The variation of NAD+-SDH gene in mutant white-fleshed loquat

The variation of NAD+-SDH gene in mutant white-fleshed loquat

Journal of Integrative Agriculture 2016, 15(8): 1744–1750 Available online at www.sciencedirect.com ScienceDirect RESEARCH ARTICLE The variation of...

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Journal of Integrative Agriculture 2016, 15(8): 1744–1750 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

The variation of NAD+-SDH gene in mutant white-fleshed loquat LI Jing1, 2, 3, WANG Yong-qing1, CHEN Dong2, 3, TU Mei-yan2, 3, XIE Hong-jiang2, 3, JIANG Guo-liang2, 3 , LIU Jia2, 3, SUN Shu-xia2, 3 1

College of Horticulture, Sichuan Agricultural University, Ya’an 611130, P.R.China Horticulture Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R.China 3 Southwestern Key Laboratory of Horticultural Crops Biology and Germplasm Enhancement, Ministry of Agriculture, Chengdu 610066, P.R.China 2

Abstract Loquat (Eriobotrya japonica Lindl.) can be divided into yellow- and white-fleshed cultivars by flesh color. However, a Dongting loquat mutant, which involved bud sport and growing white-fleshed fruit in the central region of the trunk (as wild loquat bears yellow-fleshed fruits naturally), was discovered in the preliminary study. The study cloned the coding sequence (CDS) of NAD+-dependent sorbitol dehydrogenase (NAD+-SDH ) gene from the selected materials of mutant loquat, wild loquat and other nine loquat cultivars/accessions, and found that the CDS of NAD+-SDH gene from the mutant loquat, other than the rest two types of materials, had three single nucleotide polymorphisms (SNPs) loci; in addition, the amino acid encoded at variation loci changed accordingly. NAD+-SDH plays an active role in converting sorbitol into fructose in loquat cultivars. For the mutant white-fleshed loquat, the activity of NAD+-SDH rises first and then drops, the sorbitol content decreases steadily, and its fructose content is higher than that in wild loquat from coloration to maturation stage. As demonstrated by the real-time fluorescence quantification PCR analysis, the expression level of NAD+-SDH gene at maturation stage is about 5-fold lower than wild type. It may be assumed that, the three SNPs loci might lead to excessive conversion of sorbitol into fructose under the catalytic action of NAD+-SDH of white-fleshed mutant loquat at maturation stage, resulting in the increase of fructose content and reduced expression abundance of mRNA after transcription. Besides, NAD+-SDH gene may be related to flesh color and carbohydrate variation of white-fleshed mutant loquat. Keywords: loquat, flesh color, NAD+-SDH, SNP, fructose

1. Introduction Loquat (Eriobotrya japonica Lindl.) is a subtropical ev-

Received 31 July, 2015 Accepted 15 January, 2016 Correspondence SUN Shu-xia, E-mail: [email protected]; WANG Yong-qing, E-mail: [email protected] © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61297-7

ergreen fruit tree of the Rosaceae subfamily Maloideae originated in southwest China. It falls into yellow- and white-fleshed cultivars. Where the yellow-fleshed cultivars appear orange, and have dense flesh, thick peel and good resistance to stress during storage and transportation, while the white-fleshed counterparts are milky- or light-yellow-fleshed, tender, juicy and delicious, widely received by consumers for its pleasant flavor and taste. Carbohydrate is an important index for fruit quality. Previous studies show that carbohydrate composition varies greatly among different loquat cultivars at maturation stage. The yellow- and whitefleshed loquat cultivars mainly accumulate fructose and

