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Mar 2020
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Age, Wound Size and Position of Injury – Dependent Vascular Regeneration Assay in Growing Leaves
年龄、伤口大小和损伤部位 – 生长叶片中依赖于损伤的维管再生试验   

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Abstract

Recurring damage to the aerial organs of plants necessitates their prompt repair, particularly their vasculature. While vascular regeneration assays for aerial plant parts such as the stem and inflorescence stalk are well established, those for leaf vasculature remain unexplored. Recently, we established a new vascular regeneration assay in growing leaves and discovered the underlying molecular mechanism. Here, we describe the detailed stepwise method for the incision and regeneration assay used to study leaf vascular regeneration. By using a combination of micro-surgical perturbations, brightfield microscopy, and other experimental approaches, we further show that the age of the leaf as well as the position and size of the injury determine the overall success rate of regeneration. This easy-to-master vascular regeneration assay is an efficient and rapid method to study the mechanism of vascular regeneration in growing leaves. The assay can be readily combined with cellular and molecular biology techniques.

Keywords: Arabidopsis regeneration (拟南芥再生), Leaf incision (叶切口), Vascular regeneration (维管再生), Wound size (伤口的大小), Age-dependent (年龄相关)

Background

Due to their sessile nature, plants are frequently subjected to injuries caused by biotic and abiotic factors. These injuries, when left unattended, can compromise plant immunity, growth, and even survival (Hwang et al., 2017; Radhakrishnan et al., 2020). To overcome the adversities of wounding, plants have evolved a remarkable repertoire of regenerative responses ranging from wound healing in the form of local cell proliferation to complete replacement of amputated organs, such as root tip regeneration (Ikeuchi et al., 2016; Shanmukhan et al., 2020). Although numerous studies have probed the mechanisms underlying several regenerative responses in plants, investigations regarding the regeneration potential of aerial organs are limited (Iwase et al., 2011; Kareem et al., 2015; Durgaprasad et al., 2019). Thus, despite their higher susceptibility to injury than underground organs, there is a dearth of information on regeneration in the aerial organs of plants, particularly, the leaves. Although leaves play a crucial role in plant physiology, their regeneration potential has hardly been investigated (Kuchen et al., 2012; Radhakrishnan et al., 2020).


Leaves possess an elaborate network of vascular tissue with a central midvein that transports substances back-and-forth between the main plant body. Damage to the midvein calls for prompt repair, failing which the transport of substances, and consequently the growth of the leaf and its adjacent branches, are impaired (Sachs and Hassidim, 1996; Radhakrishnan et al., 2020). Recently, a new vascular incision assay in leaves has been developed to study wound repair and tissue restoration in response to injury. The assay revealed that the mechanically disconnected parental stands are reunited by regenerating vascular tissue that bypasses the site of injury. The assay was instrumental in understanding the molecular mechanism underlying vascular regeneration in aerial organs growing in the normal developmental context. Upon injury, a coherent feed-forward loop comprising cell fate determinants, PLETHORA (PLT) and CUP-SHAPED COTYLEDON2 (CUC2), activates local auxin biosynthesis, leading to vascular regeneration in growing aerial organs (Radhakrishnan et al., 2020). Although vascular regeneration necessitates the PLT-CUC2 regulatory axis, the extent of injury acted as a limiting factor (Radhakrishnan et al., 2020). Here, we show that the regenerative ability of leaf vasculature is determined by the size of the injury, age of the leaf explant, and position of the injury along the proximodistal axis of the leaf blade.


This easy-to-master, reproducible assay can be performed using readily available laboratory supplies. The convenience of performing real-time confocal imaging and other molecular techniques, such as quantitative real-time PCR, using the injured leaves makes the assay valuable for studying the molecular players and mechanisms regulating wound-induced response and regeneration in the normal developmental context. The method will also be useful for studying the interplay between mechanisms of vein patterning during development and that of vein regeneration.


Materials and Reagents

Reagents for seed sterilization

  1. Seeds of wildtype (Columbia) Arabidopsis thaliana

    Note: For the purpose of standardization, we used wildtype plants. The assay can be performed without any modifications in Arabidopsis mutant plants to study vascular regeneration efficiency (Radhakrishnan et al., 2020).

  2. 20% sodium hypochlorite

  3. 70% ethanol

  4. Sterile autoclaved water


Reagents for sample decolorization
  1. 35 mm (diameter) round Petri dish (Himedia, catalog number: PW050)

  2. 15% ethanol

  3. 50% ethanol

  4. 70% ethanol

  5. 96% ethanol

  6. 100% ethanol

  7. Glycerol (Sigma-Aldrich, catalog number: G5516)

  8. Chloral hydrate (Sigma-Aldrich, catalog number: 23100)

  9. Sterile autoclaved water

  10. Murashige and Skoog (MS) salt (Sigma-Aldrich, catalog number: M5524)

  11. Sucrose (Sigma-Aldrich, catalog number: S0389)

  12. Plant agar (Sigma-Aldrich, catalog number: A7921)

  13. Clearing solution (see Recipes)

  14. Half-strength Murashige and Skoog (MS) medium (see Recipes)

Equipment

Equipment for in vitro culture

  1. Laminar air flow chamber (LAF)

  2. Sterile pipette tips (200 µl and 1 ml)

  3. Micropipettes

  4. 1.5 ml microcentrifuge tubes

  5. Sterile disposable square Petri dishes, size: 120 mm × 120 mm (Himedia, model: PW050-1)

  6. Plastic wrap (Himedia Phytawrap)

  7. Plant growth chamber (Percival, model: AR-100L3)


Equipment for incision and sample collection
  1. Fine-point tweezers (Dumont tweezer, model: Style 5)

  2. Sterile razor blade

  3. Forceps

  4. Micro-Vannas scissors, straight (Ted Pella, model: 1340)

  5. Gloves

  6. Face mask


Equipment for microscopy
  1. Stereozoom microscope (Zeissstemi , model: 2000) for incision and sample collection

  2. Confocal laser-scanning microscope (Leica, model: TCS SP5 II) for brightfield imaging

  3. Microscope slides (Labtech)

  4. Microscope cover glass, 22 × 22mm (Corning, model: 2850-22)

  5. Watercolor brush (with small bristles)

Software

  1. R software

Procedure

  1. Seed sterilization

    Seed sterilization should to be performed within the LAF under sterile conditions. The workspace and tools (micropipette, tip boxes, reagent bottles) required for the procedure must be thoroughly wiped with 70% ethanol and UV irradiated. Prior to commencing in vitro culture, hands should be washed using soap and wiped with 70% ethanol. A liquid surface sterilization protocol for seeds is described here.

