Overexpression of Head date 1 gene (Hd1): an adaptation of antarctic hairgrass to guano input from Macronectes giganteus colonies of Antarctica

The Antarctic biodiversity, beyond the species composition, also comprises interactions between fauna and flora. M. giganteus, is one of the species that occupy the antarctic ice-free areas for reproduction. The moss Sanionia uncinata (Hedw.) Loeske and Deschampsia antarctica Desv. gress, common in Antarctica, with other species make up vast green formations and is associated with breeding areas of seabirds. These sites are large deposits of guano, because a large amount of birds those gather in colonies. Due to this large supply of guano, the soil becomes a deposit of minerals, mainly of nitrogen available in the form of ammonium and nitrate. The problem is that not all plant species tolerate high quantities of these substances so different plant species show trends in the mechanisms of tolerance to stress by ammonium, which have been proven at the molecular level. The aim of this study was to investigate the influence of breeding colonies of seabirds on plant populations in the South Shetland Islands, Antarctica, from a molecular perspective. From the analysis of the collected samples, using the RNA-Seq and qRT-PCR approach was possible to identify a single gene differential and significantly expressed in D. antarctica. The LOC_Os06g16380 gene among the sampled treatments (control, 1m and 10m), showed higher expression coming 1m near breeding areas of M. giganteus. Our results suggest that Hd1 is associated with the plants stress related to guano input since that soil analysis demonstrated a higher concentration of mineral nitrogen available near of breeding areas of seabirds.


Introduction
The Antarctic terrestrial ecosystems are characterized by extreme abiotic conditions when compared with other continents. Around 86% of Antarctic is covered by ice, the air temperatures are low (average between the -10 ° C to -20 ° C in coastal areas) and high winds and snowstorms cover the region which also presents a short summer season (about two months).
Altogether those features hinder the establishment of terrestrial biodiversity. Besides the environmental constraints the animals influence during the short austral summer, increase the accumulation of large quantities of minerals in the soil (mainly Nitrogen) during the breeding season (Barcikowski et al., 2001;Alberdi et al., 2002;Lee et al., 2008).
Deschampsia antarctica Desv. (Figure 1a) is one of the two native flowering plant species found in Antarctica and is the only hairgrass inhabiting the region. This species is abundant mainly in the South Shetlands Islands occupying almost the entire coastal area of the Maritime Antarctic, often occurring in the vicinity of bird colonies (Edwards and Lewis-Smith et al., 1988;Parnikoza et al., 2011).  (Mendonça et al., 2011;Neufeld et al., 2015). This moss species occurs mainly in environments with constant water supply, like those close to the drain lines coming from defrosted water (Lud et al., 2002;Tojo et al., 2012). Considering that the spatial distribution of vegetation in Antarctica is closely linked to marine animals, understanding how changes in the seabird communities may affect terrestrial communities becomes necessary to determine the degree of interaction between plants and seabirds (Barcikowski, 2001). It is expected that one of the key elements is the input of In the nitrogen cycle, ammonium is replaced by an important role in living organisms that receive the nitrogen supply to its basic functions, and seabirds are responsible for large amounts of this issue (Zhu et al., 2011). This especially occurs in the Antarctic coastal regions and sub Antarctic places where the ocean has great productivity resulting from large numbers of seabirds breeding over Antarctica every year (Riddick et al., 2012).
The main nitrogen source come from the energy flow from the fish-and crustaceans-based diet of penguins, petrels and gulls (Copello et al., 2008;Hebert et al., 2009;Petry et al., 2008Petry et al., , 2010. Although the average NH4 + concentrations of soils are often 10-1000 times lower than those of NO3 - (Marchner, 1995), the difference in soil concentrations does not necessarily reflect the uptake ratio of each N source. Indeed, the role of NH4 + in plant nutrition has probably been under estimated, because most plants preferentially take up NH4 + when both forms are present. Ammonium requires less energy for uptake and assimilation than nitrate, mainly because NO3has to be reduced prior to assimilation (Bloom et al., 1992). Optimal plant growth is however usually achieved when N is supplied in both forms (Bloom et al., 1999). On the other hand, the excess of ammonium in soils, may adversely affect the growth, productivity, tolerance to drought or frost and resistance to diseases and insects, leading to long-term changes in species composition. The excess of ammonium also causes soil acidification and eutrophication (Fangmeier et al., 1994;Wilson et al., 2004), changing the atmospheric composition and land nutrient supply (Blackall et al., 2007).