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glucose at maturation stage, where the contents of fructose and glucose in white-fleshed cultivars are 32 and 23% higher than yellow-fleshed cultivars respectively; but the sucrose and sorbitol contents are very low, about 1.5–3.7% of the total carbohydrate (Chen et al. 2010). A few cultivars are dominated by sucrose, followed by fructose and glucose at maturation stage (Hirat 1980), or the contents of the three types of soluble carbohydrate are similar. However, the content of soluble carbohydrates in white-fleshed cultivars is higher than that in the yellow-fleshed cultivars (Chen et al. 2010). The carbohydrate components may be closely related to genetic and cultural environment etc. Sorbitol, the main form of carbohydrate transport in the development process of loquat fruit, declines rapidly as the fruit ripens, but sucrose, glucose and fructose accumulate constantly in contrast (Amoros et al. 2003; Chen et al. 2006). Sorbitol dehydrogenase (NAD+-SDH) with coenzyme factor NAD is the key enzyme for sorbitol metabolism, capable of catalyzing the irreversible conversion reaction of sorbitol into fructose. Bud growth rate correlated positively with the activities of NAD+-SDH, the increases of NAD+-SDH activity, both by shading and shoot bending, are suspected to be regulated at the transcriptional level (Akiko et al. 2005). NAD+-SDH activity in the seeds was found to be much higher than that in the cortex tissues of fruits during the early developmental stages of apple (Nosarzewski and Archbold 2007). NAD+-SDH was imported into the vacuoles mediated by 18 amino-acid residues as an internal sucrose phosphate (SP). And the NAD+-SDH localized in the seed protein storage vacuoles may play function by being involved in sorbitol metabolism at the early stage of seed germination (Xiu et al. 2013). Presently, the cDNA whole sequence and fragments of coded NAD+-SDH have been cloned from apple, loquat and peach fruits and pear blossom bud (Bantogn et al. 2000; Yamada et al. 2001; Akiko et al. 2005). Fructose content varies greatly in certain white- and yellow-fleshed loquat cultivars. White-fleshed cultivars are rich in fructose and have sweeter taste (Chen et al. 2010). The activity of NAD+-SDH in loquat fruit increases as the fruit ripens. The content of NAD+-SDH mRNA is very high in ripened fruit but rare in fruitlet. The activity of NAD+-SDH and protein level in loquat fruit are closely correlated with accumulation of transcription, indicating that the transcription ability of NAD+-SDH gene may be a key regulatory step for its activity (Bantog et al. 1999). In the preliminary study, a mutant loquat (bud sport emerged on a branch of a wild yellow-fleshed cultivar and bore white-fleshed fruit) was discovered and identified two polymorphic bands (UBC813/535 and OPH01/1719) thereby using a set of molecular markers. The proteins encoded by the two bands have high homology with that (EC1.1.1.14) encoded by NAD+-SDH from loquat. Based on the above

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information, the research analyzed the CDS of NAD+-SDH gene and its fluorescence quantitative expression in wild and mutant loquat, tested the activity of NAD+-SDH in loquat and the content of sorbitol and fructose in loquat using highperformance liquid chromatography-evaporative light scattering detection (HPLC-ELSD), which provides theoretical basis for study on flesh quality of loquat cultivars.

2. Materials and methods 2.1. Test materials and samples The mutant loquats described herein resulted in a change of flesh color phenotypes, which is a whitish-fleshed genotype emerged from a bud mutation of yellowish-fleshed genotype. The mutant loquats growing in the mountainous area in Wenchuan County, Aba Tibetan Autonomous Prefecture, China, are divided into mutant whitish-fleshed type (WNSC-1and WNSC-2) and wild yellowish-fleshed type (ZNSC-1and ZNSC-2). And other nine yellow- and whitefleshed loquat cultivars/accessions were also selected for test with the names Dawuxing, Huangfeng, Maomu, Daibaili, Baisha, Guifei, Xiangzhong 11, Wanzhong 518 and Xinbai 8. The mutant loquats were sampled four times respectively at 170, 175, 180 and 185 d after full bloom from coloration to maturation stage. The other cultivars/accessions were sampled once at maturation stage. The samples, which had uniform color, appearance, size and maturity, were treated with liquid nitrogen and stored in a –80°C refrigerator after peels and cores were removed immediately at fresh.

2.2. RNA extraction and cloning of CDS fragment in NAD+-SDH gene The total RNA was extracted with total RNA Extraction Kit from Huayueyang Biotechnology Co., Ltd. (Beijing, China). The flesh samples of wild, mutant and other loquat cultivars/accessions at maturation stage were taken out from refrigerator of –80°C and processed strictly as instructed. The RT-PCR amplification primers, i.e., NSC-F: 5´-ATGGGAAAGGGAGGCATGTCTGAT-3´ and NSC-R: 5´-TTACAGGTTAAACATGACCTTAATGGCA-3´ were designed and developed with Primer Premier 5.0 software according to CDS base sequence in NAD+-SDH gene (GenBank: AB042810.1) in NCBI database. RT reaction was performed using RevertAidTM First Strand cDNA Synthesis Kit of Fermentas by taking total RNA in loquat at maturation stage as the template and then PCR reaction was performed with the RT product as template and NSC-F/NSC-R as primer. RT-PCR amplification products were recovered, purified and ligated to pEASYTM-T3 (TransGen Biotech, Beijing, China) carrier after 1% agarose gel electrophoresis. The

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ligation products were transferred to competent cell of Trans1-T1 bacterial strain of Escherichia coli. After that the recombinant plasmid was obtained through white selection and determination and identification of plasmid length. The sequencing was undertaken by Shanghai Sangon Biological Engineering Technology & Services (China) and analyzed with Alignment software.

