Ecology and potential functions of plant-associated microbial communities in cold environments

ABSTRACT Complex microbial communities are associated with plants and can improve their resilience under harsh environmental conditions. In particular, plants and their associated communities have developed complex adaptation strategies against cold stress. Although changes in plant-associated microbial community structure have been analysed in different cold regions, scarce information is available on possible common taxonomic and functional features of microbial communities across cold environments. In this review, we discuss recent advances in taxonomic and functional characterization of plant-associated microbial communities in three main cold regions, such as alpine, Arctic and Antarctica environments. Culture-independent and culture-dependent approaches are analysed, in order to highlight the main factors affecting the taxonomic structure of plant-associated communities in cold environments. Moreover, biotechnological applications of plant-associated microorganisms from cold environments are proposed for agriculture, industry and medicine, according to biological functions and cold adaptation strategies of bacteria and fungi. Although further functional studies may improve our knowledge, the existing literature suggest that plants growing in cold environments harbor complex, host-specific and cold-adapted microbial communities, which may play key functional roles in plant growth and survival under cold conditions.


INTRODUCTION
Cold environments are characterized by average daily air temperatures below 5 • C throughout the year and are located in specific areas of the Earth's biosphere, such as alpine and polar (Arctic and Antarctica) regions (Zakhia et al. 2008). The term 'alpine' is used in this review to indicate regions with high elevation mountains, that include not only the Alps, but the mountain areas of Europe, Asia (e.g. Hindu Kush, Karakorum-Himalaya and Tibetan Plateau) and America (e.g. Rocky Mountains and South American Alps; Casanueva et al. 2010). Arctic regions are defined by the Arctic Circle, which include continental lands in northern Asia (e.g. Siberia), Europe (e.g. Scandinavia), North America (e.g. Alaska and northern Canada) and islands, such as Novaya Zemlya (Russia), Svalbard (Norway), Iceland and Southern Greenland (Denmark). Antarctic regions include the Antarctic continent and sub-Antarctic islands (Convey et al. 2014). Although alpine and Arctic environments have some similarities (e.g. short growing seasons with low temperatures available for plants, soils with low levels of nutrients), they are characterized by distinct features (Ives and Barry 2019). In particular, extreme wind speeds, high snowfall and well-drained soils are typically found in the alpine environments, while high annual fluctuations of solar radiation, moderate winds, low snowfall and water-logged soils due to underlying permafrost characterize Arctic environments (Ives and Barry 2019). On the other hand, the Antarctic is the coldest and driest region of the world, and it is considered among the most limiting and stressful environments for plant life (Convey et al. 2014).
Vegetation in cold regions comprises less than 7% (ca. 10 million km 2 ) of the Earth's terrestrial surface (Breen et al. 2014;Lee et al. 2017). In alpine and Arctic areas, vascular plants (including angiosperms) are prevalent below the latitudinal and altitudinal tree lines, which correspond to the limit of forest where trees naturally do not persist (Breen et al. 2014). On the other hand, only two angiosperms, namely Colobanthus quitensis and Deschampsia antarctica, can grow in the Antarctic environments (Convey 2013).
Plants are associated with complex microbial communities, whose size and taxonomic structure depend on biotic (e.g. plant species, age and type of tissue) and abiotic factors (e.g. climatic conditions and soil physiochemical characteristics; Compant et al. 2019). Moreover, members of plant-associated microbial communities interact with the host plant providing neutral, detrimental or beneficial effects (Montesinos 2003). Increasing evidences support that microbial communities can promote plant growth at low temperatures and improve plant tolerance to cold stress (Acuña-Rodríguez et al. 2020). In this review, we summarize the current knowledge on plant-associated microbial communities in alpine, Arctic and Antarctic regions, in order to discuss key factors affecting the taxonomic structure of microbial communities in cold environments and to highlight the most abundant plant-associated taxa in terms of relative abundance (dominant taxa) and their possible functional properties in such environments.
