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Experimental Design to Determine Drought Stress Response and Early Leaf Senescence in Barley (Hordeum vulgare L.)
测定大麦干旱应激响应和早期叶片衰老的实验设计   

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BMC Plant Biology
Jun 2015

Abstract

Premature leaf senescence induced by drought stress is a main factor for yield losses in barley. Research in drought stress tolerance has become more important as due to climate change the number of drought periods will increase and tolerance to drought stress has become a goal of high interest in barley breeding. However, reliable screening for drought stress tolerance is still a difficult task. This protocol describes the experimental design for the phenotyping for drought stress tolerance and early leaf senescence in the juvenile stage of barley (A) and the determination of six physiological parameters involved in drought tolerance and leaf senescence (B to G) according to Wehner et al., (2015).

Keywords: Barley (大麦), Drought stress (干旱胁迫), Leaf senescence (叶片衰老)


A. Experimental design

Materials and Reagents

  1. Sticks for labels (Hermann Meyer KG, catalog number: 180230 )
  2. Plastic sticks (Hermann Meyer KG, catalog number: 180206 )
  3. Rubber binder (Hermann Meyer KG, catalog number: 321234 )
  4. Barley seeds
  5. 70% white peat
  6. 30% clay
  7. N (nitrogen power)
  8. P2O5
  9. K2O
  10. Mixed clay soil ED73 (H. Nitsch & Sohn GmbH & Co. KG) (see Recipes)

Equipment

  1. Greenhouse facility
  2. Movable greenhouse benches (80 x 100 cm)
  3. Square pots (16 x 16 x 16 cm) (Hermann Meyer KG, catalog number: 720016 )
  4. Beaker for watering (VWR International, catalog number: 213-3402 )
  5. Labels (Baumann Industries, catalog number: 2.508.003 )
  6. Weighing scale (KERN & SOHN GmbH)
  7. Compartment dryer (Heratherm oven) (Thermo Fisher Scientific)

Procedure

  1. Adjust greenhouse by heating, ventilation and lighting for long day conditions in a temperature range from 20 to 22 °C at day (16 h) and 17 to 19 °C at night (8 h). If natural radiation is below 20 klx, additional light should be supplied from 6 am to 10 pm and above 30 klx shading should be used (light intensity of additional illumination at pot height is ~10 klx).
  2. Homogenize content of different bags of ED 73 soil to optimize comparability between the pots.
  3. In correspondence to respective humidity fill square pots with a defined weight of the soil (in our case 1,500 x g at 40% dry substance of the soil).
  4. Label pots and sow ten seeds of each barley genotype per pot (in our case three replications for control and drought stress treatment for 156 genotypes). For better visibility labels could be pinned on label stakes (Figure 1).
  5. Put 20 pots ordered by the number of genotypes at each greenhouse bench according to the experimental design chosen (in our case a split plot design). Control and stress treatment are allocated to separate benches (Figure 1).
  6. Water all pots up to 70% of the maximal soil water capacity (WC). The weight of added water is calculated out of the saturated soil weight and dry weight according to DIN ISO 11465 1996-12 (Paech and Simonis, 1952) at three exemplary pots as follows:
    1. Put gauze at the bottom, fill three pots with soil and add water till saturation.
    2. Wait for 6 h till water is drained by gravity and weight.
    3. Dry the soil (two days in compartment dryer) at 105 °C and weigh.
    4. 100% WC is calculated out of the weight of saturation minus the dry weight.
    5. The weight to which the pot is watered corresponds to 70% (control) or 20% (drought stress) WC plus the dry weight and the pot weight.
  7. Water every day to 70% WC by weighing.
  8. After germination, reduce seedlings to seven plants per pot.
  9. To minimize effects of plant position, movable benches should be moved every day.
  10. Drought stress starts at the primary leaf stage [BBCH 10, according to Stauss (1994)] seven days after sowing (das). At this time primary leaves of the juvenile barley plants are fully expanded. Stop watering of the stress variant till the pot weight reaches 20% WC. Check this by weighing ten exemplary stressed pots over the time of the experiment.
  11. Then keep this level by weighing each pot and re-water to the weight of 20% WC daily.
  12. Water control plants daily to 70% WC (weight of the plants is neglected).


    Figure 1. Pot experiment in greenhouse (10 days after sowing)

  13. At 36 das (BBCH 33) a four weeks stress period is reached and physiological parameters are determined as well as biomass is harvested according to protocol B to G below. For analysing physiological traits primary leaves should be used because at these oldest leaves drought stress induced leaf senescence occurs first (Figure 2).


    Figure 2. Primary leaves differentiating in drought stress response regarding leaf senescence

Notes

In our pot experiments it turned out, that it is necessary to measure parameters for leaf senescence in a separate pot experiment, because only in this way it was possible to separate the effects of drought stress induced leaf senescence and age-related leaf senescence from light deficiency induced leaf senescence. To achieve this, the shadowing effects of other leaves on the primary leaf are minimized by tying up all barley leaves (with rubber binder on stakes) about 14 das, except the primary leaf (Figure 3A). General settings are the same as described above, but only four plants are grown in smaller pots (12 x 12 x 12 cm with 550 g soil) and control and stress treated pots are mixed on the greenhouse benches in rows (Figure 3B).


Figure 3. Barley plants with tied up leaves. A. Single plants at 36 days after sowing under control (left) and drought stress treatment (right). B. One greenhouse bench at 20 days after sowing with four rows stress treatment (yellow sticks) and three rows control treatment (black sticks).

