The Regulation of Adaptation to Cold and Drought Stresses in Poa crymophila Keng Revealed by Integrative Transcriptomics and Metabolomics Analysis

Abstract

Poa crymophila Keng is highly adaptable to long-term low temperature and drought conditions, making it a desirable foraging grass of the Qinghai-Tibet Plateau. Here, the widely targeted metabolomics and comparative transcriptome analyses were utilized for the discovery of metabolites and genes in P. crymophila in response to cold and drought stresses. P. crymophila were exposed to -5°C for 24 h and recovered to 22°C for 48 h, as well as drought for 10 days followed by re-watering for 1 day. In total, 779 metabolic features were assigned to metabolites and 167,845 unigenes were generated. Seventeen compounds showed significant up-regulation (variable importance in project >1) under both stresses in the metabolic profiling, mainly annotated as carbohydrates, flavones, and phenylpropanoids. The genes which were positively correlated with these metabolites were assigned to pathways (sucrose-starch, raffinose, phenylpropanoid, and flavone metabolism) using the Mapman software package. Alpha-amylase, beta-fructofuranosidase, and sugar transport genes degraded the glucose and starch to small molecule sugars for the purpose of osmotic adjustment and to provide more energy for the growth of P. crymophila in an adverse environment. The induction of cinnamoyl-CoA reductase (CCR) and the MYB gene as well as the sharp increase in schizandrin, a kind of lignan, showed that this likely has the closest connection with the tolerance to both stresses. Four significantly induced flavone compounds are probably involved in reducing oxidative damage. Our results indicated that activation of the phenlypropanoid pathway plays the primary role in P. crymophila adapting to harsh environments. This study showed the mechanism of P. crymophila responding to both cold and drought stresses and showed the discovery of a new biological regulator against stresses.

Keywords: Poa crymophila Keng; carbohydrates; cold and drought; flavone; phenylpropanoids; transcriptome; widely targeted metabolomics.

Figure 1

Significantly differentially expressed genes (DEGs) in Poa crymophila Keng cv. Qinghai transcriptomes in response to cold and drought stress and recovery from the two stresses.

Figure 2

The significantly up-regulated metabolites under both cold and drought stresses.

Figure 3

The correlated genes which positively responded to cold and drought were mapped to secondary metabolism.

Figure 4

The correlated genes which positively responded to cold and drought were mapped to sucrose-starch pathway in primary metabolism.

Figure 5

The established networks between metabolites and their correlated genes. (A) The network graph of primary metabolism. (B) The network graph of secondary metabolism.

Figure 6

qRT-PCR verifying the accuracy of RNA-seq. Sixteen unigenes were selected for the qRT-PCR assay and results are from three biological replicates. Beta-actin gene from Poa crymophila Keng transcriptome was served as reference gene. The fold change (FC) of gene expression between treated samples and control sample was calculated using 2^−ΔΔCt, and ${\text{Log}}_2^{\text{FC}}$ = –ΔΔCt. the ${\text{Log}}_2^{\text{FC}}$ of each unigene in qRT-PCR and transcriptome was compared to verify the stability and accuracy of the RNA sequencing. (A) The fitting line of ${\text{Log}}_2^{\text{FC}}$ of each unigene in qRT-PCR and transcriptome. (B) The expression change of each unigene in qRT-PCR (–ΔΔCt) and transcriptome (${\text{Log}}_2^{\text{FC}}$).

Figure 7

Molecular structures of p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, and schizandrin.

Figure 8

Phenylpropanoid pathway. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase. The monolignol biosynthetic branch: CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; C3H, 4-coumarate 3-hydroxylase; COMT, caffeic acid o-methyltransferase; CCoAOMT, caffeoyl-CoA o-methyltransferase; F5H, ferulate-5-hydroxylase. The flavonoids biosynthetic branch: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase. Dash arrows refer to unspecified steps of a particular metabolic pathway. The colored boxes indicate metabolites that are significantly induced by both types of stress.

Figure 9

The response of Poa crymophila Keng to cold and drought stresses. TAC, tricarboxylic acid cycle; CCR, cinnamoyl-CoA reductase; TF, transcription factor; MAPK, mitogen-activated protein kinase.