Culturable plant-associated bacteria and fungi have been investigated in Alpine regions (Calvo et al. 2010;Sheng et al. 2011;   . Moreover, Ulloa-Muñoz and colleagues (2020) investigated the taxonomic structure of endophytic plant-growth promoting microorganisms from two wild medicinal plants (Gentianella weberbaueri and Valeriana pycnantha) in the Peruvian Andes and isolated six endophytic fungi belonging to Pyrenochaeta, Scleroconidioma, Cryptococcus and Plenodomus genera. Overall, taxonomic evidences obtained from these studies suggested that plants growing in alpine regions harbor rare and underexplored microbial species, which can be targeted for isolation and functional characterization in the future.
In Alpine regions, the plant species was one of the main factors shaping the plant-associated bacteria (Massaccesi et al. 2015;Chang et al. 2018;Wassermann et al. 2019) and fungi (Tscherko et al. 2005 (Garnica et al. 2013). Unfortunately, the plant microbiota was rarely studied in some alpine regions, such as the tropical Afroalpine mountain, Southern Alps (New Zealand), Urals, Caucasus and Pamir, indicating that further investigations on those less studied areas is required for a comprehensive understanding of the effect of the location on the taxonomic structure of plant-associated microbial communities (Ciccazzo et al. 2016). Likewise, studies on the variability of the taxonomic structure of endophytic microbial communities that considers different plant tissues in various plant species across various location may clarify the tissue-specificity in alpine plants.
Culturable plant-associated bacteria and fungi have been characterized in Arctic regions (Higgins et al. 2007;Walker et al. 2011;Nissinen, Männistö and van Elsas 2012;Poosakkannu, Nissinen and Kytöviita 2015). From three different host plants, Nissinen, Männistö and van Elsas (2012) obtained a collection of 325 endophytic bacterial isolates belonging to 56 genera of five phyla (Actinobacteria, Bacteroidetes, Firmicutes, Acidobacteria and Proteobacteria), with members of Burkholderia spp. and Sphingomonas spp. being the most abundant. Culturable endophytic microorganisms (178 bacterial and 30 fungal isolates) were also obtained from various plant tissues (leaf, root, seed and seedling) of Deschampsia flexuosa and specific taxa were isolated according to the plant tissue (Poosakkannu, Nissinen and Kytöviita 2015). For example, isolates closely related to Burkholderia sordidicola were present in leaf and root samples of both successional stages (sand and forest), while isolates closely related to Curtobacterium flaccumfaciens were present only in the leaf and root samples from the sand (Poosakkannu, Nissinen and Kytöviita 2015).
In Arctic regions, host-related factors (e.g. plant species and tissue type) and environmental-related factors (e.g. geographic location) affected the taxonomic structure of endophytic and rhizospheric bacterial communities (Nissinen, Männistö and van Elsas 2012;Kumar et al. 2016Kumar et al. , 2017Mapelli et al. 2018;Given et al. 2020; Fig. 2), indicating host-specific adaptations and environmental niche differentiation of bacterial communities. Likewise, host plants, neighboring plants (Mundra et al. 2015;Lorberau et al. 2017;Abrego et al. 2020;Botnen et al. 2020) and environmental factors (e.g. mean annual temperature and precipitation, elevation, humus content, organic matter, bedrock type, phosphorus and nitrogen content and soil pH) can affect the taxonomic structure of fungal communities associated with Arctic plants (Fujimura and Egger 2012;Timling et al. 2012;Blaalid et al. 2014;Kauppinen et al. 2014;Mundra, Bahram and Eidesen 2016;Botnen et al. 2019;Abrego et al. 2020). However, plant species have variable effect on the plant-associated fungal communities depending on the fungal group (e.g. effect on root associatedendophytes and but no effect on ectomycorrhizal fungi; Walker et al. 2011;Fujimura and Egger 2012;Timling et al. 2012;Botnen et al. 2014;Zhang and Yao 2015), indicating differential effects according to the fungal taxa (Abrego et al. 2020). Thus, further characterizations of plant-associated microbial communities are needed, particularly for some poorly studied Arctic regions, such as Norrbotten (Sweden), Iceland and Greenland (Denmark), Siberia and Novaya Zemlya (Russia), in order to better clarify the key drivers (i.e. host-and environmental-related factors) affecting the microbial community structure in the Arctic vascular plants.