Recipes

  1. Mixed clay soil ED73
    70% white peat
    30% clay with pH ~6 and 1 kg/m³
    Mineral nutrients
    1. 14% N
    2. 16% P2O5
    3. 18% K2O
    2 kg/m³ long term fertilizer
    1. 20% N
    2. 10% P2O5
    3. 15% K2O

B. Leaf senescence: Leaf colour

Materials and Reagents

  1. Barley plants grown as described above

Equipment

  1. Chlorophyll Meter SPAD-502 Plus (Konica Minolta)

Procedure

  1. Measure leaf colour directly in the greenhouse on the primary leaves at 33-34 days after sowing (das).
  2. With Minolta SPAD readings leaf colour representing the status of leaf senescence is measured non destructive.
  3. After calibration by an empty clip, take five evenly distributed readings on each of three primary leaves (three barley plants) per pot which are averaged by the SPAD-Meter.
  4. At each primary leaf, measurement should be done at the upper side of the leaf and in the middle of the leaf avoiding to clip the middle leaf-vein (Figure 4).


    Figure 4. Positions for measurement of leaf colour on three primary leaves
 

C. Leaf senescence and drought tolerance: Electron transport rate at PSII

Materials and Reagents

  1. Barley plants grown as described above

Equipment

  1. OS1p-Chlorophyll fluorometer (Opti-Sciences)

Procedure

Determine the electron transport rate (ETR) at photosystem II (PSII) as a parameter for leaf senescence at 34-35 das. With the portable OS1p (Figure 5) chlorophyll fluorescence is measured non destructive on light adapted plants. Because this analysis is very light sensitive, measurement should be conducted exactly at the position where the pot is located on the greenhouse bench and the shading of the greenhouse should be used to ensure continuous lighting conditions during all measurements. Besides, measurement should be done not before 10 am and not after 3 pm (around noon) receiving the optimum of daily photosynthesis.


Figure 5. OS1p-Chlorophyll fluorometer. The display shows three exemplary measurements down right and general settings top left. Besides this, the leaf clip for measurement is shown consisting out of the clip with the trigger button and the sensor for detecting the photosynthetic active radiation (PAR).

For measurement with the fluorometer first select the photosynthetic yield menu Y(II).
Adjust modulation intensity at 50%, saturation intensity at 100% and for the ETR calculation include the formula ETR = Y(II) * PAR * 0,84 * 0.5. Input values for constants representing light which is absorbed by the leaf (0.84) and light which is equally absorbed by PSI and PSII (0.5) in addition to the measured photosynthetic active radiation (PAR) and the quantum photosynthetic yield of PSII [Y(II)] (Krall and Edwards, 1992).
Take three measurements for each pot in the middle of three primary leaves at the upper side of the leaves. Put the leaves in the clip with the leaf margin at the bottom of the clip. Then wait till the light impulses are finished indicated by a peep.
Data dumps are stored in the memory of the fluorometer and can be read out later by USB transfer and averaged for each pot.

Notes

The Fluorometer also gives values for Y(II), but here the ETR (µmol/ m²s) is used because the PAR is included to compensate potential lighting variations.

D. Drought tolerance: Total content of soluble sugars

Materials and Reagents

  1. Pipette (1,000 µl/10 ml) (Eppendorf/Finnpipette Labsystems)
  2. Tubes (15 ml) (VWR International, catalog number: 525-0308 )
  3. Tubes (2 ml) (VWR International, catalog number: 211-2120 )
  4. Test tubes (glass) (VWR International, catalog number: 4.902.002.75 )
  5. Barley plants grown as described above
  6. Liquid nitrogen
  7. D-Glucose (VWR International, catalog number: 1.08337.0250 )
  8. Polyvinylpolypyrrolidone (PVPP) (VWR International, catalog number: 1.07302.0100 )
  9. Distilled water
  10. Sulfuric acid 100% (VWR International, catalog number: 1.00713.1000 )
  11. Borosilicate glass bottle (brown) (VWR International, catalog number: 215-2328 )
  12. Anthrone (VWR International, catalog number: 1.01468.0010 )
  13. Thiourea (VWR International, catalog number: 1.07979.0250 )
  14. Sulfuric acid 70% (see Recipes)
  15. Anthrone reagent (see Recipes)

Equipment

  1. Weighing scale (KERN & SOHN GmbH)
  2. Scissors
  3. Styropor box
  4. Freeze dryer (Christ Alpha 1-4 LD plus)
  5. Water bath (GFL)
  6. Shaker (Vortex Genie 2) (Scientific industries)
  7. Test tube racks (Geyer GmbH & Co, catalog number: 9.905 970 )
  8. Glass marbles (toy store, diameter of 15 mm)
  9. Centrifuge (23,000 x g) (Tuttlingen Germany, model: Hettich Universal 16R )
    Note: Currently, it is “Gemini BV Laboratory, model: Hettich Universal 16R”.
  10. Cuvette (VWR International, catalog number: 634-9014 )
  11. Spectrophotometer (Thermo Fisher Scientific, model: Genesys 10S UV VIS )
  12. Beaker (VWR International, catalog number: 213-1131 )

Procedure

First, prepare a standard curve for glucose content which should be repeated for every anthrone reagent.

  1. Prepare five different dilutions of D-Glucose (each 500 µl) in five test tubes: 0 µM, 50 µM, 100 µM, 150 µM and 200 µM.
  2. Slowly add for each test tube 2.5 ml anthrone reagent by vortexing till a clear solution is present.
  3. Continue with steps 12-14 together with the samples.
  4. After measuring draw the standard curve for the glucose content (Figure 6).


