How fall dormancy benefits alfalfa winter-survival? Physiologic and transcriptomic analyses of dormancy process

Abstract

Background: Fall dormancy and freezing tolerance characterized as two important phenotypic traits, have great effects on productivity and persistence of alfalfa (Medicago sativa L.). Despite the fact that one of the most limiting traits for alfalfa freezing tolerance in winter is fall dormancy, the interplay between fall dormancy and cold acclimation processes of alfalfa remains largely unknown. We compared the plant regrowth, winter survival, raffinose and amino acids accumulation, and genome-wide differentially expressed genes of fall-dormant cultivar with non-dormant cultivar under cold acclimation.

Results: Averaged over both years, the non-dormant alfalfa exhibited largely rapid regrowth compared with fall dormant alfalfa after last cutting in autumn, but the winter survival rate of fall dormant alfalfa was about 34-fold higher than that of non-dormant alfalfa. The accumulation of raffinose and amino acids were significantly increased in fall dormant alfalfa, whereas were decreased in non-dormant alfalfa under cold acclimation. Expressions of candidate genes encoding raffinose biosynthesis genes were highly up-regulated in fall dormant alfalfa, but down-regulated in non-dormant alfalfa under cold acclimation. In fall dormant alfalfa, there was a significantly down-regulated expression of candidate genes encoding the glutamine synthase, which is indirectly involved in the proline metabolism. A total of eight significantly differentially expressed transcription factors (TFs) related to CBF and ABRE-BFs were identified. The most up-regulated TFs in fall dormant alfalfa cultivar were ABF4 and DREB1C.

Conclusions: Fall dormant alfalfa drastically increased raffinose and amino acids accumulation under cold acclimation. Raffinose-associated and amino acid-associated genes involved in metabolic pathways were more highly expressed in fall dormant alfalfa than non-dormant alfalfa under cold acclimation. This global survey of transcriptome profiles provides new insights into the interplay between fall dormancy and cold acclimation in alfalfa.

Keywords: Fall dormancy; Medicago sativa; RNA-Seq; Raffinose and amino acids; qRT-PCR.

Fig. 1

Growing states 25 days after the last cutting (a), natural plant height (NPH) in September of both 2014 and 2015 (b) and average winter survival rate in two consecutive years (c) of alfalfa cultivars selected for contrasting fall dormancy. Fall dormant (FDT) alfalfa (Maverick, FD = 1) is a dormant and winter hardy cultivar, while non-dormant (NDT) alfalfa (CUF101, FD = 9) is a non-dormant and non-winter hardy cultivar. Data are presented by mean ± standard error. Lowercase represents significant differences at the 5% level of probability between the two cultivars

Fig. 2

Raffinose accumulation in taproots of cold acclimating alfalfa cultivars with contrasting fall dormancy and winter hardiness. Fall dormant (FDT) alfalfa (Maverick, FD = 1) is a dormant and winter hardy cultivar, while non-dormant (NDT) alfalfa (CUF101, FD = 9) is a non-dormant and non-winter hardy cultivar. Data are presented by mean ± standard error. Lowercase represents significant differences at the 5% level of probability between the two cultivars

Fig. 3

Concentrations of amino acids: Threonine (a), Glycine (b), Histidine (c), Arginine (d) and Proline (e) in roots of fall dormant (FDT) and non-dormant (NDT) alfalfa cultivars under cold acclimation. FDT alfalfa (Maverick, FD = 1) is a dormant and winter hardy cultivar, while NDT alfalfa (CUF101, FD = 9) is a non-dormant and non-winter hardy cultivar. Data are presented by mean ± standard error. Lowercase represents significant differences at the 5% level of probability between the two cultivars

Fig. 4

Summary of the KEGG pathways of the assembled unigenes in fall dormant and non-dormant alfalfa cultivars under cold acclimation. Blue bar represents the unigene numbers of each pathway

Fig. 5

Histogram of the eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) functional classification of all of the alfalfa root unigenes. Out of the 23,470 de novo assembled unigenes, 18,617 (79.32%) were annotated and grouped into 25 categories

Fig. 6

Fall dormant (FDT)-VS-non-dormant (NDT) differentially expressed genes (DEGs). DEGs were filtered using false discovery rate (FDR) ≤ 0.001 and |Log2Fold-Change| ≥ 2 as thresholds. The group 1 represents FDT alfalfa cultivar, and group 2 represents NDT alfalfa cultivar. The blue, red and gray spots represent the up-regulated, down-regulated DEGs and genes without obvious changes in FDT and NDT alfalfa cultivars under cold acclimation, respectively

Fig. 7

Histogram of GO Enrichment Analysis of differentially expressed genes in fall dormant (FDT) and non-dormant (NDT) alfalfa under cold acclimation. The x-axis represents the GO functional categories, grouped into Biological Process, Cellular Component and Molecular Function. The y-axis indicates the enrichment degree significance P-value calculated by hypergeometric distribution in each term. The red horizontal line represents P-value = 0.05

Fig. 8

Comparison between the gene expression ratios obtained from RNA-Seq data and qRT-PCR of 15 differentially expressed genes (DEGs) in fall dormant and non-dormant alfalfa cultivars under cold acclimation. The x-axis indicates the names of 15 DEGs, and the y-axis indicates the relative gene expression levels