ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^ СТАТЬИ
УДК 581.1
ISOLATION AND INDUCED EXPRESSION OF A FRUCTOKINASE GENE
FROM LOQUAT1 © 2014 Q. P. Qin, Y. Y. Cui, L. L. Zhang, F. F. Lin, Q. X. Lai
School of Agriculture and Food Science, Zhejiang Agriculture and Forestry University, Lin'an, Hangzhou, China The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, Zhejiang A&F University,
Lin'an, Hangzhou, China Received 29.01.2013
Fructose is essential for plant development as well as for fruit sugar composition and fruit quality. Fructose is one of the major sugars in the mature loquat (Eriobotrya japonica Lindl.) fruit, which is popular because of its flavor and availability in early summer. The elucidation of the mechanism of fructose metabolism is of great importance for fruit quality improving. Fructose is primarily phosphorylated by fructokinase (FRK). In order to understand the fructose metabolism in the loquat fruit, a putative loquat FRK full-length cDNA designated as EjFRK was isolated in this study. The EjFRK encoding FRK possesses conserved regions inherent to plant FRKs. Transient expression of 35S:EFRK-GFP fusion protein in onion epidermal cells showed that EjFRK was mainly expressed in the cytosol. The real-time RT-PCR analysis indicated that EjFRK was expressed in all loquat tissues. Monitoring the dynamic changes of EjFRK transcripts and FRK enzymatic activities demonstrated that EjFRK expression was at a relative high level during early fruit developmental stages and dropped to the lower level during maturation, similar with the changes in FRK activity, which was opposite to the fructose levels during fruit development. The results indicated that the high FRK enzymatic activity was not conducive to fructose accumulation in loquat fruit. The EjFRK transcript level in leaves of loquat seedlings was significantly enhanced after 6 h of treatment with 10 and 100 mM fructose or glucose, which indicates that EjFRK is modulated by fructose and glucose in vivo.
Keywords: Eriobotrya japonica - fructokinase - glycolysis - sugar metabolism
DOI: 10.7868/S0015330314030129
INTRODUCTION
Sugars provide energy and carbon skeletons for plant growth and also behave as signaling molecules [1, 2]. Sucrose, an essential photosynthesis product, is the primary sugar transported in various vascular plants. Sucrose is converted by sucrose synthase into UDP-glucose and fructose or by invertase into glucose and fructose to support tissue development and functioning [3]. Consequently, free fructose is phosphorylated by hexokinase (HXK, EC 2.7.1.1) or fructokinase (FRK, EC 2.7.1.4) for further metabolism. However, fructose is primarily phosphorylated by FRKs because the affinity of FRKs for fructose is much higher than that of HXKs [4].
1 This text was submitted by the authors in English.
Abbreviations'. DAF - days after flowering; FRK - fructokinase; RACE - Rapid Amplification of cDNA Ends; SDH - sorbitol dehydrogenase; S6PDH - sorbitol-6-phosphate dehydrogenase. Corresponding author. Qi Xian Lai. School of Agriculture and Food Science, Zhejiang Agriculture and Forestry University, Lin'an, Hangzhou, 311300 China. Fax. 086-571-6374-1276; e-mails. laiqixian@zafu.edu.cn, laiqixian@hotmail.com
Plant FRK enzymes have been purified from Ara-bidopsis, soybean, maize, sugarcane, etc. and analyzed [5, 6]. FRK genes have been identified from several plants, such as tomato and citrus [7]. Tomato is the first plant species, from which four FRK genes, LeFRK1-4, were isolated and characterized [8-11]. Among these genes, LeFRK2 and LeFRK3 are the major FRK-encoding genes expressed in most tissues, whereas LeFRK3 exhibits the highest expression in leaves and apices. LeFRK2 is required for stem xylem development as well as sugar metabolism [12, 13]. LeFRK1-3 and their corresponding isozymes are found in tomato fruits [11]. LeFRK4 is specifically expressed in stamens [10]. LeFRKl is located on chromosome 3, LeFRK2 - on chromosome 6, LeFRK3 -on chromosome 2, LeFRK4- on chromosome 10 [11]. Subcellular analysis showed that LeFRK3 is located within plastids, whereas LeFRKl, LeFRK2, and LeFRK4 are located in the cytosol. Hence, fructose phosphorylation is not confined to special intracellular localizations [14].
Different expression patterns were also exhibited by four apple FRK genes MdFKl-4 [15]. MdFKl,
MdFK3, and MdFK4 had similar expression patterns, whereas MdFK2 was expressed significantly higher in shoot tips than in mature leaves. MdFK2-4 were more highly expressed in young fruits than in mature fruits. MdFK2 had a significant function in the efficient utilization of fructose in shoot tips and young fruit. MdFK1 might provide fructose for sucrose synthesis during fruit maturation [15]. Similarly, the reduction of aspen FRK resulted in the accumulation of soluble neutral sugars and a decrease in hexose phosphates and UDP-glucose, which also led to the reduction of cellulose content and the thinner fiber cell walls [16].
