Tropical Plant Biology https://doi.org/10.1007/s12042-018-9200-8
Identification, Characterization and Expression Profiles of Dof Transcription Factors in Pineapple (Ananas comosus L) Syed Muhammad Azam 1 & Yanhui Liu 1 & Zia Ur Rahman 1 & Hina Ali 1 & Cheng Yan 1 & Lulu Wang 1 & S. V. G. N. Priyadarshani 1,2 & Binyan Hu 1,3 & Xinyu Huang 1 & Junjie Xiong 1 & Yuan Qin 1 Received: 19 November 2017 / Accepted: 23 February 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract DNA binding with one finger (Dof) transcription factors (TF) family is a class of plant specific transcription factors and has crucial roles in development of different tissues and response to stresses. Using GWAS we identified twenty six Dof genes located on 16 pineapple chromosomes. Dof family genes were distributed into five groups and contain highly conserved motif. High expression of AcoDof1, AcoDof12, AcoDof26 (root), AcoDof9, AcoDof22, AcoDof19 (fruit), AcoDof23, AcoDof25 (ovule), AcoDof11, AcoDof17 and AcoDof20 (petal) were found in RNA-Seq analysis. RNA-Seq data also suggest that Dof genes could have important role in female gametophyte development of pineapple. Expression analysis using qRT-PCR of pineapple Dof genes family under different abiotic stress (cold, heat, salt, drought) showed a dynamic response of Dof genes. AcoDof1 was upregulated at 48 h treatment in all four abiotic stresses, while AcoDof20 was up-regulated at 24 h of treatment. Moreover, AcoDof8, AcoDof12, AcoDof2 were up-regulated by salinity stress. Dof genes expression during abiotic stress reveals their vital role for pineapple growth and development which could be utilized agronomically. Present study provides a framework for future studies related to the functions of Dof genes in pineapple. Keywords Gene expression . Dof transcription factors . Phylogenetic analysis . RNA-Seq . Pineapple
Abbreviations TFs Transcription factors RNA-Seq RNA sequencing qRT-PCR Quantitative real-time PCR NaCl Sodium chloride ORF Open reading frame IP Iso-electric point
Introduction Transcription factors (TFs) act as key controller of major developmental processes and have vital roles in this context. KNOTTED1 (KN1) was the first reported endogenous noncell autonomous (NCA) TF in the maize (Vollbrecht et al. 1991; Lucas et al. 1995). The transcriptional regulation of
Yanhui Liu and Zia Ur Rahman contributed equally to this work. Communicated by: Yann-Rong Lin Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12042-018-9200-8) contains supplementary material, which is available to authorized users. * Yuan Qin
[email protected] 1
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Ministry of Education, Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Center for Genomics and Biotechnology, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
2
College of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
3
College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
Tropical Plant Biol.
gene expression influences many important cellular processes such as morphogenesis, environmental stress responses and signal transduction (Riechmann et al. 2000). TFs are group of proteins that influences certain cellular processes and regulate the expression of downstream target genes (Qu and Zhu 2006). Gene expression is regulated by transcription factors (TFs). Transcription factors are proteins of sequence specific DNA binding with capability of activating and/or repressing transcription (Ali et al. 2017). Using wet lab and bioinformatics analysis about 60 families of TFs are reported in plants (Zhang et al. 2011). Arabidopsis genome has about 1533 TFs which makes about 5.90% of total number of estimated genes in it (Riechmann et al. 2000). In pineapple about 1331 TFs that are identified and classified into 69 families (http://itak.feilab. net/cgi-bin/itak/index.cgi) however in plant transcription factor database there are 1277 TFs in pineapple which were classified into 57 families (http://planttfdb.cbi.pku.edu.cn/) (Zhang et al. 2011). Identification and functional depiction of TFs are vital for the transcriptional regulatory networks reconstruction (Zhang et al. 2011). The Dof belongs to a class of plantspecific TFs that are not reported in other eukaryotes such as Drosophila, yeast, humans or fish (Moreno-Risueno et al. 2007). The first Dof gene was identified in maize (ZmDof1) and was renowned to be involved in light response and transcriptional regulation of genes contributing in carbon metabolism (Yanagisawa 1998). In Arabidopsis, DAG1 and DAG2, well-characterized Dof genes are related to seed germination (Papi et al. 2002; Gualberti 2002). Moreover, three genes CDF1, CDF 2 and CDF3 are related to photo-periodic control of flowering (Imaizumi et al. 2005). Dof genes AtDof5.8, AtDof5.6/HCA2 and AtDof2.4 shows specific expression pattern in the cells at initial stage of vascular tissue growth (Guo et al. 2009). OsDof3 a rice Dof gene is reported to be involved in gibberellins regulated expression. Furthermore, maize Dof1 and Dof2 participate in the gene expression of carbohydrate metabolism and consisting of the gene encoding phosphoenolpyruvate carboxylase (Yanagisawa 2000). Wheat, Dof gene WPBF functions during seed development and vegetative growth (Dong et al. 2007). Few TaDof genes in wheat were down-regulated during drought stress however, TaDof14 and TaDof15 were highly up regulated representing that these Dof genes may have vital role in drought stress adaptation (Shaw et al. 2009). On the basis of bioinformatics analysis 36 Dof genes in the Arabidopsis, 30 Dof genes in the rice genome (Lijavetzky et al. 2003), 31 Dof genes in wheat (Shaw et al. 2009), 28 Dof genes in sorghum (Kushwaha et al. 2011), 78 Dof genes in soybeans (Guo and Qiu 2013), 76 Dof genes in Chinese cabbage (Ma et al. 2015), 38 Dof genes in pigeon peas (Malviya et al. 2015) and 36 Dof genes in cucumber (Wen et al. 2016) and 26 Dof genes in pineapple have been identified. Pineapple (Ananas comosus) a perennial monocotyledonous plants belonging from family bromeliaceae is an economically important fruit crop which is widely cultivated in tropical and
sub-tropical areas. The pineapple is a herbaceous perennial and grows to 3.3 to 4.9 ft (1.0 to 1.5 m) tall although sometimes it can be more taller (Morton J. 1987). Pineapple is an edible fruit consisting of coalesced berries also referred as pineapples. Pineapple is mostly propagated from a crown cutting of the fruit, its flowering time is 5 to 10 months and it takes six more months for fruiting after flowering. Pineapple has diploid (2n = 50) number of chromosome, it comprises a terminal inflorescence having 50 to 200 individual hermaphrodite flowers clustered together. The pineapple fruit develops from the flowers, then these flowers join together forming a cone shaped, compound in structure, fleshy and juicy, multiple fruit of approximately about 30 cm or more in height and weight about 3001000 g (Moyle et al. 2005). Various abiotic stresses pose serious threat to plant growth and productivity. Abiotic stresses are the main cause of crops failure globally, decreasing average yields of major crops by >50% (Mittler 2006). Plants are frequently exposed to plenty of stress conditions such as low and high temperatures, salt, drought, flooding and oxidative stress. Various organic evolution activities have accent the existing stress factors. These stresses limits plant to utilize their full genetic potential which ultimately affects the crop productivity. In the event of thriving concerns of uncertainties in climatic the abiotic stresses are major menace to agriculture crop production globally. Several studies pretenses that worldwide crop production needs to double by 2050 to meet the hypothetical demands for the increasing population, which seems ambitious to achieve and need deep interest (Ray et al. 2013) The genome of pineapple, sequenced recently (Ming et al. 2015), provides a powerful resource for investigating the gene functions during pineapple growth and development (Su et al. 2017). In this study, we identified 26 Dof members in pineapple based on genome sequences and divided them into seven classes on the basis of genetic resemblance. The quotient of these seven classes of Dof family genes, gene structure, protein structure, protein motifs and genes locations on chromosomes and RNA-Seq data for different tissues were also investigated. Expression profiles under four abiotic stresses (cold, heat, salt and drought) were evaluated to determine the responses of Dof genes to abiotic stresses in MD2 variety of pineapple. Our results provides novel insights into the stress responses of Dof genes in pineapple and promote a better perceptive to understand about the utility and functions of Dof genes in pineapple.
