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growth regulators in Arabidopsis

Item Type Book Chapter

Authors Alagoz, Yagiz;Mi, Jianing;Al-Babili, Salim;Dickinson, Alexandra J.;Jia, Kunpeng

Citation Alagoz, Y., Mi, J., Al-Babili, S., Dickinson, A. J., & Jia, K.-P.

(2022). Screening for apocarotenoid plant growth regulators in Arabidopsis. Methods in Enzymology. https://doi.org/10.1016/

bs.mie.2022.03.067 Eprint version Post-print

DOI

10.1016/bs.mie.2022.03.067

Publisher Elsevier

Rights Archived with thanks to Elsevier Download date 2024-01-17 23:19:20

Link to Item

http://hdl.handle.net/10754/678244

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Screening for apocarotenoid plant growth regulators in Arabidopsis

Yagiz Alagozâ, Jianing Miâ, Salim Al-Babiliâ, Alexandra J. Dickinson^b, Kun-Peng Jia^c,d,^⁎

aBiological and Environmental Sciences and Engineering Division, Center for Desert Agriculture, The BioActives Lab, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia

bSection of Cell and Developmental Biology, Dickinson Lab, University of California San Diego, La Jolla, CA, United States

cState Key Laboratory of Cotton Biology, Henan Joint International Laboratory for Crop Multi-Omics Research, School of Life Sciences, Henan University, Kaifeng, China

dSanya Institute of Henan University, Sanya, Hainan, China

⁎Corresponding author: e-mail address: [email protected]

Contents

1.Introduction 2

2.Materials 6

2.1Chemical preparation 6

2.2Plant materials and growth conditions 6

2.3Phenotyping and data processing 7

3.Methods 7

3.1Chemical preparation 7

3.2Seed sterilization and stratification 7

3.3Phenotyping and data analysis 8

4.Notes 12

Acknowledgments 14

References 14

Abstract

Apocarotenoids are bioactive metabolites found in animals, fungi and plants. Sev- eral carotenoid-derived compounds, apocarotenoids, were recently identified as new growth regulators in different plant species. Here, we introduce basic chemical screening methods, using a model plant,Arabidopsis thaliana, to elucidate the function of bioactive apocarotenoids in determining plant phenotypic traits. These short guide- lines include essential practices, such as selecting the plant growth conditions

ISBN 0076-6879

https://doi.org/10.1016/bs.mie.2022.03.067 1

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and the type of treatment, as well as phenotyping methodologies for the initial screening of novel apocarotenoid plant growth regulators.

Carotenoids are an essential group of natural C₄₀ terpenoid pig- ments involved in molecular mechanisms modulating plant phenotype and response to the environment (Dall'Osto, Fiore, Cazzaniga, Giu- liano, & Bassi, 2007; Hashimoto, Uragami, & Cogdell, 2016; Moise, Al-Babili, & Wurtzel, 2014; Polívka & Frank, 2010). The molecular structure of carotenoids consists of an extended carbon backbone con- jugated with double bonds, which is prone to oxidative cleavage by reactive oxygen species (ROS) and/or by enzymes, particularly carotenoid cleavage dioxygenases (CCDs), to yield mono‑carbonyl products called apocarotenoids (APOs) (Al-Babili & Bouwmeester, 2015; Beltran &

Stange, 2016; Giuliano, Al-Babili, & von Lintig, 2003; Moreno, Mi, Alagoz, & Al-Babili, 2021; Nisar, Li, Lu, Khin, & Pogson, 2015;

Walter & Strack, 2011). For a comprehensive explanation of apocarotenoid nomenclature, numbering, and definitions, see (Britton, 2022).