2.3. Real-time fluorescence quantification PCR analysis Two pairs of SYBR Green real-time fluorescence quantitative PCR amplification primers, NS1-F: 5´-TGAAGTTTTTTGGTTCCCCTCC-3´, NS1-R: 5´-ACCAACACTTAAGGGCTCACACA-3´ (for amplification of segments 434– 567 with amplification product of 134 bp) and NS2-F: 5´-TTTGGAAAATGGAGTGGATGTAAGC-3´, NS2-R: 5´-ATCTCTCTCTGACCCATTCCCACA-3´ (for amplification of segments 807–929 with amplification product of 123 bp) were designed respectively by using Primer Premier 5.0 software based on reported NAD+-SDH gene base sequence (GenBank: AB042810.1, 1 572 bp in total). The real-time fluorescence quantitative PCR was operated on LightCycler® 480 system (Roche Applied Science, Basel, Switzerland). β-Actin gene was selected as the reference gene (sense primer Actin-F: 5´-ATCTGCTGGAAGGTGCTGAG-3´ and anti-sense primer Actin-R: 5´-CCAAGCAGCATGAAGATCAA-3´). All reactions were performed following the procedure described by the manufacturer. The reaction volume was 20 μL, including 1 μL of each primer (10 μmol L–1), 1 μL of cDNA, 8 μL of RNase-free dH2O, and 10 μL of 2×LightCycler FastStart SYBR Green Master Mix (Roche Diagnostics, Shanghai, China). Samples were prepared in three replicates. A negative control using water as template was included in each reaction. PCR products were denatured at 95°C for 1 min, cooled to 40°C for 1 min, and then heated to 95°C at 0.02°C s–1, while continuously measuring florescence with 25 data acquisitions/°C.

2.4. Analyzing sorbitol and fructose content with HPLC-ELSD The wild and mutant loquat fruits from coloration to maturation stage were taken out from –80°C refrigerator and tested for the sorbitol and fructose content with HPLC-ELSD (Ma et al. 2014). Sample preparation: weigh 1.5 g samples, put the samples into centrifugal tube of 50 mL and add 30 mL water into the tube, homogenize with homogenizer and centrifuge at 8 000 r min–1 for 10 min. Chromatographic condition: simultaneous separation and determination of sorbitol and fructose in fruits with HPLC-ELSD. Column temperature: 30°C, mobile phase: acetonitrile:water =75:25,

flow rate: 1.0 mL min–1, tester: RID column: carbohydrate 5 μm, 4.6 mm×250 mm.

2.5. Statistical analysis The data are expressed in means±standard error (SE). Statistical analysis was carried out using the Excell 2007 by two-way analysis of variance using Tukey’s test to compare between wide yellowish and mutant whitish loquats at a probability level of 5%.

3. Results 3.1. Cloning NAD+-SDH gene from loquat cultivars RT-PCR reaction was performed with the total RNA of wild, mutant and other loquat cultivars/accessions at maturation stage as template, and then the amplification products undergone 1% agarose gel electrophoresis and obtained the bands that are consistent in size with expected segment. After that, the products were connected to T-carrier and transferred to Escherichia coli. As can be seen from the results of positive monocloning and sequencing, the CDS segment is 1 116 bp long in total, including one open reading frame of 1 023 bp, and codes 371 amino acids in total. The CDS of NAD+-SDH gene in mutant wild loquat is identical to that of the reported NAD+-SDH gene (GenBank: AB042810), of which the amino acid has 97.8% homology with that encoded by similar species, such as apple. As compared with wild type and other loquat cultivars/accessions, SNP emerges at loci 32 (from A to G), 370 (from C to T) and 461 (from A to G) of CDS (Fig. 1) in the gene from mutant loquat at maturation stage, and the encoded amino acids changed accordingly (from H to R, L to F, H to R, respectively, per locus) (GenBank: KU513551). To justify the results, nine representative white- and yellow-fleshed loquat cultivars/ accessions (see Section 2) were selected to further clone the CDS of the gene, which turned out that the test results coincide with NAD+-SDH gene of wild species and that reported in the database, except that no SNP emerges. It can be seen that, the three SNP at CDS may be unique for mutant loquat at maturation stage, this conclusion provides molecular basis for the generation of mutant from natural bud mutation to some extent.

