Background: Blueberry cultivars require a fixed quantity of chilling hours during winter endo-dormancy for vernalization. In this study, transcriptome analysis using RNA sequencing data from nonchilled, chilled, and late pink buds of southern highbush blueberry ‘Legacy’ was performed to reveal genes associated with chilling accumulation and bud break. Results: Fully chilled ‘Legacy’ plants flowered normally whereas nonchilled plants could not flower. Compared to nonchilled flower buds, chilled flower buds showed differential expression of 89% of flowering pathway genes, 86% of MADS-box genes, and 84% of cold-regulated genes. Blueberry orthologues of FLOWERING LOCUS T (FT) did not show a differential expression in chilled flower buds (compared to nonchilled flower bud) but were up-regulated in late-pink buds (compared to chilled flower bud). Orthologoues of major MADS-box genes were significantly up- regulated in chilled flower buds and down-regulated in late-pink buds. Functional orthologues of FLOWERING LOCUS C (FLC) were not found in blueberry. Orthologues of Protein FD (FD), TERMINAL FLOWER 1 (TFL1),and LEAFY (LFY) were down-regulated in chilled flower buds and in late-pink buds compared to nonchilled flower bud. Conclusions: The changes from nonchilled to chilled and chilled to late-pink buds are associated with transcriptional changes in a large number of differentially expressed (DE) phytohormone-related genes and DE flowering pathway genes. The profile of DE genes suggests that orthologues of FT, FD, TFL1, LFY, and MADS-box genes are the major genes involved in chilling-mediated blueberry bud-break. The results contribute to the comprehensive investigation of the vernalization-mediated flowering mechanism in woody plants. Keywords: Chilling requirement, Cold hardness, Flowering time control, Freezing tolerance, Vaccinium corymbosum, Vernalization, Woody plant Background Climate change in the last 40 years has caused earlier Winter dormancy (endo-dormancy) is essential for de- shifts in the onset of the growing season for trees (e.g., ciduous fruit crop survival [1, 2]. Under inductive low 2.3 days/decade in temperate Europe) and increased temperatures in the fall, deciduous woody fruit and nut temperature fluctuation . Early onset of the growing crops are acclimated to develop freezing tolerance; season causes insufficient chilling hours and prevents meanwhile, accumulation of effective chilling hours is bud-break in fruit trees. Increased temperature fluctu- stimulated . Sufficient chilling accumulation gives ation during plant bloom turns early season frosts into a plants full vernalization, which is a prerequisite for danger, with freezing injuries to flowers and young fruits bud-break in the spring. . Plant breeding to manipulate chilling requirements and develop improved freeze tolerant cultivars are con- sidered to be long-term solutions to mitigate reduced * Correspondence: email@example.com Plant Biotechnology Resource and Outreach Center, Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Song and Chen BMC Plant Biology (2018) 18:98 Page 2 of 13 winter chill, decrease freezing damages, and secure de- natural winter conditions flowered normally (Fig. 1). Tran- ciduous fruit production . scriptome comparison of the chilled to nonchilled flower Seasonal flowering plays a significant role in a plant’slife buds using Trinity and a non-annotated transcriptome cycle and is controlled by a network of flowering pathway reference (Reftrinity) of tetraploid blueberry in GenBank genes [7–9]. FLOWERING LOCUS C (FLC) is a key (Accession number: SRX2728597) [19, 20] revealed 37,000 regulator in the vernalization pathway of winter-annual differentially expressed genes (DEGs) and 47,000 DE Arabidopsis thaliana ecotypes . In winter wheat and transcripts. barley, VERNALIZATION2 (VRN2) is a major regulator of To conduct transcriptome analysis, Reftrinity (180,000 vernalization-mediated flowering . FLC and VRN2 ana- genes and 250,000 isoforms) was annotated using Trino- logs are recorded in peach (Prunus persica). These analogs tate . The annotation resulted in 14,000 known are the DORMANCY ASSOCIATED MADS-box (DAM) genes from 30,000 genes and 55,000 isoforms of Reftri- genes which are a cluster of six MADS-box transcription nity. With this annotated reference, 64% of known blue- factors. The loss of all or part of the DAMs resulted in the berry genes and isoforms showed differential expression non-vernalized peach evergrowing mutant [10, 11]. The in the comparison of chilled to nonchilled flower buds DAM genes are considered alternatives to FLC in regulat- (herein referring to DEGs/DE