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With recent progress in active research on flooding and hypoxia/anoxia tolerance in native and agricultural crop plants, vast knowledge has been gained on both individual tolerance mechanisms and the general mechanisms of flooding tolerance in plants. Research on carbohydrate consumption, ethanolic and lactic acid fermentation, and their regulation under stress conditions has been accompanied by investigations on aerenchyma development and the emergence of the radial oxygen loss barrier in some plant species under flooded conditions. The discovery of the oxygen-sensing mechanism in plants and unravelling the intricacies of this mechanism have boosted this very international research effort. Recent studies have highlighted the importance of oxygen availability as a signalling component during plant development. The latest developments in determining actual oxygen concentrations using minute probes and molecular sensors in tissues and even within cells have provided new insights into the intracellular effects of flooding. The information amassed during recent years has been used in the breeding of new flood-tolerant crop cultivars. With the wealth of metabolic, anatomical, and genetic information, novel holistic approaches can be used to enhance crop species and their productivity under increasing stress conditions due to climate change and the subsequent changes in the environment., With recent progress in active research on flooding and hypoxia/anoxia tolerance in native and agricultural crop plants, vast knowledge has been gained on both individual tolerance mechanisms and the general mechanisms of flooding tolerance in plants. Research on carbohydrate consumption, ethanolic and lactic acid fermentation, and their regulation under stress conditions has been accompanied by investigations on aerenchyma development and the emergence of the radial oxygen loss barrier in some plant species under flooded conditions. The discovery of the oxygen-sensing mechanism in plants and unravelling the intricacies of this mechanism have boosted this very international research effort. Recent studies have highlighted the importance of oxygen availability as a signalling component during plant development. The latest developments in determining actual oxygen concentrations using minute probes and molecular sensors in tissues and even within cells have provided new insights into the intracellular effects of flooding. The information amassed during recent years has been used in the breeding of new flood-tolerant crop cultivars. With the wealth of metabolic, anatomical, and genetic information, novel holistic approaches can be used to enhance crop species and their productivity under increasing stress conditions due to climate change and the subsequent changes in the environment.

Plant respiratory burst oxidase homologs (RBOHs) are plasma membrane-localized NADPH oxidases that generate superoxide anion radicals, which then dismutate to H2O2, into the apoplast using cytoplasmic NADPH as an electron donor. PaRBOH1 is the most highly expressed RBOH gene in developing xylem as well as in a lignin-forming cell culture of Norway spruce (Picea abies L. Karst.). Since no previous information about regulation of gymnosperm RBOHs exist, our aim was to resolve how PaRBOH1 is regulated with a focus on phosphorylation. The N-terminal part of PaRBOH1 was found to contain several putative phosphorylation sites and a four-times repeated motif with similarities to the Botrytis-induced kinase 1 target site in Arabidopsis AtRBOHD. Phosphorylation was indicated for six of the sites in in vitro kinase assays using 15 amino-acid-long peptides for each of the predicted phosphotarget site in the presence of protein extracts of developing xylem. Serine and threonine residues showing positive response in the peptide assays were individually mutated to alanine (kinase-inactive) or to aspartate (phosphomimic), and the wild type PaRBOH1 and the mutated constructs transfected to human kidney embryogenic (HEK293T) cells with a low endogenous level of extracellular ROS production. ROS-producing assays with HEK cells showed that Ca2+ and phosphorylation synergistically activate the enzyme and identified several serine and threonine residues that are likely to be phosphorylated including a novel phosphorylation site not characterized in other plant species. These were further investigated with a phosphoproteomic study. Results of Norway spruce, the first gymnosperm species studied in relation to RBOH regulation, show that regulation of RBOH activity is conserved among seed plants.

Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissuecultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the K-m value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.

A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls. Transcriptomics combined with single-cell metabolomics give new information on the role of rays in lignification of developing xylem in Norway spruce.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Apart from playing a key role in important biochemical reactions, molecular oxygen (O2) and its by-products also have crucial signaling roles in shaping plant developmental programs and environmental responses. Even under normal conditions, sharp O2 gradients can occur within the plant when cellular O2 demand exceeds supply, especially in dense organs such as tubers, seeds and fruits. Spatial and temporal variations in O2 concentrations are important cues for plants to modulate development (van Dongen & Licausi, 2015; Considine et al., 2016). Environmental conditions can also expand the low O2 regions within the plant. For example, excessive rainfall can lead to partial or complete plant submergence resulting in O2 deficiency in the root or the entire plant (Voesenek & Bailey-Serres, 2015). Climate change associated increases in precipitation events have made flooding a major abiotic stress threatening crop production and food sustainability. This increased flooding and associated crop losses highlight the urgency of understanding plant flooding responses and tolerance mechanisms.