(ALDH) gene superfamily in apple

Plant Physiology and Biochemistry 71 (2013) 268e282

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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus domestica Borkh.) Xiaoqin Li a, b, Rongrong Guo a, b, Jun Li a, b, Stacy D. Singer c, Yucheng Zhang d, Xiangjing Yin a, b, Yi Zheng e, Chonghui Fan a, b, *, Xiping Wang a, b, * a

State Key Laboratory of Crop Stress Biology in Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China c Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada d Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA e Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 June 2013 Accepted 26 July 2013 Available online 6 August 2013

Aldehyde dehydrogenases (ALDHs) represent a protein superfamily encoding NAD(P)þ-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. In plants, they are involved in many biological processes and play a role in the response to environmental stress. In this study, a total of 39 ALDH genes from ten families were identified in the apple (Malus domestica Borkh.) genome. Synteny analysis of the apple ALDH (MdALDH) genes indicated that segmental and tandem duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of these gene families in apple. Moreover, synteny analysis between apple and Arabidopsis demonstrated that several MdALDH genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes appeared before the divergence of lineages that led to apple and Arabidopsis. In addition, phylogenetic analysis, as well as comparisons of exoneintron and protein structures, provided further insight into both their evolutionary relationships and their putative functions. Tissue-specific expression analysis of the MdALDH genes demonstrated diverse spatiotemporal expression patterns, while their expression profiles under abiotic stress and various hormone treatments indicated that many MdALDH genes were responsive to high salinity and drought, as well as different plant hormones. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles, of the apple MdALDH genes will not only be useful for the further analysis of ALDH genes and their roles in stress response, but may also aid in the future improvement of apple stress tolerance. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Apple Aldehyde dehydrogenase Synteny analysis Phylogenetic analysis Gene expression

1. Introduction Endogenous aldehyde molecules are common intermediates of a number of catabolic and biosynthetic pathways, including amino acid, protein and carbohydrate metabolism [1], and are also Abbreviations: ABA, abscisic acid; AGNC, ALDH Gene Nomenclature Committee; ALDH, aldehyde dehydrogenase; Eth, ethylene; MeJA, methyl jasmonate; R1/2/3 d, 1/2/3-days after re-watering; R48 h, 48-h after re-watering; RT-PCR, reverse transcription PCR; SA, salicylic acid. * Corresponding authors. College of Horticulture, Northwest A&F University, Yangling, 712100 Shaanxi, China. Tel.: þ86 29 87082429; fax: þ86 29 87082613. E-mail addresses: [email protected] (C. Fan), [email protected] (X. Wang). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.07.017

produced in response to various types of environmental stress, such as exposure to salinity, drought, cold, heat and heavy metals [2,3]. Although aldehydes are indispensable to developmental growth processes, they are toxic in excessive physiological concentrations because of their inherent chemical reactivity, resulting in cytotoxicity and mutagenicity [1,4,5], and their levels must therefore be regulated within cells. The aldehyde dehydrogenase (ALDH) superfamily is composed of a wide range of divergent enzymes that catalyze the irreversible NAD(P)þ-dependent oxidation of various endogenous and exogenous aldehydes to their corresponding carboxylic acids [6,7]. In addition, under conditions that induce oxidative stress, ALDH enzymes act as ‘aldehyde scavengers’ by metabolizing reactive aldehydes derived from the

X. Li et al. / Plant Physiology and Biochemistry 71 (2013) 268e282

oxidative degradation of lipid membranes, also known as lipid peroxidation [8]. With the genomes of an ever increasing number of organisms being fully sequenced, the identification of ALDH genes from bacteria to mammals has soared in recent times. To date, more than 1000 distinct ALDH genes have been identified and categorized into 24 separate families based on protein sequence identity [9,10]. Plant ALDH proteins can be found in 14 discrete families: ALDH2, ALDH3, ALDH5, ALDH6, ALDH7, ALDH10, ALDH11, ALDH12, ALDH18, ALDH19, ALDH21, ALDH22, ALDH23 and ALDH24. Among these families, ALDH10, 12, 19, 21, 22, 23 and 24 are plant-specific, whereas the remaining families also contain mammalian orthologues. Interestingly, while the plant-specific ALDH10, 12, 21, 22 and 23 families all comprise members from multiple species, the ALDH19 family consists of only a single gene from tomato, which is thought to encode a g-glutamyl phosphate reductase that may play a role in the biosynthesis of proline from glutamate [11] and the ALDH24 family is believed to be specific to Chlamydomonas reinhardtii [12]. Of the plant ALDH genes that have been characterized to date, the majority have been implicated in diverse pathways and appear to play crucial roles in growth and development. For example, the rice ALDH7 gene has been found to be essential for seed maturation and viability [13], while the maize ALDH2 gene rf2 encodes a mitochondrial class-2 ALDH and is required for male fertility [14e 16]. In addition, many of the plant ALDH genes characterized thus far are responsive to various types of stress, including dehydration, water logging, exposure to heavy metals, high salinity, oxidative stress, and ultraviolet radiation (UVR), suggesting possible roles for these genes in improving stress tolerance [17e19]. In line with this, several studies have demonstrated that over-expression of a number of plant ALDH genes enhances plant tolerance to diverse types of abiotic and biotic stress [3,19e23]. While still limited, information concerning these genes in plants is beginning to accumulate; however, the majority of research has been carried out in model species such as Arabidopsis, with very little attention paid to woody species as of yet. Apple (Malus domestica Borkh.) is one of the most important perennial fruit crops worldwide and has been widely studied at the physiological level. However, at this time, the biological function of ALDH genes in this species has not been reported and remains unclear. Fortunately, the recent publication of the genome sequence from the diploid apple variety Golden Delicious [24] has made it possible to carry out a genome-wide identification and analysis of ALDH gene families in this species. In the present study, we systematically identified 39 ALDH genes in the apple genome (hereafter referred to as MdALDH genes) that contained a complete ALDH domain and belonged to ten different families. To gain insight into their evolutionary relationships and putative functions, we carried out structural, phylogenetic and synteny analyses, and analyzed their expression profiles in numerous tissues, as well as in response to various types of stress and phytohormone treatment. Results from this study provide a foundation for further evolutionary and functional characterization of ALDH gene families in apple and other plant species, and also yield potential target genes for the genetic improvement of apple stress tolerance. 2. Results 2.1. Genome-wide identification of ALDH gene families in the apple genome Database searches resulted in the identification of 39 unique ALDH gene sequences from the apple genome (MdALDH genes) encoding members of 10 of the 24 ALDH gene families (ALDH2,