    1. Aliquot the required number of wildtype seeds in a 1.5-ml microcentrifuge tube.

      Note: For efficient sterilization, do not take more than 300 seeds per tube and remove any debris such as parts of siliques left over from seed collection.

    2. Add 1 ml 70% ethanol. Agitate the contents by inverting the tube for 2-3 min.

      Note: Prior to centrifugation, ensure that the centrifuge and rotor surfaces are clean. Avoid touching the inside of the lid of the microcentrifuge tube while opening and closing to minimize contamination.

    3. Briefly spin the tube at 4,226 × g (Rcf) and carefully discard the ethanol without losing any seeds.

    4. Add 1 ml 20% sodium hypochlorite and shake the contents for 2-3 min. Repeat Step A3.

    5. Wash the seeds 5-7 times using 1 ml sterile autoclaved water.

    6. Stratify the seeds in 1 ml sterile autoclaved water for two days at 4°C.

    7. Pipette approximately 25 seeds using a 1-ml pipette and place on the surface of half-strength MS medium. Space the seeds out in a row using the pipette tip, leaving at least a 0.5-cm gap between each seed.

      Note: For ease of incision, avoid placing the seeds very close to each other.

    8. Incubate the Petri dishes vertically in a growth chamber under 45 μmol/m2/s continuous white light (24 h) at 22°C and 70% relative humidity.

      Note: The assay can also be performed under long day and short day conditions using 5 dpg plants.


  2. Leaf incision

    1. Leaf incision can be performed on the work bench after adopting the necessary measures to minimize contamination. Wear gloves and a face mask during the procedure. Prior to incision, wipe the surface of the dissection microscope (Zeiss stemi 2000) and gloved hands with 70% ethanol. The tweezers used for incision should be dipped in 70% ethanol and allowed to dry for a few minutes before incision. Opening the plate on multiple days for incision will increase the incidence of contamination.

      Note: Due to the fragile nature of the tweezer tips, sterilization techniques that may damage or blunt are not recommended.

    2. The plate containing the seedlings is opened under a dissection microscope to confirm the age of the seedlings and to perform an incision in 5 dpg (days post-germination) seedlings.

      Note: Due to the asynchronous nature of seed germination, the seedlings may not all be of the same age. The age of the seedlings is determined by counting the number of days post-germination (dpg). The first day of radicle emergence is counted as 0 dpg. To maintain consistency, only injure the plants that are at the desirable developmental stage. Move aside the uninjured plants to distinguish them from injured ones. The age of the seedlings is important as older seedlings display reduced regeneration efficiency, while very young seedlings are extensively damaged during the procedure. The appropriate age of incision is 4-6 dpg (Figure 1).

    3. Of the two leaves belonging to the first pair (true leaves), the leaf that faces the lid of the Petri dish is chosen for incision due to ease of access. Using the sharp tip of the tweezers, an incision is made on the lower abaxial surface of the leaf belonging to the first pair. The incision is carefully performed at the junction between the petiole and the basal end of the lamina (Figure 1a, Supplementary Figure 1, Video 1). This ensures that the injury occurs just above the first lateral vein (counted from the base of the leaf), where the regeneration efficiency is highest in comparison with other positions along the proximodistal axis of the midvein (Figure 2). The incision should be performed with just enough force so that it punctures the vascular tissue located close to the abaxial surface of the leaf without piercing through the adaxial surface. This is important as extensive damage that creates a gap exceeding 400 µm between the parental vascular strands is not repaired (Radhakrishnan et al., 2020) (Figure 1p).

      Note: Care should be taken not to inflict multiple damages on the leaves; therefore, it is not advisable to perform incision in both leaves of the first pair. During incision, a sterile 200 μl microtip or forceps may be used to restrict the movement of the plant by supporting the cotyledon or hypocotyl during incision. This will prevent the plant from submerging into the media due to the force of incision; however, avoid touching the damaged leaves as this can inflict further damage.

      Video 1. Demonstration of performing leaf incision in 5 dpg plants


    4. After incision, the plates are closed and incubated vertically under continuous light at 22°C in a growth chamber.

    5. Four days post-incision (dpi), the injured leaf is carefully cut at the petiole using Vannas scissors (Video 2) and placed in 15% ethanol in a small round Petri dish (35 mm).

      Note: Around 20-30 leaves can be treated using 2-3 ml 15% ethanol in a 35 mm Petri dish during the decolorizing procedure. Alternatively, 6-well plates can be used when handling multiple types of samples.