    Figure 6. Standard curve for glucose content

    At 36 das samples for analysis of the total content of soluble sugars are taken and analysed according to Yemm and Willis (1954).
  5. Cut 1 cm pieces from the middle of five primary leaves per pot (100 -200 mg).
  6. Weigh these (fresh weight is only used to calculate dry weight content for osmolality), fill in tubes (15 ml) as pooled samples per pot and freeze in liquid nitrogen using a styropor box.
  7. Samples are then freeze dried and reweighed (dry weight).
  8. Add 4 ml distilled water (depends on the amount of leaf tissue sampled) and boil the samples in the water bath for 30 min by 100 °C (use the test tube racks for water baths).
  9. After cooling down, add 1,000 µl of the sample solution to tubes (2 ml) filled with 0.05 g PVPP and vortex.
  10. Centrifuge the tubes for 5 min at 23,000 x g (5,000 rpm).
  11. Transfer 200 µl of the supernatant solution to test tubes and add 300 µl distilled water.
  12. By vortexing slowly add 2.5 ml of the anthrone reagent till a clear solution is present.
  13. Put on each glass tube a glass marble (Figure 7) and boil the solutions in the water bath for 15 min at 100 °C (use the test tube racks for water baths).
  14. Cool down in a cold water bath (20 °C) for 10 min.
  15. Samples (Figure 7) are measured directly (maximum in 1 h time) by a spectrophotometer at 625 nm using a cuvette. Zero calibration should be done with distilled water.


    Figure 7. Test tubes ready for measurement of total content of soluble sugars. Exemplarily, three test tubes with samples for control (light blue, left) and three test tubes with samples for drought stress (dark blue, right) treatment are shown.

  16. Total content of soluble sugars is calculated using the standard curve and set in relation to dry weight (µmol/g).

Notes

The sample volume of 200 µl taken in step D11 is varying for different developmental stages of barley and also for different crops and depends on the amount of leaf tissue sampled. Important is, that the final volume in step D11 is 500 µl. Volume should be defined for the optimum range of the standard curve.

Recipes

  1. 70% sulfuric acid (mix in a brown bottle and incubate for 12 h in dark)
    70 ml sulphuric acid (100%)
    30 ml distilled water
  2. Anthrone reagent (stable for one day)
    Sulphuric acid (70%)
    920 mg/l anthrone
    920 mg/l thiourea

E. Drought tolerance: Content of free proline

Materials and Reagents

  1. Aluminium foil
  2. Tubes (15 ml) VWR International, catalog number: 525-0308 )
  3. Stopper (VWR International, catalog number: 217-0511 )
  4. Pipette (1,000 µl/10 ml) (Eppendorf/Finnpipette Labsystems)
  5. Barley plants grown as described above
  6. Liquid nitrogen
  7. L-Proline (VWR International, catalog number: 1.07434.0010 )
  8. Toluol (VWR International, catalog number: 1.08325.1000 )
  9. Distilled water
  10. Ninhydrin (VWR International, catalog number: 1.06762.0100 )
  11. Glacial acetic acid (AppliChem, catalog number: A0820.2500 )
  12. Ninhydrin reagent (see Recipes)

Equipment

  1. Weighing scale (KERN & SOHN GmbH)
  2. Scissors
  3. Styropor box
  4. Freeze dryer (Christ Alpha 1-4 LD plus)
  5. Water bath (GFL)
  6. Test tubes (glass) (VWR International, catalog number: 4.902.002.75 )
  7. Test tube racks (Geyer GmbH & Co, catalog number: 9.905 970 )
  8. Glass marbles (toy store, diameter of 15 mm)
  9. Shaker (Scientific industries, model: Vortex Genie 2 )
  10. Magnetic mixer (RCT basic) (IKA Labortechnik)
  11. Fortuna® Optifix® Safety Dispenser (Sigma-Aldrich, catalog number: Z260207 )
  12. Fume hood
  13. Cuvette (VWR International, catalog number: 634-9014 )
  14. Spectrophotometer (Thermo Fisher Scientific, model: Genesys 10S UV VIS )
  15. Beaker (VWR International, catalog number: 213-1131 )

Procedure

First, prepare a standard curve for proline content which should be repeated for every ninhydrin reagent.

  1. Prepare five different dilutions of L-Proline (each 500 µl) in five test tubes: 0 µM, 25 µM, 50 µM, 75 µM and 100 µM.
  2. Slowly add for each test tube 2 ml ninhydrin reagent by vortexing till a clear solution is present.
  3. Continue with steps D11-16 together with the samples.
  4. After measuring draw the standard curve for the proline content (Figure 8).


    Figure 8. Standard curve for proline content

    At 36 das samples for analysis of the content of free proline are taken and analysed according to Bates et al. (1973). For this analysis, the same sample solution can be used as for the total content of soluble sugars because steps 5-8 are the same.
  5. Cut 1 cm pieces from the middle of five primary leaves per pot (100 -200 mg).
  6. Weigh these (fresh weight is only used to calculate dry weight content for osmolality), fill in tubes (15 ml) as pooled samples per pot and freeze in liquid nitrogen using a styropor box.
  7. Samples are then freeze dried and reweighed (dry weight).
  8. Add 4 ml distilled water (depends on the amount of leaf tissue sampled) and boil the samples in the water bath for 30 min at 100 °C (use test tube racks for water baths).
  9. After cooling down, 1,000 µl of the sample solution are filled in a test tube, each.
  10. By vortexing slowly add 2 ml of the ninhydrin reagent till a clear solution is present.
  11. Put on each test tube a glass marble (see Figure 7) and boil the solutions in the water bath for 15 min at 100 °C (use test tube racks for water baths).
  12. Cool down in a cold water bath (20 °C) for 10 min.
    Use a laboratory fume hood for the next steps:
  13. Remove the glass marble and add 5 ml toluol (e.g. with a dispenser).
  14. Close the test tube with a stopper and vortex for 15 sec.
  15. Incubate the samples for the next 90 min in darkness (up to 4 h are possible) at room temperature.
  16. Two phases occur (Figure 9) from which the upper one is now transferred to a cuvette for measurement with a spectrophotometer at 520 nm. Zero calibration should be done with toluol.


