A growing system closer to the natural conditions for studying root growth and response of Arabidopsis thaliana

doi:10.21203/rs.2.1737/v1

A growing system closer to the natural conditions for studying root growth and response of Arabidopsis thaliana

https://doi.org/10.21203/rs.2.1737/v1

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In natural environments, shoot was exposed to sunlight but roots are not directly exposed to light, being buried deep down in the soil and grow in darkness without sucrose addition. However, most current research in root biology has been carried out by growing the Arabidopsis root system in the presence of light and sucrose-added medium since root development is typically studied in traditional agar-plate culture system (TPG, traditional plant-growing). Here, we present a protocol for improved agar-plate culture system (IPG, improved plant-growing), allows the in vitro cultivation of Arabidopsis with the aerial part growing in normal light/dark photoperiod while roots grow in darkness without sucrose addition. IPG provides an efﬁcient system for cultivating a large number of seedlings and easily characterizing root architecture in the dark. We also provide methods for key steps all the way through to seedlings growth.We found that the primary root lengths in TPG were significantly longer than that in IPG, and the root elongation growth is dependent on auxin-triggered acidiﬁcation of the root apoplast of Arabidopsis . With this protocol, a better understanding of the mechanisms involved in root growth and responses of plants can be easily characterized.

Plant sciences

Arabidopsis

auxin

growth

root

In field conditions, the top part of the root system will grow in minimal light and the lower part will even develop in darkness, while the shoot can be exposed to various different light conditions (Smith, 1982). However, under traditional agar-plate culture system (TPG, traditional plant-growing) for Arabidopsis plants, the root system grown in the presence of light and sucrose-added medium (Xu,2013). Light affects root organogenesis (growth rate and LR production), orientation and pigmentation (Usami et al., 2004; Sassi et al., 2012; Moni et al., 2015). Root biology studies have exposed roots to light without evaluating the additional effect that light might have on them, direct perception of light by the root can be considered a non-natural condition (Silva-Navas et al., 2015).Although hydroponic-cultured or soil-cultured Arabidopsis root are grown in darkness without sucrose addition, it is inconvenient to investigate root growth and response in situ, which can be facilitated in agar-plate system (TPG) (Xu et al., 2013). For all of these reasons, studies with growing system closer to the natural one for assaying plants are essential, both in the physiological and molecular levels in higher plant.

Several solutions to these undesirable lab conditions have been postulated, keeping living roots in darkness and bring them into the light only when necessary, including dark agar plugs (Sassi et al., 2012) and the D-root system consisting of plate inserts plus cover slips (Silva-Navas et al., 2015). It should be highlighted that sucrose is added into the agar medium in these systems so that sucrose additional effect is not taken into account. Although the D-root system undoubtedly allows root to grow in the dark, it has limitations. First, the method often requires a second methacrylate comb, which adds to the time of the protocol. Second, sucrose is added into the agar medium, it is not closer to the natural one for assaying Arabidopsis plants since roots are plant organs that typically lie below the soil without sucrose addition. Third, the method also requires a auxiliary box, which increases the cost of the device.

Here we describe a simple and effective step-by-step protocol for growing the A. thaliana root in an improved agar-plate culture system (IPG, improved plant-growing), the shoot is grown outside of plates to receive light for photosynthesis ,but plates (the roots inside the in vitro plate) is black to protect the root away from light and without sucrose addition. This improved method allows to study the root growth and responses of Arabidopsis plants under the most approximate natural growing conditions.

1. Half-strength (0.5×) Murashige and Skoog (MS) plates: 2.4 g/L MS medium (Duchefa Biochemie, Haarlem, NL) containing 1 % sucrose and 0.8 % agar. The pH was adjusted to 5.8 with KOH before autoclaving.