      Video 2. Demonstration of sample collection 4 days post-leaf incision


      Figure 1. Leaf vascular regeneration upon midvein injury depends on the age of the injured leaf. (a-b) Illustration depicting the location of the incision for effective vascular regeneration. The red star represents the site of injury. (c, e) Schematic and brightfield image showing the regenerating vasculature reuniting the disconnected parental stands to form a D-loop bypassing the site of injury. (d, f) Illustration and brightfield image showing the regenerating vasculature connecting the apical cut end to the nearest lateral vein. Yellow blocks in (c) and (d) represent end-to-end connected xylem elements. (g-n) Regeneration response in leaves injured at 3 dpg (g) (*P = 0.016, n = 24), 4 dpg (h) (P = 0.426, not significant (ns), n = 45), 5 dpg (i) (n = 20), 6 dpg (j) (P = 0.605, not significant (ns), n = 40), 7 dpg (k) (*P = 0.03, n = 34), 8 dpg (l) (***P = 1.6 × 10-12, n = 43), 9 dpg (m) (***P = 2.405 × 10-9, n = 52), and 10 dpg (n) (***P = 4.8 × 10-8, n = 21). Statistical analysis using Pearson’s χ2 test. Note that the 3-7 dpg leaves are capable of reconnecting their disconnected vasculature but the regeneration efficiency declines with progressive aging of the leaves. The regenerating vasculature is indicated by the red dots. The black arrowheads indicate the site of injury. (o) Graph depicting the frequency of vascular regeneration in leaves injured at different ages. (p) Extensive damage creates a gap exceeding 400 µm between the parental vascular strands; as a result, no vascular regeneration is observed. (q) End-to-end attached xylem elements of a regenerated vascular strand. Scale bar: 50 µm.


  3. Sample decolorization and clearing

    1. After a 15-min incubation in 15% ethanol, the ethanol is drained using a micropipette with care being taken not to damage the samples.

      Note: Initially, the leaves float on the surface of the solvent. Gently submerge the leaves using a paint brush with small bristles.

    2. The tissue is gradually dehydrated by subsequent treatment with 50%, 70%, and 96% ethanol consecutively for 15 min each. After discarding 96% ethanol, the leaves are incubated for 12 h in 100% ethanol for tissue dehydration and removal of the chlorophyll pigmentation.

    3. After discarding the ethanol, the samples are consecutively incubated for 15 min each in 96%, 70%, 50%, and 15% ethanol for rehydration.

    4. After discarding the ethanol, freshly prepared clearing solution (see Recipes) containing chloral hydrate is added to the sample. The samples are incubated in the clearing solution for at least 3 h prior to mounting the slides for brightfield imaging.

      Note: Increasing the duration of clearing can enhance the contrast during brightfield imaging to some extent.


  4. Slide preparation

    Using a small paint brush, each cleared leaf is picked up from the clearing solution and placed on a clean slide with the adaxial surface of the leaf facing upward. The brush can be used to gently tease open any curled leaves without inflicting any further damage. The coverslip is mounted over the sample, taking care not to create any bubbles. Multiple leaves (6-8) can be placed under a single coverslip. Using a 200 µl pipette tip, approximately 80 µl clearing solution is added from the corner of the mounted coverslip, thereby filling the gaps between the mounted leaves.


  5. Brightfield imaging

    The regeneration of vascular strands in the cleared samples can be assessed by imaging the site of incision using the brightfield mode of a fluorescent or confocal microscope. A snapshot of the regenerating vascular strand captured by a confocal microscope is recommended to acquire high-resolution images of the regenerating xylem elements. The settings described here are for a Leica TCS SP5 II inverted microscope. An argon laser or DPSS 561 can be used to capture a snapshot at a laser power of 30%, scan speed of 200 Hz, line average of 2, and a pixel format of 1024 × 1024. Newly formed vascular strands display distinct morphology characterized by end-to-end connected xylem elements (Figure 1e, f, j, k, q). When the regenerating vein reunited the cut ends of the midvein forming a D-loop (Figure 1c, e) or connected either of the cut ends to a lateral vein (Figure 1d, f), the outcomes were scored as successful regeneration. To maintain consistency in the methodology, incisions made in locations other than the junction of the first lateral vein were not scored while studying the age dependency of regeneration. Additionally, only incisions creating a gap less than 400 µm between the detached parental strands were scored.

Data analysis

Statistical analysis was performed using the R software. The collected data were statistically analysed using Pearson’s χ2 test.


Results and Discussion

Although the regeneration ability of plants has been widely investigated, leaves have been seldom studied for their regeneration or local wound repair abilities (Kuchen et al., 2012; Radhakrishnan et al., 2020). We describe a detailed stepwise method for a novel leaf vascular regeneration assay that can be used to study regeneration of the midvein in response to local injury. Our previous studies have shown that a mechanical disconnection of the midvein, creating a gap of under 400 µm (measured after sample clearing), can be bridged by regenerating vascular strands (Radhakrishnan et al., 2020). While the injured vascular tissue degenerates, the newly synthesized vasculature can either reunite the disconnected strands or connect the cut end to the nearest lateral vein (Figure 1b-f). Either way, the reconnection ensures restoration of the leaf vascular network and transport between the leaf and the rest of the plant body. However, extensive damage generating a gap larger than 400 µm cannot be repaired, thereby denying functional restoration of the leaf vascular tissue (Radhakrishnan et al., 2020) (Figure 1p). Here, the wound-size dependency of vascular regeneration was recapitulated in silico by implementing a computational model based on the canalization hypothesis of vein formation in leaves (Rolland-Lagan and Prusinkiewicz, 2005). According to the canalization hypothesis (Sachs, 1991), positive feedback between auxin flux and auxin conductivity leads to channelized auxin flow, which in turn, promotes the differentiation of vascular tissue. Consistent with our experimental observations (Figure 1p), the computational model demonstrates that the formation of a new vascular strand is indeed dependent on the size of the opening (mimicking a wound-induced gap) created in a matrix of cells (resembling a leaf blade). The model also predicts that the failure of vascular regeneration upon extensive damage can be attributed to the disruption of auxin flux and concentration as a result of the larger wound (Video 5). Such a disruption would hinder the proper channelization of auxin and prevent the efficient differentiation of the regenerating vascular strands. Equations governing the mathematical model and other relevant details are presented in the Supplementary Information (Supplementary information 1, Videos 3-5). Our results indicate that in addition to animal cells and unicellular Dictyostelium, wound size sensitivity of the repair process is also conserved in plants (Pervin et al., 2018).