In Antarctica regions, plant species showed variable effect on the structure of microbial communities (Teixeira et al. 2010(Teixeira et al. , 2013Santiago, Rosa and Rosa 2017;Wentzel et al. 2019). Thus, additional factors can contribute to microbial community shaping, such as soil characteristics in the case of bacterial communities (Teixeira et al. 2010(Teixeira et al. , 2013Wentzel et al. 2019) or seasonal surface air temperature in the case of fungal communities (Upson, Newsham and Read 2008). However, plant-associated fungi in Antarctica environments have been mostly investigated using culture-dependent approaches and future culture-independent studies are required, in order to better assess taxonomic structure of fungal communities.

ADAPTATION STRATEGIES OF PLANT-ASSOCIATED MICROBIAL COMMUNITIES TO HARSH CONDITIONS
Although meta-analyses are required to better highlight the existence of specific microbial taxa adapted to cold environments, some dominant bacterial taxa seems to be commonly present in alpine, Arctic, Antarctic regions, such as Proteobacteria, Actinobacteria and Bacteroidetes phyla, as well as Burkholderiales, Rhizobiales, Pseudomonadales, Bacillales, Actinomycetales (particularly Microbacteriaceae, Micromonosporaceae and Micrococcaceae families), Xanthomonodales, Saprospirales (particularly Chitonophagaceae family), Sphingobacteriales, Sphingomonodales and Myxococcales King et al. 2012;Nissinen, Männistö and van Elsas 2012;Angel et al. 2016;Jorquera et al. 2016;Cid et al. 2017;Kumar et al. 2017;Chica et al. 2019;Oberhofer et al. 2019;Praeg, Pauli and Illmer 2019;Given et al. 2020;Huang et al. 2020;Zhang et al. 2020). At low taxonomic level, Bradyrhizobium, Burkholderia, Clavibacter, Clostridium, Flavobacterium, Micrococcus, Mycobaterium, Nocardia, Novosphingobium, Pedobacter, Pseudomonas, Rhizobium, Rhodoplanes, Sphingomonas and Streptomyces genera dominates the plant-associated communities of alpine, Arctic and Antarctic regions Nissinen, Männistö and van Elsas 2012;Jorquera et al. 2016;Cid et al. 2017Chica et al. 2019Kumar et al. 2017;Oberhofer et al. 2019;Wassermann et al. 2019;Given et al. 2020;Huang et al. 2020;Ma et al. 2020;Zhang et al. 2020). Furthermore, members of these bacterial taxa were also shown to be cold-adapted and tightly associated with plants, suggesting their potential importance for plant fitness and survival in cold environments (King et al. 2012;Nissinen, Männistö and van Elsas 2012;Praeg, Pauli and Illmer 2019). Likewise, Ascomycota and Basidiomycota were the dominant phyla among plant-associated fungi in cold environments and some plant-associated fungal taxa can be frequently found in alpine, Arctic and Antarctic regions, such as Cryptococcus, Fusarium, Mrakia and Rhodotorula genera (Ferreira et al. 2019;Li et al. 2018;Praeg, Pauli and Illmer 2019;Santiago, Rosa and Rosa 2017;Wassermann et al. 2019;Zhang and Yao 2015). Although some plant-associated microbial taxa have a global distribution, their relative abundance and taxonomic complexity seemed to be higher in cold environments compared to benign regions, such as mycorrhizal fungi in alpine regions (Acaulosporaceae; e.g. Acaulospora alpina and Ambisporaceae; e.g. Ambispora fennica families; Oehl et al. 2006;Liu et al. 2011;Li et al. 2014;Senés-Guerrero and Schüssler 2016;Yang et al. 2016;Casazza et al. 2017;Haug, Setaro and Suárez 2019) and Arctic regions (e.g. Thelephora, Tomentella, Inocybe, Cortinarius and Cenococcum genera; Bjorbaekmo et al. 2010;Timling et al. 2012) or Psychrobacter and Exiguobacterium genera in Arctic and Antarctic regions (Rodrigues and Tiedje 2007;Rodrigues et al. 2009;Cid et al. 2017). However, no large comparative studies have been conducted on plant-associated microbial communities across different cold environments, indicating that further quantitative studies are needed to confirm the existence of cold-adapted microbial taxa consistently associated to plants in cold environments. For example, some plant species (e.g. Bistorta, Diapensia, Dryas, Juncus, Oxyria and Saxifraga) are widely distributed in both alpine and Arctic regions and they could be suitable for a comparative analysis of microbial communities associated to the same host in different cold regions (Fisher et al. 1995;Bjorbaekmo et al. 2010;Davey et al. 2015;Kumar et al. 2017;Botnen et al. 2019). In particular, Kumar and colleagues (2017) investigated the bacterial community structure of O. digyna and S. oppositifolia in three different climatic regions (alpine, low Arctic and high Arctic), and found that both plants shared bacterial taxa (core microbiota) belonging to Burkholderiales, Actinomycetales and Rhizobiales.