APOs can be further oxidized to yield various linear dialdehyde products called diapocarotenoids (DIAPOs) (Alder, Holdermann, Beyer, &

Al-Babili, 2008; Ilg, Bruno, Beyer, & Al-Babili, 2014; Mi, Jia, Bal- akrishna, & Al-Babili, 2020; Mi, Jia, Balakrishna, Feng, & Al-Ba- bili, 2019; Scherzinger, Ruch, Kloer, Wilde, & Al-Babili, 2006). The structures of APOs are diverse and mainly determined by the cleavage site within the carbon chain, stereo-configuration (cis/trans), ring number (linear/acyclic/bicyclic), ring type (⯑ring/εring), and ring modification (i.e., epoxidation, hydroxylation) of the parent carotenoids (Fig. 1). Fur- ther modification of the primary cleavage products increases the diversity of natural apocarotenoids.

So far, various forms of endogenous APOs have been identified in different plant species (Mi et al., 2019, 2020; Mi, Jia, Wang, &

Al-Babili, 2018). However, studies to discover their bioactivity are still emerging, although early work has led to the characterization of APO-derived plant hormones, strigolactones (SLs) and abscisic acid (ABA). SLs are mainly involved in modulating plant architecture and plant-microbe rhizosphere interactions (Al-Babili & Bouwmeester, 2015; Alder et

1.

Introduction

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3

Fig. 1 Structural diversity of plant apocarotenoids. The major precursors of most identified apocarotenoids (APOs) and diapocarotenoids (DIAPOs) in plants are cyclic carotenoids, either with ⯑ or ε ring structures, produced downstream of all-trans-ly- copene. APOs and DIAPOs are produced either by ROS or CCD-mediated cleavage of carbon‑carbon double bonds in carotenoid precursors. DIAPOs are acyclic/linear forms of APOs, produced by repeated cleavage of carotenoids. Both cyclic and acyclic forms of APOs can be reduced to alcohols (blue dotted), oxidized to carboxylic acids (red dotted), and/orcis(green dotted)/transisomerized.

al., 2012; Jia, Baz, & Al-Babili, 2018), whereas ABA coordinates seed germination and dormancy, stomatal closure, and response to various abiotic and biotic stresses (Chen et al., 2020; Felemban, Braguy, Zur- briggen, & Al-Babili, 2019; Nambara & Marion-Poll, 2005).

The usage of different screening methods has led to the discovery of new members of APOs, such as⯑-cyclocitral, zaxinone, and retinal, which are also shown to be involved in plant root development and abiotic stress responses (Dickinson et al., 2019, 2021; Havaux, 2014;

Methods of screening for bioactive carotenoid derivatives in Arabidopsis

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Wang et al., 2019). Another example is the discovery of the enzymatic production of bixin and crocin, endogenous DIAPOs found inBixa orel- lanaandCrocus sativus, catalyzed by specific CCDs (Frusciante et al., 2014). Also, anchorene and isoanchorene are the most recently identified endogenous DIAPOs found in Arabidopsis, specifically regulating anchor root formation and primary root growth, respectively (Jia et al., 2019, 2021). These studies have shown that APOs are widely involved in various biological processes and are not just non-functional carotenoid degra- dation products.

Uncharacterized linearcis-carotene-derived apocarotenoids (LCDAs) have been proposed to regulate various developmental processes, including fruit (Alagoz, Nayak, Dhami, & Cazzonelli, 2018), leaf and root pigmentation (Álvarez et al., 2016; Kachanovsky, Filler, Isaacson,

& Hirschberg, 2012), plastid biogenesis during skoto- and photo-mor- phogenic development (Cazzonelli et al., 2020), and modulation of leaf morphology (Avendaño-Vázquez et al., 2014). However, the chemical structure of these uncharacterized APOs remains a mystery (Fig.

2). Despite the recent progress in uncovering novel APO regulators of plants, there remain many endogenous APOs with uncharacterized roles and mechanisms of action (Koschmieder et al., 2021).