2. Distilled and sterilized water for rinsing after disinfection of Arabidopsis seeds.

3. Sodium hypochlorite solution.

4. Ethanol.

5. SDS.

1. Square polystyrene petri dishes (12.5×12.5×1.5 cm, L×W×H).

2. Micropore surgical tape for sealing petri dishes.

3. Forceps with pointed tips (tweezers) to hold the Arabidopsis seedlings while transferring to assay system.

4. Black taps were used in oblong hole to shut out light.

5. Growth chamber.

6. Nikon camera D7100 (Tokyo, Japan).

7.Confocal microscopy.

8. The improved agar-plate system (IPG, improved plant-growing) for controlling the effect of light and sucrose on root growth. IPG has the same size as the traditional growth plate (12.5×12.5×1.5 cm, L×W×H), but IPG is black and light-tight (Fig. 1d). There are seven oblong holes (6×3.5 mm, L×W) in the medium container or upper cover of IPG (Fig. 1d). Under the IPG, shoots were illuminated, while roots were grown in darkness environment without sucrose addition (Fig. 1e and Fig. 1f).

1. Arabidopsis seeds were surface sterilized with 70% ethanol for 1 min, then with 1% sodium hypochlorite solution plus SDS for 9 min, and subsequently washed 6 times with autoclaved water (Fig. 1a).

2. Sow the seeds on half-strength Murashige and Skoog (MS) plates containing 0.8% agar and 1% sucrose, and then the plates were kept in the dark for 2 days at 4 °C for stratification (Fig. 1b).

3. Seal the plates with Micropore surgical tape and put them vertically in a growth chamber with 16/8 h photoperiod at 22°C, with the light intensity of 120 mmol photons m^-2sec^-1, and relative humidity of 70% for 5 days (Fig. 1c). Five-day-old seedlings with approximately 1.5-cm-long roots were used for the further experiments.

4. Arabidopsis seedlings were carefully transferred to TPG with half-strength MS nutrients containing 0.8% agar and 1% sucrose or IPG with half-strength MS nutrients containing only 0.8% agar (no sucrose). Under TPG, roots is grown in transparent Petri dishes in the presence of light and sucrose-added medium. Under IPG, roots will be grown at the black Petri dish without sucrose addition, black tapes was used in oblong hole to shut out light, and the top part of the seedlings will grow in light (out of oblong hole).

5. Seal the plates with Micropore surgical tape and place the plates vertically in a growth chamber at 22 °C for 10 days.

6. After 10 days, we have found that although no significant difference was found in the shoot under TPG or IPG in Arabidopsis ecotype Col-0 (Fig. 2a), the primary root lengths in TPG were significantly longer than that in IPG (Figs. 2b,e). Furthermore, the lateral root number, lateral root density or root hair density were substantially increased in IPG (Figs. 2c,d,f,g).The root meristem size in LGR is similar to TPG (Figs. 3a,c). However, the elongation zone of primary roots were increased dramatically in IPG than TPG (Figs. 3b,d). The root elongation growth is dependent on auxin-triggered acidiﬁcation of the root apoplast of Arabidopsis(Fig. 4).

STEP 4: In this transferred process for TPG or IPG, taking care not to damage roots.

STEP 5: Black taps were used in oblong hole to shut out light.

Moni, A. et al. The blue light receptor Phototropin 1 suppresses lateral root growth by controlling cell elongation. Plant Biol. 17, 34–40 (2015).

Sassi, M. et al. COP1 mediates the coordination of root and shoot growth by light through modulation of PIN1- and PIN2-dependent auxin transport in Arabidopsis. Development. 139, 3402–3412 (2012).

Silva-Navas, J. et al. D-Root: a system for cultivating plants with the roots in darkness or under different light conditions. Plant J. 84, 244–255 (2015).

Smith H. Light quality, photoreception, and plant strategy. Annual Review of Plant Physiology. 33, 481–518 (1982).

Usami, T. et al. Cryptochromes and phytochromes synergistically regulate Arabidopsis root greening under blue light. Plant Cell Physiol. 45, 1798–1808 (2004).

Xu W. et al. An improved agar-plate method for studying root growth and response of Arabidopsis thaliana[J]. Scientific Reports.3, 1273 (2013).

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A growing system closer to the natural conditions for studying root growth and response of Arabidopsis thaliana

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