    Figure 9. Test tubes ready for measurement of content of free proline. Exemplarily, three test tubes with samples for control (light red, left) and three test tubes with samples for drought stress (dark red, right) treatment are shown.

  17. Content of free proline is calculated using the standard curve and set in relation to the dry weight (µmol/g).

Notes

In step E9, 1,000 µl of the sample solution is used. This volume is varying for different developmental stages of barley and also for different crops and depends a lot on the amount of leaf tissue sampled. Volume should be defined for the optimum range of the standard curve.

Recipes

  1. Ninhydrin reagent (stable for one day, mix in dark, cover a beaker with aluminium foil and dissolve on a magnetic mixer)
    0.5 g ninhydrin
    20 ml distilled water
    30 ml glacial acetic acid

F. Drought tolerance: Osmolality

Materials and Reagents

  1. Tubes (2 ml) (VWR International, catalog number: 211-2120 )
  2. Pipette (100 µl/200 µl) (Eppendorf)
  3. Barley plants grown as described above
  4. Liquid nitrogen
  5. Distilled water
  6. Calibration standard for the osmomat (Gonotec GmbH, catalog number: 30.9.0020 )

Equipment

  1. Weighing scale (KERN & SOHN GmbH)
  2. Scissors
  3. Styropor box
  4. Tweezer
  5. Swing mill (Retsch, model: MM200 )
  6. Grinding balls (VWR International, catalog number: 412-0070 )
  7. Grinding jars (Retsch, catalog number: 22.008.0005 )
  8. Shaker (Scientific industries, model: Vortex Genie 2 )
  9. Centrifuge (21,000 x g) (Wehingen, model: Hermle Z233MK2 )
    Note: Currently, it is “HERMLE Labortechnik GmbH, model: Hermle Z233MK2”.
  10. Osmomat (Gonotec GmbH, model: Osmomat 3000 Gonotec )
  11. Tubes for the osmomat (Gonotec GmbH, catalog number: 30.9.0010 )

Procedure

At 36 das the osmolality is determined by comparative measurements of the freezing points of distilled water and the cell sap.

  1. Cut 1 cm pieces from the middle of five primary leaves per pot.
  2. Weigh these, fill pooled samples per pot in tubes (2 ml) and freeze in liquid nitrogen using a styropor box.
  3. Put also the grinding balls and grinding jars into the liquid nitrogen (into the styropor box).
  4. Take each tube and fill in two grinding balls with a tweezer.
  5. Take off the grinding jars and fill them with five tubes each.
  6. Grind the leaves in the swing mill for 3 min (30/sec).
  7. Add 200 µl distilled water into the tubes and vortex them.
  8. Take the grinding balls out of the tubes with the tweezer.
  9. Centrifuge the tubes for 15 min at 21,000 x g (15,000 rpm) by 4 °C.
  10. Pipette the supernatant cell sap in new tubes (2 ml).
    For measurement with the osmomat use the original tubes for the osmomat.
  11. Calibrate the osmomat with 15 µl calibration standard till a value of exactly 0.3 is reached.
  12. Adjust a zero value with 15 µl distilled water.
  13. Measure 15 µl cell sap each.
  14. Values (osmol/kg) can be corrected for the water content (difference of fresh and dry weight). Dry weight is calculated in relation to the dry weight content of the samples for the determination of content of free proline and the total content of soluble sugars (see above).

G. Drought tolerance: Biomass yield

Materials and Reagents

  1. Barley plants grown as described above

Equipment

  1. Weighing scale (KERN & SOHN GmbH)
  2. Crispack bags (Baumann, catalog number: 3.331.100 )
  3. Scissors
  4. Compartment dryer (Heratherm oven) (Thermo Fisher Scientific)

Procedure

  1. At 36 das cut the whole biomass above ground per pot.
  2. Put biomass for each pot with the label in one crispack bag.
  3. Close bags.
  4. Dry the biomass within the bags in a compartment dryer at 105 °C until weight doesn’t change (about two days with ventilation).
  5. By weighing the dry weight, total biomass yield (g) is detected.

Acknowledgments

The authors thank the Interdisciplinary Center for Crop Plant Research (IZN) of the Martin-Luther-University of Halle-Wittenberg for funding the project. Furthermore, some of the protocols are modified or adapted from previous work acknowledging Bates et al. (1973), Paech and Simonis (1952) as well as Yemm and Willis (1954).

References

  1. Bates, L. S., Waldren, R. P. and Teare, I. D. (1973). Rapid determination of free proline for water-stress studies. Plant Soil 39:205-207.
  2. Krall, J. P. and Edwards, G. E. (1992). Relationship between photosystem II activity and CO2 fixation in leaves. Pysiol Plantarum 86:180-187.
  3. Paech, K. and Simonis, W. (1952). Pflanzenphysiologische Praktika Band I Übungen zur Stoffwechselphysiologie der Pflanzen. Springer Verlag, Berlin Göttingen Heidelberg.
  4. Stauss, R. (1994). Compendium of growth stage identification keys for mono-and dicotyledonous plants: Extended BBCH scale. Ciba.
  5. Wehner, G. G., Balko, C. C., Enders, M. M., Humbeck, K. K. and Ordon, F. F. (2015). Identification of genomic regions involved in tolerance to drought stress and drought stress induced leaf senescence in juvenile barley. BMC Plant Biol 15: 125.
  6. Yemm, E. W. and Willis, A. J. (1954). The estimation of carbohydrates in plant extracts by anthrone. Biochem J 57(3): 508-514.

