Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

Whole-genome sequencing of tetraploid potato varieties reveals different strategies for drought tolerance.

Climate changes leading to increasingly longer seasonal drought periods in large parts of the world increase the necessity for breeding drought-tolerant crops. Cultivated potato (Solanum tuberosum), the third most important vegetable crop worldwide, is regarded as drought-sensitive due to its shallow root architecture. Two German tetraploid potato cultivars differing in drought tolerance and their F1-progeny were evaluated under various drought scenarios. Bulked segregant analyses were combined with whole-genome sequencing (BSA-Seq) using contrasting bulks of drought-tolerant and drought-sensitive F1-clones. Applying QTLseqr, 15 QTLs comprising 588,983 single nucleotide polymorphisms (SNPs) in 2325 genes associated with drought stress tolerance were identified. SeqSNP analyses in an association panel of 34 mostly starch potato varieties using 1-8 SNPs for each of 188 selected genes narrowed the number of candidate genes down to 10. In addition, ent-kaurene synthase B was the only gene present under QTL 10. Eight of the identified genes (StABP1, StBRI1, StKS, StLEA, StPKSP1, StPKSP2, StYAB5, and StZOG1) address plant development, the other three genes (StFATA, StHGD and StSYP) contribute to plant protection under drought stress. Allelic variation in these genes might be explored in future breeding for drought-tolerant potato varieties.

Different metabolic adaptation strategies after overwintering in Eutrema sp. and Arabidopsis accessions under field conditions in Germany.

Successful overwintering is a prerequisite for high fitness in temperate perennials and winter annuals and is highly dependent on increased freezing tolerance and timely balancing of deacclimation with growth resumption in spring. To assess fitness costs associated with overwintering and elucidate metabolic mechanisms underlying winter survival and the switch from acclimated freezing tolerance to growth resumption, we performed a comparative field study using 14 Eutrema salsugineum accessions, E. halophilum, E. botschantzevii and 11 Arabidopsis thaliana accessions differing in freezing tolerance. Winter survival and reproductive fitness parameters were recorded and correlated with phenological stage and metabolite status during growth resumption in spring. The results revealed considerable intraspecific variation in winter survival, but survival rates of the extremophyte Eutrema were not inherently better. In both Eutrema and A. thaliana, improved winter survival was associated with reduced reproductive fitness. Metabolic analysis by GC-MS revealed intrinsic differences in the primary metabolism of the two genera during deacclimation. Eutrema contained higher levels of several amino and chlorogenic acids, while Arabidopsis had higher levels of several sugars and sugar conjugates. In both genera, increased levels of several soluble sugars were associated with increased winter survival, whereas myo-inositol has different roles in overwintering of Eutrema and A. thaliana. In addition, differences in amino acid metabolism and polyhydroxy acids levels after winter survival were found. The results provide strong evidence for a trade-off between increased winter survival and reproductive fitness in both Eutrema and Arabidopsis and document inherent differences in their metabolic strategies to survive winter.

Specific CBF transcription factors and cold-responsive genes fine-tune the early triggering response after acquisition of cold priming and memory.

Plants need to adapt to fluctuating temperatures throughout their lifetime. Previous research showed that Arabidopsis memorizes a first cold stress (priming) and improves its primed freezing tolerance further when subjected to a second similar stress after a lag phase. This study investigates primary metabolomic and transcriptomic changes during early cold priming or triggering after 3 days at 4°C interrupted by a memory phase. DREB1 family transcription factors DREB1C/CBF2, DREB1D/CBF4, DREB1E/DDF2, and DREB1F/DDF1 were strongly significantly induced throughout the entire triggering. During triggering, genes encoding Late Embryogenesis Abundant (LEA), antifreeze proteins or detoxifiers of reactive oxygen species (ROS) were higher expressed compared with priming. Examples of early triggering responders were xyloglucan endotransglucosylase/hydrolase genes encoding proteins involved in cell wall remodeling, while late responders were identified to act in fine-tuning the stress response and developmental regulation. Induction of non-typical members of the DREB subfamily of ERF/AP2 transcription factors, the relatively small number of induced CBF regulon genes and a slower accumulation of selected cold stress associated metabolites indicate that a cold triggering stimulus might be sensed as milder stress in plants compared with priming. Further, strong induction of CBF4 throughout triggering suggests a unique function of this gene for the response to alternating temperatures.