269

ALDH3, ALDH5, ALDH6, ALDH7, ALDH10, ALDH11, ALDH12, ALDH18 and ALDH22) (Table 1). To date, plant ALDH genes have been found to be represented in 14 distinct families; however, four of these families (ALDH19, ALDH21, ALDH23 and ALDH24) do not include apple genes. All ten MdALDH gene families included more than one MdALDH member (ALDH2: thirteen genes; ALDH3: seven genes; ALDH11: three genes; ALDH18: four genes and ALDH5, 6, 7, 10, 12, 22: two genes each). 2.2. Expansion patterns of MdALDH genes in apple Segmental and tandem duplications comprise two main avenues for the expansion of gene families [37]. In apple, the MdALDH genes were mapped to chromosomes 1, 2, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15 and 16 (Table 1 and Fig. 1), and based on this map we were able to identify 12 tandemly duplicated MdALDH genes from three families (MdALDH18B1/MdALDH18B2, MdALDH2C4/MdALDH2C6/MdALDH2C7/ MdALDH2C8/MdALDH2C11/MdALDH2C12, MdALDH3H1/MdALDH3H7, and MdALDH2B12/MdALDH2B13), which were mapped to chromosomes 1, 2, 8 and 14, respectively (Fig. 1 and Supplementary Table 1). Moreover, we examined segmentally duplicated blocks within the apple genome and found that there were eight pairs of MdALDH genes that had been duplicated in this manner (Fig. 1 and Supplementary Table 1; MdALDH11A3/MdALDH11A5, MdALDH2C7/ MdALDH2C10, MdALDH18B3/MdALDH18B4, MdALDH12A1/MdALDH12 B1, MdALDH2B4/MdALDH2B12, MdALDH22A2/MdALDH22A1, MdALDH 2C9/MdALDH2C7, and MdALDH3F1/MdALDH3F2), which included members of three additional ALDH families along with two of the tandemly duplicated genes (MdALDH2C7 and MdALDH2B12). All segmentally duplicated genomic regions containing paired MdALDH genes were located on distinct chromosomes (Fig. 1 and Supplementary Table 1; chr2/chr15, chr2/chr15, chr4/chr12, chr6/ chr11, chr6/chr14, chr13/chr16, chr15/chr2 and chr15/chr2). Taken together, these results suggest that six of the ten apple ALDH gene families were associated with either segmental or tandem duplication events. It should be noted that six additional genes (MDP0000519359, MDP0000432642, MDP0000374885, MDP0000257083, MDP0000 516194 and MDP0000534784) were also located on the chromosomal distribution map of MdALDH genes even though they did not belong to the ALDH gene superfamily (Fig. 1 and Supplementary Table 1). When we examined these genes using the Pfam (http:// pfam.janelia.org/search) and SMART (http://smart.embl-heidelb erg.de/) software, we found that three of them (MDP0000519359, MDP0000432642 and MDP0000374885) had incomplete ALDH domains or active sites. In addition, MDP0000257083, MDP000051 6194 and MDP0000534784 bore no ALDH domains but did contain a PDB, SCOP or HLH domain, which were also detected in MdAL DH7B5, MdALDH12B1 and MdALDH11A4, respectively. 2.3. Evolutionary relationships of ALDH gene families between apple and Arabidopsis Genomic comparison among different organisms is a relatively easy method with which to deduce the origin, evolutionary history, and function of new genes [38]. Since Arabidopsis is a popular model plant species and the functions of a selection of Arabidopsis ALDH genes have been well-characterized, we created a comparative syntenic map between the apple and Arabidopsis genomes in an attempt to further explore the evolution and functions of the MdALDH genes (Fig. 1). Large-scale syntenies containing orthologues from six ALDH families (ALDH2, ALDH3, ALDH5, ALDH10, ALDH18 and ALDH22) in both the apple and Arabidopsis genomes, including 12 MdALDH genes from apple and 9 AthALDH genes from Arabidopsis, were

270


Table 1 The aldehyde dehydrogenase (ALDH) gene superfamily in apple. Family

Gene name

Gene locus ID

Chromo-some

Start

End

Gene length (bp)

ORF (aa)

Family 2

MdALDH2B4 MdALDH2B12 MdALDH2B13 MdALDH2B14 MdALDH2B15 MdALDH2C4 MdALDH2C6 MdALDH2C7 MdALDH2C8 MdALDH2C9 MdALDH2C10 MdALDH2C11 MdALDH2C12 MdALDH3F1 MdALDH3F2 MdALDH3H1 MdALDH3H7 MdALDH3H8 MdALDH3I1 MdALDH3I2 MdALDH5F1 MdALDH5F2 MdALDH6B7 MdALDH6B8 MdALDH7B5 MdALDH7B8 MdALDH10A8 MdALDH10A9 MdALDH11A3 MdALDH11A4 MdALDH11A5 MdALDH12A1 MdALDH12B1 MdALDH18B1 MdALDH18B2 MdALDH18B3 MdALDH18B4 MdALDH22A1 MdALDH22A2

MDP0000140980 MDP0000221713 MDP0000159395 MDP0000859857 MDP0000213640 MDP0000610753 MDP0000219155 MDP0000376347 MDP0000438458 MDP0000212975 MDP0000612909 MDP0000662387 MDP0000123081 MDP0000260947 MDP0000308856 MDP0000297821 MDP0000258979 MDP0000307809 MDP0000531301 MDP0000406860 MDP0000147030 MDP0000229588 MDP0000168881 MDP0000239328 MDP0000790166 MDP0000211987 MDP0000324559 MDP0000148461 MDP0000152497 MDP0000606021 MDP0000624696 MDP0000054636 MDP0000465381 MDP0000323330 MDP0000845591 MDP0000909091 MDP0000284559 MDP0000283106 MDP0000249620

chr6 chr14 chr14 chr13 chr13 chr2 chr2 chr2 chr2 chr15 chr15 chr2 chr2 chr15 chr2 chr8 chr8 chr15 chr15 chr12 chr5 chr10 chr15 chr9 chr15 chr2 chr14 chr6 chr2 chr2 chr15 chr6 chr11 chr1 chr1 chr4 chr12 chr16 chr13