transcripts in chilled ing vernalization-mediated chilling requirement and flow- flower buds). Chilling affected expression of numerous ering [10, 12]. However, the DAM genes show high genes simultaneously in blueberry flower buds. similarities to A. thaliana AGAMOUS-LIKE 24 (AGL24) and SHORT VEGETATIVE PHASE (SVP)genes [12, 13]. Effect of chilling accumulation on flowering pathway Additionally, functional analysis of DAMs to reveal their genes roles in chilling-mediated flowering through reverse gen- Differential expression was detected in chilled blueberry etics has not been reported in peach. To date, neither a flower buds for 89% of flowering pathway orthologues of functional FLC-LIKE nor a VRN2-LIKE gene has been known A. thaliana flowering pathway genes (Table 1; verified in Vaccinium plants . Fig. 1). One of the top two orthologues of FT (VcFT: Blueberries and cranberries are the most important hereafter Vc before any A. thaliana gene refers to the Vaccinium fruits due to their high antioxidant and blueberry orthologue gene) showed a slight decrease anti-inflammatory capacities . Deciphering the while the other showed a slight increase. Similar results mechanism of vernalization/chilling-mediated flowering were observed for CONSTANS-LIKE2 orthologues will facilitate molecular breeding of blueberry cultivars (Table 1). for low-chilling requirement. To investigate flowering Orthologues of seven major MADS-box genes in flower- responses under nonconducive conditions, functional ing pathway genes of blueberry buds, APETALA1 (AP1), analysis of a blueberry FLOWERING LOCUS T (VcFT) FRUITFULL (FUL), SUPPRESSOR OF OVEREXPRESSION gene has been conducted in highbush blueberry (Vacci- OF CONSTANS 1 (SOC1), CAULIFLOWER (CAL), FLC, nium corymbosum L.) [16–18]. Overexpression of VcFT AGL24,and SVP, were significantly up-regulated (Table 1). (about 2900-fold increase in leaf tissues) caused continu- The orthologues of Protein FD (FD), TERMINAL FLOWER ous and precocious flowering in in vitro shoots and in 1(TFL1),and LEAFY (LFY), and ACTIN-RELATED PRO- one-year old ‘Aurora’ plants . However, Overexpres- TEIN6 (ARP6) were down-regulated genes. Both VcFLC sion of VcFT was not capable of fulfilling all chilling re- and VcSVP showed a contrasting response of FLC to quirements in blueberry [17, 18]. Over 80% of the flower vernalization in A. thaliana. The down-regulated VcFD and buds in two- and three-year old VcFT-overexpressing up-regulated VcSVP support the DE VcFT results . How- plants could not bloom under greenhouse conditions ever, decreased expression of VcLFY contradicts the de- without a chilling period. To discover vernalization/chil- creased VcTFL1 and increased VcAP1, VcAGL24,and ling-responsive genes, transcriptome analyses were con- VcSOC1 expressions. The unchanged expression of VcFT ducted with blueberry flower buds. The profile of and decreased expression of VcLFY orthologues could pre- differentially expressed (DE) genes will facilitate our un- vent the flowering of chilled flower buds during plant derstanding of the role of vernalization/chilling in blue- vernalization. berry bud-break. The MADS-box transcription factor and flowering re- pressor, FLC, and FRIGIDA (FRI) are the major genes Results regulating the vernalization of A. thaliana. FRI activates Identification of DE transcripts in chilled flower buds FLC, but vernalization represses FLC . Both VcFRI and Nonchilled flower buds from the southern highbush blue- VcFLC in blueberry are present in the annotated Reftri- berry ‘Legacy’ were grown in a heated greenhouse through nity. VcFRI showed a high similarity to FRI (e = − 98) while the winter season. These buds did not flower in the fol- two VcFLC transcripts showed a lower similarity to FLC lowing spring. In contrast, flower buds fully chilled under (e = − 31). These two VcFLC transcripts were annotated to Song and Chen BMC Plant Biology (2018) 18:98 Page 3 of 13 Fig. 1 Effect of vernalization of blueberry flowering. a Nonchilled flower buds could not bloom. b Early bloom of fully chilled flower buds. c Percentages of differentially expressed (DE) genes and transcripts in chilled flower buds (in comparison to nonchilled flower buds). *DE genes including A. thaliana genes and genes annotated to other species SEP1 of A. thaliana and MADS6 of rice, respectively late-pink buds to those in chilled flower buds (hereafter (Table 1). In chilled flower buds, VcFRI did not show dif- referring to DEGs/DE transcripts in late-pink buds) re- ferential expression whereas expression of VcFLC was in- sulted in 28,000 DE isoforms, which were annotated to creased about 25-fold for the top-two