3.2. Expression of NAD+-SDH gene in mature loquat The content of NAD+-SDH mRNA is high in mature loquat but low in fruitlet (Bantog et al. 1999). Therefore, the relative expression of NAD+-SDH gene from wild and mutant loquat at maturation stage was determined and analyzed. Two pairs of primers were designed to guarantee the accuracy

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Fig. 1 The alignment of coding sequence (CDS) of NAD+-dependent sorbitol dehydrogenase (NAD+-SDH ) gene from mutant white-fleshed and wild yellow-fleshed loquat. WNSC-1 and WNSC-2 were mutant white-fleshed loquat; ZNSC-2 and ZNSC-4 were wild yellow-fleshed loquat. Wide

Mutant NAD-SDH (h g–1 FW)

Relative expression

6 5 4 3 2 1 0

NS1

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

Yellowish Whitish

Coloration

Degreening Enlargement

Maturation

Development stage

NS2 Primers

Fig. 2 The relative expression of NAD -SDH gene in wild yellowish sample ZNSC-1 and mutant whitish WNSC-1 loquat at maturation stage. The values are calculated as means±SE (n=3 independent experiments). The same as below. +

Fig. 3 Change of NAD+-SDH activity in wild yellowish sample ZNSC-1 and mutant whitish WNSC-1 loquat from fruit coloration to maturation.

nificant higher than the latter; after the enlargement stage, the former drops sharply, the latter drops slowly, the former

and stability of real-time fluorescence quantitative PCR. The determination shows that the two pairs of primers have similar expression of NAD+-SDH gene in wild and mutant fruit, which in mutant species are 4.73- and 5.00-fold lower than that in wild loquat cultivars (Fig. 2). Thus, the content of NAD+-SDH mRNA in mutant white-fleshed loquat is much lower than that in wild yellow-fleshed loquat at maturation stage.

3.4. The content of sorbitol and fructose

3.3. The activity of NAD+-SDH

In previous studies, the fructose content differs greatly

NAD -SDH facilitates the conversion of sorbitol into fructose in loquat, and its activity first rises and then drops from coloration to maturation stage in both wild and mutant loquat fruit (Fig. 3). This turning point occurs at enlargement stage. At coloration and maturation stages, NAD+-SDH activity in mutant white-fleshed loquat is lower than that in wild yellow-fleshed reference; however, at degreening and enlargement stages, the former rises abruptly and is sig-

stage, but the sorbitol content is relative low in both species

+

becomes lower than the latter around maturation stage, and becomes much lower than the latter at maturation stage. So the change level (rise or drop) of NAD+-SDH activity is much higher in mutant white-fleshed loquat than in wild yellow-fleshed loquat.

between yellow- and white-fleshed loquats at maturation (Chen et al. 2010). In this study, the contents of fructose and sorbitol in mutant loquat, wild loquat and other nine loquat cultivars/accessions from coloration to maturation were tested using HPLC-ELSD (Figs. 4 and 5). The results show that, the sorbitol content drops and the fructose content rises in both the wild and mutant loquat. The sorbitol content is remarkably higher in mutant loquat than in wild type

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4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

90

Yellowish Whitish

Yellowish Whitish

80 Fructose content (mg g–1 FW)

Sorbitol content (mg g–1 FW)

4.5

70 60 50 40 30 20 10

Coloration Degreening Enlargement Maturation Development stage

Fig. 4 Change of sorbitol content in wild yellowish sample ZNSC-1 and mutant whitish WNSC-1 loquat from fruit coloration to maturation.

in the first period (coloration), which drops fast and much more than wild type when transiting into the second period (degreening). At the enlargement and maturation stages, the sorbitol contents in both two types drop similarly and are almost the same at maturation. Overall, the fructose content in mutant white-fleshed loquat is remarkably higher than in wild loquat during the whole development process, and the difference in fructose content between the two types increases as the fruit ripens. It can be seen that mutant white-fleshed loquat enjoys higher conversion efficiency from sorbitol to other carbohydrates than wild yellow-fleshed fruit, and the fructose content of the former is remarkably higher than the latter.

