Pingping ZHANG, Xueqin KAN, Jian LI, Chi CHEN, Chengyang LUO, Jianzhong TAN

College of Architecture, Soochow University/Key Laboratory of Architecture and Urban Environment of Suzhou City, Suzhou 215123, China

Plant senescence is a developmentally regulated and genetically programmed process,and it is usually observed in many different plant tissues, such as leaves,petals, roots, seeds and other organs,ultimately leading to death of a particular organ or whole plant[1]. Flower ag ing is accompanied by typical changes in petal color, shape, fresh weight and metabolic processes[2]. Flower petals are good material for studies on plant senescence and its regulation mechanism, and are also important research content of ornamental horticulture.There have been many reports on molecular mechanism of petal senescence in carnations, petunias, roses and other ornamental plants[3]. Moreover, a variety of senescence-associated genes(SAGs)have been cloned,and they are involved in protein degradation, ethylene metabolism,lipid metabolism, cell wall modification and other physiological functions[3-5].

Previous researches on petal senescence are mostly concentrated in ethylene-sensitive flowers[2,4-6].Flower senescence is induced and regulated by the self-generated ethylene.When the amount of ethylene in plants reaches or exceeds a certain threshold, a large amount of ethylene will be induced to be generated,initiating respiratory climacterics and the whole ripening and senescence pro cesses in flowers[6]. However, in ethy leneinsensitive flowers, petal senescence is initiated by a small amount, instead of a large amount of ethylene[6]. Sothere may be different regulatory pathways for petal senescence in ethyleneinsensitive flowers[1].In this study,the ethylene-insensitive dahlia petals were used as material, and a senescenceassociated protein xyloglucan endotransglycosylase/hydrolase(XTH) was isolated from the total petal proteins by two-dimensional electrophoresis and identified by mass spectrometry. On this basis, the encoding sequence of XTH gene in dahlia was cloned using experimental techniques in bioinformatics and molecular biology, and then, the structure characteristics of XTH gene in dahlia and homology with different plant species were analyzed,providing a new experimental basis for elucidating the molecular mechanism of petal senescence in dahlia.

Materials and Methods

Plant materials

The tested material was monopetalous Dahlia (Dahlia pinnata Cav.)variety ‘Danbanhuang’.According to the degrees of development and senescence of dahlia petals, the flowering process was divided into three stages: building color stage (B), full flowering stage (F)and flower senescence stage (S).The collected petals were rinsed with double distilled water,dried, rapidly frozen by liquid nitrogen and preserved at-70 ℃.

Protein extraction and two-dimensional electrophoresis

The Dahlia petals were sampled at B,F and S stages,respectively.The proteins in Dahlia petals were extracted by phenol-methanol/ammonium acetate precipitation method[7],and the proteins concentrations were determined by Bradford method. The twodimensional electrophoresis was performed according to the operational guidelines of two-dimensional electrophoresis apparatus Ettan IPGphor(GE Healthcare).

Mass spectrometric analysis of proteins

The two-dimensional electrophoresis gel was scanned and analyzed using Image Master 5.0 (GE Healthcare). The differences in petal protein expression level among different flowering stages were compared.The in-gel digestion and mass spectrometry analysis were completed by Shanghai Applied Protein Technology Co.,Ltd.

Total RNA extraction from dahlia petals

Taking the dahlia petals at the full flowering stage as material, and the petal total RNA was extracted. The concentration of total RNA was determined by spectrophotometry, and the quality was examined by gel electrophoresis. The extracted total RNA was reverse transcribed into cDNA using reverse transcription kit produced by the Sangon Biotech (Shanghai)Co., Ltd. After the subpackage, the cDNA was preserved at -70 ℃for use.

RT-PCR

Based on the identified XTH amino acid sequence and according to Blast analysis, one pair of degenerate primers was designed. The sequence of forward primer was as follows: 5’-AAGATT (A/G) C (A/G) TTAGT (A/G)CATGG-3’;while the sequence of reverse primer was as follows: 5’-TCAA(C/T)TCAAATTCCGGC-3’. With the synthesized cDNA as template, PCR was carried out. The reaction system(25 μl) was as follows: 10× reaction buffer 2.5 μl, 2.5 mmol/L dNTP 2.5 μl,Ex Taq DNA polymerase 0.125 μl,forward primer 0.5 μl, reverse primer 0.5 μl, cDNA 1.5 μl, distilled water 17.375 μl.The reaction conditions were as follows: pre-denaturation at 94 ℃for 3 min; denaturation at 94 ℃for 1 min,annealing at 50 ℃for 2 min,extension at 72 ℃for 2 min,35 cycles;extension at 72 ℃for 10 min.The PCR products were preserved at 4 ℃.

Cloning and sequencing of PCR products

The PCR products were examined by 1.6% agarose gel electrophoresis. The target fragment was recovered by Agarose Gel DNA Extraction Kit (Sangon Biotech(Shanghai) Co., Ltd.), ligated with pUCm-T and transformed into E. coli TOP10 competent cells. The positive colons were screened on LB plates with ampicillin (60 μg/ml). The sequencing was completed by the Sangon Biotech(Shanghai)Co.,Ltd.