    Having substantiated the wound-size dependency of vascular regeneration, we next investigated whether the regeneration response is dependent on the age of the wounded plant. In many higher animals, progressive aging is associated with reduced regeneration ability (Yun, 2015). To probe how age regulates the regeneration response in leaves, we performed the incision in plants aged 3-10 dpg. Performing incisions on the miniscule leaves of 3 dpg plants was tedious and often damaged the leaves excessively. Upon comparison, leaves of 3 dpg plants showed lower regeneration efficiency than those of 5 dpg plants (Figure 1g, 1o). Leaves of plants aged 4-6 dpg displayed the highest regeneration efficiency, making this the optimal age to study vascular regeneration in leaves (Figure 1h-j, 1o). Although it is easier to perform incisions in older and larger leaves, the regeneration efficiency declined steeply, with leaves of 10 dpg plants completely failing to regenerate (Figure 1k-o). It is important to note that even when the injury-induced gap was less than 400 µm, vascular regeneration was impeded in these older leaves (Figure 1l-n); thus, our data suggest that vascular regeneration efficiency reduces with age in injured plants.

    Regeneration studies in plants and animals have demonstrated that the competence to regenerate in response to injury can vary even within a specific organ (Durgaprasad et al., 2019; Morgan, 1902); therefore, we next examined how the position of incision on the growing leaf influenced the vein regeneration efficiency. To analyze this, we made incisions at different positions along the leaf blade, namely the petiole of the leaf, the basal end (proximal to the plant body axis) of the midvein, the apical end (distal to the plant body axis) of the midvein, and the lateral veins (Figure 2a, 2c-f). The highest regeneration frequency was recorded at the basal end of the midvein, particularly between the first and second lateral vein (Figure 2b, 2d). The petiole also showed a similar regeneration efficiency upon incision and often led to the formation of multiple strands in response to injury (Figure 2b, 2c). However, since the leaf is excised at the petiole during sample collection, the incision site and regenerated vascular strand are occasionally damaged, leading to loss of valuable samples. Additionally, since incisions performed in the petiole lead to multiple stand formation instead of single-stand regeneration, it is more appropriate to make injuries in the leaf blade, as the study involves following a single regenerating strand in real-time to study recognition, communication, and reunion of vascular strands. Upon injuring other positions, we observed that the regeneration efficiency drastically declined toward the apical regions of the midvein and in the lateral veins (Figure 2b, 2e-g).

    Collectively, our data demonstrate that leaf vascular regeneration is sensitive to the size of the wound, the age of the injured leaf, and the position of incision on the leaf.



Figure 2. Vascular regeneration depends on the position of injury in the leaf. (a) Schematic depicting the positions of incision on the leaf blade and petiole. (b) The frequency of vascular regeneration at different positions (represented by colored boxes in (a)) in the leaf is represented by the same color bars in the graph (b) Petiole (n = 21, P = 0.95, not significant [ns]), basal correct position (n = 45), midvein upper end (n = 42, ***P = 0.0009), lateral vein (n = 21, ***P = 0.0002). (c-f) Images showing the incision to the vasculature in the petiole (c), base of the midvein (d), apical region of the midvein (e), and lateral vein (f). Note the multiple strand formation upon injury in the petiole (c). The red dots indicate regenerated vascular strands and the black arrowheads represent the site of incision. Scale bar: 50 µm. (g) Gradient represents the efficiency of vascular regeneration along the leaf blade with maximum regeneration (represented by red) at the base of midvein. Lateral veins and the distal end of the midvein exhibit reduced regeneration frequency.


Video 3. Formation of veins in a grid of 50 by 50 cells with no absent cells (mimicking no incision)

 

Video 4. Formation of veins in a matrix mimicking a small incision


Video 5. Formation of veins in a matrix mimicking a large incision


Conclusion

Our study reveals that 4-6 dpg leaves respond most efficiently to smaller wounds (400 µm or less in size) that are inflicted at the junction of the first lateral vein at the proximal end of the leaf blade. While adopting this assay to study regeneration in other plant species, we recommend standardization of the method with respect to the above-mentioned criteria.

    The assay will be helpful in exploring the mechanisms underlying the regeneration of vascular tissue in growing leaves. To begin with, the assay may prove tedious; however, with repeated practice, this method can be performed deftly and rapidly in a large number of samples. The short duration of the experiment (experimental data can be collected 5 days post-injury) and the dispensability of specialized equipment makes it amenable to the larger scientific community. As demonstrated previously (Radhakrishnan et al., 2020), this method can be used in combination with other cellular and molecular biology techniques with little-to-no standardization, thereby adding to its utility.

Recipes

  1. Clearing solution

    1. Dissolve 8 g chloral hydrate in 3 ml water

    2. Vortex the solution until chloral hydrate is completely dissolved

    3. Add 1 ml glycerol to the solution

    Note: The solution has to be freshly prepared for clearing the leaf samples. It is also used for mounting samples for brightfield confocal microscopy.