19919559 24724470 24725289 6018499 5998224 4419098 4410559 4389384 4417436 11957758 11959518 4421030 4402269 18305220 12219373 20347136 20330884 31364963 6918519 13176927 10104662 22998778 26836980 26642786 15976485 7137934 25057024 20226435 1617550 1609957 8301496 20739610 10687883 18309954 18359700 13325907 20199372 8710971 13837564

19922534 24727946 24728760 6022035 6003770 4421001 4415496 4391902 4420352 11961523 11963012 4423567 4404808 18307951 12221977 20353909 20336768 31370620 6922913 13183338 10111296 23005140 26845414 26653658 15980007 7141922 25061517 20235456 1620512 1622689 8313495 20744309 10693536 18315708 18365377 13331126 20204895 8715328 13841930

2976 3477 3472 3537 5547 1904 4938 2519 2917 3766 3495 2538 2540 2732 2605 6774 5885 5658 4395 6412 6635 6363 8435 10873 3523 3989 4494 9022 2963 12733 12000 4700 5654 5755 5678 5220 5524 4358 4367

539 550 551 341 538 423 501 510 501 517 444 414 414 461 483 474 469 604 552 524 538 613 1092 679 524 526 504 993 496 926 803 558 748 718 717 735 761 596 610

Family 3

Family 5 Family 6 Family 7 Family 10 Family 11

Family 12 Family 18

Family 22

identified (Fig. 1 and Supplementary Table 2). Four pairs of orthologous genes (MdALDH2B4-AthALDH2B4, MdALDH2B15AthALDH2B7, MdALDH2C9-AthALDH2C4 and MdALDH18B1-AthALDH18A1) appeared to be single apple-to-Arabidopsis ALDH gene correspondences. In all probability, these genes/families were derived from a common ancestor of apple and Arabidopsis. Interestingly, we also noted instances where a single apple gene corresponded to multiple Arabidopsis genes or where apple segmental duplications corresponded to a single Arabidopsis gene (MdALDH10A8-AthALDH10A8/AthALDH10A9, MdALDH3F1/MdALDH3F2-AthALDH3F1, MdALDH5F1/MdALDH5F2-AthALDH5F1, MdALDH10A8/MdALDH10A9-AthALDH10A8 and MdALDH22A1/MdALDH22 A2-AthALDH22A1). Interestingly, the remaining four ALDH families containing apple genes (ALDH6, ALDH7, ALDH11 and ALDH12) could not be mapped to any synteny blocks. 2.4. Phylogenetic analysis of ALDH gene families from various plant species The 39 MdALDH genes isolated from apple make up the largest ALDH superfamily identified in any plant organism to date, compared to 16 ALDH genes in Arabidopsis thaliana [26,28], 25 genes in Vitis vinifera [26,27], 23 genes in Zea mays [26,30], 20 genes in Oryza sativa [26,29], 26 genes in Populus trichocarpa, 24 genes in Selaginella moellindorffii, 19 genes in Sorghum bicolor [26], 21 genes in Physcomitrella patens, 9 genes in C. reinhardtii, 6 genes in Ostreococcus tauri [12,26] and 7 genes in Volvox carteri [26] (Table 2).

To gain further insight into the evolutionary relationship between ALDH gene families, we constructed a phylogenetic tree using the neighbor-joining algorithm (Fig. 2). The deduced protein sequences of ALDH genes from seven plant species were utilized in the analysis, including those from three eudicot species (apple, grape and Arabidopsis), two monocot species (maize and rice), and two lower plants (C. reinhardtii and P. patens). Although evolutionary relationship could not be clearly deciphered for all families, the analysis did yield some interesting results. As expected, ALDH proteins previously found to belong to the same family tended to cluster together into 13 different groupings (the ALDH19 family is not included here as our analyses did not incorporate any sequences from tomato), with the ALDH18 family being the most distantly related group. The MdALDH sequences from apple all clustered into 10 of these 13 families. Furthermore, our results indicated that the majority of apple MdALDH families were more closely related to those from grape and Arabidopsis than the other species analyzed. 2.5. Sequence and structure analysis of MdALDH genes A phylogenetic tree was also generated using the deduced protein sequences of the 39 apple MdALDH genes (Fig. 3A). The resulting topology corresponded with the phylogenetic groups obtained in the multispecies tree (Fig. 2) and, likewise, members from the same families within apple clustered together. To provide further confirmation of the evolutionary relationship among the


271

Fig. 1. Synteny analyses of MdALDH genes and ALDH genes between apple and Arabidopsis. Apple and Arabidopsis ALDH genes are indicated by vertical orange lines. Colored bars denote syntenic regions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

similar exoneintron structures, but almost all families harbored some exceptions that the gain or loss of one or more than one exons occurs in particular members in each case. This was especially prevalent in the ALDH10 family, where MdALDH10A9 was made up of 25 exons while MdALDH10A8 contained only 15. Similarly, while MdALDH11A4 and MdALDH11A5 bore 15 and 14 exons, respectively, MdALDH11A3 contained only 9. In addition, the length and structure of these three genes were also very distinct; a characteristic that was also apparent in members of the ALDH6 family in apple. Unlike all the other MdALDH gene families, all members of the ALDH18 family in apple bore the same number of exons.

MdALDH genes, we visualized the distribution of their conserved domains (Fig. 3B). All 39 MdALDH proteins identified were predicted to contain at least one ALDH domain (PF00171). In addition to their basic structures, members from the same family generally possessed further distinctive structural similarities. For example, MdALDH22A1 and MdALDH22A2, which both belonged to the ALDH22 family, contained a signal peptide near their N-terminal ends, while MdALDH18B1, MdALDH18B2, MdALDH18B3 and MdALDH18B4, which belonged to the ALDH18 family, bore an AAkinase domain (PF00696). Moreover, the MdALDH6B8 protein contained one transmembrane region and the MdALDH11A4 protein bore a HLH domain (PF00010). Since the divergence of exoneintron structure often plays an important role in the evolution of gene families, we also analyzed the exoneintron structures of all 39 MdALDH genes (Fig. 3C). Our results indicated that genes in the same family generally exhibited