简介

由干旱胁迫诱导的过早叶子衰老是大麦产量损失的主要因素。 干旱胁迫耐受性的研究变得更加重要,因为由于气候变化,干旱期的数量将增加,并且对干旱胁迫的耐受性已经成为大麦育种中高度关注的目标。 然而,对干旱胁迫耐受性的可靠筛选仍然是一项艰巨的任务。 该协议描述了针对大麦幼龄阶段(A)的干旱胁迫耐受性和早期叶衰老的表型的实验设计以及根据Wehner等人(2006)的参考干旱耐受性和叶衰老(B对G)的六个生理参数的确定 > et al。,(2015)。

关键字:大麦, 干旱胁迫, 叶片衰老

A。实验设计

材料和试剂

  1. 标签棒(Hermann Meyer KG,目录号:180230)
  2. 塑料棒(Hermann Meyer KG,目录号:180206)
  3. 橡胶粘合剂(Hermann Meyer KG,目录号:321234)
  4. 大麦种子
  5. 70%白泥炭
  6. 30%粘土
  7. N(氮气功率)
  8. P 2 O 5
  9. K 2 O
  10. 混合粘土土ED73(H.Nitsch& Sohn GmbH& Co.KG)(参见Recipes)

设备

  1. 温室设施
  2. 可移动的温室长椅(80 x 100厘米)
  3. 方形锅(16×16×16cm)(Hermann Meyer KG,目录号:720016)
  4. 用于浇水的烧杯(VWR International,目录号:213-3402)
  5. 标签(Baumann Industries,目录号:2.508.003)
  6. 称重秤(KERN& SOHN GmbH)
  7. 隔室干燥器(Heratherm oven)(Thermo Fisher Scientific)

程序

  1. 通过加热,通风和照明在温度范围为20至22°C(16 h)和17至19°C(夜间)(8 h)的长日照条件下调节温室。如果自然辐射低于20 klx,应从上午6点至下午10点提供额外的光,并应使用高于30 klx的阴影(灯泡高度附加照明的光强度约为10 klx)。
  2. 均匀化不同袋子的ED 73土壤的含量,以优化花盆之间的可比性
  3. 对应于具有限定重量的土壤(在我们的情况下,在土壤的干物质为40%时为1,500m 2/g)的相应的湿度填充方形罐。
  4. 标记盆和每盆大麦基因型的10个种子(在我们的情况下,对于156个基因型的对照和干旱胁迫处理三个重复)。为了更好的可见性,标签可以固定在标签桩上(图1)。
  5. 根据选择的实验设计(在我们的情况下是分裂图设计),在每个温室工作台上放置20个由基因型数量排序的盆。控制和压力治疗分配给单独的长椅(图1)。
  6. 将所有罐子的水量提高到最大土壤水容量(WC)的70%。根据DIN ISO 11465 1996-12(Paech和Simonis,1952),在三个示例性罐中,根据饱和土壤重量和干重计算加入的水的重量如下:
    1. 将纱布放在底部,用土填满三盆,加水至饱和。
    2. 等待6小时,直到水通过重力排出并称重
    3. 在105℃下干燥土壤(在室干燥器中两天)并称重。
    4. 100%WC由饱和度的重量减去干重计算
    5. 盆浇水的重量对应于70%(对照) 或20%(干旱胁迫)WC加干重和盆重。
  7. 每天给水70%的WC称重
  8. 发芽后,每盆减少幼苗至七株
  9. 为了最小化植物位置的影响,可移动工作台应每天移动
  10. 干旱胁迫在播种后7天(das)开始于初生叶期[BBCH 10,根据Stauss(1994)]。此时幼苗大麦植物的初生叶完全膨胀。停止应力变量的浇水,直到锅重量达到20%WC。通过在实验时间内称重十个示例性的应力锅来检查这一点
  11. 然后保持这个水平通过称重每个锅,并重新水每天的重量为20%的WC。
  12. 水控制植物每天至70%WC(植物的重量被忽略)。


    图1.温室盆栽实验(播种后10天)

  13. 在36天(BBCH 33),达到四周的胁迫期,并测定生理参数,以及根据以下方案B至G收获生物量。为了分析生理性状,应使用初生叶,因为在这些最古老的叶上,干旱胁迫诱导的叶衰老首先发生(图2)。


    图2.初级叶片在叶片衰老过程中干旱胁迫响应差异

笔记

在我们的盆栽实验中证明,有必要在单独的盆栽实验中测量叶子衰老的参数,因为只有这样才有可能分离干旱胁迫诱导的叶衰老和年龄相关叶衰老的影响缺乏诱导叶衰老。为了实现这一点,除了主叶(图3A)外,通过将所有大麦叶(在桩上具有橡胶粘合剂)捆扎约14天,使其它叶在主叶上的遮蔽效应最小化。一般设置与上述相同,但是只有四株植物在较小的盆(12×12×12cm,550g土壤)中生长,对照和胁迫处理的盆在温室长椅上成行混合(图3B) br />

