Exploration And Functional Characterization Of Stress-Related Genes From Antarctic Moss Pohlia Nutans

Antarctic terrestrial ecosystems are cold, dry and low-nutrient environments, with drastic temperature fluctuations and paradoxically low levels of water availability. Due to this harsh climate, plant growth is largely limited to the ice-free areas around the coastal fringe of continental Antarctica, which are generally small and isolated. In these regions, cryptogams (mosses and lichens) and two vascular plant species constitute the predominant (though sparse) terrestrial vegetation. Therefore, these Antarctic plants offer the exceptional opportunities for discovering new functional genes and gaining novel insights into the mechanisms of stress resistance, as well as genetic evolution, under extreme conditions. However, the mechanism of bryophytes adaptation to environmental stress remains unclear. In this study, the transcriptome analysis of the Antarctic moss, Pohlia nutans, in response to cold or salt stress using the Illumina ultra high-throughput sequencing technology were performed for discovering the stress-relevant genes as well as the mechanism of stress acclimation. In addition, the roles of the JAZ protein and flavonoid hydroxylase in Antarctic moss Pohlia nutans adapting to environmental stress were also investigated. We believe that these results will contribute to clarify the genetic mechanism of Antarctic moss in the processes of acclimatizing and adapting the rigorous climate in Antarctic continent and accelerate practical exploitation of Antarctic moss gene resources in improving the stress tolerance in agricultural crops.1. Next-generation sequencing-based transcriptome profiling analysis of Pohlia nutans reveals insight into the stress-relevant genes in Antarctic mossGenome-wide characterization of the Antarctic moss Pohlia nutans transcriptome is essential for clarifying the role of stress-relevant genes in Antarctic moss adapting to the extreme polar environment. High-throughput Illumina sequencing was firstly used to analyze the gene expression profile of P. nutans under cold treatment (4℃,6h) or salt treatment (200mM NaCl, 1h). The original data sets are available at the NCBI Short Read Archive (SRA) with the accession number SRA051595. This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GACA00000000. A total of 93488 unigenes, with an average length of 405 bp, were obtained; 18688 transcripts were longer than 500 bp. Gene annotation showed that 16781 unigenes had significant similarity to known functional protein-coding genes, most of which were annotated using the GO, KOG and KEGG pathway databases. Global profiling of the differentially expressed genes revealed that 3796 unigenes were significantly upregulated after cold treatment, while 1405 unigenes were significantly down regulated. Meanwhile, global profiling of the differentially expressed genes revealed that 820 unigenes were significantly upregulated after salt treatment, while 350 unigenes were significantly down regulated. In addition,816 receptor-like kinases and 1309 transcription factors were identified from P. nutans. Finally, we found that several receptor-like kinases, transcription factors and key enzymes of secondary metabolism may play an important role in Antarctic moss resistant to stress. This overall survey of transcripts and stress-relevant genes can contribute to understanding the stress-resistance mechanism of Antarctic moss and will accelerate the practical exploitation of the genetic resources.2. The roles of the JAZ protein in Antarctic moss Pohlia nutans adapting to environmental stress and its relevant mechanismsSalt stress is a major constraint for many crop plant. Jasmonates (JAs) and abscisic acid (ABA) are both critical players that involved in mediating salt tolerance in plants. Previous study showed that jasmonate ZIM-domain (JAZ) transcriptional repressors are key regulators of jasmonate hormonal response. Jasmonate promotes interaction between JAZ proteins and the SCFcon ubiquitin ligase, leading to JAZ degradation via the 26S proteasome. However, little is known about the roles of JAZ proteins in jasmonate-mediated adaptation of plants to salinity stress.PnJAZ9 is induced by various abiotic stresses in Antarctic moss Pohlia nutans. In the present study, we report the identification and characterization of JAZ protein from Antarctic moss. The real-time PCR analysis showed that the expression of Antarctic moss PnJAZ9 were both up-regulated after the treatments of cold (4℃), high salinity (200 mM NaCl),20%PEG, ABA (50μM) and MeJA (50μM).Multiple sequence alignment showed that PnJAZ9 was homologous to JAZs of other species. Phylogenetic analysis was used to investigate the relationship between PnJAZ9 and other JAZ proteins from A. thaliana. Phylogenetic tree revealed that PnJAZ9 protein