Results Identification and Characteristics of Dof Homologues in the Pineapple Genome To identify the Dof transcription factor coding genes in the pineapple, we used the HHM profile of the Dof domain (PF02701) as a query to perform an HMMER search (http://
Tropical Plant Biol.
hmmer.janelia.org/) against the pineapple genomes. Twentysix Dof transcription factors gene sequences were identified from the HMMER database (http://www.ebi.ac.uk/Tools/ hmmer/search/hmmsearch). The isoelectronic point or isoionic point (IP) is the pH at which the amino acid does not migrate in an electric field. IP varied from 4.89 to 11.25 for AcoDof21 and AcoDof19 respectively (Table. 1). The molecular weight ranged from 16.3 to 51.5 kD for AcoDof19 and AcoDof2 respectively. AcoDof1 has lowest ORF length (411) and lowest no. of amino acids (137) while AcoDof2 has more open reading frame length (ORF) (1437) and no. of amino acids (479) respectively. To acquire further insights into the possible gene structural study of Dof genes family in the pineapple genome, diverse exon-intron organizations of Dof family were compared. As shown in Fig. 3 and Table. 1, number of exons and introns were calculated from gene structures, indicating high structural similarity among of Dof genes family having 2 exons and 1 intron for most of genes. Four genes i.e. AcoDof25, AcoDof3, AcoDof8 and AcoDof13 exhibited only exons without introns (Table. 1).
Phylogenetic Analysis of Dof Genes To elucidate the phylogenetic relationships among 36 Arabidopsis thaliana, 30 Oryza sativa, 41 Populus trichocarpa, 28 Sorghum bicolor and 26 Ananas comosus L. Dof genes, a five species mix unrooted phylogenetic tree using MEGA 6.0 and the Neighbour-Joining method was made. The bootstrap test was performed with 1000 iterations founded on the alignment of the Dof domain sequences (Fig. 1). There were 36 Dof genes in the Arabidopsis genome and 30 in the rice genome (Lijavetzky et al. 2003). An individual tree was also constructed for Pineapple 26 Dof genes (Fig. 2). From phylogenetic tree based on neighbor joining (NJ) the Dof homologs were divided into five groups (I to V) the grouping was made according to previous studies (Lijavetzky et al. 2003; Kushwaha et al. 2011). Group III and IV have seven and five genes respectively.
Exon-intron and Motif Structure Analysis of Dof Genes To check the structural heterogeneity, we investigated the characterization of exon-intron structure in the genomic DNA sequences of individual pineapple Dof genes. The predicted numbers of exons among the Dof genes were relatively fewer, varying from one to 5, three genes have 5 exons, one gene have 3 exons while eighteen members having two exons and four genes have 1 exon. AcoDof25, AcoDof3, AcoDof8 and AcoDof13, exhibited only exons; they have no introns. Followed by AcoDof18 which have 5 introns (Fig. 3, Table. 1). Furthermore, Dof genes belonging to the same group had similar gene structures, such as introns numbers and exons lengths. These similar structural features may be
related to their functions in the pineapple genome. To reveal the diversification of Dof genes in pineapple, putative motifs were predicted by the program MEME (Multiple Em for Motif Elicitation), and a total of 10 conserved motifs were found in all the 26 Dof proteins (Fig. 4). Motif 1, 2 and 3 were commonly present in all Dof proteins and represents the conserved Dof domain. Moreover the structural information of theses motifs gives further insight to understand Dof genes in pineapple (Fig. 5).
Dof Gene’s Location on Chromosomes Dof genes location on chromosomes was identified using MapChart, which is a small program for the display and comparison of genetic linkage maps. 26 Dof genes were observed to be located on 16 chromosomes out of 25 total number of chromosomes. 11 genes were noted to be located on individual chromosomes while other genes shared the same chromosome as two or three genes on one chromosome. While three Dof genes i.e. AcoDof24, AcoDof25 and AcoDof26 were found to be located on scaffold_502, scaffold_1002 and scaffold_2504 respectively, which are still not assigned to any chromosome (Fig. 6).