VcFLC candidate 11,000 known genes. genes (Table 1). When all 26 potential VcFLC candidate genes (e < − 20) were included in the analysis, four candi- Expression of flowering pathway genes in late-pink buds dates showed a down-regulation with a fold-change of 0.6– The major DE flower pathway genes in chilled flower buds 0.8. The other 22 candidate genes showed an up-regulation were compared to those in late-pink buds (Additional file with an average of 4.9-fold (Additional file 1:Table S2).The 2: Table S1). The major flowering pathway genes VcFT, inconsistency between VcFLC and FLC response to chill- VcSOC1, VcAP1,VcFUL, VcFLC, VcSVP,and VcLFY showed ing/vernalization suggests that a different or more compli- decreased expression in late-pink buds. In A. thaliana, FT, cated mechanism is involved in vernalization-mediated SOC1, AP1, FUL,and LFY promote flowering while SVP flowering in tetraploid, blueberry plants. Additionally, all and FLC are flowering repressors [7, 21]. The expression other DEGs involved in known vernalization-mediated changes of VcFD and VcTFL1 in late-pink buds were flowering pathway were up-regulated in chilled flower buds similar to those in chilled flower buds (Table 1). The de- with the exception of VcARP6 (c49456_g2, Log FC = − 7.7) creased VcFD expression was related, at least in part, to (Additional file 2:Table S1). VcFT down-regulation. Decreased VcTFL1 expression in late-pink buds was associated with a down-regulation of Identification of DE transcripts in late-pink buds VcAP1 and VcLFY. Additionally, decreased VcSVP and Fully chilled blueberry flower buds remained dormant VcFLC expression were associated with a decrease in VcFT under chilling conditions in January until continuous expression. warm conditions in April drove dormancy release and bloom. To investigate the effect of dormancy release on Expression of MADS-box genes gene expression, RNA sequencing data was obtained Blueberry DE MADS-box and DAMs genes were identi- from late-pink buds. Comparative transcripts analysis in fied using A. thaliana MADS-box genes and Japanese Song and Chen BMC Plant Biology (2018) 18:98 Page 4 of 13 Table 1 Differentially expressed major flowering pathway genes in both chilled flower buds (CB) [vs nonchilled flower buds (NB)] and late-pink buds (LPB) (vs CB) in ‘Legacy’. LogFC for chilled buds: Log (CB/NB). LogFC for late-pink buds: Log (LPB/CB) 2 2 Arabidopsis_ID|Vc_ LogFC for LogFC for FDR for FDR for e-value_to_blast Gene_name Annotation_by_ Query_ transcript_ID chilled buds late-pink buds chilled buds late-pink buds Trinotate PmDAMs AT5G60910.1|c77424_g2_i2 1.0 2.2 6E-03 3E-19 9E-44 FUL, AGL8 AGL11_ARATH AT1G69120.1|c92021_g1_i1 −0.6 −1.8 4E-02 5E-15 1E-80 AP1, AGL7 AGL8_SOLTU AT5G60910.1|c88116_g1_i1 −0.4 0.8 4E-02 1E-10 5E-91 FUL, AGL8 AGL8_SOLTU AT1G69120.1|c88116_g8_i1 3.2 −5.4 1E-16 2E-59 4E-94 AP1, AGL7 AGL8_SOLTU AT1G69120.1|c81830_g1_i1 3.5 −2.1 2E-42 4E-24 2E-52 AP1, AGL7 AGL9_PETHY AT1G69120.1|c88116_g7_i1 3.6 −2.7 3E-33 7E-41 3E-90 AP1, AGL7 AP1_SINAL AT5G15840.1|c77980_g1_i1 −0.5 0.7 2E-03 3E-10 1E-66 CO, FG COL2_ARATH AT5G15840.1|c76265_g1_i1 2.3 −2.5 2E-09 6E-14 4E-30 CO, FG COL2_ARATH AT5G60910.1|c79125_g1_i1 −0.3 −1.1 4E-02 2E-13 2E-35 FUL, AGL8 DEFA_ANTMA AT5G60910.1|c67980_g1_i1 3.4 −4.6 7E-46 2E-70 5E-36 FUL, AGL8 DEFA_ANTMA AT5G61850.1|c96427_g2_i1 −2.4 −5.7 4E-13 4E-24 7E-102 LFY, LFY3 FLO_ANTMA AT5G61850.1|c96427_g2_i2 −2.1 −6.7 2E-13 9E-31 3E-98 LFY, LFY3 FLO_ANTMA AT1G65480.1|c84088_g2_i3 −0.9 −4.5 2E-02 7E-12 5E-95 FT HD3A_ORYSJ AT1G65480.1|c84088_g2_i5 1.1 −4.9 1E-03 9E-28 1E-94 FT HD3A_ORYSJ AT1G26310.1|c77146_g1_i1 4.9 −3.1 2E-57 9E-37 2E-56 CAL, AGL10 MADS6_ORYSJ AT2G45660.1|c89673_g3_i1 2.6 −4.0 1E-09 3E-15 3E-53 SOC1, AGL20 SOC1_ARATH AT2G45660.1|c86010_g2_i1 3.9 −4.4 1E-08 2E-09 2E-54 SOC1, AGL20 SOC1_ARATH AT2G22540.1|c91377_g1_i7 1.0 −1.6 3E-02 1E-03 5E-69 SVP, AGL22 SVP_ARATH AT2G22540.1|c90829_g2_i1 1.2 −1.2 1E-03 5E-04 4E-77 SVP, AGL22 SVP_ARATH AT4G24540.1|c91377_g1_i7 1.0 −1.6 3E-02 1E-03 1E-39 AGL24 SVP_ARATH AT5G10140.1|c77146_g1_i1 4.9 −3.1 2E-57 9E-37 2E-31 FLC, FLF, MADS6_ORYSJ AGL25 AT5G10140.1|c88116_g6_i1 4.1 −1.9 5E-91 1E-26 7E-31 FLC, FLF, SEP1_ARATH AGL25 AT4G18960.1|c77424_g2_i2 1.0 2.2 6E-03 3E-19 1E-72 MAF2, AGL31 AGL11_ARATH PmDAM1 AT2G14210.1|c80388_g1_i2 3.9 3.2 4E-02 5E-09 3E-79 MAF2, AGL31 MAD23_ORYSJ PmDAM1 AT5G60910.1|c77146_g1_i1 4.9 −3.1 2E-57 9E-37 7E-54 MAF2, AGL31 MADS6_ORYSJ PmDAM1 AT4G22950.1|c77424_g2_i2 1.0 2.2 6E-03 3E-19 1E-42 MAF3, AGL70 AGL11_ARATH PmDAM1 AT4G18960.1|c88293_g3_i1 4.7 −4.7 5E-21 5E-20 5E-59 MAF4 AG_TOBAC AT5G15800.2|c88116_g6_i1 4.1 −1.9 5E-91 1E-26 2E-87 MAF4 SEP1_ARATH PmDAM1 