Functional studies were carried out on plant-associated microorganisms of cold regions, suggesting possible adaptation strategies to the harsh conditions. For example, functional studies of plant-associated bacteria in Arctic regions demonstrated that rhizosphere communities of O. digyna and S. oppositifolia tolerate oxidative stress and produce antibiotic molecules (e.g. fusidic acid, surfactant Niaproof 4 and troleandomycin; Kumar et al. 2016). Cold treatments can also upregulate genes involved in sugar transport, protein transport, lipid biosynthesis and NADH oxidoreductase activity, as demonstrated by the transcriptional profiling of an Arctic Mesorhizobium strain N33, isolated from nodules of Oxytropis arctobia in Canada (Ghobakhlou et al. 2015). Moreover, genomic studies indicated the presence of possible adaptation strategies of plant-associated microorganisms to cold environments, as revealed by the presence of genes encoding ice-nucleation proteins in Pseudomonas isolates of Antarctica plants (Cid et al. 2018) and genes related to cold stress response, membrane transport and osmotic regulation in cold tolerant Bacillus spp. (Zubair et al. 2019). In addition, genes involved in the utilization of various carbon sources and production of antibiotics, phytohormones, pigments and antioxidants were found in microbial communities associated with Espeletia plants in alpine regions (Ruiz-Pérez, Restrepo and Zambrano 2016), suggesting an efficient nutrient acquisition and a strict microbe-host interaction.
Heterotrophy, fermentation, xenobiotic degradation, nitrogen metabolism, tryptophan metabolism and inositol metabolism were the major functional groups of plantassociated microbial communities in Antarctic regions (Peixoto et al. 2016;Zhang et al. 2020), indicating a possible adaptation strategy to harsh environmental conditions. Some other functions were found in plant-associated communities of alpine regions according to the host tissue, such as streptomycin production, biomass degradation, xylan degradation and carbon fixation in rhizosphere communities; and nitrite reduction, ammonia oxidation and chitin degradation in endosphere communities (Huang et al. 2020). Comparative and functional metagenomic analysis of rhizosphere microorganisms associated with C. quitensis, either growing alone or together with D. antarctica, revealed differences in the abundance of genes related to environmental tolerance, cellular metabolism and osmotic stress, suggesting that such microorganisms could display specific functional activities that could have an effect on plant colonization and environmental tolerance (Molina-Montenegro et al. 2019).
The above-mentioned examples indicate that plantassociated microbial communities in cold environments may have developed various adaptation strategies for cold stress tolerance, including genetic features that enables them to perform metabolic and physiological functions under cold conditions Although some studies were able to predict biological functions of microbial communities using taxonomic or genomic information, further functional analyses are required to clarify cellular mechanisms of microbial tolerance to harsh environmental conditions. Moreover, the integration of multiomic approaches (e.g. genomics, metagenomics, transcriptomics, proteomics and metabolomics) will also help to better understand microbial adaptation strategies in cold environments by identifying key genes, proteins and metabolites whose regulation is affected by cold temperatures.

