速率,因此重要的是每个板加入完全相同量的介质。倒入板后,让它们在无菌环境中固化并干燥20分钟。未使用的介质板可以在密封的容器中以4°C倒置存放数月。

致谢

美国能源基础生物科学计划,美国农业部国家研究计划竞争性资助计划以及生物科学院国家科学基金会分子生物科学部门向EV提供资助。种子萌发测定是在支持古根海姆研究金与EV的情况下开发的。这些协议由Hong和Vierling,2000和Larkindale等人,2005修改和修改。

参考

  1. Burke,JJ,O'Mahony,PJ和Oliver,MJ(2000)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/10859187”target =“_ blank”>分离缺乏获得耐热性成分的拟南芥突变体。植物生理学123(2):575-588。
  2. Charng,YY,Liu,HC,Liu,NY,Hsu,FC and Ko,SS(2006)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov / pubmed / 16500991“target =”_ blank“> 拟南芥 Hsa32是一种新型的热休克蛋白,在适应后长期恢复期间获得耐热性是必需的。植物生理学> 140(4):1297-1305。
  3. Clerkx,EJ,El-Lithy,ME,Vierling,E.,Ruys,GJ,Blankestijn-De Vries,H.,Groot,SP,Vreugdenhil,D.and Koornneef,M。(2004)。拟南芥的天然等位基因变异分析种子萌发和种子使用新的重组近交系群体,登陆Landsberg erecta和Shakdara之间的长寿特征。植物生理学135(1):432-443。
  4. Gilmour,SJ,Hajela,RK和Thomashow,MF(1988)。拟南芥中的冷驯化 植物生理学87(3):745-750。
  5. Hong,SW,and Vierling,E.(2000)。 
  6. Hong,SW,and Vierling,E.(2001)。  Hsp101对于耐热性是必需的,不需要在没有压力的情况下进行发育和发芽。 27(1):25-35。
  7. Kim,M.,Lee,U.,Small,I.,des Francs-Small,CC and Vierling,E.(2012)。< a class =“ke-insertfile”href =“http: ncbi.nlm.nih.gov/pubmed/22942382“target =”_ blank“>在拟南芥线粒体转录终止因子相关蛋白质中的突变在不存在主要分子伴侣HSP101的情况下增强耐热性。 a>植物细胞 24(8):3349-3365。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kim, M., McLoughlin, F., Basha, E. and Vierling, E. (2017). Assessing Plant Tolerance to Acute Heat Stress. Bio-protocol 7(14): e2405. DOI: 10.21769/BioProtoc.2405.
  2. McLoughlin, F., Basha, E., Fowler, M. E., Kim, M., Bordowitz, J., Katiyar-Agarwal, S. and Vierling, E. (2016). Class I and II Small Heat Shock Proteins Together with HSP101 Protect Protein Translation Factors during Heat Stress. Plant Physiol 172(2): 1221-1236.
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