Cold stress and freezing tolerance negatively affect the fitness of Arabidopsis thaliana accessions under field and controlled conditions.

MAIN CONCLUSION: Higher acclimated freezing tolerance improved winter survival, but reduced reproductive fitness of Arabidopsis thaliana accessions under field and controlled conditions. Low temperature is one of the most important abiotic factors influencing plant fitness and geographical distribution. In addition, cold stress is known to influence crop yield and is therefore of great economic importance. Increased freezing tolerance can be acquired by the process of cold acclimation, but this may be associated with a fitness cost. To assess the influence of cold stress on the fitness of plants, long-term field trials over 5 years were performed with six natural accessions of Arabidopsis thaliana ranging from very tolerant to very sensitive to freezing. Fitness parameters, as seed yield and 1000 seed mass, were measured and correlation analyses with temperature and freezing tolerance data performed. The results were compared with fitness parameters from controlled chamber experiments over 3 years with application of cold priming and triggering conditions. Winter survival and seed yield per plant were positively correlated with temperature in field experiments. In addition, winter survival and 1000 seed mass were correlated with the cold-acclimated freezing tolerance of the selected Arabidopsis accessions. The results provide strong evidence for a trade-off between higher freezing tolerance and reproductive fitness in A. thaliana, which might have ecological impacts in the context of global warming.

Subcellular Localization of Seed-Expressed LEA_4 Proteins Reveals Liquid-Liquid Phase Separation for LEA9 and for LEA48 Homo- and LEA42-LEA48 Heterodimers.

LEA proteins are involved in plant stress tolerance. In Arabidopsis, the LEA_4 Pfam group is the biggest group with the majority of its members being expressed in dry seeds. To assess subcellular localization in vivo, we investigated 11 seed-expressed LEA_4 proteins in embryos dissected from dry seeds expressing LEA_4 fusion proteins under its native promoters with the Venus fluorescent protein (proLEA_4::LEA_4:Venus). LEA_4 proteins were shown to be localized in the endoplasmic reticulum, nucleus, mitochondria, and plastids. LEA9, in addition to the nucleus, was also found in cytoplasmic condensates in dry seeds dependent on cellular hydration level. Most investigated LEA_4 proteins were detected in 4-d-old seedlings. In addition, we assessed bioinformatic tools for predicting subcellular localization and promoter motifs of 11 seed-expressed LEA_4 proteins. Ratiometric bimolecular fluorescence complementation assays showed that LEA7, LEA29, and LEA48 form homodimers while heterodimers were formed between LEA7-LEA29 and LEA42-LEA48 in tobacco leaves. Interestingly, LEA48 homodimers and LEA42-LEA48 heterodimers formed droplets structures with liquid-like behavior. These structures, along with LEA9 cytoplasmic condensates, may have been formed through liquid-liquid phase separation. These findings suggest possible important roles of LLPS for LEA protein functions.

Differentiation of the High Night Temperature Response in Leaf Segments of Rice Cultivars with Contrasting Tolerance.

High night temperatures (HNT) affect rice yield in the field and induce chlorosis symptoms in leaves in controlled chamber experiments. However, little is known about molecular changes in leaf segments under these conditions. Transcript and metabolite profiling were performed for leaf segments of six rice cultivars with different HNT sensitivity. The metabolite profile of the sheath revealed a lower metabolite abundance compared to segments of the leaf blade. Furthermore, pre-adaptation to stress under control conditions was detected in the sheath, whereas this segment was only slightly affected by HNT. No unique significant transcriptomic changes were observed in the leaf base, including the basal growth zone at HNT conditions. Instead, selected metabolites showed correlations with HNT sensitivity in the base. The middle part and the tip were most highly affected by HNT in sensitive cultivars on the transcriptomic level with higher expression of jasmonic acid signaling related genes, genes encoding enzymes involved in flavonoid metabolism and a gene encoding galactinol synthase. In addition, gene expression of expansins known to improve stress tolerance increased in tolerant and sensitive cultivars. The investigation of the different leaf segments indicated highly segment specific responses to HNT. Molecular key players for HNT sensitivity were identified.

Characterization of the Heat-Stable Proteome during Seed Germination in Arabidopsis with Special Focus on LEA Proteins.

During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.

Genome-Wide Approach to Identify Quantitative Trait Loci for Drought Tolerance in Tetraploid Potato (Solanum tuberosum L.).

