ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 5, с. 708-713
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
YM 581.1
THE EFFECT OF ELLAGIC ACID ON THE ROOT GRAVITROPIC RESPONSE
IN Arabidopsis thaliana1 © 2015 Z. Q. Yan, H. Jin, D. D. Wang, X. Y. Yang, B. Qin
Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, China
Received November 4, 2014
Gravitropism is affected by many exogenous factors such as environmental stresses. In the present study, the effect of ellagic acid (EA) on the gravitropic curvature of Arabidopsis thaliana (L.) Heynh. roots was characterized. Exogenous application of EA influenced root gravitropic responses in a concentration-dependent fashion. KI/I staining research revealed that the starch grain in the root cap was reduced by EA treatments. Simultaneously, expression of auxin-responsive reporter gene DR5::GFP showed that the auxin redistribution in roots treated with EA was less sensitive to that of control groups under gravity stimulus. Moreover, the expression of one of the auxin flux-facilitators — PIN2, was also suppressed by EA. Taken together, our results indicated that the effects of EA on root gravitropism were largely dependent on the reduction of starch grain and the disorder of lateral auxin redistribution.
Keywords: Arabidopsis thaliana — gravitropism — ellagic acid — starch grain — auxin — PIN2
DOI: 10.7868/S0015330315050188
INTRODUCTION
Plant roots have the ability to perceive gravity and then reorient their growth accordingly. This process is known as gravitropism, which contains four steps: gravity perception, signal transduction, signal transmission, and organ bending [1]. How do plant organs perceive the gravity signals? The starch-statolith hypothesis posits that the perception of gravity is mediated by the sedimentation of starch-filled plastids within columella cells of root cap [2]. Previous studies on lateral mobilization of amyloplasts [3] and the genetic or physiological depletion of starch in root tips [4, 5] both supported the hypothesis.
In plant roots, auxin is transported through the vas-culature into the root cap and then redistributed laterally to peripheral tissues and transported back to the elongation zone with a polar model [6]. Many studies suggested that auxin transport across root tip was controlled by gravity signal transduction in some way. The Kholodny—Went hypothesis proposes that gravistimu-lation promotes lateral transport of auxin to form an
1 This text was submitted by the authors in English.
Abbreviations: Col — Columbia; EA — ellagic acid; GFP — green fluorescent protein.
Corresponding author. B. Qin. Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China; fax. +86 931 8277088; e-mail. bqin@licp.cas.cn
auxin gradient across plant organs with accumulation of auxin at the bottom side, where it inhibits cellular elongation and then causes organ bending [2].
Ellagic acid (EA) is a naturally occurring polyphenol found in a variety of plant species, especially in many berries such as strawberries, raspberries, cranberries, and grapes [7, 8]. Due to its multi-biological effects such as anticancer, antimutagen, antivirus, and antibacterial [9—12], EA has been intensively studied as a novel kind of health care food and drug.
Additionally, under a bioactivity-driven fraction-ation, EA has been isolated from the root extracts of leafy spurge (Euphorbia esula L.), an invasive plant species [13]. Treatments with EA reduced the fresh weight and root elongation of Arabidopsis seedlings [13]. Therefore, EA may serve as a potential alle-lochemical that showed phytotoxic effects to receptor plants and thus contributed to the invasive behavior of leafy spurge.
The interesting preliminary studies led us to investigate the effect of EA on plant growth regulation. In this study, influence of root gravitropism by EA, as well as its effects on grain starch and auxin redistribution was studied in Arabidopsis. To our knowledge, this is the first report that EA may affect root gravitropic response in a receptor plant species.
MATERIALS AND METHODS
Plant material and growth conditions. Arabidopsis thaliana (L.) Heynh. (Columbia, Col-0) and transgenic (DR5::GFP, and PIN2::GFP) seeds were sterilized in 0.1% (w/v) HgCl2 for 7 min, followed by 5 rinses with distilled water. Then the seeds were germinated on solid MS medium supplemented with 1% sucrose at 22°C, and seedlings were grown during 4 days with 16h/8h day/night photoperiod, light intensity of ~100 |mol(m2 s).
