This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.
The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.
Plants, as sessile living organisms, have developed a highly sophisticated network of cell-to-cell signaling to address various environmental stimuli. Tropic responses are one of the phenomena by which plants respond to environmental stimuli. Plants show two main tropic responses, phototropism and gravitropism. Photosynthetic plants bend toward the light source by phototropism to harvest maximum energy. Similarly, gravitropism makes the plants to grow toward the gravity center. The fundamental mechanism leading to such tropic responses involves asymmetric gradient formation of the phytohormone auxin1. The act of local auxin gradient formation is well characterized; the genes that are involved in this mechanism provide a roadmap for hormone action2-8. The specific position of auxin efflux carriers, such as PIN-FORMED (PIN) and P-glycoproteins, executes the movement of auxin from the cytoplasm to the cell wall of donor cells9,10. Furthermore, by the active H+/IAA symport activity of auxin influx carriers, such as AUX1/LAX family proteins, auxin is finally delivered to the adjacent receiver cells2,11,12. This directional movement of auxin is known as polar auxin transport (PAT). PAT leads to a differential auxin distribution during various developmental stages and in response to different environmental stimuli13,14. Moreover, the disruption in localization or expression of any of these auxin influx or efflux carriers leads to severe alteration in PAT, which causes a disruption of the auxin gradient, leading to developmental defects. Recently, Han et al. reported that plasmodesmal regulation is also necessary to maintain the auxin gradient15. To date, more than 30 plasmodesmal proteins have been identified16. Among these proteins, AtGSL8 has been reported as a key enzyme for callose synthesis at plasmodesmata (PD) and hence plays a vital role in maintaining the PD size exclusion limit (SEL). Repressed AtGSL8 expression resulted in a distorted auxin gradient pattern leading to no tropic response in contrast to wild type seedlings15.
In this manuscript, methods to explore new candidate genes that are involved in PD regulation are provided. AtGSL8 was used as a model protein to test these methods, as it is a key enzyme contributing to PD callose biosynthesis. Due to the seedling-lethality of gsl8 knock-out mutants17, dexamethasone (dex)-inducible RNAi lines were used in accordance with a previously published report15. The strategy provided here can be adapted to screen genes that are implicated in hypocotyl tropic response controlled by PD SEL.
1. Screening of Mutants with Altered Phototropic and Gravitropic Responses
2. Screen Plant Lines with Defective Tropic Responses Due to Changes in PD SEL with an Altered PD Callose Level
In the current setup, dexamethasone (dex)-inducible RNAi lines of AtGSL8 [hereafter dsGSL8 (+dex/-dex)] were used, as homozygous gsl8 T-DNA insertion mutants are seedling lethal18. Three-day-old etiolated seedlings of dsGSL8 and wild type seedlings with ±dex were exposed to phototropic and gravitropic stimuli. We found that dsGSL8 (+dex) seedlings were defective in phototropism and gravitropism15. Figure 5 clearly shows that dsGSL8 (+dex) exhibits no bending under the influence of both phototropism and gravitropism. Furthermore, alteration in symplasmic movement in dsGSL8 (+dex) and dsGSL8 (-dex) or Col-0 was analyzed by a HPTS loading assay. Consistent with our previous finding, the HPTS dye movement in dsGSL8 (+dex) was substantially more extensive than in dsGSL8 (-dex) or Col-0, as shown in Figure 6. Callose is one of the key regulators of PD SEL, and AtGSL8 is known as a PD callose synthase16. Furthermore, callose aniline blue staining was carried out, and dsGSL8 (+dex) have persistently shown a low PD callose level before and after tropic responses, as shown in Figure 7.
Figure 1. Flow chart of the steps that are involved in the stimulation of phototropic and gravitropic responses. (A) Transfer vertically grown seedlings from different plant backgrounds to one plate but in separate lines. (B) For phototropism, keep the plates facing towards unilateral white light in a dark box that is opened on one side. For gravitropism, first wrap the plates with aluminum foil, rotate 90°, and transfer to a dark box (covered from all sides). (C) Using a scanner, scan the plates after different intervals of time, such as 1.5 hr, 3 hr, 6 hr and 12 hr. (D and E) These panels show data generation from ImageJ and data analysis using spreadsheet files, respectively. Please click here to view a larger version of this figure.