Drought represents a major abiotic stress factor negatively affecting growth, yield and tuber quality of potatoes. Quantitative trait locus (QTL) analyses were performed in cultivated potatoes for drought tolerance index DRYM (deviation of relative starch yield from the experimental median), tuber starch content, tuber starch yield, tuber fresh weight, selected transcripts and metabolites under control and drought stress conditions. Eight genomic regions of major interest for drought tolerance were identified, three representing standalone DRYM QTL. Candidate genes, e.g., from signaling pathways for ethylene, abscisic acid and brassinosteroids, and genes encoding cell wall remodeling enzymes were identified within DRYM QTL. Co-localizations of DRYM QTL and QTL for tuber starch content, tuber starch yield and tuber fresh weight with underlying genes of the carbohydrate metabolism were observed. Overlaps of DRYM QTL with metabolite QTL for ribitol or galactinol may indicate trade-offs between starch and compatible solute biosynthesis. Expression QTL confirmed the drought stress relevance of selected transcripts by overlaps with DRYM QTL. Bulked segregant analyses combined with next-generation sequencing (BSAseq) were used to identify mutations in genes under the DRYM QTL on linkage group 3. Future analyses of identified genes for drought tolerance will give a better insight into drought tolerance in potatoes.

Unravelling Differences in Candidate Genes for Drought Tolerance in Potato (Solanum tuberosum L.) by Use of New Functional Microsatellite Markers.

Potato is regarded as drought sensitive and most vulnerable to climate changes. Its cultivation in drought prone regions or under conditions of more frequent drought periods, especially in subtropical areas, requires intensive research to improve drought tolerance in order to guarantee high yields under limited water supplies. A candidate gene approach was used to develop functional simple sequence repeat (SSR) markers for association studies in potato with the aim to enhance breeding for drought tolerance. SSR primer combinations, mostly surrounding interrupted complex and compound repeats, were derived from 103 candidate genes for drought tolerance. Validation of the SSRs was performed in an association panel representing 34 mainly starch potato cultivars. Seventy-five out of 154 SSR primer combinations (49%) resulted in polymorphic, highly reproducible banding patterns with polymorphic information content (PIC) values between 0.11 and 0.90. Five SSR markers identified allelic differences between the potato cultivars that showed significant associations with drought sensitivity. In all cases, the group of drought-sensitive cultivars showed predominantly an additional allele, indicating that selection against these alleles by marker-assisted breeding might confer drought tolerance. Further studies of these differences in the candidate genes will elucidate their role for an improved performance of potatoes under water-limited conditions.

Stabilization of Dry Sucrose Glasses by Four LEA_4 Proteins from Arabidopsis thaliana.

Cells of many organisms and organs can withstand an (almost) total water loss (anhydrobiosis). Sugars play an essential role in desiccation tolerance due to their glass formation ability during dehydration. In addition, intrinsically disordered LEA proteins contribute to cellular survival under such conditions. One possible mechanism of LEA protein function is the stabilization of sugar glasses. However, little is known about the underlying mechanisms. Here we used FTIR spectroscopy to investigate sucrose (Suc) glass stability dried from water or from two buffer components in the presence of four recombinant LEA and globular reference proteins. Buffer ions influenced the strength of the Suc glass in the order Suc < Suc/Tris < Suc/NaP. LEA proteins strengthened the sugar H-bonded network and the molecular structure in the glassy state. The position of νOH peak and the wavenumber-temperature coefficient (WTCg) provided similar information about the H-bonded network. Protein aggregation of LEA proteins was reduced in the desiccation-induced Suc glassy state. Detailed knowledge about the role of LEA proteins in the stabilization of dry sugar glasses yields information about their role in anhydrobiosis. This may open the possibility to use such proteins in biotechnical applications requiring dry storage of biologicals such as proteins, cells or tissues.

Physiological and molecular attributes contribute to high night temperature tolerance in cereals.

Asymmetric warming resulting in a faster increase in night compared to day temperatures affects crop yields negatively. Physiological characterization and agronomic findings have been complemented more recently by molecular biology approaches including transcriptomic, proteomic, metabolomic and lipidomic investigations in crops exposed to high night temperature (HNT) conditions. Nevertheless, the understanding of the underlying mechanisms causing yield decline under HNT is still limited. The discovery of significant differences between HNT-tolerant and HNT-sensitive cultivars is one of the main research directions to secure continuous food supply under the challenge of increasing climate change. With this review, we provide a summary of current knowledge on the physiological and molecular basis of contrasting HNT tolerance in rice and wheat cultivars. Requirements for HNT tolerance and the special adaptation strategies of the HNT-tolerant rice cultivar Nagina-22 (N22) are discussed. Putative metabolite markers for HNT tolerance useful for marker-assisted breeding are suggested, together with future research directions aimed at improving food security under HNT conditions.

