A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought-tolerance mechanisms

Abstract

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms. Ammopiptanthus mongolicus (Leguminosae) is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions; however, its drought-tolerance mechanisms remain poorly understood. Here, we report the assembly of a reference-grade genome for A. mongolicus, describe its evolutionary history within the legume family, and examine its drought-tolerance mechanisms. The assembled genome is 843.07 Mb in length, with 98.7% of the sequences successfully anchored to the nine chromosomes of A. mongolicus. The genome is predicted to contain 47 611 protein-coding genes, and 70.71% of the genome is composed of repetitive sequences; these are dominated by transposable elements, particularly long-terminal-repeat retrotransposons. Evolutionary analyses revealed two whole-genome duplication (WGD) events at 130 and 58 million years ago (mya) that are shared by the genus Ammopiptanthus and other legumes, but no species-specific WGDs were found within this genus. Ancestral genome reconstruction revealed that the A. mongolicus genome has undergone fewer rearrangements than other genomes in the legume family, confirming its status as a "relict plant". Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed, both under normal conditions and in response to polyethylene glycol-induced dehydration. Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress, suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A. mongolicus. Ectopic expression of AmERF2, an ethylene response factor unique to A. mongolicus, can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants, demonstrating the potential for application of A. mongolicus genes in crop improvement.

Keywords: Ammopiptanthus mongolicus; cuticular wax; drought tolerance; ethylene; genome evolution; genome sequencing.

Figure 1

Morphology, karyotyping, Hi-C interaction heatmap, and genomic landscape of A. mongolicus. (A) A population of A. mongolicus grown in a field on Alxa Plateau, Inner Mongolia, China (July 2015). (B) Karyotyping of A. mongolicus by fluorescence in situ hybridization (FISH) using probes from repeat sequences of rDNAs (green, 18S rDNA; red, 5S rDNA). Scale bar, 5 μm. (C) Assembly of A. mongolicus chromosomes using Hi-C data. Bin size was set to 200 kb when plotting the Hi-C interaction heatmap. (D) Genomic landscape of A. mongolicus. i, intragenomic collinear blocks between or within chromosomes, with the most dramatic one highlighted in pink; ii, nine pseudo-chromosomes (unit, Mb); iii, GC content in each 500-kb bin of the genome; iv, gene density along the chromosomes, expressed as the number of genes per bin; v, repeat ratio of each bin; vi, number of simple short repeats (SSRs) in each bin; vii, percentage of LTR_Copia among TEs; viii, percentage of LTR_Gypsy among TEs. All density or ratio information was determined in non-overlapping 500-kb windows.

Figure 2

Synteny, orthology, and polyploidization analyses of A. mongolicus and closely related legume species. (A) Intergenomic syntenic blocks identified among A. mongolicus, M. truncatula, and G. max. The connections that are specific to chromosome 1 of M. truncatula are highlighted. The purple ribbons indicate syntenic blocks shared by M. truncatula and A. mongolicus, and the red and green ribbons denote those shared by M. truncatula (barrel medic) and G. max (soybean); each syntenic block in M. truncatula usually corresponds to two blocks in G. max. (B) Four-fold synonymous third-codon transversion (4DTv) rates of paralogous gene pairs in intragenomic collinear regions of selected plant genomes. The numbers I–III indicate WGD or triplication events during evolution. I, γ event (whole-genome triplication) shared by eudicots ∼130 million years ago (mya); II, WGD event shared by legumes 58 mya; III, WGD event specific to the genus Glycine 13 mya. Amo, A. mongolicus; Ana, A. nanus; Cca, C. cajan; Gma, G. max; Mtr, M. truncatula. (C) 4DTv distributions calculated from orthologous gene pairs in intergenomic collinear regions between different species. (D) Numbers of orthologous gene groups among selected plants.

Figure 3

Inference of divergence times and MRCAs of A. mongolicus. (A) A phylogenetic tree inferred from single-copy orthologous genes of legume species. Numbers on the nodes represent the divergence times from the present (mya). The divergence time between G. max and P. vulgaris (20.1 [19.0–21.0] mya) was used as the standard for calibration (Zheng et al., 2016). Polyploidization events are marked by red stars on the tree. The background colors of different lineages represent different legume clades. A. thaliana was used as the outgroup taxon. (B) Reconstructed ancestral genomes of six legume species. The phylogenetic tree and timescale were redrawn from (A). WGD, whole-genome duplication (2×, tetraploidy; 3×, hexaploidy); CRE ratio, completely rearranged endpoint; EA, estimated accuracy; AEA, accumulated estimated accuracy. The numbers of shuffling events (fissions and fusions) are labeled beside each branch. The shuffling events marked by an asterisk (∗) in peanut were calculated against ancestor 1.