2.6. Expression profiles of MdALDH genes in various tissues To provide further clues concerning the putative roles of the MdALDH genes in apple development, the expression patterns of all

Table 2 Number of aldehyde dehydrogenase (ALDH) family members identified in various species. e Represents the absence of the ALDH gene family in the corresponding species. Species

M. domestica A. thaliana V. vinifera Z. mays O. sativa P. trichocarpa S. moellindorffii S. bicolor P. patens C. reinhardtii O. tauri V. carteri

ALDH family

Total

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

e e e e e e e e e e e e

13 3 5 6 5 4 6 5 2 1 e 1

7 3 4 5 5 6 2 4 5 e 1 e


2 1 3 2 1 1 1 1 2 1 1 e

2 1 3 1 1 4 1 1 1 1 e 1

2 1 2 1 1 2 1 1 1 e e e



2 2 2 3 2 2 1 2 1 1 1 1

3 1 2 1 1 3 6 1 5 1 1 1

2 1 1 1 1 1 1 1 1 1 1 1






4 2 2 2 2 2 1 2 1 1 e 1



e e e e e e 1 e 1 e e e

2 1 1 1 1 1 1 1 e 1 1 1

e e e e e e 2 e 1 e e e

e e e e e e e e e 1 e e

39 16 25 23 20 26 24 19 21 9 6 7

272


Fig. 2. Phylogenetic analysis of plant ALDH proteins. The phylogenetic tree was constructed with ALDH protein sequences from apple (Md), V. vinifera (Vv), A. thaliana (Ath), Z. mays (Zm), O. sativa (Os), P. patens (Pp) and C. reinhardtii (Cr).

39 MdALDH genes were investigated by semi-quantitative RT-PCR in six different tissues (roots, stems, leaves, flowers, fruits and seeds). All 39 genes were expressed in at least one of the six tissues examined (Fig. 4). Interestingly, the majority of genes analyzed (33 genes) exhibited constitutive expression in all of the tissues tested, but with varying levels of expression in each tissue, suggesting that they may have different functions during apple’s development and ripening. For example, MdALD-H2B13 displayed high levels of expression in roots, stems, leaves and seeds, with much lower levels of expression in flowers and fruits. MdALDH11A4 showed high levels of expression in stems, leaves, flowers and fruits, but much lower expression in roots and seeds. Conversely, the remaining six genes (MdALDH2B4, MdALDH2B12, MdALDH3I1, MdALDH3I2, MdALDH7B8 and MdALD-H18B4) exhibited different tissue-specific expression. While MdAL-DH2B4 and MdALDH2B12 were not expressed in flowers or fruits, MdALDH18B4 expression was not detected in fruits or seeds. MdALDH3I1, MdALDH3I2 and MdALDH7B8 expression was detected in all tissues except seeds. 2.7. Expression profiles of MdALDH genes in response to abiotic stress conditions ALDH genes from mosses to angiosperms appear to have a common ‘stress response’ pattern [28], which is likely due to the fact that endogenous aldehydes are generated in excess in response to such environmental stresses as salinity, dehydration, cold and heat shock. To determine whether the MdALDH genes identified

here conformed to this pattern, we investigated their response to osmotic stress in leaf tissue using semi-quantitative RT-PCR and quantitative real-time RT-PCR. As shown in Figs. 5A and 6A, 26 of the 39 MdALDH genes were observed to respond to at least one of the osmotic stress treatments (drought and high salinity) when analyzed by semi-quantitative RTPCR. Among the genes affected, only five (MdALDH2C4, MdALDH3F1, MdALDH11A3, MdALDH11A4 and MdALDH22A2) were up-regulated by both treatments, whilst sixteen (MdALDH2B4, MdALDH2B12, MdALDH2B13, MdALDH2B15, MdALDH2C9, MdALDHC10, MdALDH2C11, MdALDH3F2, MdALDH3H1, MdALDH3H8, MdALDH3I1, MdALDH3I2, MdALDH7B5, MdALDH18B2, MdALDH18B3 and MdALDH22A1) were down-regulated. Of these latter 16 genes, MdALDH2B15, MdALDH2C9, MdALDH2C10, MdALDH2C11, MdALDH3I1 and MdALDH3I2 decreased gradually with time under drought stress with expression levels being restored after re-watering, while MdALDH2B4, MdALDH2B12, MdALDH2B13, MdALDH3F2, MdALDH3H1, MdALDH3H8, MdALDH7B5, MdALDH18B2, MdALDH18B3 and MdALDH22A1 transcript levels remained low even after re-hydration. Furthermore, MdALDH5F1, MdALDH6B7, MdALDH11A5 and MdALDH18B1 were affected by salt stress, but not drought, while MdALDH5F2 exhibited decreased expression under drought, but was not affected by salt treatment. The remaining genes were not regulated by either type of abiotic stress. Validation of these results was provided by further experiments in which we determined the expression levels of three randomly selected MdALDH genes under both treatments, respectively, using quantitative real-time RT-PCR (Figs. 5B and 6B).


273

Fig. 3. The ALDH gene superfamily in apple. A. Phylogenetic analysis of apple MdALDH proteins. Different families are marked by distinct colors. B. The distribution of PFAM domains in apple MdALDH proteins. The relative positions of each domain within each protein are shown in color. C. Exoneintron structures of apple MdALDH genes.

Fig. 4. Tissue-specific expression profile analysis of MdALDH genes. Tissues or organs are indicated by abbreviations: Ro e Roots; St e Stems; Le e Leaves; Fl e Flowers; Fr e Fruits; Se e Seeds.

274


Fig. 5. Expression patterns of MdALDH genes under high salinity conditions. A. Expression patterns of 39 MdALDH genes under high salt conditions as determined by semiquantitative RT-PCR analyses. For each gene, the upper seven bands represent amplified products from the leaves of ‘Fuji’ following treatment with 2 dm3 250 mM NaCl after 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h; lower bands represent amplified products from untreated leaves. B. Expression patterns of three randomly selected MdALDH genes detected by quantitative real-time RT-PCR. The apple EF-1a gene was used as an internal control to normalize the data. Each block denotes the mean relative level of expression of three replicates, while bars indicate standard errors. Asterisks indicate expression levels that are significantly increased/decreased compared to untreated controls (*P < 0.05, Student’s t test).