Expression Profiles of Dof Genes in Different Pineapple Tissues We used these RNA-Seq data for studying Dof genes expression profiles in more details. Expression profiles of Dof genes family in pineapple revealed that there is no high and specific expression in flower and leaf tissues, suggesting that this gene family has a little or no role in flower and leaf tissues development, while there is high expression of three Dof genes i.e. AcoDof1, AcoDof12 and AcoDof26 in root tissues indicating these genes cluster can be studied for expression and functions and can be implied in various studies related to root development. As pineapple is consumed as fresh fruit so studies focusing on fruit development is of vital importance for breeder as well as molecular biologists. RNA-Seq studies for fruit in various developmental stages and categorized fruit development in 7 stages was previous carried out (Ming et al. 2015). We found that for the Dof gene family, in first 3 stages there were few genes (AcoDof10, AcoDof9, AcoDof2 and AcoDof22) expressed but their expression was moderate, on the other hand neither gene was expressed in stage 4 and 5. AcoDof19 is highly expressed in stage 6 showing its very stage specific expression and can be used in very specific fruit engineering. Sepal is the outermost whorl of flower, sepal is considered as a protective organ for flower internal organs in initial stages and also provide some nutrients. Only one Dof gene i.e. AcoDof26 showed moderate expression only in stage 2 out of 4 stages of sepal development. Furthermore, ovule is a plant structure that develops into a seed when fertilized. We
Tropical Plant Biol. Table 1 Characteristics of Dof gene in pineapple
Gene ID
IP
MW (kDa)
ORF Length
No. of Amino Acids
Exons
Introns
AcoDof1
9.82
14.6
411
137
2
1
AcoDof2
5.05
51.6
1437
479
2
1
AcoDof3 AcoDof4
8.66 6.5
37.2 38.8
1086 1089
362 363
1 2
– 1
AcoDof5
6.66
35.5
1011
337
2
2
AcoDof6
5.95
30.0
858
286
2
1
AcoDof7 AcoDof8
9.81 9.01
38.4 27.2
1086 780
362 260
3 1
3 –
AcoDof9 AcoDof10
8.19 5.45
46.8 23.3
1287 639
429 213
2 2
1 1
AcoDof11
8.88
37.1
1065
355
2
1
AcoDof12 AcoDof13
9.85 8.04
19.3 38.3
543 1095
181 365
2 1
1 –
AcoDof14 AcoDof15
7.57 8.75
32.9 29.7
924 816
308 272
2 2
1 1
AcoDof16 AcoDof17 AcoDof18 AcoDof19
9.04 7.91 9.57 11.25
37.1 41.4 43.8 16.3
1092 1146 1227 441
364 382 409 147
2 2 5 2
1 1 5 1
AcoDof20 AcoDof21
8.96 4.89
22.6 28.2
642 780
214 260
2 2
1 1
AcoDof22 AcoDof23
5.99 8.93
53.7 24.8
1494 666
498 222
2 5
1 4
AcoDof24
10.12
37.8
981
327
5
4
AcoDof25 AcoDof26
8.96 9.85
37.9 19.0
1107 534
369 178
1 2
– 1
Isoelectric point (IP), molecular weight (MW), open reading frame (ORF)
divided ovule developmental processes into 7 stages, performed RNA-Seq analysis for each stage of ovule and investigated the expression profiles of Dof genes during ovule development. Collectively, 50% of Dof family genes were expressed in different stages of ovule development, but AcoDof23 and AcoDof25 were specifically expressed in ovule initiation stage showing its crucial role in early ovule development. Petal are modified leaves that surrounds the inner reproductive part of flower, petals are collectively called as corolla and are important for attracting the insects for pollination. RNA-Seq was performed for 3 stages for petal development, revealing AcoDof21 as a gene expressed in stage 1 and 2, while 5 Dof genes (AcoDof11, AcoDof17, AcoDof14, AcoDof8 and AcoDof20) were highly expressed in stage 3 of petal development. Stamen is the pollen producing organ in flower. Collectively the stamens form the androecium. A stamen typically consists of a stalk called the filament and an anther which contains spores. Androecium is male part of flower. RNA-Seq was carried out at 5 developmental stages of stamens, there was no notable expression in Dof genes but only one Dof gene AcoDof6 was moderately expressed in 5th
stage of stamen development, as a whole pineapple Dof genes are considered not to be involved in stamen development. Those Dof genes which have high RNA-Seq expression can be studied for their functional analysis and can be used in pineapple crop improvement (Fig. 7a). Overall the RNA-Seq reveals the role of Dof genes in female gametophyte development and related tissues, theses gene with high and specific expression can be utilized in gene function studies to improve pineapple crop. RNA-Seq was confirmed and validated by using qRT-PCR. Four different genes were selected randomly as they showed high RNA-Seq expression and were tested by qRT-PCR for five different tissues i.e. root, flower, fruit, leaf and stamen. The results obtained were consistent with RNASeq expression data of these genes (Fig. 7b).
Expression Patterns of Dof Genes Against Abiotic Stress To further inquire the functions and utility of Dof genes correlated with their expression, 26 Dof genes were subjected to the qRT-PCR to scrutinize the expression profiles of Dof
Tropical Plant Biol.
Fig. 1 Phylogenetic relationship of Dof gene among Pineapple (AcoDof), Rice (LOC), Populus trichocarpa (potri), Sorghum bicolor (sobic) and Arabidopsis (AT). The full-length amino acid sequences were aligned by using ClustalX and phylogenetic tree was constructed using MEGA 5.0
genes under cold, heat, salt, and drought treatments in ‘MD2’ variety of pineapple. Pineapple is cold sensitive, and almost all pineapple varieties are injured after exposure to 4 °C for 24 h. According to qRT-PCR analysis for Dof genes family expression against cold stress, a diverse response was noted. As a whole significant gene expression was observed for all gene family with some exceptions. AcoDof20 showed highly significant expression at 24 h treatment while down regulated at 48 h. For many Dof genes i.e. AcoDof11, AcoDof3, AcoDof9, AcoDof25 and AcoDof6 etc., there was a notable increase in relative expression at 48 h’ time period instead of
24 h. Only AcoDof13 was not affected by the cold stress. Genes showing high expression to cold stress may be involved in stress resistance mechanism and might also contribute to pineapple cold tolerance (Fig. 8). To investigate the pineapple Dof genes response to heat stress, plants were subjected to 45 °C temperature as heat stress. Expression profiles of pineapple Dof genes against heat stress were observed. The magnitude of variation was moderate between 24 and 48 h interval at 45 °C, while relative expression of few genes was observed to be high. Dof genes AcoDof20, AcoDof2, AcoDof3, AcoDof13 and AcoDof15
Tropical Plant Biol. Fig. 2 Phylogenetic relationship of Dof genes in pineapple genome. The full-length amino acid sequences were aligned by using ClustalX and the phylogenetic tree was constructed using MEGA 5.0
were revealed to be expressed at 24 h and down regulated at 48 h, while the rest of the genes were up regulated at 48 h. Pineapple Dof gene AcoDof23 showed maximum response to heat stress on both 24 and 48 h respectively. Generally the genes expressed at 24 h treatment were having low expression as compared with 48 h treatment. These results suggested the role of Dof genes against heat stress in pineapple (Fig. 8). We subjected pineapple to salt stress using 400 mM NaCl. qRT-PCR confessed variable Dof genes expression. Most of the Dof genes exhibited increase in gene expression in relation to time period of 48 h instead of 24 h with some exceptions. Only AcoDof18, AcoDof20 and AcoDof22 were up regulated on 24 h treatment, while the rest of the genes were highly expressed at 48 h treatment. AcoDof2, AcoDof8, AcoDof11, AcoDof12 and AcoDof24, were notably up regulated at 48 h treatment of NaCl. This dynamic response of Dof gene family against salt stress predicts the vital role of Dof genes in salt stress (Fig. 8). In order to observe the possible effect of pineapple Dof genes in drought stress we conducted drought stress trial using
mannitol 400 mM concentration. A potent gene expression was observed against drought stress for Dof genes family. Six genes i.e. AcoDof8, AcoDof11, AcoDof12, AcoDof20, AcoDof22 and AcoDof26, were up regulated at 24 h treatment, while down regulated at 48 h. AcoDof1, AcoDof5, AcoDof14 and AcoDof17 were notably up regulated at 48 h as compared to 24 h. Very high expression was noted for AcoDof1 under 48 h drought treatment. On the basis of these findings a vital role of Dof genes can be predicted against water scarcity conditions (Fig. 8).