图3.在对照(左)和干旱胁迫处理(右)播种后36天的单株植物。 B.在播种后20天的一个温室台,四行应激处理(黄色条)和三行对照处理(黑色条)。

食谱

  1. 混合粘土土ED73
    70%白泥炭
    30%粘土,pH?6和1kg/m 3 矿物营养素
    1. 14%N
    2. 16%P <2> 5
    3. 18%K 2 O
    2 kg /m3长期肥料
    1. 20%N
    2. 10%P 2 O 5
    3. 15%K <2>

B。叶衰老:叶色

材料和试剂

  1. 如上所述生长的大麦植物

设备

  1. 叶绿素仪SPAD-502 Plus(柯尼卡美能达)

程序

  1. 在播种后33-34天,在初级叶上直接在温室中测量叶色(das)。
  2. 使用Minolta SPAD读数,测量表示叶衰老状态的叶色是非破坏性的。
  3. 用空夹子校准后,取5个均匀分布的读数 ?在每盆三个主叶(三个大麦植物)的每个上 ?由SPAD-Meter进行平均。
  4. 在每个主叶,测量应在上侧进行 叶子和叶子的中间避免夹住中间 叶脉(图4)。


    图4.测量三个主叶上的叶色的位置
 

C。叶片衰老和耐旱性:PSII的电子传递速率

材料和试剂

  1. 如上所述生长的大麦植物

设备

  1. OS1p叶绿素荧光计(Opti-Sciences)

程序

确定光系统II(PSII)的电子传递速率(ETR)作为34-35天的叶子衰老的参数。使用便携式OS1p(图5),在轻适应植物上测量叶绿素荧光是非破坏性的。因为该分析非常光敏,所以应当在锅位于温室台上的位置进行精确测量,并且应该使用温室的阴影以确保在所有测量期间的连续照明条件。此外,测量应在上午10点之前进行,而不是在下午3点(中午)之后,接受每日光合作用的最佳值。


图5.OS1p-叶绿素荧光计。 显示屏向右显示三个示例测量值,左上角显示一般设置。除此之外,用于测量的叶夹被示出包括具有触发按钮和用于检测光合有效辐射(PAR)的传感器的夹子。

对于用荧光计测量,首先选择光合产量菜单Y(II)。
调整50%的调制强度,100%的饱和强度和ETR计算包括公式ETR = Y(II)* PAR * 0,84 * 0.5。除了测量的光合有效辐射(PAR)和PSII的量子光合产率(Y(II))外,叶片吸收的光的常数(0.84)和由PSI和PSII )](Krall和Edwards,1992)。
对在叶的上侧的三个主叶中间的每个花盆进行三次测量。将叶子放在夹子中,叶子边缘在夹子底部。然后等待,直到光脉冲完成由窥视指示。
数据转储存储在荧光计的存储器中,并且以后可以通过USB传输读出,并对每个罐进行平均。

笔记

荧光计还提供Y(II)的值,但在这里使用ETR(μmol/m 2),因为包括PAR以补偿潜在的照明变化。

D。耐旱性:可溶性糖的总含量

材料和试剂

  1. 移液管(1,000μl/10ml)(Eppendorf/Finnpipette Labsystems)
  2. 管(15ml)(VWR International,目录号:525-0308)
  3. 管(2ml)(VWR International,目录号:211-2120)
  4. 试管(玻璃)(VWR International,目录号:4.902.002.75)
  5. 如上所述生长的大麦植物
  6. 液氮
  7. D-葡萄糖(VWR International,目录号:1.08337.0250)
  8. 聚乙烯聚吡咯烷酮(PVPP)(VWR International,目录号:1.07302.0100)
  9. 蒸馏水
  10. 硫酸100%(VWR International,目录号:1.00713.1000)
  11. 硼硅酸盐玻璃瓶(棕色)(VWR International,目录号:215-2328)
  12. Anthrone(VWR International,目录号:1.01468.0010)
  13. 硫脲(VWR International,目录号:1.07979.0250)
  14. 硫酸70%(见配方)
  15. 蒽酮试剂(见配方)

设备

  1. 称重秤(KERN& SOHN GmbH)
  2. 剪刀
  3. Styropor框
  4. 冷冻干燥机(Christ Alpha 1-4 LD plus)
  5. 水浴(GFL)
  6. 振动器(Vortex Genie 2)(科学工业)
  7. 试管架(Geyer GmbH& Co,目录号:9.905 970)
  8. 玻璃弹珠(玩具店,直径15毫米)
  9. 离心机(23,000×g)(Tuttlingen Germany,型号:Hettich Universal 16R)
    注意:目前,它是"Gemini BV Laboratory,型号:Hettich Universal 16R"。
  10. Cuvette(VWR International,目录号:634-9014)
  11. 分光光度计(Thermo Fisher Scientific,型号:Genesys 10S UV VIS)
  12. 烧杯(VWR国际,目录号:213-1131)

程序

首先,准备一个葡萄糖含量的标准曲线,应该为每个蒽酮试剂重复。

  1. 在五个试管中制备五种不同稀释度的D-葡萄糖(每种500μl):0μM,50μM,100μM,150μM和200μM。
  2. 通过涡旋将每个试管缓慢加入2.5ml蒽酮试剂直到存在澄清溶液
  3. 继续执行步骤12-14以及样品。
  4. 测量后绘制葡萄糖含量的标准曲线(图6)