2.8. Expression profiles of MdALDH genes in response to various hormone treatments Since phytohormones are known to play important roles in the ability of plants to respond to stress, we endeavoured to analyze the effect of the plant hormones, abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), and ethylene (Eth) on the expression of MdALDH genes in apple. Analysis of transcript levels from the leaves of apple trees treated with exogenous ABA, which is a key messenger in the response of plants to various stress conditions [39], indicated that 12 of the 39 MdALDH genes exhibited increased expression at different time points following treatment (Fig. 7A). While MdALDH3F1, MdALDH6B8, MdALDH7B5, MdALDH10A9, MdALDH11A4 and MdALDH12A1 exhibited increased expression at every time point, MdALDH3I1 and MdALDH11A5 were up-regulated only from 0.5 h to 6 h post-treatment. In contrast, MdALDH3F2, MdALDH10A8, MdALDH11A3 and MdALDH18B4 increased in expression at least at 0.5 h and 1 h post treatment. Of the remaining genes,

MdALDH5F1, MdALDH5F2, MdALDH18B2 and MdALDH18B3 exhibited decreased expression following ABA treatment, while the remainder showed no obvious changes in transcript levels in response to ABA. The plant hormones SA, MeJA and Eth are known to play particularly vital roles in the response to biotic stress, such as wounding [39,40]. Following SA treatment, MdALDH2C6, MdALDH3F1, MdALDH7B8, MdALDH11A3, MdALDH11A4, MdALDH12A1 and MdALDH22A1 were up-regulated, while MdALDH2B12, MdALDH2B14, MdAL DH3F2, MdALDH3H1, MdALDH3H8, MdALDH3I1, MdALDH5F2, MdALDH10A8, MdALDH11A5, MdALDH12B1, MdALDH18B3 and MdALDH18B4 were down-regulated. No obvious changes were noted in the expression of the remaining 20 MdALDH genes analyzed (Fig. 8A). Following treatment with MeJA, 8 MdALDH genes (MdALDH2B13, MdALDH2C6, MdALDH3F1, MdALDH7B8, MdALDH-11A3, MdALDH11A4, MdALDH12A1 and MdALDH22A1) exhibited increased expression (Fig. 9A), while 16 of the 39 genes were down-regulated. Of particular note was the fact that all seven genes that were up-


275

Fig. 6. Expression patterns of MdALDH genes under drought conditions. A. Expression patterns of 39 MdALDH genes under drought conditions as determined by semi-quantitative RT-PCR analyses. For each gene, the upper ten bands represent amplified products from the leaves of ‘Fuji’ under drought stress after 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, as well as after rehydration 1 d (R1), 2 d (R2), 3 d (R3); lower bands represent amplified products from untreated leaves. B. Expression patterns of three randomly selected MdALDH genes detected by quantitative real-time RT-PCR. The apple EF-1a gene was used as an internal control to normalize the data. Each block denotes the mean relative level of expression of three replicates, while bars indicate standard errors. Asterisks indicate expression levels that are significantly increased/decreased compared to untreated controls (*P < 0.05, Student’s t test).

regulated following SA treatment were also up-regulated following treatment with MeJA. As was the case for SA treatment, analysis of the transcript levels from leaves sprayed with Eth indicated that the majority of genes did not respond to treatment with this hormone (Fig. 10A). Of those genes that were responsive to Eth, MdALDH2C4, MdALDH2C7, MdALDH3F1, MdALDH7B5, MdALDH11A3, MdALDH11A4, MdALDH12A1, MdALDH22A1 and MdALDH22A2 exhibited increased expression following treatment, whereas MdALDH2C9, MdALDH2C10, MdALDH3I1 and MdALDH5F1 were down-regulated. Support for these results was obtained by additional experiments in which we determined the expression levels of three randomly selected MdALDH genes following each of the hormone treatments, respectively, using quantitative real-time RT-PCR (Figs. 7Be10B). 3. Discussion Aldehyde dehydrogenases, which play essential roles in metabolism and are critical for both development and responses to environmental changes, are found in both prokaryotic and eukaryotic organisms, and are well-represented within all plant species analyzed thus far. However, virtually nothing was known

concerning the ALDH gene family in apple, which is one of the most important fruit crops worldwide. Since various forms of stress have serious impacts on the production and quality of apple crops, we aimed to identify and characterize the structure, evolution, expression and stress-related responses of MdALDH genes in this species. 3.1. Evolution of MdALDH genes in apple In this study, a total of 39 genes were identified in the apple genome. Phylogenetic analysis based on 153 protein sequences from 13 ALDH gene families in apple and six additional plants (the tomato-specific ALDH19 family was not included in our analysis) confirmed previous findings in that ALDH genes from the same family tended to group together. Furthermore, most MdALDH genes were found to be more closely related to grape or Arabidopsis ALDH genes than rice or maize ALDH genes, which is consistent with the fact that apple, grape and Arabidopsis are all eudicots and diverged more recently than from the lineage leading to monocots (Fig. 2). While plant ALDH genes can be grouped into 14 families, the apple MdALDH genes were grouped within only ten of these (ALDH2, ALDH3, ALDH5, ALDH6, ALDH7, ALDH10, ALDH11, ALDH12,

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Fig. 7. Expression patterns of MdALDH genes following ABA treatment. A. Expression patterns of 39 MdALDH genes following ABA treatment as determined by semi-quantitative RTPCR analyses. For each gene, the upper seven bands represent amplified products from the leaves of ‘Fuji’ under treatment with 300 mM ABA after 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h; the lower bands represent amplified products from untreated leaves. B. Expression patterns of three randomly selected MdALDH genes detected by quantitative real-time RT-PCR. The apple EF-1a gene was used as an internal control to normalize the data. Each block denotes the mean relative level of expression of three replicates, while bars indicate standard errors. Asterisks indicate expression levels that are significantly increased/decreased compared to untreated controls (*P < 0.05, Student’s t test).