Natural Variation among Arabidopsis Accessions in the Regulation of Flavonoid Metabolism and Stress Gene Expression by Combined UV Radiation and Cold.

Plants are constantly exposed to stressful environmental conditions. Plant stress reactions were mainly investigated for single stress factors. However, under natural conditions plants may be simultaneously exposed to different stresses. Responses to combined stresses cannot be predicted from the reactions to the single stresses. Flavonoids accumulate in Arabidopsis thaliana during exposure to UV-A, UV-B or cold, but the interactions of these factors on flavonoid biosynthesis were unknown. We therefore investigated the interaction of UV radiation and cold in regulating the expression of well-characterized stress-regulated genes, and on transcripts and metabolites of the flavonoid biosynthetic pathway in 52 natural Arabidopsis accessions that differ widely in their freezing tolerance. The data revealed interactions of cold and UV on the regulation of stress-related and flavonoid biosynthesis genes, and on flavonoid composition. In many cases, plant reactions to a combination of cold and UV were unique under combined stress and not predictable from the responses to the single stresses. Strikingly, all correlations between expression levels of flavonoid biosynthesis genes and flavonol levels were abolished by UV-B exposure. Similarly, correlations between transcript levels of flavonoid biosynthesis genes or flavonoid contents, and freezing tolerance were lost in the presence of UV radiation, while correlations with the expression levels of cold-regulated genes largely persisted. This may indicate different molecular cold acclimation responses in the presence or absence of UV radiation.

Cell wall modification by the xyloglucan endotransglucosylase/hydrolase XTH19 influences freezing tolerance after cold and sub-zero acclimation.

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze-thaw cycle. Many plant species increase their freezing tolerance during exposure to low, non-freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub-zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodelling may be particularly important for sub-zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub-zero acclimation, compared to the Col-0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centres of rosette indicated that epitopes of the xyloglucan-specific antibody LM25 were highly abundant in the vasculature of Col-0 plants after sub-zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodelling for the increased freezing tolerance observed after low temperature acclimation.

Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L.

The wide natural variation present in rice is an important source of genes to facilitate stress tolerance breeding. However, identification of candidate genes from RNA-Seq studies is hampered by the lack of high-quality genome assemblies for the most stress tolerant cultivars. A more targeted solution is the reconstruction of transcriptomes to provide templates to map RNA-seq reads. Here, we sequenced transcriptomes of ten rice cultivars of three subspecies on the PacBio Sequel platform. RNA was isolated from different organs of plants grown under control and abiotic stress conditions in different environments. Reconstructed de novo reference transcriptomes resulted in 37,500 to 54,600 plant-specific high-quality isoforms per cultivar. Isoforms were collapsed to reduce sequence redundancy and evaluated, e.g., for protein completeness (BUSCO). About 40% of all identified transcripts were novel isoforms compared to the Nipponbare reference transcriptome. For the drought/heat tolerant aus cultivar N22, 56 differentially expressed genes in developing seeds were identified at combined heat and drought in the field. The newly generated rice transcriptomes are useful to identify candidate genes for stress tolerance breeding not present in the reference transcriptomes/genomes. In addition, our approach provides a cost-effective alternative to genome sequencing for identification of candidate genes in highly stress tolerant genotypes.

Can Metabolite- and Transcript-Based Selection for Drought Tolerance in Solanum tuberosum Replace Selection on Yield in Arid Environments?