    图6.葡萄糖含量的标准曲线

    在36 das样品用于分析可溶性糖的总含量 并根据Yemm和Willis(1954)进行分析。
  5. 从每盆五个原叶中间切下1厘米的块(100-200毫克)
  6. 称重这些(鲜重仅用于计算干重 重量克分子渗透浓度的含量),填充管(15ml)作为每盆的合并样品 ?并使用styropor盒在液氮中冷冻
  7. 然后将样品冷冻干燥并重新称重(干重)
  8. 加入4毫升蒸馏水(取决于叶组织的量 取样),并将样品在水浴中在100℃下煮沸30分钟 (使用试管架水浴)。
  9. 冷却后,将1,000μl样品溶液加入填充有0.05g PVPP的管(2ml)中并涡旋
  10. 在23,000×g(5000rpm)下离心管子5分钟
  11. 将200μl上清液转移到试管中,加入300μl蒸馏水
  12. 通过缓慢涡旋加入2.5ml蒽酮试剂,直到存在澄清溶液
  13. 在每个玻璃管上放一块玻璃大理石(图7)并煮沸 溶液在100℃水浴中15分钟(使用试管 水浴架)。
  14. 在冷水浴(20℃)中冷却10分钟
  15. 通过a直接测量样品(图7)(最大1小时时间) ?分光光度计在625nm使用比色杯。零点校准应该 ?用蒸馏水

    图7.试管准备就绪 测量可溶性糖的总含量。示例性地,三次测试 管具有对照样品(浅蓝色,左)和三个试管 用于干旱胁迫(深蓝色,右)处理的样品
  16. 使用标准曲线计算可溶性糖的总含量,并相对于干重(μmol/g)设定。

笔记

在步骤D11中取出的200μl样品体积对于大麦和不同作物的不同发育阶段而变化,并且取决于取样的叶组织的量。重要的是,步骤D11中的最终体积为500μl。体积应定义为标准曲线的最佳范围。

食谱

  1. 70%硫酸(在棕色瓶中混合并在黑暗中孵育12小时) 70ml硫酸(100%) 30 ml蒸馏水
  2. 蒽酮试剂(稳定一天)
    硫酸(70%)
    920 mg/l蒽酮
    920mg/l硫脲

E。耐旱性:游离脯氨酸含量

材料和试剂

  1. 铝箔
  2. 管(15ml)VWR International,目录号:525-0308)
  3. 塞子(VWR国际,目录号:217-0511)
  4. 移液管(1,000μl/10ml)(Eppendorf/Finnpipette Labsystems)
  5. 如上所述生长的大麦植物
  6. 液氮
  7. L-Proline(VWR International,目录号:1.07434.0010)
  8. Toluol(VWR International,目录号:1.08325.1000)
  9. 蒸馏水
  10. 茚三酮(VWR International,目录号:1.06762.0100)
  11. 冰醋酸(AppliChem,目录号:A0820.2500)
  12. 水合试剂(见配方)

设备

  1. 称重秤(KERN& SOHN GmbH)
  2. 剪刀
  3. Styropor框
  4. 冷冻干燥机(Christ Alpha 1-4 LD plus)
  5. 水浴(GFL)
  6. 试管(玻璃)(VWR International,目录号:4.902.002.75)
  7. 试管架(Geyer GmbH& Co,目录号:9.905 970)
  8. 玻璃弹珠(玩具店,直径15毫米)
  9. 振动器(科学工业,型号:Vortex Genie 2)
  10. 磁力搅拌器(RCT basic)(IKA Labortechnik)
  11. Fortuna Optifix安全分配器(Sigma-Aldrich,目录号:Z260207)
  12. 通风橱
  13. Cuvette(VWR International,目录号:634-9014)
  14. 分光光度计(Thermo Fisher Scientific,型号:Genesys 10S UV VIS)
  15. 烧杯(VWR国际,目录号:213-1131)

程序

首先,制备脯氨酸含量的标准曲线,应该对每种茚三酮试剂重复。

  1. 在五个试管中制备五种不同稀释度的L-脯氨酸(各500μl):0μM,25μM,50μM,75μM和100μM。
  2. 通过涡旋慢慢地向每个试管中加入2ml茚三酮试剂,直至存在澄清溶液
  3. 继续执行步骤D11-16以及样品。
  4. 测量后绘制脯氨酸含量的标准曲线(图8)。


    图8.脯氨酸含量的标准曲线

    在36天时,取用于分析游离脯氨酸含量的样品 ?并根据Bates等人(1973)进行分析。对于这个分析 可以使用相同的样品溶液作为可溶性的总含量 因为步骤5-8是一样的。
  5. 从每个盆中五个初级叶的中部切割1cm片(100-200mg)。
  6. 称重这些(鲜重仅用于计算干重 重量克分子渗透浓度的含量),填充管(15ml)作为每盆的合并样品 ?并使用styropor盒在液氮中冷冻
  7. 然后将样品冷冻干燥并重新称重(干重)
  8. 加入4毫升蒸馏水(取决于叶组织的量 取样),并将样品在100℃水浴中煮沸30分钟 (使用试管架水浴)。
  9. 冷却后,将1000μl样品溶液各自填充在试管中
  10. 通过缓慢涡旋加入2ml茚三酮试剂,直至存在澄清溶液
  11. 在每个试管上放置玻璃大理石(见图7)并煮沸 溶液在100℃水浴中15分钟(使用试管架 水浴)。
  12. 在冷水浴(20℃)中冷却10分钟 使用实验室通风柜进行下一步骤:
  13. 取出玻璃大理石,加入5毫升甲苯(例如用分配器)。
  14. 用塞子关闭试管,涡旋15秒
  15. 在室温下在黑暗中孵育样品(可能长达4小时)。
  16. 发生两个阶段(图9),从上面的阶段开始 转移到用于在520处用分光光度计测量的比色皿中 ?nm。零点校准应使用甲苯进行。