Abiotic stress caused by cold, drought and increased salinity (e.g. excess of ammonium), generated a selective pressure for plants to develop mechanisms that would enable their development in environments where these factors reach extremes. This adaptive process resulted in a gene pool facing to a successful survival strategy for climate change, especially those of unexpected and extreme level (Lee et al., 2013). Lee et al. (2008), using the Expressed Sequences Tag (EST) approach, generated by large scale single-pass sequencing of cDNA clones, found novel genes of D. antarctica, related to the differential response of the species abiotic stress in Antarctic environment, demonstrating the strong selective pressure under the Antarctic plants, but the action of these genes have not experimentally clarified.
Thus, the biological question involving the breeding influence under the adaptation of plants to the Antarctic environment is which genes (if so) are differentially expressed when those plants are exposed to the higher nitrogen compounds input, as guano. Within this work we attempted to answer this question by using the transcriptome-based analysis sativa annotation of genes were downloaded from Phytozome V10.1 (phytozome.jgi.doe.gov). Statistics and graphical analysis of the differential expression on the transcripts detected were performed in the program R (version 3.1.1) with CummeRbund extension (Goff et al., 2012).
qRT-PCR analysis. The candidate gene selected from the RNA Sequence analysis had the expression patterns analyzed by qRT-PCR using SYBR® Green detection system (Applied Biosystems®, California, USA). The quantitative variation between different samples was evaluated using the comparative CT method (ΔΔCT), and the data of target gene expression, normalized to the level of expression of TIP41-like genes, used as standard reference in internal control (endogenous) (Caldana, et al., 2007, Jain et al., 2006. The qRT-PCR reactions were performed in triplicate techniques from: 2.0 ul 10x buffer; 1.2 uL of 50 mM MgCl2; 0.4 uL of 5 mM dNTPs; 1 ul of each oligonucleotide (10 mM); 0.05 uL Taq Platinum -DNA polymerase (5 U / uL); 2 uL of Syber Green (1x); 0.4 uL ROX, the first tape 1 ul cDNA (diluted 1: 5) and water to make a final volume of 20 uL. The cycling conditions used for amplification were 50 °C for 2 minutes, 95 °C for 10 minutes, and 40 cycles of 95 ° C for 30 seconds, 60 °C for 1 minute and 72 °C for 1 minute, occurring reading fluorescence in this last step. Finally, a cycle of 72 °C for 5 minutes.
Soil mineral nitrogen analysis. In each GHG sampling event, sSoil samples were taken for analysis of mineral nitrogen (nitrate -NO3 and ammonium -NH4 by Kjeldahl distillation), determined according to Tedesco et al. (1995).

Results and Discussion
Differential expression analysis. As expected the gene expression analysis distribution across treatments were distinct for each species tested. The moss species presented lower differential expression detected within treatments ( Figure   2a). Furthermore Kernel distribution of FPKM scores for overall genes detected across the Antarctic hairgrass D. antarctica indicated a similar distribution of transcripts within the treatments (Figure 2b), meaning that only this plant was affected by the guano from the Southern Giant Petrel. Following the kernel analysis Cufflink tool was applied in order to determine which genes were differentially expressed in S. uncinata and D. antarctica were identified a single significant gene expressed and only for the grass species. The LOC_Os06g16380 gene had its expression in D. antarctica, and among the three treatments (control, 1m and 10m), with higher expression close to the M. giganteus colonies (1m treatment). The moss species does not show a significant gene expressed for both treatments.

Confirmation of differentially expressed genes by qRT-PCR analysis.