Figure 4

Classification of repetitive sequences in legumes and evolution of TEs in A. mongolicus. (A) Percentages of transposable elements (TEs): Ty1/Copia, Ty3/Gypsy, other TEs (unknown types), non-TE repeats (microsatellites, low-complexity sequences, rRNAs, snRNAs, etc.), and non-repeat sequences in the genomes of the respective species. (B) Insertion times of long-terminal-repeat retrotransposons (LTR-RTs), calculated as T = K/(2r), where T is insertion time, K is the number of base substitutions per site between the two LTRs of an LTR-RT, and r is the synonymous nucleotide substitution rate, expressed as the number of synonymous mutations per site per year. For legumes, r is 7e−9 (Jing et al., 2005). (C and D) Clade-level classification of LTR-RTs using TEsorter (https://github.com/zhangrengang/TEsorter). (C) Ty1/Copia. (D) Ty1/Gypsy. (E) Classification of Ty1/Copia into sub-lineages. (F) Classification of Ty3/Gypsy into sub-lineages. The phylogenetic trees in (E) and (F) were constructed from the RT domains of the LTR-RTs.

Figure 5

Genes involved in cuticular wax biosynthesis and transport in A. mongolicus and their responses to simulated drought stress. Heatmaps show the expression levels of genes involved in the cuticular wax biosynthesis and transport pathways upon drought treatment simulated by polyethylene glycol (PEG) at 0, 2, 24, and 48 h in leaves and roots of 2-month-old A. mongolicus plants. Gene expression values were normalized as log10(FPKM + 1). LACS, long-chain fatty acid acyl-CoA synthetase; FAE, fatty acid elongase; VLFCA, very-long-chain fatty acids; FAR, fatty acyl-CoA reductase; CER, eceriferum; WSD, wax ester synthase and diacylglycerol acyltransferase; MAH, midchain alkane hydroxylase.

Figure 6

Ethylene-related genes play important roles in stress resistance of A. mongolicus. (A) Both transcriptomic and qRT–PCR analyses indicated marked induction of the ethylene biosynthetic genes EFE1/ACO and EFE2/ACO. Data are from 2-month-old A. mongolicus plants treated with 15% PEG for 24 h. The housekeeping gene AmEIF was used as an internal control in qRT–PCR analysis, and the significance of changes was evaluated by Student’s t-test (∗∗p < 0.01). (B) Expression profiles of genes related to ethylene biosynthesis and signaling in leaves of 2-month-old A. mongolicus plants during PEG treatment. The expression levels of each gene at different treatment time points are displayed in the heatmaps by converting FPKM values into Z scores. ACS, ACC synthase; ACO, ACC oxidase; ETR, ethylene receptor; EIN, ethylene insensitive; EIL, EIN3-like; ERF, ethylene response factors; ERT, ethylene-responsive transcriptional coactivator-like protein; EGY, ethylene-dependent gravitropism-deficient and yellow-green-like; AP2, ethylene-responsive element-binding protein 2; ERS, ethylene response sensor 1. (C) Microsynteny analysis of collinear regions of the ERF2 gene and its flanking regions among A. mongolicus, A. nanus, M. truncatula, C. cajan, G. max, and V. vinifera. Blue and green boxes represent gene loci in the chromosome regions. Gray ribbons represent orthologous gene relationships among the six species. A red ribbon highlights the ERF2 locus in A. mongolicus and A. nanus. ALA2, Aminophospholipid ATPase 2 (a homolog of Arabidopsis AT5G44240); TAT2, Tyrosine aminotransferase 2 (a homolog of AT5G36160). (D) Western blot validation of 35S::AmERF2-6Myc transgenic Arabidopsis plants using an anti-Myc antibody. Ponceau S staining of the Rubisco large subunit band was used as a loading control. WT, wild-type (Col-0); OE1, OE2, and OE3, three independent AmERF2-overexpressing lines. (E) Drought stress assay of 35S::AmERF2-6Myc transgenic A. thaliana plants. Ten-day-old seedlings were transplanted to soil and subjected to drought treatment for 16 days without watering in a 22°C growth chamber with a 16-h light/8-h dark photoperiod. After the drought treatment, the plants were rewatered for 2 days to allow for recovery.