  2. Half-strength MS medium

    1. Add 2.165 g MS salt and 10 g sucrose to about 850 ml Milli-Q water

    2. Adjust pH to 5.7 with 1 N KOH and make up the volume to 1 L

    3. Add 8 g plant agar

    4. Autoclave the medium (121°C for 20 min) and cool to about 45-50°C.

    5. Add 1 ml 100 mg/ml filter-sterilized ampicillin (final concentration in medium: 100 µg/ml) to 1 L medium and pour 50 ml into each sterile square Petri dish within the LAF

    6. Allow to cool and solidify

Acknowledgments

K.P. acknowledges grants from the Department of Biotechnology (DBT), Government of India [grant BT/PR12394/AGIII/103/891/2014] and the Department of Science and Technology, Science and Engineering Research Board (DST-SERB), Government of India [grant EMR/2017/002503/PS], and also acknowledges the Indian Institute of Science Education and Research-Thiruvananthapuram (IISER-TVM) for infrastructure and financial support. D.R. and M.M.M acknowledge University Grants Commission (UGC) fellowships. A.K. was supported by an Indian Institute of Science Education and Research-Thiruvananthapuram fellowship. A.P.S. and V.V. are recipients of Council of Scientific and Industrial Research (CSIR) fellowships. M.A. acknowledges the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India for granting the DBT-Post Doctoral Fellowship (DBT-RA Program). K.R.M. and R.K.R acknowledge funding by the Department of Biotechnology (DBT). A.S. acknowledges support from the Science and Engineering Research Board, Government of India through EMR grant No. EMR/2016/007221. We are thankful to Aswathy Syam, Aleesha Jaleel, and Kaustuv Ghosh for assistance with preliminary experiments. We are also grateful to Bejoy Manoj for technical help with video preparation.

Competing interests

The authors declare no competing or financial interests.

References

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简介

[摘要]植物的气生器官经常受到损伤,需要及时修复,特别是脉管系统。虽然气生植物的茎和花序茎等部分的维管再生分析已经建立,但叶片脉管系统的维管再生分析仍然没有被探索。最近,我们建立了一种新的叶片维管再生方法,并发现了其分子机制。在这里,我们描述了详细的分步法切割和再生试验用于研究叶片维管束再生。通过结合显微手术扰动、布莱特菲尔德显微镜和其他实验方法,我们进一步证明了叶片的年龄以及损伤的位置和大小决定了再生的总体成功率。这是一种快速有效的研究叶片维管再生机制的方法。这种分析方法可以很容易地与细胞和分子生物学技术相结合。


[背景]由于固着性,植物经常受到生物和非生物因素的伤害。当无人看管时,这些伤害会损害植物的免疫力、生长,甚至生存(Hwang等人,2017;Radhakrishnan等人,2020年)。为了克服创伤带来的不利影响,植物已经进化出一系列显著的再生反应,从局部细胞增殖形式的伤口愈合到截肢器官的完全替换,如根尖再生(Ikeuchi et al.,2016;Shanmukhan等人,2020年)。尽管许多研究探讨了植物几种再生反应的机制,但有关气生器官再生潜力的研究有限(Iwase et al.,2011;Kareem等人,2015年;Durgaprasad等人,2019年)。因此,尽管它们比地下器官更容易受到伤害,但有关植物地上器官,特别是叶片再生的信息却很少。尽管叶片在植物生理中起着至关重要的作用,但它们的再生潜力却很少被研究(Kuchen et al.,2012;Radhakrishnan等人,2020年)。

叶子有一个复杂的维管组织网络,中央中脉在植物主体之间来回运输物质。中脉的损伤需要及时修复,否则物质的运输,从而影响叶片及其相邻枝条的生长(Sachs和Hassidim,1996;Radhakrishnan等人,2020年)。近年来,一种新的叶片维管切口试验被开发出来,用于研究创伤修复和组织修复对损伤的反应。试验表明,机械断开的亲本支架通过绕过损伤部位的再生血管组织重新结合。该分析有助于理解在正常发育环境下生长的气生器官血管再生的分子机制。损伤后,由细胞命运决定因子、过多(PLT)和杯状子叶2(CUC2)组成的连贯前馈环激活局部生长素生物合成,导致生长中的空中器官中的血管再生(Radhakrishnan et al.,2020)。尽管血管再生需要PLT-CUC2调节轴,但损伤程度是一个限制因素(Radhakrishnan等人,2020)。在这里,我们证明了叶片脉管系统的再生能力是由损伤的大小、叶片外植体的年龄和损伤沿叶片近轴的位置决定的。

这种易于掌握、可重复的分析方法可以使用现成的实验室用品进行。利用受损叶片进行实时共焦成像和其他分子技术(如定量实时PCR)的便利性,使得该分析对于研究正常发育环境下伤口诱导反应和再生的分子参与者和机制具有重要价值。该方法也将有助于研究静脉发育过程中的模式形成机制和静脉再生机制之间的相互作用。

关键字:拟南芥再生, 叶切口, 维管再生, 伤口的大小, 年龄相关

材料和试剂

种子灭菌试剂

1.    野生型(哥伦比亚)拟南芥种子

注:为了标准化,我们使用了野生型植物。为了研究维管再生效率,可以在没有任何修改的情况下对拟南芥突变体植物进行分析(Radhakrishnan等人,2020)。

2.    20%次氯酸钠

3.    70%乙醇

4.    无菌高压灭菌水



样品脱色试剂

1.    35mm(直径)圆形培养皿(Himedia,目录号:PW050)

2.    15%乙醇

3.    50%乙醇

4.    70%乙醇

5.    96%乙醇

6.    100%乙醇

7.    甘油(Sigma-Aldrich,目录号:G5516)

8.    水合氯醛(Sigma-Aldrich,目录号:23100)

9.    无菌高压灭菌水

10.  Murashige和Skoog(MS)盐(Sigma-Aldrich,目录号:M5524)

11.  蔗糖(Sigma-Aldrich,目录号:S0389)

12.  植物琼脂(Sigma-Aldrich,目录号:A7921)

13.  清除溶液(见配方)

14.  半强度Murashige和Skoog(MS)培养基(见配方)



设备


体外培养设备

1.     层流空气室

2.     无菌移液管头(200µ1毫升和1毫升)

3.     微量移液管

4.     1.5毫升微量离心管

5.     无菌一次性方形培养皿,尺寸:120mm× 120毫米(Himedia,型号:PW050-1)