Discussion In recent years, gene family analysis has become an important approach to understand gene structure, function and evolution. Dof genes are ubiquitous plant-specific transcription factors that participate in various biological processes. The function and evolution of Dof genes have been thoroughly studied in
Tropical Plant Biol.
AcoDof 17 AcoDof 9 AcoDof 2 AcoDof 22 AcoDof 25 AcoDof 1 AcoDof 16 AcoDof 3 AcoDof 20 AcoDof 11 AcoDof 15 AcoDof 8 AcoDof 23 AcoDof 21 AcoDof 12 AcoDof 26 AcoDof 6 AcoDof 10 AcoDof 7 AcoDof 5 AcoDof 18 AcoDof 13 AcoDof 19 AcoDof 24 AcoDof 4 AcoDof 14
Fig. 3 Intron-Exon structure of 26 Dof genes in pineapple genome. Yellow bars indicates exon (CDS), Blue bars indicated UTR while plain lines showing introns
various plant species. Increasing evidence suggests that the Dof genes play important role in a series of plant-specific physiological phenomena. To date, most of the research on the functions of the Dof genes has been focused in Arabidopsis with 36 AtDof genes (Yanagisawa 2002; Lijavetzky et al. 2003), rice having 30 OsDof genes (Lijavetzky et al. 2003), soybean with 78 GmDof genes (Guo and Qiu 2013), Chinese cabbage with 76 BraDof genes (Ma et al. 2015), potato with 35 StDof genes (Venkatesh and Park 2015), pigeonpea with 38 CcDof genes (Malviya et al. 2015), tomato with 34 SlDof genes (Cai et al. 2013), Chrysanthemum morifolium with 20 Dof genes (Song et al. 2016), pepper (Wu et al. 2016), cucumber (Wen et al. 2016) and in Barrel clover (Shu et al. 2015). We think there is dire need to study Dof transcription factors gene family genomewide identification and transcriptional profiles in response to
abiotic stresses in an important fruit pineapple. In the following study we used genome wide data to identify 26 Dof genes in pineapple genome.
Phylogenetic Analysis, exon/intron Structure Analysis Pineapple Dof genes were shown along with Arabidopsis, rice, sorghum and populus Dof genes family showing high similarity and conservation. Pineapple Dof were divided in 5 clusters on basis of their structural similarity and were compared with model plant Arabidopsis. The intron-exon organizations and intron numbers of Dof genes in pepper genome were quite similar to Arabidopsis (Yanagisawa 2002), rice (Yanagisawa 2002) and tomato (Cai et al. 2013). Like other Dof genes in Arabidopsis and rice, Dof genes in pineapple also showed few introns ranging from zero intron to five introns in one gene
Fig. 4 Multiple sequence alignment of Dof proteins in pineapple. Motifs with specific colors can be find on respective genes
Tropical Plant Biol.
Motif 1
Motif 2
Motif 3
Motif 4
Motif 5
Motif 6
Motif 7
Motif 8
Motif 9
Motif 10
Fig. 5 Motif structure of Dof proteins in pineapple. MEME search tool was used to make motif structures
Tropical Plant Biol.
Fig. 6 Distribution of Dof genes in pineapple genome. MapChart was used to located genes on chromosomes. Gene start point is shown on chromosome while genes size are shown in Mbs against each gene
(Table 1). Number of introns were reported to be very low in soybean with 0 to 1 only (Guo and Qiu 2013) and Barrel clover (Shu et al. 2015). Cucumber Dof genes also exhibit 0 to 2 introns (Wen et al. 2016), predicting structural resemblance with other species. Another property of intron-less genes can be associated with pineapple Dof gene family, containing 4 genes with no introns (Fig. 3). This feature was also reported in tomato Dof gene containing 21 genes with no introns out of 33 Dof genes (Kang et al. 2016) while Dof proteins (Fig. 4) were observed to be highly conserved and comparable with previous studies conducted on Dof genes in other species.
Identification of Conserved Motifs and their Structure Pineapple protein structure and motifs provide evolutionary resemblance with Arabidopsis and rice. Pineapple proteins were identified in 10 motifs with high conservation. The structural variation among motifs provides more information for its involvement in many biological phenomenon. Dof genes were
observed highly scattered in pineapple genome, having different locations on 16 different chromosomes, while 3 genes were still not assigned to any chromosome according to the genome data available (Fig. 6). As 11 pineapples Dof were located on individual chromosomes, the same phenomenon was observed in barrel clover (Shu et al. 2015).
Expression Profiles of Dof Genes in Various Tissues of Pineapple Since high-throughput sequencing and gene expression analyses have been performed on many plant species for different tissues at various developmental stages that are publicly available. RNA-Seq data is thought to be a useful resource for studying gene expression profiles. There is lack of RNA-Seq study on recently sequenced pineapple genome. We carried out RNA-Seq for various tissues at different developmental stages including root, flower, root, six stages for fruit (Ming et al. 2015), four stages of sepal, seven stages of ovule, three
Tropical Plant Biol.
Tropical Plant Biol.
Fig. 7
a Heat-map of tissue-specific expression profiles of Dof genes in pineapple. RNA-Seq expression level can be understood using the given scale and roman numbers on right-side shows clusters based on gene expression. b Heat-map for validation of Dof genes RNA-Seq. Validation of four genes at five different tissues through qRT-PCR. Heat-map was constructed from relative gene expression in different tissues (qRT-PCR) data and FPKM values (RNA-Seq) data for these tissues
stages of petal and five stages of stamen development (Fig. 7a). Some of the Dof genes showed distinct tissue-specific expression patterns across the eight tissues examined. All of the pineapple Dof ’s having expression profiles were clustered into six groups/clusters based on their expression patterns. None of the Dof genes were expressed in flower and leaf tissues. Our results for these tissues expression are in line with the findings of Guo and Qiu 2013. They also observed no expression of Dof genes in leaves tissues in soybean but contrary to our findings they observe moderate expression of Dof genes in flowering bud. Wu et al. 2016., also observed low expression of most of Dof genes in flower in pepper with some exception. Only one gene AcoDof26 was moderately expressed in sepal while the rest family did not expressed in sepal (Fig. 7a). Cluster I genes were expressed in fruit developmental stage 1–3, suggesting that this cluster plays vital role in fruit development. Cluster II showed high expression in root tissue, predicting its vital role in root development, while these results were consistent with the observation of Dof genes
Fig. 8 Heat-map of expression profiles of Dof genes under abiotic stresses [cold (4 °C), heat (45 °C), Salt (NaCl) and drought (Mannitol)]. qRT-PCR was used to analyze the relative expression level of each Dof gene. The expression level of pineapple Actin was used as the internal control to standardize the RNA samples for each reaction, and the expression at 0 h was set as 1 (data not shown). Expression level can be understand using the given scale
high expression in root in soybean (Guo and Qiu 2013). According to Kang et al. 2016, four genes showed high RNA-Seq expression in root in pepper, which confirms our recent findings. Cluster III showed high expression in petal; interestingly these Dof genes expressions was very specific to stage 3 in petal development. As petal is important part of flower and helps in pollination can be also considered to improve pineapple beauty, so these genes likely to be used as pineapple crop flower improvement and beautification. Cluster IV was moderately expressed in ovule varied from stage 1 to 5, with very high expression of two genes AcoDof23 and AcoDof25 in stage 1 of ovule development. These two Dof genes can be employed in transformation studies for their expression and functions in ovule development. Shu et al. 2015, also observed Dof gene expression in flower tissues in barrel clover. AcoDof19 in cluster IV was highly expressed in fruit, showing its role for fruit related improvement in pineapple, while this gene have no specific expression in other tissues. Cluster IIV showed low expression for some tissues but only two genes AcoDof7 and AcoDof6 were expressed in stamen developmental stage 1 and 5 respectively (Fig. 7a). While the RNA-Seq validity was checked by qRTPCR, to confirm whether RNA-Seq data for Dof genes was reliable or not. RNA-Seq was confirmed and validated by using qRT-PCR. All genes tested by qRT-PCR for five different tissues i.e. root, flower, fruit, leaf and stamen have consistent results with RNA-Seq (Fig. 7b). Data for tissue specific
Tropical Plant Biol.