Having found the LOC_Os06g16380 gene differentially expressed in D. Antarctica by RNA-Seq, the next step was to perform a qRT-PCR on total mRNA to confirm the expression patterns. A higher and significant concentration of the target gene fragments was observed in D. Antarctica at 1m of distance from M. giganteus colonies in both sampling places (Copacabana and Stinker Point) (Figure 3). At 5m and 10m of distance from the breeding colonies, the expression patterns were similar to each other but smaller than at 1m of distance ( Figure 3). A higher amount of ammonium in the soil near the breeding colonies (e.g. 1m) in both sites (Copacabana -1.8 mg/dm 3 and Stinker Point -1.5 mg/dm 3 ; Figure 4) was detected, suggesting the influence of nitrogen in the differential expression of LOC_Os06g16380 gene.  The LOC_Os06g16380 gene was previously found and described by Zhang et al. (2012) as belonging to a region related with the Heading date gene I (Hd1) found in rice. This gene is an orthologous gene of CONSTANS gene identified in Arabidopsis model species (Takakashi & Shimamoto, 2011), and regulates the expression of florigen gene Hd3, responsible for controlling the mechanism involved with the transition from the vegetative to the reproductive phase in flowering plants (Kojima et al., 2002;Sonoda et al., 2003;Park et al., 2008). In rice, the Hd1 gene is reported as the major quantitative trait locus (QTL) controlling response to photoperiod (Yano et al., 2000) that determines the regional and seasonal adaptation of rice crops (Zhang et al., 2012). This trait conferring short or long vegetative phase, susceptible to use in breeding program to increase the yield in distinct latitudes (Takakashi & Shimamoto, 2011;Zhang et al., 2015). However, the pleiotropic effect from Hd1 expression on the productivity/yield and growth in rice was already observed (Zhang et al., 2012), although this gene did not affect these characteristics, and thus expression was not detected in roots. Our results in D. antarctica suggests that the higher input of NH4 + close to the seabirds colonies induced an increase in the LOC_Os06g16380 expression and that this region can be related with the capacity of this grass species to respond to high contents of ammonium in soil and even be related to the transport of these mineral in grass roots. These findings corroborate the theory of guano input from sea mammals and birds enables in which nutrients can change the chemical and organic characteristics of the soil and in turn can determine the spatial distribution of D. antarctica (Smykla et al., 2007;Park et al., 2012).
A review of works that investigate the effect of seabirds on continental plant communities in tropical and subtropical regions carried out by Ellis (2005), analyzed fifty seven studies, which indicate evidence that seabird nests affect the biomass of seabirds. Aboveground plants and significantly alters population richness and plant community composition. These data are confirmed for the plant communities of ice-free areas, in which a significant number of species, mainly mosses and lichens, are classified as ornithocoprophiles or ornithocoprophobes (Victoria et al., 2013;Schmitz et al., 2018).
Studies on impacts of seabird colonies on plant communities in ice-free areas of Antarctica are rare, for example Zwolicki (2015), which does not assess stress but the occurrence of plant populations grouped in six zones that were classified based on the occurrence of Prasiola crispa (Lightfoot) Menegh, Deschampsia antarctica Desv., Colobanthus quitensis (Kunth.) Bart. and a lichen identified as Usnea sp.
The higher and significant concentration of the target gene fragments was observed in D. Antarctica at 1m of distance from M. giganteus colonies in both sampling places (Copacabana and Stinker Point) (Figure 3). This can be explained by the fact that the passage of nitrate (NO3-) and ammonium (NH4+) through the cell wall of leaf epidermis cells and after its entry into the cell, nitrate can be reduced to nitrite (NO2-), in the cytosol, through the enzyme nitrate reductase (RN) and, soon after, converted to ammonium (NH4+) in the plastid, through the enzyme nitrite reductase (RNi). Ammonium is then incorporated into amino acids by the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT), forming glutamine (GLN), glutamate (GLU) and other amino acids and their metabolites. Therefore, the greater proximity of the nests, the concentration of guano is greater mainly due to the fact that the strong winds that occur in Antarctica take the guano that is released on the plant populations. (Crawford, 1995;Bredemeier et al., 2000;Victoria et al., 2013;Schmitz et al., 2018).
Regarding S. uncinata, no significant results relating to differential gene expression in the three treatments suggest that this is not an ideal moss to this type of analysis. This is probably due to their morphological plasticity associated with its great ecological amplitude (Gimingham & Smith, 1971;Hebel et al., 2012.), being the mechanisms that control these adaptations remains unknown up to date. The ecological amplitude of S. uncinata is because this species does not suffer a significant effect from the presence of seabird colonies, occurring near or far from them. Among many other species of mosses, such as Polytrichastrum alpinum Hedw. are considered ornithocoprophiles because they occur in areas that have guano input, whereas Polytrichum juniperinum Hedw. It is classified as ornithocoprophobic because it does not occur in areas that have guano input (Victoria et al., 2013, Schmitz et al., 2018. Another ecological characteristic that explains the non-expression of the studied gene is the fact that the rhizoids of S. uncinata do not have contact with the soil. This species grows mainly on rock fragments forming mats, which is very characteristic in the plant communities of ice-free areas. Its use in this research is because it is the species with the highest Index of Ecological Importance (IES), and with large areas of occurrence (Raven & Edwards, 2001;Victor & Dolan, 2012).