AT1G24260.2|c68983_g1_i1 2.5 0.0 3E-06 0E + 00 3E-42 MAF5, AGL68 . AT5G15800.2|c81830_g1_i1 3.5 −2.1 2E-42 4E-24 3E-89 MAF5, AGL68 AGL9_PETHY PmDAM1 #N/A: No differential expression apricot (Prunus mume) DAMs (PmDAMs) (Additional VcSOC1 and VcFLC could have multiple functions in file 1: Table S2; Table 1). Orthologues of 62 A. thaliana blueberry. MADS-box gene were identified in blueberry (unpub- Three DE PmDAM orthologues (VcPmDAM1, lished data). DE orthologues of 53 and 44 MADS-box VcPmDAM2, and VcPmDAM5) were identified and showed genes were detected in chilled flower buds and late-pink high similarities to four A. thaliana MADS-box genes, buds, respectively. These orthologues include the major MADS AFFECTING FLOWERING 2 (MAF2), MAF4, flowering pathway genes VcFLC, VcSOC1, VcSVP, VcAP1, MAF5 and FOREVER YOUNG FLOWER.In A. thaliana, VcFUL, VcCAL,and VcAGL24 (Table 1). The annotated MAF2, MAF4,and MAF5 are FLC paralogs. MAF2, MAF5 VcSOC1 (c86010_g2_i1) showed high similarities to 25 and FLC are down-regulated and MAF5 is up-regulated A. thaliana MADS-box genes. Similarly, the annotated during vernalization . In contrast, the blueberry ortho- VcFLC homologues were similar to 23 A. thaliana logues VcFLC, VcMAF1, VcMAF2, VcMAF4, and VcMAF5 MADS-box genes (Table 1). The results suggest that were up-regulated while VcMAF3 was repressed in chilled Song and Chen BMC Plant Biology (2018) 18:98 Page 5 of 13 flower buds (Table 1; Additional file 1: Table S2). Add- phyotohormone orthologues than the chilled buds itionally, three DE VcPmDAMs were annotated to the (Fig. 2). TheDE phytohormonegenes suggest thepo- homologues VcSOC1, VcSVP, VcAP1,and VcSEP1.The tential involvement of these phytohormones during up-regulated VcPmDAM1 homologues were the only DE chilling and flowering. orthologue in chilled flower buds. In late-pink buds, 75% Due to the tetraploid nature of ‘Legacy’, orthologues of of DE VcPmDAM1 homologues and all DE VcPmDAM5 each A. thaliana gene used for query often have more homologues were down-regulated while DE VcPmDAM2 than one homologue (Fig. 2). Thus, an average of the Fold Change was up-regulated (Additional file 1:Table S2).These blue- changes (Log ) for all the DE transcripts that berry MADS-box genes showed significant changes in re- are orthologues derived from a single A. thaliana query sponse to both chilling and flower bud breaking (Table 1). gene was used to represent the overall change of each However, during vernalization, the responses of VcFLC, phytohormone-related gene (Fig. 3). Increased expres- VcMAFs, and VcPmDAMs diverges from FLC’s response sion of ABA1, ABA2 and NINE-CIS-EPOXYCAROTE- to vernalization in A. thaliana. NOID DIOXYGENASE 3 (NCED3) in the ABA biosynthesis pathway were seen in chilled flower buds. Response of phytohormone-related genes in chilled and ABA1 and ABA2 continued to increase and NCED3 de- late-pink buds creased in late-pink buds (Fig. 3). The increased expres- For both chilled buds and late-pink buds, DE transcripts sion of these orthologues indicates a potential increase showed high similarities to the pathway genes for five in ABA biosynthesis during vernalization. Regardless of major phytohormones (Additional file 3: Table S3). Over decreased ABA1 expression, increased NCED3 expres- 50% of the DE blueberry orthologues were related to sion suggests that there is an increase in ABA biosyn- abscisic acid (ABA), ethylene, auxin, and gibberellin thesis during floral bud break (Fig. 3). (GA) genes while 25% were related to cytokinin genes DE orthologues of ethylene signaling pathway genes (Fig. 2). The late-pink bud showed more DE were all up-regulated in chilled buds and down-regulated in flowering buds (Fig. 3). These orthologues are consid- ered regulators for freezing tolerance in A. thaliana. Indole-3-acetic acid (IAA) and GA biosynthesis pathway orthologues were up-regulated in chilled flower buds and decreased in late-pink buds (Fig. 3). GIBBERELLIN 3-BETA-DIOXYGENASE 2 (GA3OX2) in the GA pathway and AUX1 in the IAA pathway were major DEGs with high expression changes. The DE ARABIDOPSIS RE- SPONSE REGULATORS (ARRs) orthologues included two A-type, two B-type and five ARR-like genes in chilled buds and flowering buds. One B-type orthologue (ARR10) was suppressed only in chilled buds (Fig. 3). Gene networks of DEGs in chilled flower buds and late-pink buds Over-represented Gene Ontology (GO) terms (P <0.05) were grouped to visualize gene networks of the annotated DE transcripts using the GOslim_Plant as the selected GO file and A. thaliana annotation as the reference. The DE transcripts were classified in 70 and 73 over-represented