Arabidopsis is an ideal model system to screen for bioactive metabolites due to its small size and rapid growth rate, which shortens the time required to observe how APOs shape plant phenotypes. Chemical screens in this model plant have revealed several endogenous carotenoid-derived metabolites, including ⯑-cyclocitral, anchorene, retinal, and β-apo-11-carotenoids. Each of these compounds were identified as bioactive signals involved in specific physiological functions (Dickinson et al., 2019, 2021; Jia et al., 2019, 2022). Further studies exploring small molecule candidates for the discovery of new carotenoid-derived phytohormones and the enzymes involved in their metabolism are expected to open new avenues in plant carotenoid research. Using a systematic set of robust protocols for screening bioactive APOs is essential for advancing the understanding of how APOs regulate plant architecture and responses to biotic and abiotic stress. Therefore, we describe comprehensive protocols to screen for novel bioactive APOs.

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Fig. 2 Apocarotenoid phytohormones and carotenoid-derived growth regulators are small bioactive molecules involved in many aspects of plant life (A) Major APOs and APO-derived phytohormones and regulatory metabolites. (B) APOs are involved in several biological processes, including seed dormancy, hypocotyl and cotyledon development, leaf morphology and pigmentation, as well as overall modulation of root and shoot architecture in the model plant Arabidopsis thaliana. Abbreviations: abscisic acid (ABA);

all-trans-β-apo-11-carotenal (APO11); linear cis-carotene-derived apocarotenoids (LCDAs).

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2.1 Chemical preparation

1. APO library (custom synthesis or commercial products from Sigma Aldrich and Buchem) (Mi et al., 2021)

2. HPLC-grade acetone (Sigma Aldrich, catalog number: 34850)

3. Dimethyl sulfoxide (DMSO) (Sigma Aldrich, catalog number: D8418) 4. Analytical precision balance (0.1–0.001 mg)

5. 1.5 mL microcentrifuge tube (black or amber colored) (Note 1) 2.2 Plant materials and growth conditions

1. Freshly harvestedArabidopsis thalianaCol-0 (Columbia) seeds Col-0 seeds (stock number: CS70000) can be obtained from the Ara- bidopsis Biological Resource Center (ABRC).

2. Seed sterilization solution: 20% (v/v) household bleach + 0.02% (v/v) Triton X-100

3. Autoclaved distilled water (dH₂O) 4. 1.5 mL microcentrifuge tubes 5. Laminar flow hood

6. A plant growth chamber equipped with LED lights (Note 2) 7. Plant growth medium

Prepare 0.5 × Murashige and Skoog (MS) agar by dissolving 2.15 g MS basal medium salts (Sigma Aldrich, catalog number: M5519), 10 g plant culture tested sucrose (1%) (Sigma Aldrich, catalog number: S5390), and 1 g ultra-pure β-(N-Morpholino) ethanesulphonic acid (MES) (Goldbio, catalog number: M-090) (0.1%, w/v) in 900 mL dH₂O and adjust the pH to 5.8 using 1 M KOH (Sigma Aldrich, catalog number: 484016) and add dH₂O up to 1 L of the final volume.

(Note 4).

8. Parafilm / micropore tape (Note 5) 9. Square petri dishes (12 × 12 cm)

10.Round petri dishes (100 mm × 20 mm diameter) (Note 6) 11.Aluminum foil

2.

Materials

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2.3 Phenotyping and data processing 1. High-resolution flatbed scanner (Epson V600) 2. Confocal microscope (Zeiss LSM 510) 3. Plant growth chamber (Percival AR-36 L3)

4. Propidium iodide (C₂₇H₃₄I₂N₄) solution (1 μg/mL dissolved in water) (Invitrogen, catalog number: P3566)

5. Sterile tissue culture hood (Thermo Scientific 1300 Series Class II, Type A2)

6. Sterile surgical blades with safety handle (Fine Science Tools 1031512)

7. ImageJ software (https://imagej.nih.gov/ij)

3.1 Chemical preparation

1. Synthesize or purchase APOs designed according to Fig. 1 2. Weigh 1–5 mg APOs and DIAPOs using the analytical balance 3. Store the APOs in a 1.5 mL black/amber microcentrifuge tube

