^^^^^^^^^^^^ ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^
СТАТЬИ
581.1
EXPRESSION OF OsAMTl (1.1-1.3) IN RICE VARIETIES DIFFERING
IN NITROGEN ACCUMULATION1
© 2014 S. P. Zhao*, **, X. Z. Ye*, W. M. Shi*, ***
* Zhejiang Province Key Lab for Food Safety, Institute of Quality and Standard for Agroproducts, Zhejiang Academy of Agricultural Sciences, Hangzhou, P.R. China ** Graduate School of Chinese Academy of Sciences, Beijing, P.R. China *** State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Science,
Nanjing, P.R. China Received November 6, 2013
OsAMT is a high-affinity ammonium transporter responsible for NH+ uptake by rice plants. To investigate the expression patterns of OsAMT in different genotypes in relation to nitrogen accumulation, we measured the expression of OsAMTl.1, OsAMT1.2, and OsAMT1.3 using Real-Time PCR (RT-PCR) in GD (higher N accumulation) and NG (lower N accumulation) seedlings of the Oryza sativa L. cultivar treated with 0.1 mM NH4NO3 and 2 mM NH4NO3. We found that the expression level of OsAMT1.1 was significantly higher than those of OsAMT1.2 and OsAMT1.3 in the roots treated with 0.1 mM NH4NO3, suggesting that OsAMT1.1 contributed the most to N accumulation among the three genes. In GD root, OsAMT1.1 had significantly higher expression levels when it was up-regulated by 0.1 mM NH4NO3 than when down-regulated by 2 mM NH4NO3. OsAMT1.1 was mainly found in GD roots treated with 0.1 mM NH4NO3. We conclude that the OsAMT1.1 in GD roots, which was significantly up-regulated by low N and down-regulated by high N, was the dominating factor in determining the higher N acquisition in GD than in NG at 0.1 mM Nh4NO3.
Keywords: Oryza sativa - OsAMT1 - transporter - nitrogen
УДК
DOI: 10.7868/S0015330314040228
INTRODUCTION
OsAMT is a high-affinity ammonium transporter mainly responsible for NH+ uptake by rice plants. Rice (Oryza sativa L.) is one of the most important crop species on Earth, which provides for staple food for 70% of the world's human population. NH+ is the major form of nitrogen available for growth of rice
plants in paddy fields and requires NH+ transport systems at the root plasma membrane [1]. Ninnemann et al. [2] first identified the gene encoding a high-affinity NH+ transporter, AtAMTl.1, from Arabidopsis thaliana using functional complementation of a yeast mutant defective in NH+ uptake. Since then, the isolation of further AMT1 homologs from A. thaliana [3-6], Brassica napus [7], Lotus japonicus [8, 9], Lycopersicon esculentum [10, 11], and Oryza sativa [1, 12] have shown that the AMT gene family in plants consists of at least three to five members. Li et al. [13] reported
1 This text was submitted by the authors in English.
Corresponding author. S. P. Zhao. Zhejiang Province Key Lab for Food Safety; Institute of Quality and Standard for Agroproducts, Zhejiang Academy of Agricultural Sciences; Hangzhou 310021, China; fax. +86 0571 86419052; e-mail. zhaosppaper@163.com
that there are 12 putative OsAMT genes in rice. three each for OsAMTl, OsAMT2, and OsAMT3 and one for OsAMT4. Deng et al. [14] and Li et al. [13] identified OsAMT5.1 and OsAMT5.2 as two members of OsAMT5. In the present study, three members of the OsAMTl gene family, OsAMTl.1, OsAMT1.2, and OsAMT1.3, were cloned and analyzed.
Kumar et al. [15] proved that the expression patterns of OsAMT 1.1, OsAMT 1.2, and OsAMT1.3 were the same as AtAMT in Arabidopsis [3]. The expression levels of OsAMT1.1 in roots decreased several folds within 48 h when plants acclimated to 10 ^M NH+ for 3 weeks were transferred to 10 mM NH+ . Likewise, when plants acclimated to 10 mM NH+ were transferred to 10 ^M NH+ , there was an equally rapid up-regulation of OsAMT1.1-mediated ammonium influx in the roots. Changes in the transcript abundance of OsAMT1.2 following these treatments were approximately 50% less than those of OsAMT1.1, while OsAMT1.3 increased approximately threefold late in the photostage during the cycle of dark and light. In contrast, the study by Sonoda et al. [16] showed that OsAMT1.1 was expressed constitutively in shoots and roots; expression of OsAMT1.2 was root-specific and ammonium-inducible; and OsAMT1.3 expression was
root-specific and nitrogen-derepressible. These two studies, however, did not take different cultivars into account. Although much effort has been spent studying the expression patterns of OsAMT, no one has studied the differences in their expression between cultivars, especially in relation to the genetic differences of N accumulation. Moreover, semi-quantitative PCR or northern blotting was used to detect gene expression in previous studies, the methods, which cannot detect accurately the low expression levels of OsAMT.
In our study, we detected the expression of OsAMTl (1.1-1.3) genes under high and low N treatment by RT-PCR in two rice genotypes with different N accumulation. Our purpose was to investigate the molecular basis of different N accumulation in rice and provide for a foundation for breeding new cultivars with high N accumulation using molecular biology techniques.