GO terms for chilled flower buds and late-pink buds, respectively (Fig. 4). The over-represented GO terms for chilled flower buds and late-pink buds were identical except for two GO terms (Fig. 4), suggesting that the same tran- Fig. 2 Response of phytohormone-related genes and transcripts in scripts responded to temperature changes in these buds. chilled flower buds (vs. nonchilled flower buds) and late-pink buds (vs. Thedifferencein “biological_process” was two additional chilled flower buds). a Percentage of differentially expressed (DE) orthologues of A. thaliana genes (The number of DE A. thaliana genes over-represented GO terms (GO:0007610-behavior and ÷ total number of A. thaliana genes × 100). b Percentage of DE GO:040029-regulation of gene expression, epigenetic) transcripts of transcripts of blueberry phytohormone-related genes found in late-pink buds but not in chilled flower (The number of DE transcripts ÷ total number of transcripts × 100) buds. (Fig. 4). Song and Chen BMC Plant Biology (2018) 18:98 Page 6 of 13 Fig. 3 Average fold changes (Log FC) of differentially expressed homologues for each of the phytohormone-related orthologue of A. thaliana in chilled flower buds (CB) [vs. nonchilled flower buds (NB)] and late-pink buds (LPB) [vs. chilled flower buds (CB)]. LogFC for chilled buds: Log (CB/ NB). LogFC for late-pink buds: Log (LPB/CB). a Abscisic acid biosynthesis pathway genes . b Ethylene biosynthesis and signaling pathway genes [45, 47]. c Gibberellin biosynthesis pathway genes . d Two-component ARABIDOPSIS RESPONSE REGULATORS (ARR) [49, 50]. e Auxin biosynthesis pathway genes . The bars represent standard deviation Over-represented GO terms in chilled flower buds terms related to growth, response to stress, and revealed the impact of chilling on GO terms in three reproduction (Fig. 4). Thegenenetwork basedon categories (Fig. 4). The over-represented GO terms over-represented GO terms facilitate our understand- in “biological_process” revealed that vernalization/ ing of the role DEGs in both chilled flower buds and chilling affected expression of genes in multiple GO late-pink buds (Fig. 4). Song and Chen BMC Plant Biology (2018) 18:98 Page 7 of 13 cd Fig. 4 (See legend on next page.) Song and Chen BMC Plant Biology (2018) 18:98 Page 8 of 13 (See figure on previous page.) Fig. 4 Gene networks of differentially expressed genes (DEGs) in chilled flower buds and late-pink buds. DEGs in chilled flower buds were identified in comparison to nonchilled flower buds while DEGs in late-pink buds were identified in comparison to chilled flower buds. The ontology file of GOSlim_Plants in BiNGO was used to identify over-represented GO terms (P < 0.05). a Comparison of the gene network in chilled flower buds to late-pink buds; white nodes and black edges are present in both gene networks; red nodes and edges are present only in the chilled buds; and green nodes and edges are present only in the late-pink buds. The number in each circle is a GO identity number. A gene network in chilled flower buds (b “Biological_process” c “Cellular component” d “Molecular function”). I, II, and III in b show GO terms related to stress, plant growth, and reproduction, respectively Validation of the expression of selected genes For blueberries, expressed sequence tags have been gener- In chilled flower buds and late-pink buds, qRT-PCR were ated from blueberry flower buds . However, compara- used to validate DE transcripts of VcFD, VcTFL1,and tive transcriptome analyses of different stage floral buds VcARP6 (Fig. 5). The results suggested high-reliability of have not been documented. the RNA-seq data. The roles of VcFT, VcLFY and VcARP6 in vernalization- Discussion mediated blueberry flowering Transcriptome analysis is an effective approach to study Overexpression of VcFT (expression level > 2000-fold flowering pathway genes [23, 24]. Using this approach, in leaf tissues) resulted in precocious flowering [16, DEGs in response to vernalization have been identified in 17]. However, the high expression level did not com- Japanese pear (Pyrus pyrifolia Nakai) and oriental lily . pletely reverse the need for chilling for normal plant Fig. 5 Comparison of RNA sequencing and qRT-PCR analysis of three differentially expressed genes in (a) chilled flower buds (compared to nonchilled flower buds) (b) and late-pink buds (compared to chilled flower buds). Eukaryotic translation initiation factor 3 subunit H is the internal control. Log fold-change was calculated by -ΔΔCt = − [(Ct – Ct ) – (Ct – Ct ) ]. Average Log fold-change ± standard deviation of three 2 GOI nom tissue 1 GOI nom tissue 2 2 biological