4. Dissolve the APOs in acetone or DMSO to make a 10 mM stock solution

5. Keep the stock solution at − 80 °C for short-term use 3.2 Seed sterilization and stratification

1. Place Col-0 Arabidopsis seeds in a 1.5 mL microcentrifuge tube 2. Add 1 mL seed sterilization solution to the tube with seeds and incu-

bate for 10–15 min by vortexing every 3–4 min

3. Wash the sterilized seeds using 1 mL autoclaved dH₂O. Repeat 5 times in a laminar hood

4. Remove 500 μL dH₂O, so that there is about 500 μL remaining in the tube (Note 7).

5. Keep the sterilized seeds in the dark at 4 °C for 3 days for seed stratification. The stratification process breaks the seed dormancy and promotes synchronized germination of Arabidopsis seeds

3.

Methods

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3.3 Phenotyping and data analysis 3.3.1 Determining effects on seed germination 1. Stratify seeds according to Section 3.2.

2. Add APO stock solution to 100 mL of 0.5 × MS agar medium while it is still warm (around 45 °C). The final concentration of the APO should be between 0.2 and 20 μM (Note 8). For the control treatment, add an equivalent volume of solvent to 100 mL of 0.5 × MS agar media.

3. Pour 25 mL 0.5 × MS agar media supplemented with individual APOs. Pour at least 3–4 circular petri dishes per treatment to ensure that there are sufficient replicates for statistical analysis (Fig. 3).

4. Working in the laminar hood, sow a minimum of 100 seeds on each circular petri dish for each treatment.

5. Seal the dishes using parafilm/micropore tape.

6. Incubate the petri dishes in an upright position in the plant growth chamber set to 22 °C, 50% humidity, 16 h light/8 h darkness photoperiod, 120 μmol m^{− 2}s^{− 1}light density (Note 9).

7. At 24 h after incubation, use a high-resolution flatbed scanner to collect images of the petri dishes at ≥ 600 dpi every 12–24 h for the next 72 h (Note 10).

8. Analysis: Quantify the percentage of germinated seedlings for each treatment (germinated seedlings have a visible radicle emerged from the seed coat), and make a germination curve using any available software of choice (we recommend GraphPad Prism).

9. Perform statistical analysis to evaluate the significance of the impact of the treatment on seed germination.

3.3.2 Characterizing primary root development and anchor root number

1. Stratify seeds according to Section 3.2.

2. Add APO stock solution to 200 mL of 0.5 × MS agar medium while it is still warm (around 45 °C). The final concentration of APOs should be between 0.2 and 20 μM. For the control treatment, add an equivalent volume of solvent to 200 mL of 0.5 × MS agar medium.

3. Pour 50 mL 0.5 × MS agar medium supplemented with individual APOs. Pour at least three square petri dishes per treatment to ensure there are sufficient replicates for statistical analysis (Fig. 3).

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Fig. 3 Screening methods for bioactive apocarotenoids (APOs). Based on the research ob- jective, screening can be performed either in a light or dark environment. The physical state of the APOs (volatile/non-volatile) and the type of treatment (direct/indirect) should be considered while choosing the correct screening method. Bioactive APOs can alter different plant phenotypic traits, including seed germination rate, hypocotyl and cotyledon development, root length and branching, biomass, and leaf pigmentation, which can be uncovered only with the correct choice of a screening method that has been designed for the specific purpose.

4. Working in a laminar hood, sow 25 seeds per square petri dish. Plate seeds along a line 1 cm below the top of the petri dish.

5. Seal the petri dishes using parafilm/micropore tape.

6. Incubate the square petri dishes vertically (with approximately a 90-degree angle) in the plant growth chamber set to 22 °C, 50% humidity, 16 h light/8 h darkness photoperiod, 120 μmol m^{− 2}s^{− 1} light density.

7. 7 days after incubation, use a high-resolution flatbed scanner to image the petri dishes at ≥ 600 dpi.