RNA-Seq and RNA-Seq validation through qRT-PCR is presented in heat-map (Fig. 7a and b). A heat-map is a graphical representation of data where the individual values contained in a matrix are represented as colors, which helps the readers to quickly understand the data.
Response of Dof Gene Family to Abiotic Stress Dof TFs have been shown to play crucial roles in the regulatory networks of plant defense, including responses to diverse biotic and abiotic stresses (Corrales et al. 2014; Ma et al. 2015). In plant, Dof family transcription factors play very important roles in development as well as in hormonal regulation and stress responses. However, there is no evidence of such studies in pineapple for Dof transcription factors. Arabidopsis, OBP2 (AtDof1.1) expression level increased within 4–6 h after treating with MeJA and mechanical injury (Skirycz et al. 2006). Homologs of Arabidopsis CDFs, in tomato SlCDF1–5, were reported to be significantly induced against salt, heat, cold and osmotic stresses (Corrales et al. 2014; Ma et al. 2015) studied BraDof in Chinese cabbage, where most of the genes were up-regulated by abiotic stresses. In potato, most StDof genes were up-regulated in various abiotic stresses and ABA treatment. Furthermore, chrysanthemum, CmDofs were expressed when exposed to abiotic stress and mechanical injury (Song et al. 2016). In our study, most genes showed up-regulated expression profiles under these four stresses with few exceptions. In response to cold stress treatment, only two Dof genes were up regulated at 24 h treatment while the rest twenty four genes were up regulated at 48 h treatment (Fig. 8). The same phenomenon was also observed for Dof genes family in Chinese cabbage under cold stress (Ma et al. 2015). The same variable results were also reported for Dof genes family under cold stress in pepper (Wu et al. 2016). These findings show that Dof genes response to cold stress at very specific cold temperature predicts its role against cold stress in pineapple. However, because its shortest production cycle is 14 months, the crop must undergo cold stress at least once in its life cycle, especially in subtropical regions (Chen et al. 2016). As pineapple is considered as tropical and subtropical plant there the temperature is ranged from 25 to 40°Cin summer. We observed no significant gene expression in Dof genes family when exposed to heat stress of 45°Cfor 24 and 48 h respectively. Six Dof genes were observed to be up-regulated at 24 h’ time point, while the rest were highly expressed at 48 h treatment (Fig. 8). Contrary to our results low Dof genes expression at maximum time interval during the heat treatment at 38 °C for pepper (Wen et al. 2016). This may be the difference of heat treatment or difference of species that is why Dof genes responded differently, but Wu et al. 2016 also noted high gene expression for little time interval, which supports our recent findings in pineapple.
The findings of Song et al. 2016, are in agreement with our recent observation. They found high response of many genes in regarding heat stress for Chrysanthemum leaves at high temperature. Pineapple Dof gene were observed to have highly influenced by salinity stress (NaCl at 400 mM). A significant up-regulation of genes was observed against salt stress. AcoDof2, AcoDof24 and AcoDof8 were very significantly expressed at 48 h treatment (Fig. 8). Similar response of Dof genes was observed in Chrysanthemum (Song et al. 2016). They found six genes were highly expressed in response to salinity stress. Wu et al. 2016, also reported upregulated Dof genes response to salinity stress in pepper. Drought stress (Mannitol at 400 mM) was applied to pineapple, which showed dynamic gene expression. Most of the Dof genes were up-regulated due to drought stress while only two genes expression was very low (Fig. 8). High Dof genes expression for almost all genes in Chinese cabbage (Ma et al. 2015), which supports our current findings. Briefly, Dof genes of pineapple were responsive to all abiotic stresses as reported by other researchers in different species. In short, nine pineapple Dof genes i.e. AcoDof19, AcoDof6, AcoDof11, AcoDof17, AcoDof20, AcoDof1, AcoDof12, AcoDof25 and AcoDof23 were having high and more specific RNA-Seq expression at specific tissues including, root, ovule, sepal, stamen, petal and fruit and these genes were up-regulated in response to abiotic stress also. Collectively these findings suggesting the vital role of Dof genes in pineapple.
Conclusions We accomplished a comprehensive analysis of Dof transcription factors in pineapple. A total of 26 genes encoding Dof family transcription factor were identified in pineapple genome. Further, Dof transcription factors were characterized according to the conserved amino acid residues within the Dof domain, the conserved motifs, gene organization and phylogenetic analysis. High-throughput sequencing RNA-Seq expression profile among different tissues revealed candidate genes with high expression profile. A vital role of Dof genes was observed in female gametophyte development in pineapple that can be utilized to study pineapple plant reproduction for its improvement. Also some basic characteristics of Dof genes (molecular weight, iso-electric point) were identified and exposure of pineapple MD2 variety to various abiotic stresses (cold, heat, salt and drought) and Dof genes expression was analyzed by using qRT-PCR. The results obtained from this study provide useful evidence to understand the molecular background of the Dof gene family in the pineapple. We obtained fruitful results as we were expected on the basis of information already published regarding Dof gene family studies in different species. Our study has the utility
Tropical Plant Biol.
for understanding how Dof transcription factors respond to abiotic stresses and the interaction of these genes for abiotic stress response mechanism and how pineapple crop can be improved by utilizing Dof genes.