Figure 2. Measurement of the bending angle by ImageJ. (A) View the Image J program by double clicking. (B) The major tools that were used in the angle measurement. (C) Open the "File" option followed by the sub-option "open" in the Image J toolbar to open microscopy pictures that were saved in JPEG file format. (D) Use the magnifying tool to zoom in or out on the picture by clicking the left or right mouse button, respectively. (E) Use the angle tool to measure the angle of bending. (F) Outline of the angle measurement: abc defines a fixed A, and the true bending angle is calculated as B, which is equal to 180°-A. (G) Line representing the distance from point a to point b. (H) Continuity of line a-b to point c making A. (I) Result file of Image J showing A value. (J) Representative spreadsheet file displaying the average bending angle measurements, standard deviations, standard errors and t-test calculations. (K) Bar graph diagram displaying the bending angle measurements between two different plant backgrounds Col-0 and dsGSL8 EMS mutant. Error bars are SEM (standard error of the mean +) and *** represents the significance by T-test (p value >0.001). Please click here to view a larger version of this figure.
Figure 3. Outline of the steps that are involved in the HPTS loading assay. (A) Panel showing the preparation of HPTS agarose blocks. (B) Panel representing the excision and transfer of seedlings and the loading of HPTS agarose blocks. (C) Panel displaying the steps that are involved in sample preparation for confocal imaging. Please click here to view a larger version of this figure.
Figure 4. Measurement of the callose intensity by ImageJ. (A) View the ImageJ program by double clicking. (B) The major tools that were used in the callose intensity measurement. (C) Open the "File" option followed by the sub-option "open" in the ImageJ toolbar to open microscopy pictures that were saved in JPEG file format. (D) The rectangular icon on ImageJ toolbar. (E) The selected area of the image for analyzing the callose intensity. (F) ImageJ result file displaying the callose intensity value as "Mean". (G) Representative spreadsheet file displaying the average callose intensity measurements, standard deviations, standard errors and t-test calculations. (H) Bar graph diagram representing the callose intensity difference between the two plant backgrounds. Error bars are SEM (standard error of the mean +) and *** represents the significance by T-test (p value >0.001). Please click here to view a larger version of this figure.
Figure 5. Reduction of AtGSL8 expression leads to defective tropic responses. (A) Phototropic response showed by dsGSL8 (±dex) along with Col-0. (B) Gravitropic response shown by dsGSL8 (±dex) along with Col-0. dsGSL8 (+dex) shows no phototropic or gravitropic responses. dsGSL8 (+dex) and dsGSL8 (-dex) symbolize dexamethasone-treated and untreated dsGSL8-inducible RNAi lines, respectively. Scale bar represents 0.2 cm. Please click here to view a larger version of this figure.
Figure 6. The symplasmic movement increased after the suppression of AtGSL8. Fluorescence images were taken after HPTS loading to dsGSL8±dex hypocotyl cut surfaces along with wild type Col-0. dsGSL8 (+dex) showed more extensive movement of HPTS dye, demonstrating increased symplasmic movement. Scale bar represents 0.2 mm. Please click here to view a larger version of this figure.
Figure 7. AtGSL8 suppressed lines showed symmetric PD callose distribution between illuminated and shaded sides of the hypocotyl. (A) Fluorescence image showing aniline blue callose staining for dsGSL8 (-dex) at 0 hr. (B) Fluorescence image showing aniline blue callose staining for dsGSL8 (-dex) after 6 hr of phototropism. (C) Fluorescence image showing aniline blue callose staining for dsGSL8 (+dex) after 6 hr of phototropism. Yellow arrows indicate the callose accumulation on shaded region of hypocotyl. Scale bar represents 50 µm. (D) Bar graph diagram representing the amount of PD callose that accumulated in dsGSL8 (±dex) hypocotyls before and after 3 hr and 6 hr of phototropism. Fluorescence foci intensities were measured from ten independent hypocotyls (data are mean ± SD; Student's t-test, *p <0.01). This figure has been modified from (Han et al., 2014) 15. Please click here to view a larger version of this figure.