6.     保鲜膜

7.     植物生长室(Percival,型号:AR-100L3)



切口和样本采集设备

1.     细尖镊子(Dumont镊子,型号:Style 5)

2.     无菌刀片

3.     镊子

4.     微型Vannas直剪(Ted Pella,型号:1340)

5.     手套

6.     面罩



显微镜设备

1.     用于切割和样品采集的立体变焦显微镜(Zeisstemi,型号:2000)

2.     用于亮场成像的共焦激光扫描显微镜(徕卡,型号:TCS SP5 II)

3.     显微镜载玻片(Labtech)

4.     显微镜盖玻璃,22× 22mm(康宁,型号:2850-22)5。水彩刷(带小刷毛)



软件软件



1R软件



程序


答。种子灭菌

种子灭菌应在无菌条件下在LAF内进行。操作所需的工作区和工具(微量移液管、尖端盒、试剂瓶)必须用70%乙醇和紫外线彻底擦拭。在开始体外培养之前,应使用肥皂洗手并用70%乙醇擦拭。本文介绍了一种用于种子的液体表面灭菌方法。

1.    在1.5毫升微型离心管中等分所需数量的野生型种子。

注意:为了有效的灭菌,每管不要取超过300粒种子,并清除种子收集过程中留下的任何碎片,如部分西力克。

2.    加入1毫升70%乙醇。倒置试管搅拌内容物2-3分钟。

注意:离心前,确保离心机和转子表面清洁。打开和关闭时,避免接触微型离心管盖的内部,以尽量减少污染。

3.    在4226转一圈× g(Rcf),小心地丢弃乙醇,不要丢失任何种子。

4.    加入1毫升20%次氯酸钠,摇匀2-3分钟,重复步骤A3。

5.    用1毫升无菌高压灭菌水清洗种子5-7次。

6.    将种子在1毫升无菌高压灭菌水中分层2天,温度为4℃°C。

7.    用1毫升移液管吸取大约25粒种子,放在半强度MS培养基表面。用移液管尖将种子排成一行,每个种子之间至少留有0.5厘米的间隙。

注意:为了便于切割,避免将种子放得很近。

8.    将培养皿垂直放置在45℃以下的生长室中培养μmol/m2/s连续白光(24小时)22°C和70%相对湿度。

注:也可以在长日和短日条件下使用5个dpg植物进行分析。



B。切叶

1.    在采取必要措施尽量减少污染后,可在工作台上进行切叶。操作过程中戴手套和面罩。切割前,用70%乙醇擦拭解剖显微镜(蔡司stemi 2000)表面和戴手套的手。用于切口的镊子应在70%乙醇中浸泡,并在切口前干燥几分钟。多天切开钢板会增加污染的发生率。

注意:由于镊子尖易碎,不建议使用可能损坏或钝的灭菌技术。

2.    在解剖显微镜下打开装有幼苗的平板,以确认幼苗的年龄,并对5 dpg(发芽后天数)的幼苗进行切割。注意:由于种子萌发的非同步性,幼苗的年龄可能不完全相同。幼苗的年龄是通过计算发芽后的天数(dpg)来确定的。胚根出苗第一天按0dpg计。为了保持一致性,只伤害处于理想发育阶段的植物。把没有受伤的植物移到一边,以区别它们和受伤的植物。幼苗的年龄很重要,因为年龄较大的幼苗表现出较低的再生效率,而非常年轻的幼苗在这个过程中受到广泛的损害。切口的适宜年龄为4~6dpg(图1)。

3.    在属于第一对(真叶)的两片叶子中,选择面向培养皿盖的叶子进行切割,以便于接近。使用镊子的尖头,在第一对叶片的下背面做切口。在叶柄和叶片基端之间的连接处小心地进行切口(图1a,补充图1,视频1)。这确保了损伤发生在第一侧脉的正上方(从叶的基部开始计算),与中脉近中轴的其他位置相比,此处的再生效率最高(图2)。切口应以足够的力进行,以使其刺穿靠近叶片背面的维管组织,而不刺穿近轴面。这一点很重要,因为大范围的损伤会造成超过400的间隙µ亲本血管链之间的m没有修复(Radhakrishnan等人,2020年)(图1p)。

注意:注意不要对叶子造成多重伤害;因此,不建议在第一对的两片叶子上进行切口。在切开过程中,无菌的200μ在切割过程中,可以用微尖或镊子支撑子叶或下胚轴来限制植物的运动。这将防止植物因切口力而浸入培养基中;但是,避免触摸受损的叶子,因为这会造成进一步的伤害。





视频1。5株dpg植物切叶试验示范



4.    切开后,关闭平板,在22℃连续光照下垂直孵育°在生长室里。

5.    切口(dpi)后4天,用Vannas剪刀小心地在叶柄处切割受伤的叶片(视频2),并将其放置在小圆形培养皿(35 mm)中的15%乙醇中。

注意:大约20-30片叶子可以用2-3毫升处理脱色过程中,在35 mm培养皿中加入15%乙醇。或者,当处理多种类型的样品时,可以使用6孔板。





视频2。切叶后4天样本采集演示



图1。中脉损伤后叶片维管束的再生取决于损伤叶片的年龄。(a-b)描述有效血管再生切口位置的图示。红星代表受伤部位(c、 e)示意图和brightfield图像,显示再生血管系统重新连接断开的亲本支架,形成绕过损伤部位的Dloop(d、 f)图示和brightfield图像显示连接顶端切端和最近侧静脉的再生血管系统。(c)和(d)中的黄色方块代表端到端连接的木质部元素(在3dpg(g)(*P=0.016,n=24)、4dpg(h)(P=0.426,不显著(ns),n=45)、5dpg(i)(n=20)、6dpg(j)(P=0.605,不显著(ns),n=40)、7dpg(k)(*P=0.03,n=34)、8dpg(l)条件下叶片的再生反应

