ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 5, с. 730-738
^ ЭКСПЕРИМЕНТАЛЬНЫЕ
СТАТЬИ
УДК 581.1
AUXIN REDISTRIBUTION AND SHIFTS IN PIN GENE EXPRESSION
DURING Arabidopsis GRAFTING1
© 2014 J. Wang*, Z. Jin*, H. Yin*, B. Yan*, Z. Z. Ren*, J. Xu*, C. J. Mu*, Y. Zhang**, M. Q. Wang**, H. Liu*
*Institute of Cell Biology, School of Life Science, Lanzhou University, Lanzhou, P.R. China **Lanzhou Institute of Biological Products, Lanzhou, P.R. China Received November 28, 2012
Auxin is important in the development of plant vascular tissues. Reconnection of vascular bundles between scion and stock is a primary aim of grafting, and polar auxin transport greatly affects the formation of a continuous vascular model. The role of auxin in the process of graft-union development was studied by grafting the seedlings of Arabidopsis thaliana Heynh. DR5:GUS marker plants, which exerts the auxin-specific responses. Auxin induced the DR5:GUS expression in the vascular bundles around graft surface and stimulated the formation of multiple vascular bundle reconnections on the third day after grafting (DAG). DR5:GUS expression was delayed for one day in both scion and stock and dramatically declined by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Vascular bundle reconnection was observed only on the fourth DAG. These results suggest that auxin stimulates the reconnection of the vascular bundles, whereas NPA inhibits it. We studied the role of PIN proteins in graft development by grafting seedlings of PIN:GUS plants. PIN had different expression patterns in the graft process. Expression levels of PIN genes were analyzed by real-time PCR. All PIN genes had the higher expression level at the third DAG. We conclude that auxin stimulates the development of graft unions, and the patterns of expressions of PIN family genes can affect the development of graft union by controlling the auxin flow.
Keywords: Arabidopsis thaliana — graft development — micrografting — DR5:GUS marker — grafting — vascular reconnection — auxin accumulation — PIN gene family — stock—scion interaction
DOI: 10.7868/S0015330314050157
INTRODUCTION
Auxin participates in all aspects ofplant growth and development, especially in the development of vascular tissues [1, 2]. The mechanisms, by which auxin controls the development of vascular tissues, are not very clear yet [3]. New vascular bundles are formed, being induced by auxin. Vascular tissues tremendously increase in plants treated with auxin [2, 4].
The decisive function of auxin in the plant development depends on its distribution pattern [5, 6]. Auxin transport and distribution depend largely on PIN proteins as output carriers, and eight PIN genes have been identified [7]. The corresponding proteins, including PIN1, PIN2, PIN3, PIN4, and PIN7, are described in the literature [8]. Among these PIN proteins, PIN1 has a remarkable effect on the development of the leaf
1 This text was submitted by the authors in English.
Abbreviations'. DAG — day(s) after grafting; NPA — N-1-naphthyl-phthalamic acid; WT — wild-type.
Corresponding author. Heng Liu. Institute of Cell Biology, School of Life Science, Lanzhou University, Lanzhou 730000, P.R. China; e-mail. dewy1021@sina.com
and apical hook [9, 10]. To our knowledge, previous studies were only focused on changes in PIN1 distribution in the developing roots, but few correlation studies were conducted on hypocotyls. In pea hypoco-tyls, Sauer et al. [11] found that cut injury increased the expression level of PIN1, leading to the changes in the polarity of PIN1 protein distribution in the cell, thereby affecting the reconstruction of the vascular tissue. A disruption of the normal PIN1 protein accumulation at the basal ends of hypocotyl cells in the mdr mutants changed their tropic responses [12]. PIN3 proteins in the roots are repositioned to the bases of endodermis cells and promote auxin transport driven by gravity. PIN3 protein is important to the regulation of lateral auxin translocation, because PIN3 single mutants show defects in phototropism and is asymmetrically localized in response to phototropic stimulation [13]. ProPIN7.PIN7 -GUS activity could be observed at the node and throughout hypocotyl in dark-grown seedlings [14]. The abundance of PIN7 in leaf cells may contribute to substrate specificity seen in protoplast transport assays. Current study demonstrates that both PIN3 and PIN7 are required for the regulation of preferential auxin flow from adaxial to
abaxial side of the hook during continuous light-induced and positive phototropism [14]. PIN2 expression is mainly located in the Arabidopsis roots, but not in hypocotyls [15, 16]. However, the intracellular transport and proteolysis of Arabidopsis auxin efflux facilitator PIN2 are involved in the root gravitropism. AtPIN4 is involved in the auxin down transport to the quiescent center of the root meristem, being essential for auxin distribution and patterning [17].