In this manuscript, a strategy to screen mutant/over-expression lines that are defective in phototropic and gravitropic responses due to altered PD callose and, hence, PD SEL is described in detail. PD callose synthesis and degradation is mainly accomplished by callose synthases and β-1,3-Glucanases, but regulation of these enzymes is controlled by many upstream factors. To search for such upstream factors or candidates which are directly involved in PD regulation, we have set up this method for screening. This set up has some limitations as mutants of some key enzymes regulating PD callose biosynthesis (gsl8) are lethal15 and to sort out such mutants would be limited by this set up. Also, if the candidate gene shows faster or slower tropic response along with open or closed PD, but the callose level remains unchanged, then detailed analysis is needed to further characterize such candidates. The critical factors and troubleshooting processes in each step are also elaborated. First, in medium preparation, a critical factor that can affect the plant growth is the pH of the media, which should range from 5.7-5.8. Second, appropriate surface drying of agar plates is important; it would be difficult to lift the 3 day-old seedlings without any damage from agar plates with high water content. However, too much drying of agar plates may cause some cracks in the media during incubation and result in asymmetric seedling growth. During the surface sterilization of seeds using sodium hypochlorite solution, care must be taken to remove sodium hypochlorite completely by repetitive washing with autoclaved ddH2O, as it is a strong bleaching agent. Autoclaved ddH2O should be prepared freshly and handled aseptically, as old stocks might cause fungal contamination in MS plates. After sterilization, seeds are stratified by cold treatment to synchronize germination. This process can be performed either by transferring sterilized wet seeds directly to a cold room in dark conditions or first dotting the seeds onto MS plates and then transferring the plates to a cold room in dark conditions. However, we prefer the latter because it gives a more uniform growth of seedlings. While dotting seeds, the distance between two adjacent seeds should be at least of 0.1 cm because very closely dotted seeds would result in the growth of overlapping seedling, which are very difficult to transfer in subsequent experiments.
For proper phototropic and gravitropic responses, seedlings should not be exposed to light and should remain undamaged. To avoid the background effects of white light, carefully select the seedlings under a green light source in the dark room as plants do not respond to green light. Green light source can be generated by covering the white light source using transparent green vinyl sheets. For selecting the seedlings, a sterilized toothpick is the best option. Touch the very lower part of the hypocotyl with the toothpick to lift the seedlings so that they can be easily transferred to a new plate. All of the seedlings should be similar in size and hook orientation. During our experiments, we saw that if the hook direction is on the opposite side of the light source, plants will show a faster tropic response than plants with the same hook direction as the light source. Normally, in the case of phototropism, one can see a clear difference in the bending angle between true candidates and Col-0 within 3 hr, while in the case of gravitropism, it usually takes 6-12 hr. Finally, to have statistically relevant data, a minimum of three biological replicates and 20 seedlings are needed each time.
To test the extent of symplasmic movement between different plant backgrounds, HPTS dye loading is a convenient technique, as it does not need sophisticated tools and instrumentation. For the HPTS loading assay, the percentage of gel in HPTS agarose blocks is of great importance, as a very brittle or hard gel will not give optimum results. It is better to use a 50 ml conical flask with loose rubber and not to use a large-volume flask for boiling agarose with 10 ml of water. HPTS agarose blocks can be used for one week after preparation if kept wrapped with aluminum foil at 4 °C. Another key factor in the HPTS loading assay is the excision of the hypocotyl hook region. Excision should be performed with sharp dissection scissors and at a similar position for all of the seedlings. Excision with blunt scissors can cause unwanted damage to seedlings and can hinder HPTS loading. Additionally, the seedling growth conditions and developmental stages might play a crucial role in dye movement, as callose accumulation is more prone to respond to such changes, which finally account for the hindrance in dye movement. Thus, growing the seedlings under optimum conditions is necessary. Callose at PD plays a key role in the tropic responses of etiolated seedlings by regulating PD SEL, which is important for auxin gradient formation15. Callose levels can be detected by staining with aniline blue or immunogold labeling. Staining with aniline blue is a simple and rapid method for detecting the callose level. Because hypocotyl cells have high turgor pressure and an epi-cuticle layer, it is not easy for the dye to penetrate the cells. Therefore, we developed the cut-staining method to allow aniline blue dye to penetrate easily. However, there is a risk of synthesis of new callose that can influence the staining results, which is a limitation of the normal callose staining method. To avoid such effects, the staining protocol is modified by adding the callose synthase inhibitor 2-D-deoxy glucose (DDG) to the staining buffer. Thus, this protocol involves the measurement of pre-existing callose only in the lower region of the hypocotyl that is exactly below the cut site. Avoid the use of freshly prepared aniline blue, and keep the aniline blue solution at RT for a minimum of 48 hr prior to use. We noticed that staining with fresh aniline blue solution does not give optimum callose staining results.