replicates. Significant average fold-change determined using a Student’s t-test is denoted. An asterisk (*) indicates p <0.001 Song and Chen BMC Plant Biology (2018) 18:98 Page 9 of 13 Fig. 6 Response of major flowering pathway genes in chilled flower buds (compared to nonchilled flower buds). The relationships among the listed genes are drawn according to the diagram for A. thaliana by Fornara et al. 2010 , although not all DE genes of blueberry align perfectly with the correlations proposed for A. thaliana. All the listed genes in this diagram showed down-regulation in late-pink buds (compared to chilled flower buds) (Additional file 2: Table S1) flowering which suggests that chilling requirement is The interaction of FD and FT promotes flowering in not replaceable by VcFT manipulation. When the re- A. thaliana while TFL1 is a negative regulator of FT [27, sults were aligned to the flowering pathway of A. 28]. In this study, reduced VcFD and VcTFL1 expres- thaliana , the acquired chill did not change VcFT sions did not changed VcFT expression in chilled flower expression in blueberry flower buds (Fig. 6). This re- buds. However, increased VcFD and decreased VcTFL1 sult was also observed in woody pear but not in in late-pink buds were associated with increased VcFT. herbaceous lily . The inactive VcFT expression in TFL1 was considered a repressor of both LFY and AP1 response to chilling may be one major reason that in A. thaliana until recent evidence suggest that TFL1 chilled flower buds remain dormant prior to exposure transcription was suppressed by AP1 but promoted by to bud-breaking temperatures since VcFT increased in LFY [29, 30]. For chilled blueberry flower buds, in- late-pink buds (Table 1). creased expression of VcAP1 and decreased expression Overexpression of VcFT in leaves promoted expression of VcLFY were associated with decreased expression of of downstream genes VcSOC1, VcFUL, VcAP1, and VcTFL1. This result is consistent with the recent report VcLFY . In this study, expression of VcSOC1, VcFUL, about the interactions among these three genes . and VcAP1 were up-regulated but VcLFY was repressed During flower bud break, repressed VcTFL1 associated regardless of VcFT expression in flower buds (Table 1: with decreased VcLFY and VcAP1 supports the theory Fig. 5). The expression of VcFT-downstream genes was that TFL1 represses LFY . Although some DEGs of regulated independently of VcFT in chilled flower buds. flowering pathway genes in blueberry match the pro- Additionally, repressed VcLFY response in chilled flower posed interactions in A. thaliana , VcFD and VcTFL1 buds is similar to results observed in grapevine (Vitis vi- seem to be playing a more significant role in blueberry nifera). These results suggest that VcLFY supression (Fig. 4). may play a role in chilling-mediated flowering by main- FLC interacts with SVP and both are repressed during taining bud dormancy before bud break. vernalization in A. thaliana . In chilled blueberry Song and Chen BMC Plant Biology (2018) 18:98 Page 10 of 13 flower buds, both VcFLC and VcSVP homologues tolerance, and chilling-mediated flowering in blueberries. showed decreased expression but increased in late-pink For example, chilled blueberry buds showed higher buds (Table 1). In A. thaliana, ARP6 activates FLC, freezing tolerance than nonchilled buds and flower tis- MAF4, and MAF5, which are all repressors of plant sue . Increased DE orthologues of ethylene genes in flowering in vernalization pathway . In blueberry, the chilled blueberry buds were responsible for the en- decreased VcARP6 in chilled flower buds was not associ- hanced freezing tolerance in chilled buds while decreased ated with decreased expression of VcFLC, VcMAF4,or expression of DE ethylene orthologues in late-pink buds VcMAF5.However,increased VcARP6 (c49456_g2, Log FC reduced freezing tolerance (Fig. 3). = 8.1) was associated with an increase in these genes in late-pink buds (Table 1;Additional file 2: Table S1). Due Conclusions to ARP6’srole in A. thaliana vernalization, DE VcARP6 The changes from nonchilled to chilled and chilled to may significantly contribute to chilling-mediated flowering late-pink buds are associated with transcriptional of blueberryflowerbuds. changes in a large number of DE phytohormone-related genes and DE flowering pathway genes. The DE flower- Expression of blueberry MADS-box genes in chilled ing pathway genes suggest that orthologues of FT, FD, flower buds and late-pink buds TFL1, LFY, and MADS-box genes are the major genes The major flowering pathway genes SOC1, FLC, AP1, involved in chilling-mediated blueberry bud-break. The FUL, SVP, and