4. Discussion Loquats are classified into yellow- and white-fleshed loquats by peel color, where all orange-red-, orange- and yellow-fleshed loquats are clustered into yellow-fleshed loquats; and all light color-, milky- or off-white-fleshed loquats are clustered into white-fleshed loquats. The two types of loquat not only differ in peel color, but also in carbohydrate accumulation, cellulose content, taste and flavor. A mutant of Dongting loquat was discovered in this study, which bears yellow-fleshed fruit in natural state but is subject to bud sport on one branch and bears white-fleshed fruit. In addition to determination with molecular markers in the preliminary stage, the CDS of NAD+-SDH gene from mutant loquat was analyzed in this study. The results show that, mutant white-fleshed material has three SNPs on the genetic sequence and variance in corresponding encoded amino acid as compared with wild loquat. However, this variance has not been detected in the nine yellow- or white-fleshed

0

Coloration

Degreening Enlargement Maturation Development stage

Fig. 5 Change of fructose content in wild yellowish sample ZNSC-1 and mutant whitish WNSC-1 loquat from fruit coloration to maturation.

loquat cultivars/accessions (including Dawuxing and Guifei). The influence of the three SNPs on genetic functions needs to be explored further. Through HPLC-ELSD analysis in the study, it is found that sorbitol content decreases and the fructose content increases from coloration to maturation stage in both the wild and mutant loquat fruit, the fructose content in mutant white-fleshed fruit remains higher than that in wild yellow-fleshed counterpart throughout all stages. The results agree with the findings by Chen et al. (2010). The difference in carbohydrate content among loquat cultivars/accessions has a lot to do with the activity level of metabolic enzymes. Metabolic enzymes facilitate the conversion of sorbitol into fructose and glucose with its catalytic action, promoting the enlargement and quality formation of fruit. The NAD+-SDH in the loquat catalyzes the conversion of sorbitol into fructose. NAD+-SDH is a main enzyme for sorbitol degradation in most rosaceous plants. It is found in the study that NAD+-SDH activity changes similarly in both wild and mutant loquat fruit, which rises rapidly from coloration to enlargement, drops from enlargement to maturation. In addition, the change level of NAD+-SDH activity in mutant white-fleshed loquat is much greater than that in wild control during the development process, this may be the reason for the higher fructose content in mutant white-fleshed loquat than in wild control. At maturation stage, NAD+-SDH activity in mutant whitefleshed loquat is lower than wild loquat, which coincides with the performance of relative expression abundance of mRNA in NAD+-SDH gene from the test samples at maturation stage and indicates that NAD+-SDH activity is closely related to mRNA expression abundance. The results agree with the findings by Bantog et al. (1999). According to the

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research by Chen et al. (2010), Ninghaibai white-fleshed loquat has higher fructose content than that in Dahongpao yellow-fleshed loquat, but has low SDH activity, complying with the findings in this study. There are several reasons that lead to high fructose content and low NAD+-SDH activity in loquat fruit. One reason may be that NAD+-SDH is not the only enzyme that decides the level of fructose content, since Sizuki et al. (2001) found in apple and Asian pear that high level of fructose content may be relevant to that fact that low fructokinase activity leads to less fructose metabolism. Since loquat, apple and pear are clustered into maloideae of rosaceae, high fructose content in mutant white-fleshed loquat may be relevant to low fructokinase activity at maturation stage, which needs to be studied further. Compared with wild loquat, mutant loquat has 3 SNPs in the CDS of NAD+-SDH gene, and the NAD+-SDH activity rises and then drops from coloration to maturation stage, while the relative expression abundance of mRNA at maturation stage is 5-fold lower than that of the wild type. Thus it can be concluded that the 3 SNPs may have led to the over-efficient catalytic action of NAD+-SDH in converting sorbitol into fructose in mutant white-fleshed loquat at maturation stage, resulting in increase of fructose content and relative low expression abundance of mRNA after transcription; in addition, NAD+-SDH gene might be a hub for the variation in flesh color and carbohydrate in mutant white-fleshed loquat. The study not only enriches the biological study on fruit quality but also provides new basis and direction for research on genetic improvement of fruit quality.

5. Conclusion A white-fleshed loquat mutant of Dongting has caused our concern for a long time, and was used to explore the variation of fruit quality in yellow- and white-fleshed samples. In the current study, we discovered three SNPs (where the encoded amino acid was changed accordingly) in NAD+SDH CDS of mutant fruit, detected the gene expression of maturation loquat, NAD+-SDH activity, and sorbitol and fructose content of wide and mutant loquat at different development stages from fruit coloration to maturation. Results from the above were assumed that, the variation of NAD+-SDH in white-fleshed mutant might accelerate the conversion of sorbitol into fructose, resulting in the increase of fructose content and reduced expression abundance of mRNA after transcription.

Acknowledgements This study was supported by the Key Laboratory Program of the Ministry of Agriculture of China (2013JCYJ-004) and its supplementary items (2015JSCX-036, 2015LWJJ-010);

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the Program of Modern Agriculture Technology Innovation and Demonstration of Provincial Finance Department, China (2014CXSF-015).

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