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8. Use the scanned images to count the number of emerged anchor roots.

Anchor roots emerge from the junction between the hypocotyl and the primary root. Typically, untreated Col-0 seeds have between 0 and 2 anchor roots at 8 days after stratification.

9. For primary root length measurement, using the “freehand line” or

“segmented line” function in ImageJ software, quantify the primary root length in each treatment (Note 11).

10.Perform statistical analysis and evaluate the effects of APOs on anchor root emergence and primary root length.

11.For treatments where further investigation of primary root development is required, collect images of the roots using a confocal microscope.

12.Incubate roots with propidium iodide (C₂₇H₃₄I₂N₄) solution for 1–5 min.

13.Use a confocal microscope to collect images of a section in the center of the root tip (the meristem). At the right focal plane, a full single file of cortex cells should be visible, starting with the cortex/endodermal initial (CEI) cell.

14.For each root, count the number of cells in a single file of cortex cells in the root meristem, starting with the CEI and ending with the first cell that is twice as long as the CEI. Quantify this number for at least 10 roots per treatment.

15.Use the confocal microscope to collect images of the differentiated cortex cells (any section of the root where root hairs are fully devel- oped). Image at least 4 full-length cortex cells per root.

16.Quantify the length of the differentiated cortex cells in each treatment (at least 10 roots per treatment).

17.Comparing the effect of the APO treatment on meristematic cell number and differentiated cell length reveals how cell divisions and growth changed during the treatment.

3.3.3 Measuring lateral root capacity, density, and emergence 1. Stratify and incubate seeds according to Section 3.3.2, steps 1–6.

2. At 8 days after incubation, use a high-resolution flatbed scanner to image the petri dishes at ≥ 600 dpi. Make sure the petri dishes are sealed to remain sterile.

3. Use the images to count the number of emerged lateral roots. This number can provide information about whether the treatment affects the entire course of lateral root development.

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4. After scanning the roots, bring the petri dishes into a sterile tissue culture hood. Using a sterile scalpel blade held parallel to the surface of the agar medium, excise the lower 1 mm tip of the root.

5. Reseal the petri dishes and incubate for another 4 days. The lateral root primordia should emerge from the primary root during this time.

6. After 4 days (12 days after incubation), use the scanner to collect images of the petri dishes again.

7. Quantify the number of lateral roots that have emerged after excision.

This number can reveal whether the treatment affects lateral root initiation.

8. Calculate the density of lateral roots by dividing the number of lateral roots by the primary root length.

9. Perform statistical analysis and evaluate the effects of APOs on lateral root capacity. The type of statistical tests (i.e.,t-test, ANOVA) analysis depends on the experimental setup. We recommend using Graph- Pad Prism software while performing statistical analysis.

3.3.4 Quantifying effects on biomass

1. Stratify and incubate seeds according to Section 3.3.2, steps 1–6.

2. At 7 days after incubation, use an analytical balance to measure the mass of two weigh boats per petri dish. It is critical to keep track of the exact mass of each weigh boat.

3. Unless this is the phenotype, discard the ungerminated seeds from each plate before taking the measurements. We recommend at least 20 individual plants used for the analysis.

4. Excise the junction between the hypocotyl and the root for the remaining plants. This step will separate each plant into shoot and root samples.

5. For each petri dish, use an analytical balance to weigh the fresh biomass of at least 20 shoots in one weigh boat of known mass (determined in step 2). Then, measure the roots in another weight boat. Keep each sample in its own weigh boat. Repeat for all petri dishes.

6. Place all samples in their weigh boats for a week, uncovered, in a 30 °C incubator.

7. To measure the dry biomass, remove the samples from the incubator, cool them to room temperature, and weigh each dried sample using an analytical balance.

8. Perform statistical analysis to evaluate the effect of APOs on fresh and dry biomass (see Section 3.3.3, step 9).

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3.3.5 Phenotyping the hypocotyl length and cotyledon opening 1. Stratify seeds according to Method 3.2.