ALDH18 and ALDH22). Conversely, MdALDH genes were lacking in the ALDH19, 21, 23 and 24 families. To date, the ALDH21 and 23 families have been found to contain only genes from primitive terrestrial plants [41], and ALDH24 appears to be unique to C. reinhardtii [12], which suggests that these three families might have played important roles in the evolution of lower plants and were subsequently lost in higher plants. Furthermore, the ALDH19 family consists of a single gene from tomato at present, suggesting that it may have evolved specifically in this lineage [11]. Interestingly, our phylogenetic analyses also indicated that the ALDH18 family was the most phylogenetically distant group in relation to the remaining families (Fig. 2). Consistent with this, the structures of MdALDH genes from this family were distinct from those in other families, bearing additional AA-kinase domains (Fig. 3B). Additionally, previous research in rice also indicated that the two OsALDH18 proteins exhibited the greatest degree of

sequence divergence from other ALDH families and did not contain conserved ALDH active sites [42], which provides further evidence that the ALDH18 family is more distantly related to the other families. 3.2. Expansion of the apple ALDH gene superfamily As plants moved onto land there was a concomitant loss of many genes associated with aquatic life and expansion of gene families required for adaptation to terrestrial conditions. One such group of genes may have been the ALDH superfamily due to their purported roles in plant responses to various environmental stresses. Support for this notion comes from the fact that there appears to be far more ALDH genes in land plants than in their algal ancestors (Table 2). Intriguingly, there also appears to have been a substantial expansion of this superfamily in the apple genome compared to other


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Fig. 8. Expression patterns of MdALDH genes following SA treatment. A. Expression patterns of 39 MdALDH genes following SA treatment as determined by semi-quantitative RTPCR analyses. For each gene, the upper seven bands represent amplified products from the leaves of ‘Fuji’ following treatment with 100 mM SA after 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h; the lower bands represent amplified products from untreated leaves. B. Expression patterns of three randomly selected MdALDH genes detected by quantitative real-time RTPCR. The apple EF-1a gene was used as an internal control to normalize the data. Each block denotes the mean relative level of expression of three replicates, while bars indicate standard errors. Asterisks indicate expression levels that are significantly increased/decreased compared to untreated controls (*P < 0.05, Student’s t test).

plant species analyzed to date (Table 2). While the majority of the ALDH families present in apple comprised more genes than in other species, this was especially the case in the ALDH2 family, which consisted of 13 genes (compared to 2e6 genes in other land plant species). It has been proposed that gene duplications, including tandem, segmental and whole genome duplications, have played crucial roles in the evolutionary novelty of various organisms, including land plants [43]. Based on a comprehensive analysis of chromosomal locations, gene lengths, gene structures and sequence similarities, 25 of the 39 MdALDH genes appeared to be associated with either tandem or segmental duplication events (Fig. 1 and Supplementary Table 1), indicating that both types of duplication played an important role in the expansion of this gene superfamily in apple. In addition, it has been reported that a relatively recent (>50 million years ago) whole genome duplication has contributed to the formation of the 17 chromosomes currently found in apple from an initial nine ancestral chromosomes [24]. Thus, it is

plausible that this genome duplication may also have contributed to the expansion of the MdALDH genes. Although we found that many of the MdALDH genes resulted from segmental or tandem duplication events, it was often not possible to accurately conclude what the ancestral functions and expression patterns of these duplicated ALDH gene pairs may have been due to a high level of divergence between them. For example, while the MdALDH2B12/MdALDH2B13 tandemly duplicated gene pair yielded consistent results under hormone treatments and abiotic stress, the segmentally duplicated MdALDH18B3/MdALDH18B4 gene pair exhibited highly divergent responsiveness to ABA treatment, as well as salt and drought stress (Figs. 5e7). A number of duplicated MdALDH genes (MdALDH2B12/MdALDH2B13, MdALDH2C4/MdALDH2C6/MdALDH2C7/MdALDH2C8/MdALDH2C11/MdALDH2C12, MdALDH2C7/MdALDH2C10, MdALDH2C9/MdALDH2C7, MdALDH18B3/MdALDH18B4) also exhibited distinct tissue-specific expression patterns (Fig. 4), suggesting that the gene duplications supplied opportunities for the duplicates to be free from functional

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Fig. 9. Expression patterns of MdALDH genes following MeJA treatment. A. Expression patterns of 39 MdALDH genes following MeJA treatment conditions as determined by semiquantitative RT-PCR analyses. For each gene, the upper seven bands represent amplified products from the leaves of ‘Fuji’ following treatment with 50 mM MeJA after 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h; lower bands represent amplified products from untreated leaves. B. Expression patterns of three randomly selected MdALDH genes detected by quantitative real-time RT-PCR. The apple EF-1a gene was used as an internal control to normalize the data. Each block denotes the mean relative level of expression of three replicates, while bars indicate standard errors. Asterisks indicate expression levels that are significantly increased/decreased compared to untreated controls (*P < 0.05, Student’s t test).

constraints, allowing them to undergo changes in their structures and/or regulatory mechanisms, and thus gain novel roles [44]. 3.3. MdALDH genes play important roles in various biological processes The functions of ALDH gene families have been well-studied in other plant species. For example, ALDH2 family members in plants metabolize acetaldehyde generated as a consequence of ethanolic fermentation [45], ALDH5 genes participate in the GABA ‘shunt’ pathway, which allows organisms to metabolically bypass the tricarboxylic acid pathway [26], and ALDH10 members have been linked to polyamine catabolism [46]. Furthermore, in addition to their important roles in various metabolic pathways, many of these genes have been shown to be stress responsive, underscoring their importance in a plant’s ability to respond to these types of events. In line with this, ALDH7B, ALDH10, ALDH18 and ALDH22A family members have also been found to be up-regulated in response to

many stressors, including ultraviolet radiation, dehydration, increased salinity, low temperature, and heat shock [20e22,46,47]. Genomic comparison is considered to be a relatively rapid and effective way to transfer genomic knowledge acquired from one taxon for which there is a better understanding of genome structure, function and evolution to a less well-studied taxon [38]. Since Arabidopsis is an important model plant species that has been studied relatively extensively in terms of ALDH gene function, it is theoretically possible to understand the functions of apple MdALDH genes through a genomic comparison between Arabidopsis and apple and the subsequent identification of orthologous genes. To this effect, we carried out synteny analysis of duplicated blocks between the apple and Arabidopsis genomes. Twelve apple MdALDH genes and nine Arabidopsis AthALDH genes from six ALDH families (ALDH2, 3, 5, 10, 18 and 22) were located in syntenic genomic regions. Four pairs of single apple-to-Arabidopsis ALDH genes (MdALDH2B4-AthALDH2B4, MdALDH2B15-AthALDH2B7, MdALDH2C9-AthALDH2C4 and MdALDH18B1-AthALDH18A1) were identified,