Grafting combines different plant genotypes to generate their chimera. Micrografting, firstly reported by Turnbull et al. [18], was applied to Arabidopsis seedlings germinated for three to four days. The micro -grafting technique is widely used for studying longdistance signaling in plant development, especially during flowering [19]. Auxin is believed to be important for the reconnection between vascular tissue of the scion and the root stock. In the interface fusion of graft, PIN proteins influence auxin distribution by the accumulation of auxin in specific cells.
In our study, the Arabidopsis DR5:GUS marker plants were treated with IAA and N-1-naphthylphtha-lamic acid (NPA). Treatment with IAA could promote interface healing of the graft. NPA application could cause the inhibition of auxin downward transport and suppress the connection of vascular bundles. The expression distribution of PIN:GUS was also observed in this work, and these findings indicated that various PIN genes participated in the graft. The changes in the expression levels of PIN proteins are detected in the first three days of grafting. The PIN protein family is highly expressed on the third day ofgrafting, when vascular bundles are connected, and mediates auxin transport in the newly formed plant.
The objective of this study was to investigate the effects of PIN proteins on auxin redistribution by mi-crografting technique. Arabidopsis DR5:GUS marker plants were treated with IAA and NPA.
MATERIALS AND METHODS
Plant materials and micrografting procedure. Arabidopsis thaliana Heynh. ecotype Col-0 was used as a wildtype (WT) plant. Transgenic lines pro-DR5:GUS that express GUS under the control of DR5 (a synthetic auxin response element) promoter were purchased from Nottingham Arabidopsis Stock Centre (NASC European Arabidopsis Stock Centre, http://arabidopsis. info/). The transgenic lines pro-PIN (1, 2, 3, 4, and 7):GUS were a kind gift from Dr. Benkova.
Micrografting of Arabidopsis was carried out as described by Yin et al. [19]. Plates were tilted using a glass rod 0.7 to 1 cm in diameter; thus, after solidifying the medium has thin-layer side and thick-layer side. Seeds were sown on 1% agar-solidified PNS medium containing the macronutrients (5 mM KNO3, 2 mM Ca(NO3) • 2H2O, 2 mM MgSO4 • 7H2O, 2.4 mM KH2PO4, 0.1 mM K2HPO4, 50 |M Fe-EDTA, 50 |M
FeSO4 • 7H2O, and 50 |M EDTA-Na2 • 2H2O), as well as the micronutrients (0.5 |M KI, 10 |M H3BO3, 10 |M MnSO4 • 4H2O, 3 |M ZnSO4 • 7H2O, 0.1 |M Na2MoO4 • 2H2O, 0.01 |M CuSO4 • 5H2O, and 0.01 |M CoCl2 • 6H2O) at pH 5.7-5.8. The plates were incubated in the dark at 4°C for two to three days and then placed vertically in a growth room (temperature of 22-25°C; light intensity of 6000 lx; a photope-riod of 16 h) with the thin-layer side pointing downward and the thick-layer side pointing upward. Grafting was performed by using a dissecting microscope at 4 days after germination. Seedlings with long straight hypocotyls were chosen, and the hypocotyls were cut transversely, when seedlings were on the agarized medium. The stock donor hypocotyl was cut near the shoot apical meristem, and the scion donor was cut halfway from the base of the hypocotyl. Forceps were used to keep the seedling stable while cutting the hy-pocotyl with razor blade under a dissecting microscope. Quick cutting and pushing rather than pulling the blade are important, so that the cut surface is clean and smooth. The scion was lifted and brought closer to the stock, and the stock was carefully adjusted to connect to the scion. Note that it is important to raise the graft union up away from the agar surface (the oblique surface will provide enough space to make it possible). The scion or stock was carefully and slightly pushed to adjust the relative positions of the two parts, and the graft was inspected from all sides of the graft junction to ensure that the two parts are thoroughly connected and supported. The plate was returned to the growth room to the same vertical position with the thin side pointing downward and the thick side pointing upward.
For plant grafting in culture medium containing hormones, we artificially created a double-deck medium as shown in fig. 1. During grafting, only root of stock part touched the PNS medium with hormones, and in this way, exogenous IAA and NPA were applied only to the root stock.
GUS staining. GUS staining was carried out as described for Arabidopsis by Jefferson et al. [21, 22]. Collected tissues were fixed in cold 90% acetone at 4°C for 10 min and then rinsed five to six times with staining buffer (50 mM Na-phosphate buffer, 0.1% Triton X-100, 2 mM potassium ferrocyanide, and 10 mM EDTA-Na2). The staining buffer was removed from the samples, and the staining solution containing 2 mM 5-bromo-4-chloro-3-indolyl-P-D-glucuronide was added. Samples were incubated at 37°C. The exact time course for incubation depended on the GUS activity in the chosen line (for example, 24 h for DR5/DR5 grafted after one
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