Materials and Methods Genome-wide Identification of Dof Homologue Sequences The conserved Dof domain based on a Hidden Markov Model (HMM) (PF02701) were downloaded from the Pfam protein family database (http://pfam.sanger.ac.uk/). The amino acid sequence of the Dof domain was used to search for possible Dof-domain homolog hits in the whole-genome sequence of Dof protein sequence of Arabidopsis, Rice and Pineapple, from Phytozome (http://www.phytozome.net/arabidopsis.php) for arabidopsis Dof genes protein sequences, (http://www. phytozome.net/rice.php) for rice Dof proteins sequences, (http://www.phytozome.net/sorghum.php) for sorghum Dof protein sequences and (http://www.phytozome.net/populus. php) for populus Dof protein sequences, while (http://www. phytozome.net/pineapple.php) for pineapple Dof gene protein sequences respectively (Kawahara et al. 2013). To identify the Dof transcription factor coding genes of Ananas comosus L, we used the HHM profile of the Dof domain as a query to execute a HMMER search (http://hmmer.janelia.org/) in pineapple genome. All non-redundant sequences encoding complete Dof domains were well thought out to be putative Dof genes. Each non-redundant sequences of Dof gene was double restrained for the presence of the conserved Dof domain by utilization of SMART search (http://smart.emblheidelberg.de/).
Characteristics of Dof Genes and their Location on Chromosomes Individual Dof genes length, number of amino acids and open reading frame (ORF) length were calculated manually from genome sequences obtained from phytozome (http://www. phytozome.net/pineapple.php). While isoionic point (IP) and molecular weights of Dof genes family for pineapple were computed using the ExPASy server (http://web.expasy.org/ compute_pi/) (Gasteiger 2003). Genes start point and stop points were calculated from pineapple genome for Dof genes and MapChart was used to locate 26 Dof genes on 25 chromosomes according to the start site (first nucleotide) of gene.
Phylogenetic Characterization of Pineapple Dof Homologs Multiple sequence alignments were carried out on the amino acid sequences of Dof proteins from Pineapple, Arabidopsis,
Rice, Sorghum and Populus using MUSCLE with default settings. Subsequently, MEGA 5.0 software was employed to make an unrooted phylogenetic tree based on NeighbourJoining (NJ) procedure with the following parameters: JTTmodel, pairwise gap deletion and 1000 bootstraps (Tamura et al. 2011). Furthermore, maximum likelihood, minimal evolution and PhyML methods were also practiced for the tree construction to validate the results of the NJ method.
Gene Structure Analysis and Conserved Motif Identification The exon-intron arrangements of the genes were determined using the Gene Structure Display Server (http://gsds.cbi.pku. edu.cn) through a comparison of their full-length predicted coding sequence (CDS) (Guo et al. 2007) with their corresponding genomic DNA sequences from Phytozome (http:// www.phytozome.net/pineapple.php). (Guo et al. 2007). The deduced amino-acid sequences of the 26 AcoDofs were analyzed and statistically identified by MEME (Multiple EM for Motif Elicitation) (http://meme-suite.org/tools/meme) with the motif length set to 6–100 and motif sites to 2–120. The maximum number of motifs was set to 10, the statistical distribution of one single motif was Bany number of repetitions^ and the other trait was Bsearch given strand only^.
Plant Material and Growth Conditions Pineapple (Ananas comosus) variety MD2 was provided by Qin Lab, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fujian, China (www. qinlab.net). Plant crowns were grown on soil mix [peat moss:perlite,2:1(v/v)] in plastic pots placed in greenhouse at ~30 °C with light availability of 60–70 umol m−1 s−1 photons, under 70% humidity with 16-h light/8-h dark photoperiod (Ali et al. 2017; Rahman et al. 2017).
RNA-Seq for Different Tissues Healthy plants were selected from MD2 variety; samples were collected from different developmental stages of ovule, petal, sepal and stamen. Collected samples were quickly stored in liquid nitrogen prior to total RNA extraction. RNA was extracted using RNA extraction Kit (Omega Bio-Tek, Shanghai, China) following manufacturer’s protocol and as described by Cai et al. 2017, Su et al. 2017. Total RNA was diluted with nuclease free water and then mRNA was isolated, followed by fragmentation and priming. First and second Strand cDNA Synthesis were synthesized. Then purify the Doublestranded cDNA Using 1.8X Agencourt AMPure XP Beads. Perform End Repair/dA-tail of cDNA Library followed by adaptor ligation using Blunt/TA Ligase Master Mix and diluted NEBNext Adaptor. Purify the ligation reaction and
Tropical Plant Biol.
approximate insert size was kept 25-400 bp with final library was set to 300-500 bp. Performed PCR Library construction followed by purity of the PCR reaction using Agencourt AMPure XP Beads and assessed library quality on a Bioanalyzer® (Agilent high sensitivity chip) and send to company for sequencing (NEB next Ultra RNA Library Prep Kit for Illumina Biolabs) (Biolabs). The RNA-Seq data was analyzed following (Trapnell et al. 2012). Details of RNA-Seq quality and sequence depths are attached as supplementary material (supplementary Table. 1) RNA-Seq for different tissues i.e. seven stages of ovule (AC (Archesporial cell), MMC (Megaspore mother cell), meiosis, FG1-FG4 (Female gametophyte), three stages of petal (petal without bud, with bud before flower, flowering petal), five stages of stamen (AC (Archesporial cell), MMC (Megaspore mother cell), meiosis, MG1-MG2 (male gametophyte), four stages of sepal (calyx without bud, with bud before flower, during flowering, after flowering) was determined. While leaf, root, flower and six fruit developmental stages RNA-Seq data was obtained from Ming et al. 2015. Furthermore, RNA-Seq was validated through qRT-PCR for different genes i.e. AcoDof4, AcoDof6, AcoDof9 and AcoDof12 were tested at five different tissues i.e. leaf, root, flower, fruit and stamen.
Heatmap for Dof genes RNA-Seq A gene expression heat map’s visualization features helping users to immediately make sense of the data by assigning different colors to each gene. R software was employed to construct the heat-map using FPKM values of RNA-Seq for each gene. Heatmaps created by the metaseq python program (Dale et al. 2014; Dai et al. 2017). Expression Heat map was constructed for 26 Dof genes for RNA-Seq of reported tissues in pineapple. FPKM values of RNA-Seq for each tissues in each genes were used to construct heat-map (Fig. 7a). FPKM values of RNA-Seq and relative expression of four Dof genes in five different tissues were presented in heatmap (Fig. 7b), while relative expression values of 26 Dof genes against abiotic stress treatment were shown as heatmap (Fig. 8).
Abiotic Stress Treatment Abiotic stresses (cold, heat, drought and salt) were applied to fully grown (with well developed roots and shoot) healthy and disease free pineapple plants and one control (3 biological repeats). For cold treatment (cold stress) healthy plants were placed in growth chamber and temperature was set at 4 °C and samples were collected at 24 h and 48 h time period, for heat stress at plants were kept 45 °C and samples were collected at 24 h and 48 h interval (Ali et al. 2017). For drought stress mannitol in concentration of 400 mM and salt stress as 400 mM NaCl were applied and samples were collected at 24 h and 48 h time intervals respectively. For Mannitol and
salt treatment plants were selected with no fertilizer for 15 days, followed by application of mannitol and salt solutions in clean pots according to plant requirements. Samples were collected from both stressed and normal plants that were not exposed to stress treatment were used as control; collected samples were immediately stored in liquid nitrogen prior to total RNA extraction (Rahman et al. 2017).