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Fig. 10. Expression patterns of MdALDH genes following Eth treatment. A. Expression patterns of 39 MdALDH genes following Eth treatment conditions as determined by semiquantitative RT-PCR analyses. For each gene, the upper seven bands represent amplified products from the leaves of ‘Fuji’ following treatment with 0.5 g/L Eth after 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h; lower bands represent amplified products from untreated leaves. B. Expression patterns of three randomly selected MdALDH genes detected by quantitative real-time RT-PCR. The apple EF-1a gene was used as an internal control to normalize the data. Each block denotes the mean relative level of expression of three replicates, while bars indicate standard errors. Asterisks indicate expression levels that are significantly increased/decreased compared to untreated controls (*P < 0.05, Student’s t test).

indicating that these genes/families were likely present in the genome of the last common ancestor of apple and Arabidopsis (Fig. 1 and Supplementary Table 2). The remaining gene combinations constituted a more complex situation, with 4 cases of two MdALDH genes corresponding to one AthALDH gene (MdALDH3F1/ MdALDH3F2-AthALDH3F1, MdALDH5F1/MdALDH5F2-AthALDH5F1, MdALDH10A8/MdALDH10A9-AthALDH10A8, MdALDH22A1/MdALDH22A2-AthALDH22A1), and one instance of a single MdALDH gene corresponding to multiple AthALDH genes (MdALDH10A8-AthALDH10A8/AthALDH10A9). Moreover, while the remaining four families present in these two species (ALDH6, 7, 11 and 12) could not be mapped into any syntenic blocks, we cannot conclude that orthology does not exist as it is possible that their genomes have undergone significant chromosomal rearrangement and fusions, followed by selective gene loss, which can severely obscure the identification of chromosomal syntenies. To gain further insight into the functions of the MdALDH genes, we also analyzed spatiotemporal expression patterns of these genes in apple, as well as their responsiveness to various types of

abiotic stress and hormone treatments. Our results indicated that all 39 MdALDH genes were expressed, but often varied in their levels of expression in each tissue (Fig. 4), suggesting that they are likely all functional and some may have distinct roles in particular organs. One such example is MdALDH3F1 and MdALDH10A8, which are expressed at high levels in fruits and thus may play a role in their development. Moreover, we also demonstrated that seven of the MdALDH genes exhibited substantial increases in their levels of expression in response to dehydration and/or high salinity (Figs. 5 and 6), five of which (MdALDH2C4, MdALDH3F1, MdALDH11A3, MdALDH11A4 and MdALDH22A2) were up-regulated by both treatments. In line with this, it has been reported previously that plant ALDH3 genes are involved in stress-regulated detoxification pathways and that the ALDH22A gene from Z. mays was up-regulated in response to dehydration and high salinity [20]. Conversely, while ALDH11A3 has been found to encode a non-phosphorylating glyceraldehyde-3phosphate dehydrogenase (GAPDH), which generates NADPH required for biosynthetic processes in Arabidopsis [28], its exact

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function during dehydration and salt stress remains unclear, as well as ALDH2C4. Our results suggest that like a number of ALDH genes studied previously in other plant species, these MdALDH genes may play important roles in protecting apple from damage caused by various types of abiotic stress. Since plant hormones such as ABA, SA, MeJA and Eth have been reported to be involved in the ability to withstand various types of stress, we also analyzed the responsiveness of all 39 MdALDH genes to treatment with these compounds (Figs. 7e10). Our results demonstrated that many of the apple genes exhibited differential expression in response to the plant hormones, which correlates well with previous findings from other plant species. For example, ALDH7B has been found to be significantly up-regulated in response to ABA in Arabidopsis [20e22]. In a similar fashion, we found that MdALDH7B5 exhibited increased expression in apple after treatment with exogenous ABA. Taken together, these results indicate that a number of MdALDH genes may represent important targets for increasing the resistance of apple to stress conditions. 4. Conclusion We identified a total of 39 ALDH genes grouped into 10 families in the apple genome, and found that segmental and tandem duplications, as well as whole genome duplications, have contributed to the expansion of this superfamily in this species. Comparative synteny analysis between apple and Arabidopsis indicated that a number of apple and Arabidopsis ALDH genes were located in syntenic regions, suggesting that these genes are likely orthologues. Expression analysis of the MdALDH genes implied that all 39 genes were functional and that a selection of them may have specific roles in particular tissues. Furthermore, a number of these genes were also found to be responsive to abiotic stress and/or hormone treatments. Based on our findings, we speculate that the MdALDH genes likely possess a variety of functions in apple, including a key role in stress response. This new information not only provides a basis for further investigations concerning the function of the MdALDH genes, but may also prove to be useful for the future improvement of the ability of apple to respond to stress. 5. Materials and methods 5.1. Identification and annotation of apple MdALDH genes All apple MdALDH genes were identified within the GenBank non-redundant protein database and Apple Genome Database (http://www.rosaceae.org/projects/apple_genome) using Pfam domains PF00171 (ALDH family), PS00070 (ALDH cysteine active site), PS00687 (ALDH glutamic acid active site), KOG2450 (aldehyde dehydrogenase), KOG2451 (aldehyde dehydrogenase), KOG2453 (aldehyde dehydrogenase) and KOG2456 (aldehyde dehydrogenase) as queries. Protein motifs were additionally queried against Pfam, PROSITE, and CDD (Conserved Domain Database) databases [25]. Nomenclature of the putative MdALDH genes was based on criteria established by the ALDH Gene Nomenclature Committee (AGNC) [6]. Briefly, deduced amino acid sequences that shared more than 40% identity to previously identified ALDH sequences were considered to belong to the same family, while those exhibiting at least 60% identity were grouped into the same subfamily. Sequences sharing less than 40% identity with previously identified ALDH sequences were grouped into novel families. 5.2. Tandem duplication and synteny analysis Tandem duplications of MdALDH genes in the apple genome were identified via an analysis of their physical chromosomal