    图9.测试 管准备测量游离脯氨酸的含量。示例性地, 三个试管,具有对照样品(浅红色,左)和三个 带有用于干旱胁迫(深红,右)处理的样品的试管 显示。

  17. 使用标准曲线计算游离脯氨酸的含量,并相对于干重(μmol/g)设定。

笔记

在步骤E9中,使用1000μl的样品溶液。该体积对于大麦的不同发育阶段以及对于不同作物是不同的,并且取决于取样的叶组织的量。体积应定义为标准曲线的最佳范围。

食谱

  1. 水合试剂(稳定一天,在黑暗中混合,用铝箔覆盖烧杯并在磁力搅拌器上溶解)
    0.5克茚三酮
    20ml蒸馏水
    30ml冰醋酸

F。耐旱:渗透压

材料和试剂

  1. 管(2ml)(VWR International,目录号:211-2120)
  2. 移液管(100μl/200μl)(Eppendorf)
  3. 如上所述生长的大麦植物
  4. 液氮
  5. 蒸馏水
  6. osmomat的校准标准(Gonotec GmbH,目录号:30.9.0020)

设备

  1. 称重秤(KERN& SOHN GmbH)
  2. 剪刀
  3. Styropor框
  4. 镊子
  5. 摇摆磨(Retsch,型号:MM200)
  6. 研磨球(VWR International,目录号:412-0070)
  7. 研磨罐(Retsch,目录号:22.008.0005)
  8. 振动器(科学工业,型号:Vortex Genie 2)
  9. 离心机(21,000×g )(Wehingen,型号:Hermle Z233MK2)
    注意:目前,它是"HERMLE Labortechnik GmbH,型号:Hermle Z233MK2"。
  10. Osmomat(Gonotec GmbH,型号:Osmomat 3000 Gonotec)
  11. 用于osmomat的管(Gonotec GmbH,目录号:30.9.0010)

程序

在36天,重量克分子渗透压浓度通过蒸馏水和细胞液的凝固点的比较测量来确定。

  1. 从每盆五个原叶的中间切下1厘米的块。
  2. 称量这些,将每盆中的合并样品填充在管(2ml)中,并使用styropor箱在液氮中冷冻。
  3. 也将研磨球和研磨罐放入液氮(进入styropor盒)
  4. 取每个试管,用镊子填入两个研磨球
  5. 取出研磨罐,每个填充五个管。
  6. 在摇摆磨中研磨叶子3分钟(30 /秒)
  7. 向管中加入200μl蒸馏水并涡旋
  8. 用镊子将研磨球从管中取出。
  9. 以21,000×g(15,000rpm)离心管4分钟15分钟。
  10. 吸取上清液细胞液在新管(2毫升) 使用osmomat进行测量时,请使用osmomat的原始管。
  11. 使用15μl校准标准品校准osmomat,直到达到准确的0.3的值。
  12. 用15μl蒸馏水调整零值。
  13. 每个测量15微升细胞液。
  14. 可以对水含量校正值(osmol/kg) (鲜重和干重的差异)。干重计算为 与用于测定的样品的干重含量的关系 的游离脯氨酸含量和可溶性糖的总含量 以上)。

G。耐旱性:生物量产量

材料和试剂

  1. 如上所述生长的大麦植物

设备

  1. 称重秤(KERN& SOHN GmbH)
  2. Crispack包(Baumann,目录号:3.331.100)
  3. 剪刀
  4. 隔室干燥器(Heratherm oven)(Thermo Fisher Scientific)

程序

  1. 在36天,每盆切割整个生物量高于地面。
  2. 将每个锅的生物量与标签放在一个包装袋中
  3. 关闭行李。
  4. 在105℃的室干燥器中干燥袋内的生物质,直到重量不变(通风约两天)。
  5. 通过称重干重,检测总生物量产量(g)

致谢

作者感谢Halle-Wittenberg马丁路德大学作物植物研究跨学科中心(IZN)为该项目提供资金。此外,一些协议是从先前承认Bates等人(1973),Paech和Simonis(1952)以及Yemm和Willis(1954)的工作修改或调整的。

参考文献

  1. Bates,L.S.,Waldren,R.P.and Teare,I.D。(1973)。 快速测定水分胁迫研究中的游离脯氨酸。 植物土壤 39:205-207。
  2. Krall,J.P。和Edwards,G.E。(1992)。 光系统II活性与CO 2 固定之间的关系 Pysiol Plantarum 86:180-187。
  3. Paech,K。和Simonis,W。(1952)。 Pflanzenphysiologische Praktika Band Iübungenzur Stoffwechselphysiologie der Pflanzen。 Springer Verlag,BerlinG?ttingenHeidelberg。
  4. Stauss,R。(1994)。 单子叶植物和双子叶植物的生长阶段识别密钥摘要:扩展BBCH标度 Ciba。
  5. Wehner,G.G.,Balko,C.C.,Enders,M.M.,Humbeck,K.K.and Ordon,F.F.(2015)。 涉及对干旱胁迫和干旱胁迫的耐受性的基因组区域的鉴定诱导幼苗大麦叶片衰老。/a> BMC Plant Biol 15:125
  6. Yemm,E.W。和Willis,A.J。(1954)。 蒽醌在植物提取物中碳水化合物的估计。生物化学杂志em> 57(3):508-514。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wehner, G., Balko, C. and Ordon, F. (2016). Experimental Design to Determine Drought Stress Response and Early Leaf Senescence in Barley (Hordeum vulgare L.). Bio-protocol 6(5): e1749. DOI: 10.21769/BioProtoc.1749.
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当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。