(***P=1.6)× 10-12,n=43),9 dpg(m)(***P=2.405)× 10-9,n=52)和10 dpg(n)(***P=4.8)× 10-08,n=21)。统计分析采用皮尔逊检验。注意,3-7 dpg叶片能够重新连接其断开的脉管系统,但随着叶片的逐渐老化,再生效率下降。再生的脉管系统由红点表示。黑色箭头表示受伤部位(o) 描述不同年龄受害叶片的维管再生频率的图表(p) 大范围的伤害会造成超过400的缺口µ在亲本血管束之间;因此,没有观察到血管再生。χ2

(q) 再生维管链的端到端附着的木质部成分。比例尺:50µm。



C。样品脱色和清除

1.    在15%乙醇中培养15分钟后,使用微量移液管排空乙醇,注意不要损坏样品。

注意:最初,叶子漂浮在溶剂表面。用带有小鬃毛的油漆刷轻轻地浸没叶子。

2.    随后用50%、70%和96%乙醇连续处理15分钟,使组织逐渐脱水。丢弃96%乙醇后,叶片在100%乙醇中培养12h,以进行组织脱水和去除叶绿素色素沉着。

3.    丢弃乙醇后,样品分别在96%、70%、50%和15%乙醇中连续培养15分钟,以进行再水化。

4.    丢弃乙醇后,将新制备的含有水合氯醛的清洁溶液(见配方)添加到样品中。在安装载玻片进行brightfield成像之前,样品在清洁溶液中培养至少3小时。

注:增加清除时间可以在一定程度上提高亮场成像的对比度。



D。载玻片制备

用一把小刷子,从清洗液中取出每片清洗过的叶子,放在干净的载玻片上,叶子的正面朝上。刷子可以用来轻轻地挑开任何卷曲的叶子,而不会造成任何进一步的伤害。盖玻片安装在样品上,注意不要产生任何气泡。多片叶子(6-8)可以放在一张盖玻片下面。使用200µl移液管尖端,约80µl从安装好的盖玻片的拐角处添加清洁液,从而填充安装好的叶片之间的间隙。



E。亮场成像

通过荧光显微镜或共焦显微镜的亮场模式对切口部位成像,可以评估清除标本中血管链的再生情况。建议用共焦显微镜拍摄再生维管链的快照,以获得再生木质部成分的高分辨率图像。这里描述的设置适用于徕卡TCS SP5 II倒置显微镜。氩激光器或DPSS 561可用于以30%的激光功率、200 Hz的扫描速度、2的行平均值和1024像素格式捕获快照× 1024

新形成的维管链显示出端到端连接木质部元素的独特形态(图1e、f、j、k、q)。当再生静脉重新连接中脉切端形成D形环(图1c,e)或将任一切端连接到侧静脉(图1d,f)时,结果被认为是成功再生。为了保持方法的一致性,在研究再生的年龄依赖性时,在第一侧静脉连接处以外的位置进行的切口没有评分。此外,只有切口的间隙小于400µ分离的亲本链之间的m被评分。



数据分析


用R软件进行统计分析。收集的数据用Pearson检验进行统计分析。χ2



结果与讨论

尽管植物的再生能力已被广泛研究,但叶片的再生或局部伤口修复能力却很少被研究(Kuchen et al.,2012;Radhakrishnan等人,2020年)。我们描述了一种新的叶片维管再生测定的详细的逐步方法,可用于研究中脉对局部损伤的反应。我们以前的研究表明,中脉的机械性断开,造成400以下的间隙µm(样品清除后测量),可通过再生血管链桥接(Radhakrishnan等人,2020)。当受伤的血管组织退化时,新合成的血管系统可以重新连接断开的血管束或将切端连接到最近的侧静脉(图1b)-

f) 是的。无论哪种方式,重新连接确保恢复叶片维管网络和运输之间的叶片和植物体的其余部分。但是,广泛的损害产生的差距大于400µm无法修复,从而否定了叶维管组织的功能恢复(Radhakrishnan等人,2020年)(图1p)。在这里,血管再生的伤口大小依赖性通过实施基于叶片中静脉形成的管道化假设的计算模型在硅片中重新得到(Rolland Lagan和Prusinkiewicz,2005)。根据导管化假说(Sachs,1991),生长素流量和生长素电导率之间的正反馈导致导管化生长素流量,进而促进维管组织的分化。与我们的实验观察结果(图1p)一致,计算模型表明,新血管链的形成确实取决于细胞基质(类似于叶片)中形成的开口(模拟伤口诱导的间隙)的大小。该模型还预测,大面积损伤后血管再生的失败可归因于较大创伤导致的生长素通量和浓度的中断(视频5)。这种破坏会阻碍生长素的适当通道化,并阻止再生维管链的有效分化。有关数学模型的方程式和其他相关细节见补充资料(补充资料1,视频3-5)。我们的结果表明,除了动物细胞和单细胞网柄菌外,修复过程对伤口大小的敏感性在植物中也是保守的(Pervin et al.,2018)。

在证实了血管再生对伤口大小的依赖性之后,我们接下来研究了再生反应是否依赖于受伤植株的年龄。在许多高等动物中,逐渐老化与再生能力降低有关(Yun,2015)。为了探讨年龄如何调节叶片的再生反应,我们对年龄为310dpg的植株进行了切割。在3株dpg植物的微小叶片上进行切割是一项繁琐的工作,经常会对叶片造成过度损伤。经比较,3株dpg植株的叶片再生效率低于5株dpg植株(图1g,1o)。4-6 dpg龄植株的叶片表现出最高的再生效率,这是研究叶片维管再生的最佳年龄(图1h-j,1o)。虽然在较老和较大的叶片上进行切割比较容易,但再生效率急剧下降,10株dpg植株的叶片完全无法再生(图1k-o)。值得注意的是,即使损伤引起的间隙小于400µm、 这些老叶的维管再生受阻(图1l-n);因此,我们的数据表明,受伤植物的维管再生效率随着年龄的增长而降低。