MATERIALS AND METHODS
Plant materials. The Oryza sativa L. cultivars GD and NG were used in our experiment. Our previous field experiment had investigated the high and low N use efficiency of GD and NG, respectively, and found that GD could obtain significantly higher yield, biomass, and N accumulation than NG when low fertilizer is supplied, while NG can obtain high yield only under high N fertilizer treatment. Furthermore, GD was more sensitive to N enhancement than NG [17, 18].
Growth conditions. Hydroponically grown rice plants were used for all experiments. Rice seeds were surface-sterilized in 1% NaOCl for 30 min, rinsed with deionized water several times, and left to imbibe in aerated water at 37°C for 48 h in a water bath in the dark. The germinating seeds were then placed onto plastic mesh mounted on plastic containers. The containers were filled with tap water just above the level of the seeds and were placed in a controlled-environ-ment growth room with a temperature of 25 ± 2°C, relative humidity of 70%, and an irradiance of 300 ^E/(m2 s) under fluorescent lighting and a 14-h photoperiod. After about two days (seed radicle length of 2 cm), rice plants were grown hydroponically in 0.5 Kimura B solution composed of 65.9 mg/L MgSO4, 40.18 mg/L CaCl2, 21.88 mg/L NaH2PO4, 40.61 mg/L KCl, 5.89 mg/L C2H4N4, 5.6 mg/L P, 21.4 mg/L K, 14.6 mg/L Ca, 13.3 mg/L Mg, 17.58 mg/L S, 1.12 mg/L Fe, and microelement solution (A-Z), in which the N source was 0.5 mM NH4NO3. The nitrification inhibitor C2H4N4 (5.89 mg/L) was also added to prevent the conversion of ammonium into nitrate by nitrification. Generally, 1 L/day of nutrient solution was supplied (total solution of 18 L) in the morning and the nutrient solution was renewed every 4 days. The pH of the growth media was maintained at 5.5 ± 0.5 by adding diluted NaOH or HCl once or twice daily.
Growth of plants. Fifteen-day-old rice plants of uniform size were divided into the two groups, each in triplicate, based on the concentration source of N: 0.1 mM NH4NO3 and 2 mM NH4NO3 (N concentration was determined based on our own experimental preparation; data not published).
Preparation and analysis of N accumulation by the two cultivars. The roots and leaves of the rice plants were harvested 5 and 10 days later, weighed quickly, dried at 80°C for several days, and the biomass of shoots and roots and N concentration in tissues were determined. N concentrations in shoots and roots of every cultivar/line were determined by the Kjeldahl method. The N accumulation was calculated by biomass x N concentration.
Preparation for analysis of OsAMT mRNA expression levels. The roots and leaves of the rice plants were harvested 10 days later, frozen quickly in liquid nitrogen, and then stored at -80°C until total RNA extraction, cDNA synthesis, and RT-PCR analysis.
Preparation and analysis of up-regulation and down-regulation of OsAMTl.1 mRNA levels. 10 days later, plants acclimated to 0.1 mM NH4NO3 were transferred to 2 mM NH4NO3 for 0, 2, and 6 h, and plants acclimated to 2 mM NH4NO3 were transferred to 0.1 mM NH4NO3 for 0 and 6 h. The roots and leaves of the rice plants then were harvested, frozen quickly in liquid nitrogen, and stored at -80°C until total RNA extraction, cDNA synthesis, and RT-PCR analysis.
Total RNA extraction, cDNA synthesis, preparation of primers, and verification of RT-PCR products. Total RNA samples were isolated using the guanidine isothio-cyanate method. Total RNA (5 ^g) was used to synthesize cDNA by reverse transcriptase powerscript™ following the manufacturer's protocol ("Takara Biotechnology", China). The cDNA samples were used as a template to quantify the target gene expression levels. We designed the primers for each gene according to the rice cDNA database (NCBI/GenBank accession nos. AF289477 for OsAMTl.1; AF289478 for OsAMT1.2; AF289479 for OsAMT1.3; and XM469569 for OsActin). The products of cDNA segments for OsAMT1.1, OsAMT1.2, OsAMT1.3, and OsActin were 1295~1595, 785~1347, 776~1170, and 801~997 on the full cDNA sequence, and the product sizes were 304, 562, 395, and 196 bp, respectively. The specific primers for the genes were as follows: OsAMT1.1 forward 5'-tctcttc-tacgggctcaagaagc-3', reverse 5'-caaatttatgacgtgacgatc-gaga-3'; OsAMT1.2 forward 5'-gatctacggcgagtcgggcac-gat-3', reverse 5'-ttccatctctgtcgaggtcgagacg-3'; OsAMT1.3 forward 5'-tcaaatcctacggcccgcccggtag-3', reverse 5'-gccgaagatctggtccacgtactcctt-3'; and OsActin forward 5'-cttcataggaatggaagctgcgggta-3', reverse 5'-cgaccaccttgatcttcatgctgcta-3'. All five specific RT-PCR products were cloned, sequenced, and compared with the sequence of the respective AMT genes to confirm the specificity of the RT-PCR products.
®H3HO.roratf PACTEHHH TOM 61 № 5 2014
Table 1. The biomass, nitrogen concentration, and nitrogen accumulation in GD and NG und
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