locations. Tandemly duplicated genes were defined as adjacent homologous MdALDH genes on the same chromosome, with no more than one intervening gene. For synteny analysis, syntenic blocks within the apple genome, as well as between apple and Arabidopsis genomes, were downloaded from the Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication) and those containing MdALDH genes were identified and analyzed. All databases used to analyze the expansion patterns of MdALDH genes are shown in Supplementary Table 1. 5.3. Sequence alignment and phylogenetic analysis Pairwise alignments of amino acid sequences were performed using the EMBOSS global alignment software along with the NeedlemaneWunsch algorithm (http://www.ebi.ac.uk/Tools/psa/). ALDH protein sequences from apple, grape [26,27], Arabidopsis [26,28], rice [26,29], maize [26,30], P. patens and C. reinhardtii [12] were used to create multiple sequence alignments using ClustalX [31]. Phylogenetic trees were constructed with the MEGA 5.0 software using the neighbor-joining (NJ) method and the bootstrap test replicated 1000 times [32]. 5.4. Exoneintron structure analysis of MdALDH genes The PFAM domain and signal peptide of the MdALDH genes were obtained using the Simple Modular Architecture Research Tool (SMART; http://smart.embl-heidelberg.de/smart/set_mode.cgi? NORMAL¼1) [33]. DOG 1.0 software (http://dog.biocuckoo.org/) [34] was utilized to construct diagrams of protein structures. Exone intron structures were determined from alignments of their coding sequences with corresponding genomic sequences using the est2genome program [35]. Diagrams of exoneintron structures were generated using the online Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn) [36]. 5.5. Plant materials and treatments M. domestica cv. ‘Fuji’ were obtained from an experimental field of the College of Horticulture, Northwest A&F University, Yangling, China (34 200 N, 108 240 E), and were used throughout the study. Tissues, including roots (newly-growing lateral roots with 1e2 mm in diameter), stems (near the apices of newly-growing shoots and with 3e4 mm in diameter), leaves (the third to fifth fully expanded young leaves beneath shoot apices when newly-growing shoots were 40e60 cm in length), flowers, fruits (ripe fruits with about 10 cm in diameter) and seeds of mature fruit, were harvested from ‘Fuji’ trees in their adult phase (aged 9e10 years). In the case of stress and hormone treatments, two year-old ‘Fuji’ seedlings were utilized that had previously been planted in pots. Salt stress was conducted by irrigating seedlings with 2 dm3 250 mM NaCl, followed by the collection of leaves at 0.5, 1, 3, 6, 12, 24 and 48 h post-treatment. Plants irrigated with the same volume of water were utilized as controls. Drought stress was carried out by withholding water from seedlings, with leaves subsequently being harvested at 1, 2, 3, 4, 5, 6 and 7 d post-treatment. Drought-stressed plants were then re-watered to soil saturation and leaves were collected at 1, 2 and 3 d after re-watering. Plants watered every three days were used as a control in the case of the drought stress experiment. Hormone treatments were performed by spraying leaves with 300 mM abscisic acid (ABA), 100 mM salicylic acid (SA), 50 mM methyl jasmonate (MeJA), or 0.5 g/L ethylene (Eth)-releasing ethephon followed by sampling at 0.5, 1, 3, 6, 12, 24 and 48 h posttreatment. As controls for hormone responsive expression analysis, leaves were sprayed with sterile distilled water and collected at the same time periods. In every instance, plant samples were


immediately frozen in liquid nitrogen and stored at 80 C for subsequent RNA isolation and expression analysis. 5.6. Expression analysis of MdALDH genes Total RNA was isolated from apple tissues using the E.Z.N.A.Ô Plant RNA Kit according to the manufacturer’s instructions (OMEGA, China). Five hundred ng DNase-treated total RNA were used for first-strand cDNA synthesis, using a mixture of Poly(dT) and random hexamer primers along with PrimeScriptÔ RTase (TaKaRa Biotechnology, Dalian, China). The resulting cDNA products were then diluted to a 1/6 dilution and stored at 40 C for the subsequent semi-quantitative RT-PCR and quantitative real-time PCR analyses. For semi-quantitative RT-PCR, primers were designed to be specific to each MdALDH gene using the Primer 5.0 software (Supplementary Table 3). An apple EF-1a gene fragment (GenBank accession DQ341381), amplified with primers F (50 - ATT CAA GTA TGC CTG GGT GC -30 ) and R (50 - CAG TCA GCC TGT GAT GTT CC -30 ), was used as an internal control. PCR assays were carried out with 2 Taq Master Mix (CWBIO) in a 20 ml reaction volume, and included 1.0 ml diluted cDNA template along with 0.8 ml of each gene-specific primer (10 pmol L1). Thermal parameters were as follows: 94 C for 2 min, 28e38 cycles at 94 C for 30 s, 55e62 C for 30 s and 72 C for 30 s, with a final extension of 72 C for 2 min. Three biological replicates were carried out for each PCR assay. In each case, 5 ml samples of the resulting semi-quantitative RT-PCR products were resolved on a 1.5% (w/v) agarose gel and visualized using ethidium bromide. The results of semi-quantitative RT-PCR were quantified using the Gene Tools software, the log-transformed values of the relative expression levels of MdALDH genes under abiotic stress and hormone treatment compared to the control were used for hierarchical cluster analysis with Genesis software. Quantitative real-time PCR analyses were performed using an IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA), with denaturation at 95 C for 30 s, followed by 40 cycles of 95 C for 10 s and 60 C for 30 s. To ensure specificity of the amplification reactions, melting curve analyses were carried out at 95 C for 15 s, followed by a constant increase from 60 C to 95 C at a 2% ramp rate. Three biological replicates were utilized for each assay with SYBRÒ Premix Ex TaqÔ RTase (TaKaRa Biotechnology, Dalian, China) in a final volume of 20 ml, using 1.0 ml diluted cDNA template and 1.6 ml gene-specific primers (10 pmol L1). The apple EF-1a gene was utilized as an internal control. Relative expression levels were determined using the IQ5 software and the normalized expression method. Paired t tests were performed using the SPSS Statistics 17.0 software (IBM China Company Ltd., Beijing, China) to assess significant differences between negative controls and treated samples. Acknowledgments This work was supported by the National Natural Science Foundation of China (31071782), Chinese Universities Scientific Fund (QN2011056), as well as the Program for Innovative Research Team of Grape Germplasm Resources and Breeding (2013KCT-25). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2013.07.017. References [1] E. Schauenstein, H. Esterbauer, H. Zollner, Aldehydes in Biological Systems: Their Natural Occurrence and Biological Activities, Pion, London, 1977.

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