Root gravitropism assay. Ellagic acid (EA, "Sigma", United States) was dissolved in DMSO and applied to solid MS medium. The concentrations of EA were 10, 50, and 200 |M, and a corresponding amount (1%, v/v) of DMSO without EA was used as the control. Seeds of Col-0 were sowed into the medium and then incubated vertically under standard conditions. After 5 d, images were captured with a digital camera ("Panasonic", Japan). Root length and gravitropic angle were measured according to Zou et al. [14] with Image J software ("NIH", United States). Root length refers to the entire length of the root. The gravitropic angle is the angle of the root tip with respect to the gravity vector.
For gravistimulation analyses, four-day-old Col-0 seedlings were transferred into the medium with or without EA and root tips were marked, and after that rotated 90° to initiate lateral gravistimulation. Images were captured at 1, 2, 3, 4, 6, 8, and 12 h. Gravitropic root tip angle measurements were made with Image J software following Perbal et al. [15] and defined as:
AaT(t-t0) = aTt - aT(t = 0),
where a — root tip curvature angle, Tt and T(t=0) — time of the end and the beginning of experiment. Root elongation was measured as root length increased after transfer to treatment medium.
Starch analyses. Roots of four-day-old Col-0 seedlings cultured in EA-containing and control medium were stained in KI/I (10%/5%) solution and then observed under a bright-field microscopy ("Shanghai", China). MacScope software ("Mitani Co.", Japan) was used to determine the amount of amyloplasts by measuring the area of the stained amyloplasts.
Confocal microscopy. Four-day-old Arabidopsis DR5::GFP and PIN2::GFP seedlings were transferred into the medium with 200 |M of EA. DR5::GFP seedlings were incubated vertically for 12 h, and then rotated 90° for 3 h. PIN2::GFP seedlings were incubated vertically for 24 h. Fluorescent images of treated roots were captured with a laser-scanning confocal microscope ("Olympus", Japan).
Data analyses. All treatments were replicated three times and at least 30 seeds or seedlings were used per treatment. SPSS software ("SPSS Inc.", United States) were used for data statistical analysis. The differences in root growth and gravitropism and amount
of amyloplasts between EA treatments and control were analyzed by ANOVA with Dunnett's two-tailed comparisons.
RESULTS AND DISCUSSION
Gravitropism of Arabidopsis root was disrupted by EA
It has been demonstrated that the presence and concentration of certain compounds in the medium affect root gravitropic response. For instance, exoge-nously applied ascorbic acid and dehydroascorbic acid both stimulated root gravitropic responses in a concentration-dependent fashion [16]. In this study, Arabidopsis was used to determine the effect of EA on plant gravitropic responses. After grown vertically for 5 days in EA-containing medium, the root gravitropic angles of Arabidopsis seedlings first decreased and then increased with the rise of EA concentration and the root growth was slightly inhibited by EA treatments (fig. 1a).
To better understand the influence of EA on root gravitropism, an additional experiment was carried out after lateral (reoriented 90°) gravistimulation. Results showed that gravitropic bending of root tips was delayed relative to control within 12 h, especially at the first 6 h after gravistimulation (fig. 1b). Slower or delayed gravity responses after EA treatments were in a concentration-dependent manner, the highest concentration (200 | M) had the strongest effects at every time point (fig. 1b). Root elongation of Arabidopsis treated with EA was similar to that of control (fig. 1b).
In plants, influences on root elongation may affect the root gravitropism responses. Our results showed that EA only has slightly inhibitory effects on root elongation, and gravitropism was strengthened at 10 and 50 |M, and weakened with EA concentration rising to 200 |M (fig. 1a). Moreover, the dynamic results obtained with variations in time and concentration showed that with time rising, the gravitropic bending was delayed in a concentration-dependent manner, however, root elongations were almost same as the control (fig. 1b). These results suggested that the influence of EA on the gravitropic bending angle was not a secondary effect of its inhibition of root elongation and these two biological processes under EA stress may be regulated independently.
EA reduced the starch grain in Arabidopsis root caps
According to starch-statolith hypothesis, starch-dense amyloplasts within the columella cells of the root cap play an important role in gravitropic response [2]. Therefore, the morphology and amount of col-umella amyloplasts in root cap after EA treatments were investigated. Results showed that starch levels in root caps under 10 |M EA were similar to that of control (figs. 2a, 2b). However, with the increase of EA concentration to 50 |M, starch levels in root caps were
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