Deschampsia antarctica É.Desv.
Creators: name: Cuadra, Pedro
name: Guajardo, Joselin
name: Carrasco-Orellana, Cristian
name: Stappung, Yazmina
name: Fajardo, Víctor
name: Herrera, Raúl
Publisher: name: Zenodo
Year: 2020
Subjects: Biodiversity
Taxonomy
Plantae
Tracheophyta
Liliopsida
Poales
Poaceae
Deschampsia
Deschampsia antarctica

description: 2.1. Analysis of CHS gene from D. antarctica
DaCHS full-length cDNA was obtained using the partial sequence of an EST as the template, designing primers for 5′- and 3′-RACE-PCR reactions. A sequence of 1741 bp with a poly (A) tail, containing 143 bp and 407 bp of 5′- and 3′ -UTR sequences respectively, was obtained as well as an ORF of 1191 bp with a deduced polypeptide sequence of 397 amino acids. This sequence was deposited in GenBank (accession number MG766286). Analysis of the predicted DaCHS protein demonstrated the typical conserved structural features among CHSs. The mature protein has an estimated molecular weight of 43.53 kDa (pI 6.44). Important residues for the active site motif can be observed in DaCHS (C167, F218, H306 and N339). Moreover, the active site motif is formed by a variety of residues typical of the chalcone synthase family, which are also observed in the Deschampsia protein sequence. It has two domains: the N-terminal domain ranges from amino acid 8 to 231 and the C-terminal domain ranges from amino acid 241 to 391. Multiple alignment analysis with fourteen other CHS sequences of representative monocotyledons showed a highly conserved pattern among sequences (Fig. 1). All these findings suggest that DaCHS belongs to the CHS family.
2.2. Phylogenetic analysis of DaCHS
Fifteen other CHS amino acid sequences were considered for phylogenetic analysis of DaCHS, including proteins from O. sativa L, Z. mays L. and H. vulgare L. According to these results, DaCHS can be grouped together with H. vulgare CHS 1 (Fig. 2). Acetate kinase A from Streptococcus equi was used as the outlier.
2.3. DaCHS homology model
Analysis of the DaCHS 3D-structure showed one pocket in the middle zone of the protein. Substrates interact with the catalytic amino acids in this location. It contains the typical residues C167, F218, F268, H306 and N339. The protein is composed of 12 α helices, 8α helices 310, 13β sheets and 23 loops (Fig. 3). The best coincidence in the pairwise alignment analysis was found with O. sativa template (4YJY chain A). Both sequences have a high homology index (92% sequence identity) and highly conserved secondary structures (Fig. 4). A similar degree of conservation is observed with protein sequences from other monocotyledons (Suppl. Fig. 1) This high degree of structure conservation suggests that the spatial volume and amino acid position inside pockets of these two structures are similar (Fig. 5A). Structural alignment analysis and an RMDS value of 0.25 corroborated this assumption and confirmed a high degree of structural conservation among both superposed proteins (Fig. 5A). MDS analysis showed more fluctuations in DaCHS’ structural stability at the beginning of the trajectory. It reaches a plateau after 1.5 ns. Similar behaviour was observed in template 4YJY. However, template values were always lower than the DaCHS model (Fig. 5B). RMSF plotting of Cα-atom residues was generated in order to examine the flexible regions of model and template during the MD simulations. Residues in the protein loops showed the greater fluctuations (Fig. 5C).
2.4. UV-B treatments
Accumulation of transcripts was determined on UV-B irradiated plants. Non-irradiated plants were used as controls. After 3.5 days of UV-B irradiation the accumulation of transcripts was higher in the treated group compared to non-irradiated plants. The maximum difference between both groups was reached after 7 days of treatment (Fig. 6).
2.5. Chemical analysis
Analysis by LC-DAD and LC-MS showed several peaks at 3.41, 5.57, 7.05, 7.50, 8.41, 8.98, 11.71 and 11.94 min, among others. LC-MS analysis showed signals at m/z 328.8 and 283, among others (see Supplementary data 1. and Supplementary data). The peak at 11.94 min observed in the LC-DAD traces was tentatively identified as luteolin by comparing its retention time with that of a reference standard. LC-MS traces showed a signal at m/z 328.8 which was attributed to the base peak in the negative ion mode of tricin molecule. A tiny signal at m/z 283 was similarly assigned to luteolin after some fragment losses of two H. EIMS spectra showed the molecular ion for two peaks at m/z 330 and 286, one as for a trihydroxy-dimethoxyflavone and a tetrahydroxyflavone, respectively. The UV spectra indicated a 5,7,4′- oxygenated system with a hydroxyl group at C-5. The bathochromic shift (5 nm) observed after addition of NaOAc in both compounds suggests a free hydroxyl at C-7. Classical purification and spectroscopic analyses led to the isolation from the EtOH extract of two major compounds. These were identified by comparing their UV, EIMS and NMR spectral properties to literature values as 5,7,4′-trihydroxy-3′,5′-dimethoxyflavone (tricin 1), and 3′,4′,5,7-tetrahydroxyflavone (luteolin 2).
Apart from these compounds there is no evidence of any flavonoid glucosides in the EtOH extract (the absence of signals at m/z 466 and at m/z 510 for luteolin and tricin glucosides respectively).