Sequence analysis

The sequencing results were compared with the corresponding nucleic acid and protein sequences in GenBank using NCBI BLAST. The multiple comparisons and phylogenetic analysis were performed using DNAMAN Version 6.

Results

Isolation and mass spectrometric identification of senescence-associated protein XTH in dahlia petals

As shown in Fig.1, the protein components from dahlia petals were mainly distributed in the PI range of 5-8 and molecular weight range of 14-90 kDa. A total of 432, 421 and 387 protein spots were detected at B, F and S stages, respectively. And there were 44 protein spots among which the differences in expression level were more than two times. From the 44 protein spots, total 15 spots with different expression levels and clear separation were selected for in-gel digestion and MALDI-TOF/TOF mass spectrometry.The results showed that the No.D11 protein spot was identified as xyloglucan glycosyltransferase/hydrolase (XTH);the expression level of XTH was increased with the development process of dahlia petals, and the expression levels at full flower and flower senescence stages were in-creased by 1.17 and 2.83 times respectively compared with that at building color stage. According expression characteristics of senescence-associated genes in plants[1], XTH in dahlia could be considered as a kind of petal senescence-associated protein.

Cloning of cDNA sequence of XTH gene in dahlia petals

The extracted total RNA from dahlia petals was reverse transcribed into cDNA, which was amplified by PCR using a pair of degenerate primers. An amplified band in full length of 900 bp was obtained (Fig.2).The recovered objective band was ligated with pUCm-T vector and then transformed. The screening of bluewhite spots was carried out on LB plates containing ampicillin. The positive clones were cultured and then verified by PCR. The results showed that a clear band, in the same molecular weight with target band,was finally amplified(not shown).

Analysis of cDNA sequence of XTH gene in dahlia petals

The sequencing results showed that the size of cloned fragment was 882 bp; the target fragment encoded 293 amino acid residues and 1 stop codon (Fig.3) and was named as DpXTH1(accession number: HM053613.1). The Blast analysis of encoding sequence in DpXTH1 showed that the homologies between DpXTH1 and other 100 XTH gene sequences ranged from 82% to 91%.Most of the plants belonged to Compositae. The XTH gene derived from Helianthus petiolaris showed the highest homology(91%)with DpXTH1,indicating that the genetic relationship between the two XTH genes was closest.

Homology analysis of XTH amino acid sequences among different plants

XTH is a kind of protein that has a highly conserved structure and is encoded by a very large family of genes.To analyze the homology in XTH between dahlia and other plants, the cluster analysis of XTH amino acid sequences was carried out among dahlia and other 28 plants. As shown in Fig.4, the 29 XTH gene sequences were divided into 4 groups. The DpXTH1 (HM053613) from dahlia,XET (DQ235255) from gerbera, XET4(AF093507) from alfalfa, EXGT(D88413) from upland cotton and AtXTH (NM119942)from Arabidopsis belonged to one group. Among them,DpXTH1 showed the highest homology with TpXTH (JN164663) from tagetes, followed by CmXET(HM752243) from chrysanthemum,GeXET (DQ235255) from gerbera,GeXET (D884135) from gossypium and the like.However, the homologies in XTH amino acid sequence between dahlia and monocots, such as corn(U15781), and barely (X93174,X93175), were relatively low, and the homologies between DpXTH and three AtXETs from arabidopsis and TmXG1 (X68254) from nasturtium were lowest.

Discussion

Xyloglucan endotransglycosylases/hydrolases(XTHs)are a class of important cell wall modifying enzymes associated with plant cell growth and development[7-8].Their function is to cut glycosidic bond in glucan constituting the cell wall microfibers and transfer and link the cut oligosaccharide molecules to ends of similar molecules. XTHs also have the function of hydrolytic enzymes, leading to relaxation and reconstruction of plant cell walls, thereby participating in a variety of metabolic processes of plant growth and development[8-9]. Corollas are a vital organ for ornamental plants, and they are also involved in cell wall modification during petal senescence[10].Toikkanen et al.cloned a XTH gene from gerbera, and they determined the activity of XTH enzyme[11]. Sane et al. found that the expression level of RbEXPA1 gene was all up-regulated during the opening and shedding of rose petals, so they speculated that XTH was involved in petal cell wall modification, resulting in petal falling[12]. In this study, it was detected that the expression level of XTH was increased with the senescence process of petals on the two-dimensional electrophorogram of proteins from dahlia petals. According to expression characteristics of senescence-associated genes and their definitions in plants,DpXTH1 can be considered as a senescence-associated protein in dahlia petals.

DpXTH1 shows high homologies among different species. Most of the plant-derived XTH proteins contain a highly conserved amino acid residue region DEIDFEFLG and two double and half cystine sites[8-9].In current reports about plant XTH, the encoding sequences of XTH genes are ultimately obtained with their conserved sequences combined with RACE technique.In this study,from the perspective of differential proteomics, the differences in XTH expression level during flower senescence were detected.Based on plant-derived XTH amino acid sequences,XTH gene sequences were obtained by electronic cloning.In addition, one pair of degenerate primers locating between the start codon and stop codon was designed,and the cDNA sequence of XTH gene coding region in Dahlia petals was directly cloned by RT-PCR technology.All the above will lay a foundation for further study on expression characteristics of XTH gene at the protein and mRNA levels.

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