Integrative transcriptomic and metabolomic analyses reveal the flavonoid biosynthesis of Pyrus hopeiensis flowers under cold stress

Low temperature is among the most restrictive factors to limit the yield and distribution of pear. Pyrus hopeiensis is a valuable wild resource.PCA showed that P. hopeiensis had strong cold resistance. In this study, the mRNA and metabolome sequencing of P. hopeiensis flower organs exposed to different low temperatures were performed to identify changes of genes and metabolites in response to low-temperature stress. A total of 4 851 differentially expressed genes(DEGs) were identified. Trend analysis showed that these DEGs were significantly enriched in profiles 19, 18, 7, 14, 1, 4 and 11. And the KEGG enrichment analysis showed that the DEGs in profile 18 were significantly enriched in flavone and flavonol biosynthesis. Besides, the expressed trends as well as GO and KEGG functional enrichment analyses of DEGs under cold and freezing stress showed significantly difference. Analyses of flavonoid-related pathways indicated that flavonoid structural genes had undergone significant changes. Correlation analysis showed that b HLH and MYB TFs may affect flavonoid biosynthesis by regulating structural gene expression. And PhMYB308 and PhMYB330 were likely candidate repressors of flavonoid biosynthesis by binding to a specific site in bHLH proteins. In total, 92 differentially accumulated metabolites(DAMs) were identified in P. hopeiensis flowers including 12 flavonoids. WGCNA results showed that coral 1, pink and brown 4 modules were closely associated with flavonoids and 11 MYBs and 15 bHLHs among the three modules may activate or inhibit the expression of 23 structural genes of flavonoid biosynthesis. Taken together, the results of this study provided a theoretical basis for further exploration of the molecular mechanisms of flavonoid biosynthesis and cold resistance of P. hopeiensis flower organs and our findings laid a foundation for further molecular breeding in cold-resistant pear varieties.

Identification and characterization of CBF/DREB1-related genes in Populus hopeiensis

The dehydration-responsive element-binding factor （DREB） is a plant-specific family of transcription factors and plays an important role in plant response and adaptation to abiotic stress. In the present work, two highly similar CBF/DREB l-like genes, designated as PhCBF4a and PhCBF4b, were identified from Populus hopeiensis. These two genes contain all conserved domains known to exist in other CBF/DREB1 genes. In the AP2 domain, there is only one different amino acid residue between PhCBF4a and PhCBF4b, alanine or valine, a nonpolar amino acid, suggesting that PhCBF4a and PhCBF4b may have similar DNA binding ability. Their expression is induced by water-loss treatment and their expression patterns are similar. Moreover, with a genomic DNA as template, the presence of the same bands in PCR products as those in expression pattern analysis indicated that PhCBF4a and PhCBF4b exist in the genome ofP. hopeiensis. Their detailed functions are discussed and will need further study.

Differential expression of PeSOS1, PeNhaD1 and PeNHX1 genes between the salt-tolerant Populus euphratica and salt-sensitive P. hopeiensis calli

Populus euphratica Oliv. is of high salinity tolerance and used as a model species for investigating molecular mechanisms of trees＇ responses to salt stress. In the work presented here we found that calli of P. euphratica grew more rapidly and accumulated less Na＋, but more K＋, under salt stress than those of salt-sensitive poplar, Populus hopeiensis. Different types of Na＋/H＋ antiporters （SOS1, NhaD1 and NHX1） were isolated from P. euphratica; all of these genes have been shown to play important roles in plant salt tolerance mechanism in previous studies. Expression profiles of these three genes were compared between P. hopeiensis and P. euphratica in the presence and absence of salt stress by real-time PCR. The three genes were induced in both P. euphratica and P. hopeiensis by salt. Transcript levels of PeNHX1 were lower in P. euphratica than in P. hopeiensis under 150 mM NaCl stress. In addition, transcript levels of PeNhaD1 were lower, while PeSOS1 were higher in P. euphratica than in P. hopeiensis under both stressed and unstressed conditions. The results indicated that P. euphratica up-regulates different genes and consistently maintains both effluxes of Na＋ and high K＋ levels. Our data suggests that differences in gene expression patterns may contribute to the dif-ference in salt tolerance between these two poplars.

Construction and Characterization of cDNA Library from Water-Stressed Plantlets Regenerated in vitro of Populus hopeiensis

In order to isolate and clone water-stress-responsive genes, total RNA was extracted from water-stressed plantlets regenerated in vitro of Populus hopeiensis using a QIAGEN RNeasy Plant Mini Kit. CDNA, synthesized by LD-PCR with the SMART cDNA Library Construction Kit, was in vitro packaged into a phage λTriplEx2 vector. The resulting primary library and amplified library have a titer of 1.68×10^6 and 1.69×10^9 pfu·mL^-1 respectively. The combination ratio reached 98.8% and the average size of inserts was about 800 bp. In addition, the percentage of inserted fragments （〉400bp） was approximately 90%. The results indicate that a cDNA library has been successfully constructed.