Climate models predict an increased likelihood of drought, demanding efficient selection for drought tolerance to maintain yield stability. Classic tolerance breeding relies on selection for yield in arid environments, which depends on yield trials and takes decades. Breeding could be accelerated by marker-assisted selection (MAS). As an alternative to genomic markers, transcript and metabolite markers have been suggested for important crops but also for orphan corps. For potato, we suggested a random-forest-based model that predicts tolerance from leaf metabolite and transcript levels with a precision of more than 90% independent of the agro-environment. To find out how the model based selection compares to yield-based selection in arid environments, we applied this approach to a population of 200 tetraploid Solanum tuberosum ssp. tuberosum lines segregating for drought tolerance. Twenty-four lines were selected into a phenotypic subpopulation (PPt) for superior tolerance based on relative tuber starch yield data from three drought stress trials. Two subpopulations with superior (MPt) and inferior (MPs) tolerance were selected based on drought tolerance predictions based on leaf metabolite and transcript levels from two sites. The 60 selected lines were phenotyped for yield and drought tolerance in 10 multi-environment drought stress trials representing typical Central European drought scenarios. Neither selection affected development or yield potential. Lines with superior drought tolerance and high yields under stress were over-represented in both populations selected for superior tolerance, with a higher number in PPt compared to MPt. However, selection based on leaf metabolites may still be an alternative to yield-based selection in arid environments as it works on leaves sampled in breeder's fields independent of drought trials. As the selection against low tolerance was ineffective, the method is best used in combination with tools that select against sensitive genotypes. Thus, metabolic and transcript marker-based selection for drought tolerance is a viable alternative to the selection on yield in arid environments.

Introduction: Plant Cold Acclimation and Winter Survival.

This introductory chapter provides a brief overview of plant freezing tolerance, cold acclimation, including subzero acclimation, and the subsequent deacclimation when plants return to warm conditions favoring growth and development. We describe the basic concepts and approaches that are currently followed to investigate these phenomena. We highlight the multidisciplinary nature of these investigations and the necessity to use methodologies from different branches of science, such as ecology, genetics, physiology, cell biology, biochemistry, and biophysics to gain a complete understanding of the complex adaptive mechanisms ultimately underlying plant winter survival.

Measuring Freezing Tolerance of Leaves and Rosettes: Electrolyte Leakage and Chlorophyll Fluorescence Assays.

Quantitative assessment of freezing tolerance is essential to unravel plant adaptations to cold temperatures. Not only the survival of whole plants, but also impairment of detached leaves or small rosettes after a freeze-thaw cycle can be used to accurately quantify plant freezing tolerance in terms of LT50 values. Here we describe two methods to determine the freezing tolerance of detached leaves or rosettes using a full or selected set of freezing temperatures and an additional method using chlorophyll fluorescence as a different physiological parameter. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus.

Analysis of Changes in Plant Cell Wall Composition and Structure During Cold Acclimation.

The cell wall has a crucial influence on the mechanical properties of plant cells. It therefore has a strong impact on the freezing behavior and very likely also the freezing tolerance of plants. However, not many studies have addressed the question how cell wall composition and structure impact plant freezing tolerance and cold acclimation. In this chapter, we describe a comprehensive workflow to extract total cell wall material from leaves of Arabidopsis thaliana and to separate this material into fractions enriched in crystalline cellulose, pectins, and hemicelluloses by sequential fractionation. We further describe methods for the analysis of chemical structure, monosaccharide composition, and cellulose and uronic acid contents in the total cell wall material and the fractions in response to cold acclimation. Structural properties of cell wall material are analyzed by attenuated total reflectance-Fourier-transform infrared spectrometry (ATR-FTIR) and monosaccharide composition by gas chromatography-mass spectrometry (GC-MS) after isolation of alditol acetate derivatives of the sugars.

Season Affects Yield and Metabolic Profiles of Rice (Oryza sativa) under High Night Temperature Stress in the Field.

Rice (Oryza sativa) is the main food source for more than 3.5 billion people in the world. Global climate change is having a strong negative effect on rice production. One of the climatic factors impacting rice yield is asymmetric warming, i.e., the stronger increase in nighttime as compared to daytime temperatures. Little is known of the metabolic responses of rice to high night temperature (HNT) in the field. Eight rice cultivars with contrasting HNT sensitivity were grown in the field during the wet (WS) and dry season (DS) in the Philippines. Plant height, 1000-grain weight and harvest index were influenced by HNT in both seasons, while total grain yield was only consistently reduced in the WS. Metabolite composition was analysed by gas chromatography-mass spectrometry (GC-MS). HNT effects were more pronounced in panicles than in flag leaves. A decreased abundance of sugar phosphates and sucrose, and a higher abundance of monosaccharides in panicles indicated impaired glycolysis and higher respiration-driven carbon losses in response to HNT in the WS. Higher amounts of alanine and cyano-alanine in panicles grown in the DS compared to in those grown in the WS point to an improved N-assimilation and more effective detoxification of cyanide, contributing to the smaller impact of HNT on grain yield in the DS.