AGL24 are MADS-box genes encoding DE phytohormone genes reveal the potential roles of MIKC (classical MIKC) proteins . Similar to A. thali- phytohormone genes in cold acclimation, dormancy, ana, multiple blueberry MADS-box genes are present freezing tolerance, and chilling-mediated flowering in and activated at different flowering stages. VcSOC1, blueberries. The results contribute to the comprehensive VCAP1 and VcFUL are responsive to VcFT overexpres- investigation of the chilling-mediated flowering mechan- sion . Additionally, constitutively expressed VcSOC1 ism in woody plants. or Keratin-like (K) domain of VcSOC1 promoted blue- berry flowering . In this study, the functional ortho- Method logues of FLC and AGL24 were not detected in Plant materials blueberry, suggesting that the vernalization/chilling-me- The tetraploid southern highbush blueberry ‘Legacy’ needs diated flowering pathway of blueberry is different from over 800 chilling units (CU) for normal flowering. Twelve A. thaliana. VcSVP showed differential expression in 4-year old ‘Legacy’ plants were obtained through micro- chilled and late-pink buds (Table 1). propagation of in vitro cultured shoots. All plants were In woody fruit crops, functional FLC orthologues have grown in 4-gal pots in a secured courtyard under natural not been identified. The peach DAM genes mimic FLC light conditions at Michigan State University, East Lansing, response in A. thaliana under dormancy . The DAM Michigan (latitude 42.701847, longitude − 84.482170). The genes are the orthologues of A. thaliana AGL24 and average low and high temperatures in January 2016 SVP genes [12, 33]. In this study, the DE DAMs showed were − 11 °C and − 1.8 °C, respectively (http://www.u- similarity to several MADS-box genes (VcAP1, VcSVP, sclimatedata.com/climate/east-lansing/michigan/united VcSOC1, and VcSPL3) (Table 1). Therefore, it is possible -states/usmi0248). In September 2015, six plants were that the interaction of multiple MADS-box genes moved to a heated greenhouse with a 12-h photo- co-regulates chilling-mediated flowering in blueberry as period and a minimum temperature of 23 °C in order well as other woody plants. to keep the plants from any chilling hour accumula- tion. The remaining six plants were kept in the se- Response of phytohormone genes during vernalization cured courtyard. In November, three plants were and devernalization selected from the greenhouse and 30–50 flower buds Phytohormones are involved in plant flowering and dor- were harvested per plant. These flower buds did not mancy. In A. thaliana, cold acclimation, dormancy, and receive any chilling temperatures and were labeled as plant flowering are affected by phytohormone gene ex- nonchilled flower buds. At the end of January 2016, pression [7, 34, 35]. The gibberellin pathway interacts three plants were selected from the courtyard and with the flowering pathway through SOC1 [7, 36]. Ethyl- 30–50 flower buds were harvested per plants. These ene signaling pathway genes are considered regulators flower buds experienced natural chilling conditions for freezing tolerance in A. thaliana. In this study, DE through mid-winter and were labeled as chilled flower phytohormone genes were identified in both chilled buds buds. In April, 20–30 flower buds per plant were ob- and late-pink buds of blueberry (Fig. 2; Additional file 3: tained from a second harvest of the same three plants Table S3). These DE phytohormone genes reveal their in the courtyard. These flower buds experienced nat- potential roles in cold acclimation, dormancy, freezing ural chilling conditions and began to flower in early Song and Chen BMC Plant Biology (2018) 18:98 Page 11 of 13 spring. The buds selected were at early-pink-stage Identification of the selected pathway genes and were labeled as late-pink buds. All tissues col- Representative protein sequences of selected genes of A. lected were frozen immediately in liquid nitrogen and thaliana were downloaded from the TAIR server (https:// stored at − 80 °C. Three plants for each bud type www.arabidopsis.org/tools/bulk/sequences/index.jsp). The were used as the three biological replicates for tran- retrieved sequences were used to search the blueberry scriptome analysis. transcriptome reference (refTrinity) using the tblastn command of BLAST+. The resultant transcripts that show e-value lower than − 20 were used to screen the DE tran- RNA preparation, sequencing, and de novo transcriptome script list of nonchilled floral buds. assembly The blueberry floral genes identified in the previous Total RNA of each blueberry sample (from individual study  were used to analyze