植物和动物的再生研究表明,即使在特定器官内,对损伤的再生能力也可能不同(Durgaprasad et al.,2019;摩根,1902年);因此,我们接下来研究了切口在生长叶片上的位置对叶脉再生效率的影响。为了分析这一点,我们沿着叶片的不同位置进行了切割,即叶柄、中脉的基端(靠近植物体轴)、中脉的顶端(远离植物体轴)和侧脉(图2a,2c-f)。最高的再生频率记录在中脉底端,尤其是第一和第二侧静脉之间(图2b,2d)。叶柄在切割时也表现出相似的再生效率,并且经常导致在损伤时形成多股(图2b,2c)。然而,由于样品采集过程中叶柄处的叶片被切除,切口部位和再生的维管束偶尔会受到损伤,导致有价值的样品丢失。此外,由于在叶柄上进行的切割导致多个林分的形成而不是单个林分的再生,因此更适合在叶片上进行损伤,因为研究涉及实时跟踪单个再生链以研究维管链的识别、通信和重聚。在损伤其他部位后,我们观察到再生效率向中静脉和侧静脉的顶端区域急剧下降(图2b,2e-g)。

总的来说,我们的数据表明,叶片血管再生对伤口的大小、受伤叶片的年龄和切口在叶片上的位置是敏感的。





图2。维管再生取决于叶片损伤的位置。(a) 叶片和叶柄切口位置示意图(b) 叶片中不同位置(用(a)中的彩色框表示)的维管再生频率用图中相同的色条表示(b)叶柄(n=21,P=0.95,不显著[ns])、基部正确位置(n=45)、中脉上端(n=42,***P=0.0009)、侧脉(n=21,***P=0.0002)(c-f)图像显示叶柄(c)、中脉基部(d)、中脉顶端(e)和侧脉(f)血管的切口。注意叶柄损伤后的多股形成(c)。红点表示再生血管束,黑色箭头表示切口位置。比例尺:50µm(g) 梯度代表叶片的维管再生效率,中脉基部再生最大(以红色表示)。侧静脉和中静脉远端的再生频率降低。







视频3。在50×50个细胞的网格中形成静脉,没有缺失的细胞(模拟没有切口)





视频4。在基质中形成类似小切口的静脉





视频5。在基质中形成类似大切口的静脉



结论

我们的研究表明,4-6 dpg叶片对较小的伤口反应最有效(400)µ在叶片近端第一侧脉的连接处造成的。在采用这种方法研究其他植物物种的再生时,我们建议按照上述标准对方法进行标准化。

本实验将有助于探索叶片维管组织再生的机制。首先,分析可能会被证明是乏味的;然而,通过反复实践,这种方法可以在大量样本中灵活快速地进行。实验持续时间短(实验数据可在受伤后5天内收集)以及专用设备的可有可无性使其适合更大的科学界。如前所述(Radhakrishnan et al.,2020),该方法可与其他细胞和分子生物学技术结合使用,几乎没有标准化,从而增加了其实用性。



食谱


1.     清除溶液

答。在3毫升水中溶解8克水合氯醛

b。旋转溶液直到水合氯醛完全溶解

c。向溶液中加入1毫升甘油

注:溶液必须是新鲜制备的,以清除叶样品。它也用于安装样品的亮场共焦显微镜。

2.     半强度MS中等

答。向约850 ml Milli-Q水中添加2.165 g MS盐和10 g蔗糖

b。用1 N KOH将pH值调节至5.7,并补充至1 L

c。加入8g植物琼脂

d。高压灭菌介质(121°C冷却20分钟)并冷却至45-50℃左右°C。

e。加入1ml 100mg/ml过滤灭菌氨苄西林(培养基终浓度:100)µg/ml)至

1 L培养基,将50 ml倒入LAF内的每个无菌方形培养皿中

f。使冷却和凝固



致谢


K.P.承认印度政府生物技术部(DBT)的拨款[拨款]

BT/PR12394/AGIII/103/891/2014]和印度政府科学和技术部、科学和工程研究委员会(DST-SERB)[grant EMR/2017/002503/PS],并感谢印度科学教育和研究所Thiruvananthapuram

(IISER-TVM)用于基础设施和财政支持。D.R.和M.M.M.承认大学资助委员会(UGC)的研究金。A.K.得到了印度科学教育和研究所Thiruvananthapuram奖学金的支持。A.P.S.和V.V.是科学和工业研究委员会(CSIR)研究金的获得者。M.A.感谢印度政府科技部生物技术部(DBT)授予

DBT博士后奖学金(DBT-RA计划)。K.R.M.和R.K.R.承认生物技术部(DBT)的资助。A.S.通过EMR批准号EMR/2016/007221,感谢印度政府科学与工程研究委员会的支持。我们感谢Aswathy Syam、Aleesha Jaleel和Kaustuv Ghosh对初步实验的帮助。

我们也非常感谢Bejoy Manoj在视频制作方面提供的技术帮助。



相互竞争的利益相互竞争的利益



提交人声明没有竞争或经济利益。



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引用:Radhakrishnan, D., Shanmukhan, A. P., Kareem, A., Mathew, M. M., Varaparambathu, V., Aiyaz, M., Radha, R. K., Mekala, K. R., Shaji, A. and Prasad, K. (2021). Age, Wound Size and Position of Injury – Dependent Vascular Regeneration Assay in Growing Leaves. Bio-protocol 11(9): e4010. DOI: 10.21769/BioProtoc.4010.
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