Physiological, transcriptomic and metabolomic insights of three extremophyte woody species living in the multi-stress environment of the Atacama Desert.

MAIN CONCLUSIONS: In contrast to Neltuma species, S. tamarugo exhibited higher stress tolerance, maintaining photosynthetic performance through enhanced gene expression and metabolites. Differentially accumulated metabolites include chlorophyll and carotenoids and accumulation of non-nitrogen osmoprotectants. Plant species have developed different adaptive strategies to live under extreme environmental conditions. Hypothetically, extremophyte species present a unique configuration of physiological functions that prioritize stress-tolerance mechanisms while carefully managing resource allocation for photosynthesis. This could be particularly challenging under a multi-stress environment, where the synthesis of multiple and sequential molecular mechanisms is induced. We explored this hypothesis in three phylogenetically related woody species co-occurring in the Atacama Desert, Strombocarpa tamarugo, Neltuma alba, and Neltuma chilensis, by analyzing their leaf dehydration and freezing tolerance and by characterizing their photosynthetic performance under natural growth conditions. Besides, the transcriptomic profiling, biochemical analyses of leaf pigments, and metabolite analysis by untargeted metabolomics were conducted to study gene expression and metabolomic landscape within this challenging multi-stress environment. S. tamarugo showed a higher photosynthetic capacity and leaf stress tolerance than the other species. In this species, a multifactorial response was observed, which involves high photochemical activity associated with a higher content of chlorophylls and ß-carotene. The oxidative damage of the photosynthetic apparatus is probably attenuated by the synthesis of complex antioxidant molecules in the three species, but S. tamarugo showed the highest antioxidant capacity. Comparative transcriptomic and metabolomic analyses among the species showed the differential expression of genes involved in the biosynthetic pathways of key stress-related metabolites. Moreover, the synthesis of non-nitrogen osmoprotectant molecules, such as ciceritol and mannitol in S. tamarugo, would allow the nitrogen allocation to support its high photosynthetic capacity without compromising leaf dehydration tolerance and freezing stress avoidance.

Preferential freezing avoidance localised in anthers and embryo sacs in wintering Daphne kamtschatica var. jezoensis flower buds visualised by magnetic resonance imaging.

To explore diversity in cold hardiness mechanisms, high resolution magnetic resonance imaging (MRI) was used to visualise freezing behaviours in wintering Daphne kamtschatica var. jezoensis flower buds, which have naked florets and no bud scales. MRI images showed that anthers remained stably supercooled to the range from -14 to -21°C or lower while most other tissues froze by -7°C. Freezing of some anthers detected in MRI images between -14 and -21°C corresponded with numerous low temperature exotherms and also with the 'all-or-nothing' type of anther injuries. In ovules/pistils, only embryo sacs remained supercooled at -7°C or lower, but slowly dehydrated during further cooling. Cryomicroscopic observation revealed ice formation in the cavities of calyx tubes and pistils but detected no ice in embryo sacs or in anthers. The distribution of ice nucleation activity in floral tissues corroborated the tissue freezing behaviours. Filaments likely work as the ice blocking barrier that prevents ice intrusion from extracellularly frozen calyx tubes to connecting unfrozen anthers. Unique freezing behaviours were demonstrated in Daphne flower buds: preferential freezing avoidance in male and female gametophytes and their surrounding tissues (by stable supercooling in anthers and by supercooling with slow dehydration in embryo sacs) while the remaining tissues tolerate extracellular freezing.

A model for extracellular freezing based on observations on Equisetum hyemale.

In frost hardy plants, the lethal intracellular formation of ice crystals has to be prevented during frost periods. Besides the ability for supercooling and pre-frost dehydration of tissues, extracellular ice formation is another strategy to control ice development in tissues. During extracellular ice formation, partially large ice bodies accumulate in intercellular spaces, often at preferred sites which can also be expandable. In this contribution, the physico-chemical processes underlying the water movements towards the sites of extracellular ice formation are studied theoretically, based on observations on the frost hardy horsetail species Equisetum hyemale, with the overall aim to obtain a better understanding of the physical processes involved in extracellular ice formation. In E. hyemale, ice accumulates in the extensive internal canal system. The study focuses on the processes which are triggered in the cellular osmotic-mechanic system by falling, and especially subzero temperatures. It can be shown that when the temperature falls, (1) water flow out of cells is actuated and (2) "stiff-walled" cells lose less water than "soft-walled" cells. Furthermore, (3) cell water loss increases with increasing (= less negative) turgor loss point. These processes are not related to any specific activities of the cell but are solely a consequence of the structure of the cellular osmotic system. On this basis, a directed water flow can be initiated triggered by subzero temperatures. The suggested mechanism may be quite common in frost hardy species with extracellular ice formation.