Quantitative Real-time PCR To determine the relative transcript levels of selected Dof genes real-time PCR was performed with gene specific primers according to the manufacturer’s instructions on the Bio-Rad Real-time PCR system (Foster City, CA, USA). Total RNA was extracted using RNA extraction Kit (Omega Bio-Tek, Shanghai, China) following manufacturer’s protocol. First-strand cDNA synthesis was carried out using cDNA extraction kit (Trans Gen, Beijing, China) with gDNA remover. A 10-fold dilution of the resultant cDNA was done. Performed qRT-PCR by using qRT-PCR kit (Trans Gen, Beijing, China). Real-time PCR system in a total volume of 20-μL volume with the following program: 95 °C for 30s; 95 °C for 5 s and 60 °C for 34 s; with 39 cycles (Zhao et al. 2018). Amplification of the target genes was observed during each cycle by SYBR green fluorescence. The Ct (threshold cycle), is defined as the real-time PCR repeat at which a statistically significant increase in reporter fluorescence is first perceived, was used as a measure for the starting copy numbers of the reference genes. Three technical and 3 biological repeats were performed for each experiment as described by Chen et al. 2017. Data were analyzed by the Livak method (Livak and Schmittgen 2001) and explicit as a normalized relative expression level (2-ΔΔCT) of the respective genes. The relative transcript levels of the analyzed Dof genes were normalized to the transcript levels of AcoActin. In each case, three technical replicates were performed for each of at least three independent biological replicates. Primer Quest Tool was used to design primers for Dof gene family (http:// www.idtdna.com/PrimerQuest/Home/Index?Display= AdvancedParams). The actin gene (house keeping gene) from pineapple was used as a reference (supplementary Table. 2). Acknowledgments This work was supported by NSFC (U1605212; 31522009 to Y.Q.), Fujian Innovative Center for Germplasm Resources and Innovation Project of Characteristic Horticultural Crop Seed Industry (KLA15001D) and FAFU international collaboration project (KXb16006A). Author Contribution S.M. Azam performed experiments, data analysis and manuscript writing, Y.L carried out bioinformatics assistance, Z.R and H.A worked on qRT-PCR, L.W, B.H, X.H and X.J.J helped and observed RNA-Seq analysis. We are thankful to M. Aslam for reviewing the manuscript.. YQ conceived the study.
Tropical Plant Biol.
Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interests.
References Ali H, Liu Y, Azam SM et al (2017) Genomic Survey, Characterization, and Expression Profile Analysis of the SBP Genes in Pineapple (Ananas comosus L.) Int J Genomics 2017:1032846. https://doi. org/10.1155/2017/1032846 Cai X, Zhang Y, Zhang C et al (2013) Genome-wide Analysis of Plantspecific Dof Transcription Factor Family in Tomato. J Integr Plant Biol 55:552–566. https://doi.org/10.1111/jipb.12043 Cai H, Zhao L, Wang L et al (2017) ERECTA signaling controls Arabidopsis inflorescence architecture through chromatinmediated activation of PRE1 expression. New Phytol 214:1579– 1596. https://doi.org/10.1111/nph.14521 Chen C, Zhang Y, Xu Z et al (2016) Transcriptome Profiling of the Pineapple under Low Temperature to Facilitate Its Breeding for Cold Tolerance. PLoS One 11:e0163315. https://doi.org/10.1371/ journal.pone.0163315 Chen P, Li Y, Zhao L et al (2017) Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development. Front Plant Sci 8:2150. https://doi.org/10.3389/fpls.2017.02150 Corrales A-R, Nebauer SG, Carrillo L et al (2014) Characterization of tomato Cycling Dof Factors reveals conserved and new functions in the control of flowering time and abiotic stress responses. J Exp Bot 65:995–1012. https://doi.org/10.1093/jxb/ert451 Dai X, Bai Y, Zhao L et al (2017) H2A.Z Represses Gene Expression by Modulating Promoter Nucleosome Structure and Enhancer Histone Modifications in Arabidopsis. Mol Plant 10:1274–1292. https://doi. org/10.1016/j.molp.2017.09.007 Dale RK, Matzat LH et al (2014) metaseq: a Python package for integrative genome-wide analysis reveals relationships between chromatin insulators and associated nuclear mRNA. Nucleic Acids Res 42: 9158–9170 Dong G, Ni Z, Yao Y et al (2007) Wheat Dof transcription factor WPBF interacts with TaQM and activates transcription of an alpha-gliadin gene during wheat seed development. Plant Mol Biol 63:73–84. https://doi.org/10.1007/s11103-006-9073-3 Gasteiger E (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788. https:// doi.org/10.1093/nar/gkg563 Gualberti G (2002) Mutations in the Dof Zinc Finger Genes DAG2 and DAG1 Influence with Opposite Effects the Germination of Arabidopsis Seeds. PLANT CELL ONLINE 14:1253–1263. https://doi.org/10.1105/tpc.010491 Guo Y, Qiu LJ (2013) Genome-Wide Analysis of the Dof Transcription Factor Gene Family Reveals Soybean-Specific Duplicable and Functional Characteristics. PLoS One. https://doi.org/10.1371/ journal.pone.0076809 Guo A-Y, Zhu Q-H, Chen X, Luo J-C (2007) GSDS: a gene structure display server. Yi chuan = Hered 29:1023–1026 Guo Y, Qin G, Gu H, Qu L-J (2009) Dof5.6/HCA2, a Dof transcription factor gene, regulates interfascicular cambium formation and vascular tissue development in Arabidopsis. Plant Cell 21:3518–3534. https://doi.org/10.1105/tpc.108.064139 Hina A, Yanhui L, Syed MA et al (2017) Genomic Survey, Characterization, and Expression Profile Analysis of the SBP
