Investigations to uncover the versatility of carbohydrate active enzymes belonging to the auxiliary activity family 3 (AA3) have focused on microbial enzymes. Plant genomes also harbor AA3 encoding genes, and reverse genetics approaches have shown their importance in plant development. To date, however, detailed biochemical characterizations and structures of plant AA3 enzymes have not been reported. Here, we describe screening AA3 encoding genes from Norway spruce and the first in-depth biochemical and structural characterization of a plant AA3 oxidase, PaAOX1. PaAOX1 was crystallized, and the structure was solved by X-ray crystallography. PaAOX1 demonstrated highest oxidase activity against glycerol. It oxidized glycerol to glyceraldehyde, then further to glyceric acid, and showed preference for L-glyceraldehyde over D-glyceraldehyde. Despite the low sequence similarity with fungal alcohol oxidases, it had all the structural features of the typical AA3 enzyme. PaAOX1 has a novel activity among the characterized AA3s, and it showed a new structural arrangement of the region surrounding the catalytic center. Our findings contribute to a deeper understanding of the structural and functional aspects of enzymes in the GMC superfamily, and especially of alcohol oxidases, expanding the knowledge from fungi to plants. ### Competing Interest Statement The authors have declared no competing interest.

SUMMARY The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn‐deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin‐like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn‐depleted conditions. DEFLs are small cysteine‐rich peptides involved in defense responses, development and excess metal stress in plants. However, the functions of DEFLs in the Zn‐deficiency response are largely unknown. Here, phylogenetic tree analysis revealed that seven DEFLs (DEFL202–DEFL208) were categorized into one subgroup. Among the seven DEFL s, the transcripts of five (not DEFL204 and DEFL205 ) were upregulated by Zn deficiency, consistent with the presence of cis ‐elements for basic‐region leucine‐zipper 19 (bZIP19) or bZIP23 in their promoter regions. Microscopic observation of GFP‐tagged DEFL203 showed that DEFL203‐sGFP was localized to the apoplast and plasma membrane. Whereas a single mutation of the DEFL202 or DEFL203 genes only slightly affected root growth, defl202 defl203 double mutants showed enhanced root growth under all growth conditions. We also showed that the size of the root meristem was increased in the double mutants compared with the wild type. Our results suggest that DEFL202 and DEFL203 are redundantly involved in the inhibition of root growth under Zn‐deficient conditions through a reduction in root meristem length and cell number.

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.

Large protein families are a prominent feature of plant genomes and their size variation is a key element for adaptation in plants. Here we infer the evolutionary history of a representative protein family, the DOMAIN OF UNKNOWN FUNCTION (DUF) 26-containing proteins. The DUF26 first appeared in secreted proteins. Domain duplications and rearrangements led to the emergence of CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES (CRKs) and PLASMODESMATA-LOCALIZED PROTEINS (PDLPs). While the DUF26 itself is specific to land plants, structural analyses of Arabidopsis PDLP5 and PDLP8 ectodomains revealed strong similarity to fungal lectins. Therefore, we propose that DUF26-containing proteins constitute a novel group of plant carbohydrate-binding proteins. Following their appearance, CRKs expanded both through tandem duplications and preferential retention of duplicates in whole genome duplication events, whereas PDLPs evolved according to the dosage balance hypothesis. Based on our findings, we suggest that the main mechanism of expansion in new gene families is small-scale duplication, whereas genome fractionation and genetic drift after whole genome multiplications drive families towards dosage balance.

In the version of this article initially published, there was a mistake in the calculation of the nucleotide mutation rate per site per generation: 1 × 10 −9 mutations per site per generation was used, whereas 9.5 × 10 −9 was correct. This error affects the interpretation of population-size changes over time and their possible correspondence with known geological events, as shown in the original Fig. 4 and supporting discussion in the text, as well as details in the Supplementary Note. Neither the data themselves nor any other results are affected. Figure 4 has been revised accordingly. Images of the original and corrected figure panels are shown in the correction notice.

Programmed cell death regulates developmental and stress responses in eukaryotes. Golgi anti-apoptotic proteins (GAAPs) are evolutionarily conserved cell death regulators. Human and viral GAAPs inhibit apoptosis and modulate intracellular Ca fluxes, and viral GAAPs form cation-selective channels. Although most mammalian cell death regulators are not conserved at the sequence level in plants, the GAAP gene family shows expansion, with five paralogues () in the Arabidopsis genome. We pursued molecular and physiological characterization of AtGAAPs making use of the advanced knowledge of their human and viral counterparts. Structural modeling of AtGAAPs predicted the presence of a channel-like pore, and electrophysiological recordings from purified AtGAAP3 reconstituted into lipid bilayers confirmed that plant GAAPs can function as ion channels. AtGAAP1 and AtGAAP4 localized exclusively to the Golgi within the plant cell, while AtGAAP2, AtGAAP3 and AtGAAP5 also showed tonoplastic localization. Gene expression analysis revealed differential spatial expression and abundance of transcript for paralogues in Arabidopsis tissues. We demonstrate that AtGAAP1-5 inhibit Bax-induced cell death in yeast. However, overexpression of AtGAAP1 induces cell death in leaves and lesion mimic phenotype in Arabidopsis. We propose that AtGAAPs function as Golgi-localized ion channels that regulate cell death by affecting ionic homeostasis within the cell.