Overall, we have presented a set of strategies that can be used for the rapid screening of mutants/over-expression lines with a defective or enhanced tropic response by altering PD SEL by directly or indirectly modulating PD callose. An earlier fact about tropic responses was majorly restricted to the action of auxin carriers and signaling players, such as PINIOD2-14. In addition, we have also established a critical role of symplasmic movement of auxin in maintaining an auxin gradient in the hypocotyl system15. The simplicity and versatility of this method undoubtedly reinforce its utility in investigating candidate genes for their regulation of PD SEL, thereby playing a vital role in plant development, including tropic responses.
The authors have nothing to disclose.
This research was supported by the National Research Foundation of Korea (NRF-2015R1A2A1A10053576), and by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant PJ01137901), Rural Development Administration, Republic of Korea. RK, WS, ABB and DK were supported by Brain Korea 21 Plus program (BK21+).
HPTS (8-Hydroxypyrene -1,3,6-trisulfonic acid trisodium salt) | Sigma | H1529-1G | Fluorescent dye as symplasmic tracer |
LE Agarose | Dongin-Genomic | GEL001-500G | Used for HPTS agarose block |
Microwave oven | LG-Goldstar | Machine for boiling agarose gel | |
100 mL glass conical flask | Dong Kwang | A0205 | Used to boil HPTS agarose gel |
Petri Dish (35×10 mm) | SPL life sciences | SPL10035 | Used to make HPTS agarose blocks and wash plant samples |
Microscope cover slides and glass slides (24 x 50 mm) | Marienfeld Laboratory Glassware | 101222 | Used for HPTS agarose blocks and microscopic sample preparation |
MS medium plates 125 x 125 x 20 mm | SPL life sciences | SPL11125 | Plates to make MS agar medium |
Scissors | Germany Stainless | HSB 942-11 | Used to excise hook region of plant samples |
Murashige and Skoog (MS) basal salt mixture | Duchefa | P10453.01 | MS medium including vitamins. |
(N-morpholino) ethanesulfonic acid (MES) monohydrate | Bioshop | 3G30212 | To make MS media. |
Plant agar | Duchefa | P1001.1000 | To solidify MS media. |
Autoclave | ALP | CL-40L | |
Shaker | Wise Mix | SHO-1D | To wash off the aniline blue staining buffer and HPTS dye in a placid way. |
1 ml Blue tips | Sorenson | 10040 | |
1 ml pipette | BioPette | L-1101-2 | |
Surgical tape | MIcropore | 1530-0 | To seal the MS plate |
Aniline blue (Methyl blue) | Sigma | M5528-25G | Used to prepare aniline blue staining buffer. |
Glycine | Bioshop | GLN001 | Used to prepare aniline blue staining buffer. |
DDG | Sigma | D8375-1G | Used for the inhibition of callose synthases. |
Confocal microscope | Olympus | FV1000MPE SIM | To check aniline blue staining and HPTS dye loading result. |
Stirrer | I lab | K400 | To mix media solution. |
Aluminium foil | SW cooking foil | To wrap plates in a dark condition. | |
Sodium hypochlorite | Samjin Industry | To surface-sterilized seeds |