flowering pathway plants) was isolated from 200 mg of bud tissues using genes. The pathway genes of major phytohormones a separate CTAB method  and was purified using (gibberellin , abscisic acid , cytokinin/Arabi- RNeasy Mini Kit (Qiagen, Valencia, CA, USA). All RNA dopsis Responsive Regulator , indole-3-acetic acid samples were purified using On-Column DNase digestion , and ethylene ) in A. thaliana were retrieved with the RNase-free DNase Set (Qiagen). The integrity of from TAIR_10 server based on published gene iden- the RNA samples was assessed using the Agilent RNA 6000 tities (Additional file 3: Table S3). Additionally, se- Pico Kit (Agilent Technologies, Inc., Germany). All samples quences of A. thaliana MADS-box proteins were had an RNA quality score greater than 8.0 prior to submis- used to analyze blueberry MADS-box genes. Percent- sion for sequencing (100-bp pair end reads) using the Illu- ages of DE phyotohormone genes were calculated mina HiSeq2500 platform at the Research Technology based either on the number of orthologues to A. Support Facility at Michigan State University (East Lansing, thaliana genes or on the number of DE blueberry Michigan, USA). The FastQC program (www.bioinforma- transcripts. tics.babraham.ac.uk/projects/fastqc/) was used to assess the quality of sequencing reads for the per base quality scores RT-PCR of DE transcripts ranging from 30 to 40. Reliability of DE genes or transcripts identified through RNA-seq was evaluated through qRT-PCR analysis of six Differential expression analysis and transcriptome selected transcripts (Additional file 4: Table S4). These annotation transcripts are from the representative DE genes in RNA-seq reads of three biological replicates for non- auxin, ethylene, cytokinin, and gibberellin pathways. chilled, chilled, and late-pink buds were analyzed. Two They have high fold changes (> 2) and sequence specifi- technical replicates were sequenced for each biological city (based on alignment result of different isoforms) for replicate and were combined together for analysis. The PCR amplification. Eukaryotic translation initiation paired reads, two sets for each biological replicate, were factor 3 subunit H was the internal control (Additional aligned to the transcriptome reference Reftrinity file 4: Table S4). developed for ‘Legacy’  and the abundance of each The same RNA samples used for RNA-sequencing, read was estimated using the Trinity command including samples of three biological replicates, were “align_and_estimate_abundance.pl”. The Trinity com- used for cDNA preparation. Reverse transcription of mand “run_DE_analysis.pl –method edgeR” was used RNA to cDNA was performed using SuperScript II re- for differential expression analysis. The DE transcripts verse transcriptase (Invitrogen, Carlsbad, CA, USA). The with false discovery rate (FDR) values below 0.05 were resulting cDNA of one micro gram of RNA was diluted used for further analyses. Comparison of transcriptome (volume 1: 4) in water and a 1 μl/sample (25 ng) was in chilled to nonchilled flower buds resulted in DE used for PCR reactions. transcripts/genes in chilled flower buds. Comparison of Integrated DNA Technologies, Inc. (https://www.idtdna. transcriptome in late-pink buds to chilled flower buds com/Primerquest/Home/Index) provided the online tool resulted in DE transcripts/genes in late-pink buds. DE for primer design and synthesized the primers (Additional transcripts in chilled buds were annotated using Trinota- file 4: Table S4). Three qRT-PCR analyses were performed te_v2.0 (https://trinotate.github.io). on an Agilent Technologies Stratagene Mx3005P (Agilent Technologies, Santa Clara, CA) using the SYBR Green sys- Gene network construction tem (Life Technologies, Carlsbad, CA). In each 25 μl reac- Annotated transcripts were imported to Cytoscape 3.5.0 tion mixture, 25 ng of cDNA, 200 nM of primers, and under BiNGO’s default parameters with selected ontol- 12.5 μl of 2× SYBR Green master mix were included. The ogy file ‘GOSlim_Plants’ and selected organism A. thali- reaction conditions for all primer pairs were 95 °C for ana [39, 40]. 10 min, 40 cycles of 30 s at 95 °C, 60 s at 60 °C and 60 s at Song and Chen BMC Plant Biology (2018) 18:98 Page 12 of 13 72 °C, and followed by one cycle of 60 s at 95 °C, 30 s at Received: 5 January 2018 Accepted: 15 May 2018 55 °C and 30 s at 95 °C. The specificity of the amplification reaction for each primer pair was determined by the melt- ing curve. 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BMC Plant Biology – Springer Journals
Published: May 31, 2018
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