2. Under a laminar flow hood, sow at least 100 seeds per each circular petri dish (for statistical analysis) and seal the petri dishes with parafilm or micropore tape. Ensure there are at least 3–4 petri dishes per treatment (Note 12).

3. Cover the petri dishes with two layers of aluminum foil to completely protect the petri dishes from light exposure and place in a growth chamber adjusted to 22 °C, 50% humidity and 24 h continuous dark (Note 13).

4. After 4–7 days, pick at least 100 dark-grown seedlings and place them horizontally onto a transparent plastic sheet. Use a high-resolution flatbed scanner to collect images of the petri dishes at ≥ 600 dpi (Note 14).

5. Measure the hypocotyl length and cotyledon angle using the ImageJ software (Note 15).

6. Perform statistical analysis to evaluate the effects of APOs on hypocotyl length and cotyledon opening.

1. Many APOs are light sensitive. Black/amber colored microtubes help prevent light exposure to chemicals.

2. APOs are more stable under white LED light than white fluorescent light.

3. The type of solidifying/gelling reagent (e.g., agarose, phytagel) may alter the root phenotype due to the difference in consistency. We recommend consistently using a specific gelling reagent throughout each experimental replicates or any experiments associated with phenotypic screening.

4. KOH is preferable to NaOH for adjusting the pH value to avoid salt stress caused by sodium.

5. Sealing the petri dishes with parafilm is preferable to micropore tape if the APOs are volatile. However, the use of parafilm may cause ethylene accumulation, and so parafilm is not recommended for applica- tions of non-volatile APOs. Also, micropore tape helps air circulation without propagating pathogen growth in the plant growth medium.

4.

Notes

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6. Round petri dishes are placed horizontally in the growth chamber, and square petri dishes are placed vertically. We recommend that square petri dishes be used only to screen the root-associated phenotypes.

7. Retaining approximately 500 μL of water helps in storing and plating the seeds.

8. The concentration may differ depending on the bioactivity of APOs.

Therefore, applying a wide range of concentrations is required to opti- mize the assay before collecting phenotypic data related to bioactivity.

9. Treatment with different light intensities, spectra, and duration corresponding to photoperiod is crucial in uncovering the bioactivity of APOs. Different lighting options should be checked and optimized, if necessary.

10.The emergence of the radicle is usually considered the endpoint of seed germination. Arabidopsis seed germination occurs after 18–24 h.

Timepoints after 72 h after sowing seeds are usually ignored in seed germination studies unless working with an inhibitor such as ABA. In studies with Arabidopsis mutant lines with defects in phytohormone biosynthesis or with altered levels/ratios of phytohormones (including ABA, gibberellic acids, and cytokinins), the time for germination screening should be extended. If germination takes longer than 48 h in untreated Col-0 seeds, the seed viability needs to be checked before performing any further experiments.

11.Primary root length and anchor root number can be measured using the same image.

12.Square petri dishes should not be used for hypocotyl length and cotyledon opening analysis unless there is a specific reason. Seedlings with hypocotyls touching the surface of the medium and to the cover/lid of the petri dish should be avoided while collecting data as this physical interaction may perturb ethylene signaling and may give false results.

13.Ensure that the petri dishes are entirely sealed and kept away from light exposure. Also, make sure that there is no light leakage into the growth chamber, which may trigger photomorphogenesis. Commer- cially available, heavy-duty aluminum foils, which are light-proof, can be used to assure that seedlings are not exposed to light during periods of dark growth.

14.The duration required for the dark-growth might differ depending on the nature of the research.

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15.Hypocotyl length and cotyledon angle can be measured using the same image. A ruler should be added next to the seedling for length compar- isons.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (31900245 & 32170271, to K.P.J), and King Abdullah University of Science and Technol- ogy (KAUST) baseline funding and a Competitive Research Grant (CRG4) (to S.A.B).

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