Loquat (Eriobotrya japonica Lindl.) belongs to the family Rosaceae and subfamily Maloideae and is an evergreen fruit tree origined from southeastern China. The tree is mainly cultivated in China, Japan, Israel, and Brazil. Loquat fruit is popular not only because of its succulent, rich, and acidic flavor, but also because it ripens in spring or early summer, during which time limited fresh fruits are available. The sugar and acid contents of loquat fruits are responsible for its characteristic flavor. Fruit acids are degraded during ripening, resulting in the increased sugar content and sugar/acid ratio. Sweetness is the basic taste for fruits, with sweeter loquat fruits preferred [17]. Therefore, the mechanism of loquat fruit sugar metabolism is important to elucidate. Several mechanisms of loquat fruit sugar accumulation were proposed. Sucrose, glucose, fructose, and sorbitol are the major sugar components of most loquat fruits [18-21]. Enzymes related to loquat sucrose accumulation were reported. Sor-bitol-6-phosphate dehydrogenase (S6PDH), sorbitol dehydrogenase (SDH), cell wall-bound acid invertase, soluble acid invertase, sucrose synthase, and sucrose phosphate synthase are evidently related to sugar accumulation [19, 20]. The sugar content increase occurs simultaneously with enzymatic activity during fruit maturation. S6PDH and NAD-SDH regulate sugar accumulation mainly at the transcriptional level [19, 20]. Considering that FRK is essential in fructose metabolism, in this study we isolated a transcript of a putative FRK from loquat and investigated its regulated expression mechanism. The results may help elucidate fructose metabolism in loquat and other horticultural fruits.
MATERIALS AND METHODS
Plant material and cultivation. The experimental material for gene isolation and expression analysis was obtained from seven-year-old Eriobotrya japonica Lindl. (loquat) trees in the botanical garden of Zhe-jiang Agriculture and Forestry University, Hangzhou, China. Leaves, flowers, stems, and fruits at different developmental stages (60, 83, 100, 117, 125, and 143 days after flowering (DAF)) were collected, corre-
sponding to the slow development stage (60 DAF), fast development stage (83, 100, and 117 DAF), color breaking stage (125 DAF), and maturation stage (143 DAF). All the tissues were selectively collected from three trees, respectively mixed, immediately frozen in liquid nitrogen, and stored at —80°C until analysis.
cDNA cloning and sequence analysis. Total RNA was extracted from loquat tissues using a modified CTAB (cetyltrimethyl ammonium bromide) method [22]. RNA quality was verified by spectrophotometry and gel electrophoresis. First-strand cDNA was synthesized using a RevertAid first-strand cDNA synthesis kit ("Fermentas", Canada). To isolate the FRK gene from loquat, degenerate primers were designed according to the conserved region by aligning of putative FRK genes from GenBank. The forward degenerate primer was 5'-TTCGG(T/C/G)GAGATG(C/T)T(G/A)ATC-GA(C/T)TTCGT(C/T)CC-3', and the reverse primer was 5'-CA(G/C/T)CAGT(G/T/A)TCCCCA(T/G) -ACTC(A/G)AAATTTCTT-3'. A ~550-bp fragment was amplified using this primer pair. For amplification of the unknown 5' and 3' ends of the putative loquat FRK gene, specific primers were designed according to the sequenced fragment. A 3'-Full Rapid Amplification of cDNA Ends (RACE) Core Set, and a 5'-Full RACE kit ("TaKaRa", Japan) were used for 3'- and 5'-RACE. The primers for 3'-RACE were 5'-GAGGAGT-GATGGGGAACGTGAGTTC-3' and 5'-CAGCAA-CAAAAGCTGCAAAGGACGCT- 3'; the primers for 5'-RACE were 5' - GAACTCACGTTCCCCAT-CACTCCTC-3' and 5'-ATGCCAACTGCAACATT-AGCAGGCGC-3'. PCRproducts were extracted and cloned into a pUC18-T vector ("Sangon", China), transformed into Escherichia coli DH5a competent cells, and sequenced by "Sangon". The sequences were analyzed using NCBI BLASTX and ClustalX. To construct a phylogenetic tree, putative plant FRK sequences were retrieved from NCBI or Swiss-Prot. The phylogenetic tree was drawn using the Maximum Likelihood method of MEGA5 (http://megasoftware.net/).
Transcript detection by quantitative real-time RT-PCR.
Real-time RT-PCR primers were designed using the online real-time PCR Primer design tool (http:// www.genscript.com/). The forward primer used for loquat FRK was 5'-TAATGTTGCAGTTGGCATAG- 3'; the reverse primer was 5'-CTTGAAGCAACATAT-CAGCA-3'. The primer pair generated an amplicon at 236 bp in length. The loquat actin gene (GenBank ID: FJ481118) was used as the internal standard. The forward primer for actin was 5'-TGGTCGTACAACAG-GTAT-3'; the reverse primer was 5'-GGGCAA-CATATGCAAGCT-3'. QuantiTect SYBR Green PCR Kits ("Qiagen") and Applied Biosystems 7300 Real-time PCR System (ABI) were used to analyze transcript levels. Three replicates were performed for each sample. All da-
ta were analyzed using the statistical software SPSS 14.0 (http://www.spss.com).
Sugar treatment of loquat seedlings. Loquat seeds we
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