3. Discussion
In this study the cloning and characterization of the Chalcone synthase gene from D. antarctica (DaCHS1) is reported. Four amino acid residues were identified in the active motif site of DaCHS (C167, F218, H306 and N339). The presence of these residues in the active site of proteins tallies with previous reports (Zhou et al., 2011; Go et al., 2015; Wannapinpong et al., 2015). However, in these studies the main role at the catalytic site has been assigned to just three residues (Jez et al., 2000; Wang et al., 2017b; Sanmugavelan et al., 2018). These three residues are involved in decarboxylation and condensation reactions. Furthermore, phenylalanine plays a more structural role in the formation of the active site by giving specificity to CHS (Sun et al., 2015). On the other hand, DaCHS1 showed a variety of conserved residues in the Chalcone synthase family (P141, G166, G166, L217, D220, G265, P307, G308, G309, G338, G377, P378 and G379). DaCHS protein contains two domains: the N-terminal domain ranges from amino acid 8 to 231 and the C-terminal domain ranges from amino acid 241 to 391, as previously reported (Wannapinpong et al., 2015).
The phylogenetic tree grouped both sequences: DaCHS1 and HvCHS 1 in the same branch. CHS from other monocotyledons was used in this analysis because monocotyledons and dicotyledons species are constituted by two different clades that form a monophyletic group (Nakatsuka et al., 2003; Zhou et al., 2011). A high degree of identity for all sequences was indicated by the predicted DaCHS1 protein. Furthermore, the estimated molecular weight is within the range for this protein but DaCHS exhibited a slight difference from the closest HvCHS1 (Zhou et al., 2011; Wannapinpong et al., 2015).
The DaCHS1 expression profile was analysed in response to UV-B radiation. After seven days of UV-B exposure, a high level of transcripts was observed in the irradiated plants compared to controls. These results agreed with those reported in Astragalus membranaceus. In this study, CHS and other enzymes of the flavonoid pathway were expressed at their maximum level after 8 days of UV-B treatment (Xu et al., 2011). On the other hand, a rapid response within the first 3 h was reported from D antarctica subjected to UV-B radiation (Köhler et al., 2017). These findings may be regarded as a plant response to this harmful radiation. Although the enzymatic content was not measured in our study, the increased transcriptional activity and accumulation of transcripts presumably results in more traduced products and/or an increased content of end-metabolites. This latter assumption may be partially supported by the presence of two flavones, luteolin 1 and tricin 2, identified in the EtOH extract of the Patagonian D. antarctica. The presence of flavonoids in the specialised metabolite profile can be seen as part of a plant response to UV-B radiation (Rozema et al., 2001, 2002; Berg et al., 2008). Chalcones, flavonols, anthocyanins and flavones have often been involved in many ecological interactions and their production at increased levels has been regarded as a response to environmental stress (Flück, 1963; Hoffmann et al., 1983; Di Ferdinando et al., 2012; Petrussa et al., 2013; Mouradov and Spangenberg, 2014; Daniels et al., 2015). One of the most important roles of flavonoids might be that of absorbing compounds, screening the UV solar radiation as a natural filter. Given their absorption spectra (λ max 270–346 nm), these compounds are likely to be involved in protecting plants against UV-B radiation. As pointed out by Kunz et al. (2006), plants counteract cell damage by attenuating the received UV-B dose through the accumulation of UV-absorbing secondary metabolites. Another piece of experimental evidence relating to the role of phenylpropanoids in plant UV-B protection is found in Burchard et al. (2000). The contribution of these metabolites to the epidermal shielding of rye primary leaves was studied. Good correlation between the epidermal UV-A and UV-B absorbance and the content of flavonoids was found. All these plant responses to UV-B radiation are based on the absorption bands of hydroxycinnamic esters within the UV-B range. Tricin 2 was previously reported in Antarctic specimens of D. antarctica (Webby and Markham, 1994) and in other Deschampsia species (Harborne and Williams, 1976). The other metabolite (luteolin 1) is a well-known flavone which has previously been isolated from many other plant families (Ulubelen et al., 1979; Barberan et al., 1985; Williams et al., 1996; Zhang et al., 2007).
Flavonoids are derived from the phenylpropanoid pathway after deamination of aromatic amino acids (phenylalanine or tyrosine). A critical role is played by Chalcone synthase, the key enzyme for the synthesis of flavonoids and anthocyanins (Beritognolo et al., 2002). The expression of the gene is modulated by UVR8 when Arabidopsis plants are subjected to sunfleck (Moriconi et al., 2018), although this gene is not modulated by a MYB transcription factor (Salvatierra et al., 2013). Furthermore, UVR8 has been involved in several plant protective responses under UV-B inducing growth, accumulation of flavonoids and DNA repair (Kliebenstein et al., 2002; Brown et al., 2005; Heijde and Ulm, 2012; Singh et al., 2014). All of this evidence reported in the literature is in agreement with the results described here.
description: Published as part of Cuadra, Pedro, Guajardo, Joselin, Carrasco-Orellana, Cristian, Stappung, Yazmina, Fajardo, Víctor & Herrera, Raúl, 2020, Differential expression after UV-B radiation and characterization of chalcone synthase from the Patagonian hairgrass Deschampsia antarctica, pp. 1-10 in Phytochemistry (112179) (112179) 169 on pages 2-6, DOI: 10.1016/j.phytochem.2019.112179, http://zenodo.org/record/8293717