Genes in Pineapple (Ananas comosus L.) Int. J. of Genomics 2017:1032846 Imaizumi T, Schultz TF, Harmon FG et al (2005) FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science 309:293–297. https://doi.org/10.1126/ science.1110586 Kang W-H, Kim S, Lee H-A et al (2016) Genome-wide analysis of Dof transcription factors reveals functional characteristics during development and response to biotic stresses in pepper. Sci Rep 6:33332. https://doi.org/10.1038/srep33332 Kawahara Y, de la Bastide M, Hamilton JP, et al (2013) Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice (N Y) 6:4. https://doi.org/ 10.1186/1939-8433-6-4 Kushwaha H, Gupta S, Singh VK et al (2011) Genome wide identification of Dof transcription factor gene family in sorghum and its comparative phylogenetic analysis with rice and Arabidopsis. Mol Biol Rep 38:5037–5053. https://doi.org/10.1007/s11033-010-0650-9 Lijavetzky D, Carbonero P, Vicente-Carbajosa J (2003) Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families. BMC Evol Biol 3:17. https://doi.org/10.1186/14712148-3-17 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001. 1262 Lucas WJ, Bouché-Pillon S, Jackson DP et al (1995) Selective trafficking of KNOTTED1 homeodomain protein and its mRNA through plasmodesmata. Science 270:1980–1983. https://doi.org/10.1126/ science.270.5244.1980 Ma J, Li M-Y, Wang F et al (2015) Genome-wide analysis of Dof family transcription factors and their responses to abiotic stresses in Chinese cabbage. BMC Genomics 16:33. https://doi.org/10.1186/ s12864-015-1242-9 Malviya N, Gupta S, Singh VK et al (2015) Genome wide in silico characterization of Dof gene families of pigeonpea (Cajanus cajan (L) Millsp.) Mol Biol Rep 42:535–552. https://doi.org/10.1007/ s11033-014-3797-y Ming R, VanBuren R, Wai CM et al (2015) The pineapple genome and the evolution of CAM photosynthesis. Nat Genet 47:1435–1442. https://doi.org/10.1038/ng.3435 Mittler R (2006) Abiotic stress, the field environment and stress combination. Trends Plant Sci 11:15–19. https://doi.org/10.1016/j.tplants. 2005.11.002 Moreno-Risueno MÁ, Díaz I, Carrillo L et al (2007) The HvDOF19 transcription factor mediates the abscisic acid-dependent repression of hydrolase genes in germinating barley aleurone. Plant J 51:352– 365. https://doi.org/10.1111/j.1365-313X.2007.03146.x Morton J (1987) Pineapple. In: Fruits of warm climates. In: Pineapple. J.F. Morton, p 505 Moyle R, Fairbairn DJ, Ripi J, et al (2005) Developing pineapple fruit has a small transcriptome dominated by metallothionein. J Exp Bot 56: 101–112. https://doi.org/10.1093/jxb/eri015 Papi M, Sabatini S, Altamura MM et al (2002) Inactivation of the Phloem-Specific Dof Zinc Finger Gene DAG1 Affects Response to Light and Integrity of the Testa of Arabidopsis Seeds. Plant Physiol 128:411–417. https://doi.org/10.1104/pp.010488 Qu L-J, Zhu Y-X (2006) Transcription factor families in Arabidopsis: major progress and outstanding issues for future research. Curr Opin Plant Biol 9:544–549. https://doi.org/10.1016/j.pbi.2006.07. 005 Rahman ZU, Azam SM, Liu Y et al (2017) Expression Profiles of Wuschel-Related Homeobox Gene Family in Pineapple (Ananas comosus L). Trop Plant Biol 10:204–215. https://doi.org/10.1007/ s12042-017-9192-9
Tropical Plant Biol. Ray DK, Mueller ND, West PC, Foley JA (2013) Yield Trends Are Insufficient to Double Global Crop Production by 2050. PLoS One 8:e66428. https://doi.org/10.1371/journal.pone.0066428 Riechmann JL, Heard J, Martin G et al (2000) Arabidopsis Transcription Factors: Genome-Wide Comparative Analysis Among Eukaryotes. Science 290(80):2105–2110. https://doi.org/10.1126/science.290. 5499.2105 Shaw LM, McIntyre CL, Gresshoff PM, Xue G-P (2009) Members of the Dof transcription factor family in Triticum aestivum are associated with light-mediated gene regulation. Funct Integr Genomics 9:485– 498. https://doi.org/10.1007/s10142-009-0130-2 Shu YJ, Song LL, Zhang J et al (2015) Genome-wide identification and characterization of the Dof gene family in Medicago truncatula. Genet Mol Res 14:10645–10657. https://doi.org/10.4238/2015. September.9.5 Skirycz A, Reichelt M, Burow M et al (2006) DOF transcription factor AtDof1.1 (OBP2) is part of a regulatory network controlling glucosinolate biosynthesis in Arabidopsis. Plant J 47:10–24. https://doi. org/10.1111/j.1365-313X.2006.02767 Song A, Gao T, Li P et al (2016) Transcriptome-Wide Identification and Expression Profiling of the DOF Transcription Factor Gene Family in Chrysanthemum morifolium. Front Plant Sci 7:199. https://doi. org/10.3389/fpls.2016.00199 Su Z, Wang L, Li W et al (2017) Genome-Wide Identification of Auxin Response Factor (ARF) Genes Family and its Tissue-Specific Prominent Expression in Pineapple (Ananas comosus). Trop Plant Biol 10:86–96. https://doi.org/10.1007/s12042-017-9187-6 Tamura K, Peterson D, Peterson N et al (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 28:2731–2739. https://doi.org/10.1093/molbev/ msr121 Trapnell C, Roberts A, Goff L et al (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and
Cufflinks. Nat Protoc 7:562–578. https://doi.org/10.1038/nprot. 2012.016 Venkatesh J, Park SW (2015) Genome-wide analysis and expression profiling of DNA-binding with one zinc finger (Dof) transcription factor family in potato. Plant Physiol Biochem 94:73–85. https://doi. org/10.1016/j.plaphy.2015.05.010 Vollbrecht E, Veit B, Sinha N, Hake S (1991) The developmental gene Knotted-1 is a member of a maize homeobox gene family. Nature 350:241–243. https://doi.org/10.1038/350241a0 Wen C, Cheng Q, Zhao L, et al (2016) Identification and characterisation of Dof transcription factors in the cucumber genome. Sci Rep 6: 23072. https://doi.org/10.1038/srep23072 Wu Z, Cheng J, Cui J et al (2016) Genome-Wide Identification and Expression Profile of Dof Transcription Factor Gene Family in Pepper (Capsicum annuum L.) Front Plant Sci 7:574. https://doi. org/10.3389/fpls.2016.00574 Yanagisawa S (1998) Involvement of Maize Dof Zinc Finger Proteins in Tissue-Specific and Light-Regulated Gene Expression. PLANT CELL ONLINE 10:75–90. https://doi.org/10.1105/tpc.10.1.75 Yanagisawa S (2000) Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize. Plant J 21:281–288. https://doi.org/10.1046/j.1365-313x. 2000.00685.x Yanagisawa S (2002) The Dof family of plant transcription factors. Trends Plant Sci 7:555–560. https://doi.org/10.1016/S13601385(02)02362-2 Zhang H, Jin J, Tang L et al (2011) PlantTFDB 2.0: update and improvement of the comprehensive plant transcription factor database. Nucleic Acids Res 39:D1114–D1117. https://doi.org/10.1093/nar/ gkq1141 Zhao L, Cai H, Su Z et al (2018) KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis. Proc Natl Acad Sci 115:E526–E535. https://doi.org/ 10.1073/pnas.1716054115
