In this study, using RNA-Seq technology, a ‘Suli’ pear cDNA library and four DGE libraries (from samples collected on Nov. 15, Dec. 15, Jan. 15, and Feb. 15, 2010–2011) were constructed and used to screen DEGs during dormancy. Surprisingly, we obtained 69,393 unique sequences, and of which 51,448 could be annotated, in total 48,725 genes, including 14,531 clusters and 34,194 singletons. Until 23 May, 2012, there were only 4,339 expressed sequence tags (ESTs) and 2,837 nucleotide sequences of Pyrus plants deposited in GenBank. Several recent studies have used traditional EST analyses to study dormancy in other species. Horvath et al. (2008) and Mazzitelli et al. (2007) identified nearly 1,000 genes and 327 ESTs associated with bud dormancy in leafy spurge and raspberry, respectively, using cDNA microarrays
[5, 22]. Leida et al. (2010) identified nearly 400 ESTs associated with bud dormancy in peach by constructing four subtracted-cDNA libraries
. These ESTs participated in different metabolic pathways related to photoperiod, temperature, circadian clocks, water, energy, reactive oxygen species, and hormones
[5, 22, 46]. To our knowledge, this is the first report to use RNA-Seq technique to identify large numbers of genes involved in different metabolic pathways in pear bud dormancy. Compared with traditional EST analyses, RNA-Seq was less expensive, more efficient, and allowed faster gene discovery in bud dormancy studies.
Through RNA-seq analysis, we found that the numbers and expression profiles of DEGs differed at different times during dormancy. A total of 1,978, 1,024, and 3,468 genes were differentially expressed between Nov. 15 and Dec. 15, Dec. 15 and Jan. 15, and Jan. 15 and Feb. 15, respectively. These results showed that the number of DEGs was fewer in the endodormant stage (Nov. 15 and Dec. 15) than in the ecodormant stage (Jan. 15 and Feb. 15), increased with endodormancy-release to reach a maximum by Feb. 15. Hedley et al. (2010) reported that gene activity was lowest in the early stages of dormancy and peaked around the time of bud break in blackcurrant (Ribes nigrum L.)
. By analyzing KEGG pathways, we found DEGs that participated in several different pathways. Some pathways (such as starch and sucrose metabolism, circadian rhythm, and flavonoid biosynthesis) had been previously correlated to bud break in other species
[5, 17, 22, 46], and some like phenylpropanoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, zeatin biosynthesis, ether lipid metabolism, endocytosis, and glycerophospholipid metabolism were associated with bud break for the first time in this study. These data may suggest new research directions for understanding bud dormancy.
Some of the genes found in this work had been previously identified in other perennial plants. The DAM genes, widely described in perennial species such as leafy spurge
, Japanese apricot
, and Japanese pear
, are candidates for internal factors controlling endodormancy. In this study, we also found two DAM genes, and phylogenetic analysis revealed that CL 1161.contig2 was more closely related to PpMADS13-1 of Pyrus pyrifolia, whereas CL 1161.contig5 was more similar to PpMADS13-2 of Pyrus pyrifolia.Changes in the expression of CL 1161.contig2 and CL 1161.contig5 decreased with endodormancy release in lateral flower buds were consistent with the findings of earlier work comparing PpMADS13-1 and PpMADS13-2 gene expression in lateral leaf buds of Japanese pear
, PpDAM5 and PpDAM6 in lateral vegetative buds and lateral flower buds of peach
[10, 11], and all PmDAMs (PmDAM1
PmDAM6) in lateral vegetative buds of Japanese apricot
. Our study, along with previous studies, suggested that DAM genes might play significant roles in the regulation of bud dormancy in ‘Suli’ pear.
The accumulation of dehydrin (DHN) is known to be associated with freezing tolerance in plants
. Recent studies have reported that the accumulation of DHNs in woody plants correlates with seasonal transitions in dormancy and cold acclimation stages during winter
[16, 48], but characterizations of DHN genes expressed during dormancy are limited. Yakovlev et al. (2008) found a reduction in the transcript levels of most of the 15 DHNs that they cloned as Norway spruce neared bud-burst
. Garcia-Bañuelos et al. (2009) reported that transcripts of apple DHN were highly expressed in bark and bud tissues when the plant was dormant and cold hardy in midwinter, but were not expressed in early spring when cold hardiness was lost and buds were growing
. Recently, several studies have identified DHN genes that were activated by ABA and C- repeat binding factor (CBF) in response to abiotic stresses
[14, 50–52]. Intriguingly, in leafy spurge, ABA levels were elevated during endodormancy but dropped following the transition to ecodormancy
. Horvath et al. (2008) found that CBF genes involved in cold regulated were up-regulated during the transition from para- to endo-dormancy
. In the present study, one gene (CL 9148) encoding dehydrin showed significantly higher expression during the transition from endo- to eco-dormancy; thereafter, the expression level of this gene rapidly decreased, as indicated by DGE analysis and Q-PCR data. Based on previous studies, we speculated that DHN genes may act as signals and offer some protection for ‘Suli’ pear after the end of endodormancy, when pear often encounters unfavorable environmental conditions, such as cold. Therefore, more attention should be paid to ABA and CBF, which activate DHN genes, in future studies of transcriptional regulation related to the pear dormancy process.
Generally, sugar transport is thought to occur via H+/sugar symports that depend on a pH gradient generated by a plasma membrane H+-ATPase
. Gevaudant et al. (2001) examined expression of the four H+-ATPase genes and reported that the levels of three H+-ATPase gene mRNAs increased, whereas the level of one H+-ATPase gene decreased in vegetative buds of peach trees after dormancy release
. Mazzitelli et al. (2007) demonstrated that the plasma membrane H+-ATPase gene was highly expressed during the dormancy transition
. Our results showed that the expression of a plasma membrane H+-ATPase gene (CL 1729) was up-regulated in pear buds during the endodormant maintenance period, down-regulated during endodormancy-release, and then up-regulated again. The expression patterns of plasma membrane H+-ATPase in ‘Suli’ pear were different from those of peach and raspberry buds, perhaps due to species-level or tissue-level differences.
In the present study, some genes encoding galactinol synthase (CL 9475), plastocyanin A (CL 1227), chlorophyll A/B binding protein (CL 7129, CL 514, CL 4948, CL 9178, CL 9961, CL 3279), and S-adenosylemethionine decarboxylase (CL 2122) were differentially expressed. Of these, chlorophyll A/B binding protein and S-adenosylemethionine decarboxylase were previously reported in other perennial plants
[46, 55]. The expression levels of these genes changed significantly during the dormancy process. Thus, these genes may play roles in the regulation of bud dormancy in ‘Suli’ pear.
In addition, differentially-regulated transcription factors were identified in this study, including AP2 (unigene 1554), Zn-finger (unigene 16353), NAC (CL 7187), WRKY (unigene 19749), SPL (CL 3589), and bHLH (CL 12548). Of these, AP2, Zn-finger, and NAC were previously reported in leafy spurge
 and peach
. Based on DGE analysis, the expression levels of the genes encoding these transcription factors significantly changed during dormancy in ‘Suli’ pear.
Although the molecular mechanisms associated with dormancy transitions in pear trees remain largely unknown, the present transcriptome analysis provided valuable information that could facilitate future studies on the detailed molecular functions of genes related to pear bud dormancy.