AnDHN, a Dehydrin Protein From Ammopiptanthus nanus, Mitigates the Negative Effects of Drought Stress in Plants

Dehydrins (DHNs) play crucial roles in a broad spectrum of abiotic stresses in model plants. However, the evolutionary role of DHNs has not been explored, and the function of DHN proteins is largely unknown in Ammopiptanthus nanus (A. nanus), an ancient and endangered legume species from the deserts of northwestern China. In this study, we isolated a drought-response gene (c195333_g1_i1) from a drought-induced RNA-seq library of A. nanus. Evolutionary bioinformatics showed that c195333_g1_i1 is an ortholog of Arabidopsis DHN, and we renamed it AnDHN. Moreover, DHN proteins may define a class of proteins that are evolutionarily conserved in all angiosperms that have experienced a contraction during the evolution of legumes. Arabidopsis plants overexpressing AnDHN exhibited morpho-physiological changes, such as an increased germination rate, higher relative water content (RWC), higher proline (PRO) content, increased peroxidase (POD) and catalase (CAT) activities, lower contents of malondialdehyde (MDA), H2O2 and O2–, and longer root length. Our results showed that the transgenic lines had improved drought resistance with deep root system architecture, excellent water retention, increased osmotic adjustment, and enhanced reactive oxygen species (ROS) scavenging. Furthermore, the transgenic lines also had enhanced salt and cold tolerance. Our findings demonstrate that AnDHN may be a good candidate gene for improving abiotic stress tolerance in crops.Key Message: Using transcriptome analysis in Ammopiptanthus nanus, we isolated a drought-responsive gene, AnDHN, that plays a key role in enhancing abiotic stress tolerance in plants, with strong functional diversification in legumes.

Population Genetics and Evolution Analysis Reveal Diversity and Origin of Ammopiptanthus in China.

Background：Elucidating and revealing the population genetic structure, genetic diversity and recombination is essential for understanding the evolution and adaptation of species. Ammopiptanthus, which is an endangered survivor from the Tethys in the Tertiary Period, is the only evergreen broadleaf shrub grown in Northwest of China. However, little is known about its genetic diversity and underlying adaptation mechanisms. Results：Here, 111 Ammopiptanthus individuals collected from fifteen natural populations in estern China were analyzed by means of the specific locus amplified fragment sequencing (SLAF-seq). Based on the single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) detected by SLAF-seq, genetic diversity and markers associated with climate and geographical distribution variables were identified. The results of genetic diversity and genetic differentiation revealed that all fifteen populations showed medium genetic diversity, with PIC values ranging from 0.1648 to 0.3081. AMOVA and Fst indicated that a low genetic differentiation existed among populations. Phylogenetic analysis showed that NX-BG and NMG-DQH of fifteen populations have the highest homology，while the genetic structure analysis revealed that these Ammopiptanthus germplasm accessions were structured primarily along the basis of their geographic collection, and that an extensive admixture occurred in each group. In addition, the genome-wide linkage disequilibrium (LD) and principal component analysis showed that Ammopiptanthus nanus had a more diverse genomic background, and all genetic populations were clearly distinguished, although different degrees of introgression were detected in these groups. Conclusion：Our study could provide guidance to the future design of association studies and the systematic utilization and protection of the genetic variation characterizing the Ammopiptanthus.

Genome-Wide Identification and Expression Analyses of AnSnRK2 Gene Family under Osmotic Stress in Ammopiptanthus nanus

Sucrose non-fermenting-1 (SNF1)-related protein kinase 2’s (SnRK2s) are plant-specific serine/threonine protein kinases and play crucial roles in the abscisic acid signaling pathway and abiotic stress response. Ammopiptanthus nanus is a relict xerophyte shrub and extremely tolerant of abiotic stresses. Therefore, we performed genome-wide identification of the AnSnRK2 genes and analyzed their expression profiles under osmotic stresses including drought and salinity. A total of 11 AnSnRK2 genes (AnSnRK2.1-AnSnRK2.11) were identified in the A. nanus genome and were divided into three groups according to the phylogenetic tree. The AnSnRK2.6 has seven introns and others have eight introns. All of the AnSnRK2 proteins are highly conserved at the N-terminus and contain similar motif composition. The result of cis-acting element analysis showed that there were abundant hormone- and stress-related cis-elements in the promoter regions of AnSnRK2s. Moreover, the results of quantitative real-time PCR exhibited that the expression of most AnSnRK2s was induced by NaCl and PEG-6000 treatments, but the expression of AnSnRK2.3 and AnSnRK2.6 was inhibited, suggesting that the AnSnRK2s might play key roles in stress tolerance. The study provides insights into understanding the function of AnSnRK2s.

Antifreeze protein from Ammopiptanthus nanus functions in temperature-stress through domain A

Temperature stress restricts plant growth and development. Antifreeze protein (AFP) can improve plants antifreeze ability. In our previous study, the AnAFP gene cloned from Ammopiptanthus nanus was confirmed to be an excellent candidate enhancing plant cold resistance. But, AnAFP protein shared similar structures with KnS type dehydrins including K, N and S domains except ice crystal binding domain A. Here, we generated AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, and transformed them into ordinary and cold sensitive strains of E. coli, and Arabidopsis KS type dehydrin mutant to evaluate their function. Expression of AnAFPΔA decreases cold and heat tolerance in E. coli, meanwhile, AnAFP enhances heat tolerance in Arabidopsis, suggesting that domain A is a thermal stable functional domain. AnAFP, AnAFPΔA and AnAFPΔS localize in whole cell, but AnAFPΔK and AnAFPΔN only localizes in nucleus and cytoplasm, respectively, exhibiting that K and N domains control localization of AnAFP. Likewise, K domain blocks interaction between AnAFP and AnICE1. The result of RT-qPCR showed that expression of AnAFP, AnICE1 and AnCBF genes was significantly induced by high-temperature, indicating that the AnAFP is likely regulated by ICE1-CBF-COR signal pathway. Taken together, the study provides insights into understanding the mechanism of AnAFP in response to temperature stress and gene resource to improve heat or cold tolerance of plants in transgenic engineering.

Genome-Wide Identification and Analysis of the WRKY Gene Family in the Xerophytic Evergreen Ammopiptanthus nanus

The WRKY family of transcription factors plays important roles in plant growth and responses to biotic and abiotic stresses. Ammopiptanthus nanus, the only evergreen broadleaf shrub endemic to the desert and semi-desert regions of northwestern China, is highly tolerant to various stresses. However, a systematic study of WRKY proteins in A. nanus has not been reported. In the present study, we identified 63 WRKY genes in the A. nanus genome. Based on the conserved WRKY domains, zinc finger structures, and phylogenetic relationships in their encoded proteins, we classified these genes into four groups (group I–IV) and several subgroups (subgroup IIa–IIe). Conserved motif analysis showed that all motifs except those within the WRKY domains had a subfamily-specific distribution. Expression analysis revealed that the AnWRKY genes had distinct expression patterns, with some being more responsive to herbivory and drought stresses than others. Based on the results of our current study, we speculate that AnWRKY40 and AnWRKY48 are positive regulators of the plant’s response to drought and herbivory stresses, respectively. Our results indicate that AnWRKY genes contribute to the ability of A. nanus plants to withstand harsh, dry conditions.