Freezing resistance, safety margins, and survival vary among big sagebrush populations across the western United States.

PREMISE: Physiological responses to temperature extremes are considered strong drivers of species' demographic responses to climate variability. Plants are typically classified as either avoiders or tolerators in their freezing-resistance mechanism, but a gradient of physiological-threshold freezing responses may exist among individuals of a species. Moreover, adaptive significance of physiological freezing responses is poorly characterized, particularly under warming conditions that relax selection on cold hardiness. METHODS: Freezing responses were measured in winter and again for new foliage in spring for 14 populations of Artemisia tridentata collected throughout its range and planted in a warm common garden. The relationships of the freezing responses to survival were evaluated in the warm garden and in two colder gardens. RESULTS: Winter and spring freezing resistance were not correlated and appeared to be under differing selection regimes, as evident in correlations with different population climate of origin variables. All populations resisted considerably lower temperatures in winter than in spring, with populations from more continental climates showing narrower freezing safety margins (difference in temperatures at which ice-nucleation occurs and 50% reduction in chlorophyll fluorescence occurs) in spring. Populations with greater winter freezing resistance had lower survivorship in the warmest garden, while populations with greater spring freezing resistance had lower survivorship in a colder garden. CONCLUSIONS: These survivorship patterns relative to physiological thresholds suggest excess freezing resistance may incur a survival cost that likely relates to a trade-off between carbon gain and freezing resistance during critical periods of moisture availability. This cost has implications for seed moved from cooler to warmer environments and for plants growing in warming environments.

Ice Nucleation Activity in Plants: The Distribution, Characterization, and Their Roles in Cold Hardiness Mechanisms.

Control of freezing in plant tissues is a key issue in cold hardiness mechanisms. Yet freeze-regulation mechanisms remain mostly unexplored. Among them, ice nucleation activity (INA) is a primary factor involved in the initiation and regulation of freezing events in plant tissues, yet the details remain poorly understood. To address this, we developed a highly reproducible assay for determining plant tissue INA and noninvasive freeze visualization tools using MRI and infrared thermography. The results of visualization studies on plant freezing behaviors and INA survey of over 600 species tissues show that (1) freezing-sensitive plants tend to have low INA in their tissues (thus tend to transiently supercool), while wintering cold-hardy species have high INA in some specialized tissues; and (2) the high INA in cold-hardy tissues likely functions as a freezing sensor to initiate freezing at warm subzero temperatures at appropriate locations and timing, resulting in the induction of tissue-/species-specific freezing behaviors (e.g., extracellular freezing, extraorgan freezing) and the freezing order among tissues: from the primary freeze to the last tissue remaining unfrozen (likely INA level dependent). The spatiotemporal distributions of tissue INA, their characterization, and functional roles are detailed. INA assay principles, anti-nucleation activity (ANA), and freeze visualization tools are also described.

Frost resistance in alpine woody plants.

This report provides a brief review of key findings related to frost resistance in alpine woody plant species, summarizes data on their frost resistance, highlights the importance of freeze avoidance mechanisms, and indicates areas of future research. Freezing temperatures are possible throughout the whole growing period in the alpine life zone. Frost severity, comprised of both intensity and duration, becomes greater with increasing elevation and, there is also a greater probability, that small statured woody plants, may be insulated by snow cover. Several frost survival mechanisms have evolved in woody alpine plants in response to these environmental conditions. Examples of tolerance to extracellular freezing and freeze dehydration, life cycles that allow species to escape frost, and freeze avoidance mechanisms can all be found. Despite their specific adaption to the alpine environment, frost damage can occur in spring, while all alpine woody plants have a low risk of frost damage in winter. Experimental evidence indicates that premature deacclimation in Pinus cembra in the spring, and a limited ability of many species of alpine woody shrubs to rapidly reacclimate when they lose snow cover, resulting in reduced levels of frost resistance in the spring, may be particularly critical under the projected changes in climate. In this review, frost resistance and specific frost survival mechanisms of different organs (leaves, stems, vegetative and reproductive over-wintering buds, flowers, and fruits) and tissues are compared. The seasonal dynamics of frost resistance of leaves of trees, as opposed to woody shrubs, is also discussed. The ability of some tissues and organs to avoid freezing by supercooling, as visualized by high resolution infrared thermography, are also provided. Collectively, the report provides a review of the complex and diverse ways that woody plants survive in the frost dominated environment of the alpine life zone.