POTENTIAL FUNCTIONS OF PLANT-ASSOCIATED MICROORGANISMS FROM COLD REGIONS
To mitigate the effect of climate changes in agriculture, new ecofriendly and sustainable strategies are needed, particularly to limit negative effects of cold stress on crops. In particular, global warming is expected to promote earlier spring-related phenological events in plants, and, as a consequence, it will increase the risk and severity of spring frosts (Menzel et al. 2006;Gu et al. 2008). The use of plant-associated microorganisms and their compounds could be one of the most promising solution against cold stresses, but most microbial products for crop protection commonly used in agriculture are based on mesophilic microorganisms, which are unable to exert positive effects on plant growth in cold conditions (Wu et al. 2019;Torracchi et al. 2020). Conversely, plant-associated microorganisms isolated from cold environments, including bacteria (e.g. Bacillus spp., Brevibacterium spp., Clavibacter spp. and Pseudomonas spp.) and fungi (e.g. Geomyces spp., Lecanicillium spp. and Neotyphodium spp.; mycorrhizal fungi: Glomus spp.; and DSE: Mollisia spp., Phialocephala spp. and Tapesia spp.), could be used to promote plant growth under cold stress (Upson, Read and Newsham 2009b;Haselwandter and Read 1982;Ruotsalainen and Kytoviita 2004;Wäli et al. 2008;Ding et al. 2011;Mishra et al. 2011;Berríos et al. 2013;Suyal, Shukla and Goel 2014;Molina-Montenegro et al. 2015;Yarzábal et al. 2018;Hill et al. 2019;Tiryaki, Aydın and Atıcı 2019;Wu et al. 2019;Zubair et al. 2019;Tapia-Vázquez et al. 2020). Some other microorganisms isolated from cold environments (e.g. Achromobacter, Enterobacter, Exiguobacterium, Pseudomonas, Rahnella and Stenotrophomonas) promoted plant growth under normal conditions (16-25 • C), although their effect on cold-stressed plants was not yet investigated (Selvakumar et al. 2009(Selvakumar et al. , 2011Vyas et al. 2010;Ghyselinck et al. 2013;Ogata-Gutiérrez et al. 2017;Castellano-Hinojosa et al. 2018;Chumpitaz-Segovia et al. 2020). For example, 37 psychrophilic isolates of Bacillus spp. (including B. pumilus, B. safensis and B. atrophaeus able to grow at below 10 • C) obtained from the plant rhizosphere in the Qinghai-Tibetan plateau (2788-4780 m a.s.l.) promoted the growth of winter wheat seedlings at 10 • C (Wu et al. 2019). Likewise, Pseudomonas, Bacillus and Enterobacter isolates obtained from native potato varieties in the high Andes promoted plant growth, but also reduced Rhizoctonia solani severity (Ghyselinck et al. 2013). Root-associated microorganisms positively influenced Plantago major growth in a plant ecotypedependent manner: plant ecotypes growing with their local rootassociated microorganisms performed better than when growing with foreign microorganisms (Formenti et al. 2020). Thus, the development of microbial biostimulants based on indigenous cold-adapted microorganisms could be a promising approach to protect crop plants from cold stress, but further functional studies are required to better characterize the modes of action and possible limitations due to host specificity of psychrotolerant plant-growth promoting bacteria.

CONCLUSIONS
Plants growing in cold environments harbor complex, hostspecific and cold-adapted microbial communities that may play key functional roles in plant growth and survival. However, most studies investigated the taxonomic structure and potential functions of plant-associated bacterial and fungal communities in cold environments, while deeper taxonomic and functional studies are required on plant-associated Archaea and protists. Although microbial communities of plant rhizosphere and vegetative organs (e.g. roots, stems and leaves) were studied, taxonomic structure and functional properties of those associated with reproductive organs (e.g. flowers, fruits and seeds) needs further investigations. Future comparative studies and metaanalyses on plant microbiota in cold and temperate environments are also required, in order to identify possible microbial taxa specifically adapted to cold environments.