was most closely related to sequences of AtJAZ9 (TIFY7). The location of the fluorescent signal produced by the transient expression of a PnJAZ9::GFP fusion in Arabidopsis mesophyll protoplasts indicated that PnJAZ9 were mainly localized in nucleus and only a little in cytoplasm.JAZ proteins act as negative regulators for JA signaling in Arabidopsis. Overexpression of PnJAZ9 inhibited the jasmonic acid signaling pathway. After 100 μM MeJA treatment, Arabidopsis PnJAZ9 overexpression (OE) lines have higher germination rate than the WT plants. The real-time PCR analysis confirmed that the transcription of jasmonic acid pathway genes MYC2 and VSP1 were significantly lower, in the AtOE lines. Finally, the yeast two-hybrid assays showed that either the full-length of JAZ9 (03918) or the JAZ9 (03918)ΔNΔC could interact with AtMYC2, AtMYC3, and AtMYC4, respectively.Overexpression of Antarctic moss JAZ9 (03918) increases transgenic Arabidopsis resistance to salt stress during germination and early developmental stages. In the presence of 100 mM NaCl, the germination rates of the AtOE1, AtOE2 and AtOE3 lines were 47.06%,44.71% and 56.47% (6 days’ seed germination), whereas for wild-type Col-0, the germination proportion was almost equal to zero.JAZ9 (03918) also contributes to the osmotic tolerance of transgene Arabidopsis. The seed germination rate of AtOE seedlings was significantly higher than that of the wild type under 0.2 or 0.3 M mannitol stress conditions (6 days’ seed germination).Overexpression of PnJAZ9 (03918) to improve plant resistance to ABA stress during germination and early developmental stages. In the presence of 0.25 μM ABA, the germination rates of the AtOE1, AtOE2 and AtOE3 lines were 94.87%,84.64%, 92.31% respectively (6 days’ seed germination), whereas for wild-type Col-0, the germination proportion was only 25.64%.Overexpression of Antarctic moss JAZ9 (03918) increases transgenic Arabidopsis resistance to auxin IAA and glucose during germination and early developmental stages, which minics the phenotypes of abi4 or abi5 mutants. These results demonstrate that, in the ABA signaling pathway, the function of a certain key positive transcription factor such as ABI4 or ABI5 was possibly inhibited by PnJAZ9 in transgenic plants.Overexpression of JAZ9 (03918) inhibited the gene expression of ABA pathway related to the seed germination and seedling growth in transgenic plants. Real-time PCR analysis showed that the transcription levels of AtABI4, AtABI5, ABF3 and RD29a were substantially down-regulated in the AtOE lines when compared the WT plants.Therefore, we speculated that PnJAZ9 protein might physically interact with a certain positive regulator of ABA signaling and mediate the complex network of interactions between jasmonates and ABA signaling pathways under salt stress.3. Phylogenetic analysis of the flavonoid hydroxylase from Antarctic moss and their expression under different abiotic stressFlavonoids are ubiquitous plant secondary metabolites with diverse functions in defense, UV protection, auxin transport inhibition, allelopathy, and flower coloring. Flavonoids may have served a significant role in the early evolution of plants shifted from aquatic to terrestrial environments. The hydroxylation pattern of flavonoids determines their color, stability, and antioxidant capacity. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of cytochrome P450 protein (P450) family, the flavonoid 3’-hydroxylase (F3’H) and flavonoid 3’,5’-hydroxylase (F3’,5’H).However, they are still not well documented in lower plants such as bryophytes. We report the identification of gene encoding F3’H, F3’,5’H from Antarctic moss Pohlia nutans and their transcriptional regulation under different stress conditions. Totally, sixteen cDNAs were isolated from P. nutans by RT-PCR and RACE techniques, all of which were predicted to code for F3’Hs or F3’,5’Hs based on their annotations of Blast results. Amino acid alignment showed that they possessed the featured conserved domains of flavonoid hydroxylase, including proline-rich "hinge" region, EXXR motif, oxygen binding pocket motif, heme binding domain and substrate recognition sites. Phylogenetic analysis indicated that moss F3’Hs and F3’5’Hs were highly conserved and have independent evolution from the monocots, dicots and ferns. Real-time PCR analysis revealed that the expression profiling of flavonoid hydroxylase genes was influenced by diverse abiotic stresses including cold, salinity, drought or UV-B radiation and plant hormone abscisic acid (ABA) or jasmonic acid (JA) treatment. Since 3’,4’,5’-hydroxylated flavonoid-derivatives may serve a multitude of functions, including antioxidant activity and UV filters, the evolution and expression profile of flavonoid hydroxylase reflect the adaptive value of Antarctic moss in the acclimation of polar environment.