The use of draft genomes of different species and re-sequencing of accessions and populations are now common tools for plant biology research. The de novo assembled draft genomes make it possible to identify pivotal divergence points in the plant lineage and provide an opportunity to investigate the genomic basis and timing of biological innovations by inferring orthologs between species. Furthermore, re-sequencing facilitates the mapping and subsequent molecular characterization of causative loci for traits, such as those for plant stress tolerance and development. In both cases high-quality gene annotation-the identification of protein-coding regions, gene promoters, and 5'- and 3'-untranslated regions-is critical for investigation of gene function. Annotations are constantly improving but automated gene annotations still require manual curation and experimental validation. This is particularly important for genes with large introns, genes located in regions rich with transposable elements or repeats, large gene families, and segmentally duplicated genes. In this opinion paper, we highlight the impact of annotation quality on evolutionary analyses, genome-wide association studies, and the identification of orthologous genes in plants. Furthermore, we predict that incorporating accurate information from manual curation into databases will dramatically improve the performance of automated gene predictors.

Comparing related organisms with differing ecological requirements and evolutionary histories can shed light on the mechanisms and drivers underlying genetic adaptation. Here, by examining a common set of hundreds of loci, we compare patterns of nucleotide diversity and molecular adaptation of two European conifers (Scots pine and maritime pine) living in contrasted environments and characterized by distinct population genetic structure (low and clinal in Scots pine, high and ecotypic in maritime pine) and demographic histories. We found higher nucleotide diversity in Scots pine than in maritime pine, whereas rates of new adaptive substitutions (ωa), as estimated from the distribution of fitness effects, were similar across species and among the highest found in plants. Sample size and population genetic structure did not appear to have resulted in significant bias in estimates of ωa. Moreover, population contraction–expansion dynamics for each species did not affect differentially the rate of adaptive substitution in these two pines. Several methodological and biological factors may underlie the unusually high rate of adaptive evolution of Scots pine and maritime pine. By providing two new case studies with contrasting evolutionary histories, we contribute to disentangling the multiple factors potentially affecting adaptive evolution in natural plant populations.

Analysis of gene families and identification of homologous genes are important for phylogenetic analysis and for translating results from model to crop species. While numerous plant genomes have been sequenced and made available, the identification of gene models can be difficult, in particular for large gene families arranged in tandem repeats or encoding proteins with a variable number of internal repeats. Thus, correct annotation of plant receptor kinases (PRK) is a challenge. Here, we describe a workflow for the semi-manual extraction, annotation, and verification of genes from annotated gene models as well as from non-annotated DNA regions. This protocol allows the efficient identification of gene family member of PRK from most available plant genomes.

Poly(ADP-ribos)ylation, originally described as a mechanism of DNA break repair, is now considered as part of a complex regulatory system involved in dynamic reorganization of chromatin structure, transcriptional control of gene expression and regulation of metabolism. In plants poly(ADP-ribos)ylation has received surprisingly little attention. It has been implicated in abiotic and biotic stress responses, cell cycle control and development; however, the molecular mechanisms and proteins involved are largely unknown. In this review we summarize current knowledge on plant PARP, PARG and PARP-like domain containing proteins and discuss their possible roles in plant development, immune responses, programmed cell death and stress responses in general. The genome of the model plant Arabidopsis contains three genes encoding PARP proteins, two of which have been shown to be active PARPs, and two genes encoding PARG proteins, one of which was shown to possess enzymatic activity. In addition, SROs (Similar to RCD One) represent a plant specific family of proteins containing a PARP-like domain. Although bioinformatics and biochemical data suggest that the PARP-like domain in SRO proteins does not have PARP activity, these proteins play a significant role in stress response as revealed by mutant analyses. SRO proteins interact with transcription factors involved in various stress and developmental responses and are suggested to serve as hubs in many signaling pathways. Altogether current data imply that poly(ADP-ribos)ylation plays significant regulatory role in many aspects of plant biology.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

Poly(ADP-ribos)ylation, originally described as a mechanism of DNA break repair, is now considered as part of a complex regulatory system involved in dynamic reorganization of chromatin structure, transcriptional control of gene expression and regulation of metabolism. In plants poly(ADP-ribos)ylation has received surprisingly little attention. It has been implicated in abiotic and biotic stress responses, cell cycle control and development; however the molecular mechanisms and proteins involved are largely unknown. In this review we summarize current knowledge on plant PARP, PARG and PARPlike domain containing proteins and discuss their possible roles in plant development, innate immune responses, programmed cell death and stress responses in general. The genome of the model plant Arabidopsis contains three genes encoding PARP proteins, two of which have been shown to be active PARPs, and two genes encoding PARG proteins, one of which was shown to possess enzymatic activity. In addition, SROs (Similar to RCD One) represent a plant specific family of proteins containing a PARP-like domain. Although bioinformatics and biochemical data suggest that the PARP-like domain in SRO proteins does not have PARP activity, these proteins play a significant role in stress response as revealed by mutant analysis. SRO proteins interact with transcription factors involved in various stress and developmental responses and are suggested to serve as hubs in many signaling pathways. Altogether current data imply that poly(ADP-ribos)ylation plays significant regulatory role in many aspects of plant biology.