Abstract Oaks ( Quercus L.) are among the most ecologically important tree genera in the northern hemisphere, with an intricate evolutionary history reflected in a reticulated phylogeny. Oak diversity has been profoundly shaped by introgression and diversification, yet the Iberian Peninsula remains an understudied natural laboratory for understanding these evolutionary processes. We used RAD-seq to characterize 38 taxa (including nothotaxa) and investigate the evolutionary history of the Iberian white oaks, with an emphasis on hybrid swarms. Results led to a readdressing of Iberian white oak species, expanding our current understanding of the phylogeography of the European Section Quercus . Furthermore, molecular evidence led to the circumscription of two new subsections, reflecting the contrast between typical temperate and Atlantic distributed species (Group A), and the submediterranean marcescent oaks (Group B). The former unveiled the recovery of Q. estremadurensis and a Northwestern Iberian lineage represented by Q. broteroana and Q. orocantabrica as southwestern representatives of the broad European pedunculate oaks ( Q. robur s.l. ). The latter led to the validation of hybrid swarms, emphasizing the Iberian oak syngameon and the importance of gene flow to oak evolution. Ultimately, our approach advances the understanding of European white oak evolution across different evolutionary scales, establishing the Iberian Peninsula as an important reservoir of oak diversity.

Abstract Background and Aims The development of unisexual flowers relies on the tight coordination of flower organ identity and sex determination. The genus Quercus is typically considered strictly monoecious, bearing fully segregated male and female flowers within the same individual tree. However, several reports of atypical flowering across the genus challenge this canonical view, suggesting that flowering in oaks may be more flexible than traditionally assumed. In this work, the dynamics of flower development in Quercus orocantabrica were examined to correlate contrasting floral morphologies with divergent molecular profiles. Methods The flowering phenology of Q. orocantabrica trees was closely monitored over several individuals and years, together with a detailed floral morphological analysis of male, female and atypical flowers. Key floral homeotic gene homologues were identified, and their expression assayed in the development of different flowers. Key Results Recurrent and widespread hermaphroditic flowering was detected in several Q. orocantabrica trees, frequently associated with unseasonal flowering events. Gene expression analysis of male, female and hermaphroditic flowers revealed a sex-biased expression of Q. orocantabrica B- and C-class genes, with the B-class gene QoPI in particular being tightly associated with the presence of fully-developed stamens. In addition, the expression of the C-class gene QoSHP contrasted with reports in other Fagaceae, highlighting a potential functional divergence of the C/D-class lineage within the family. Conclusions The results here depicted indicate that the dynamics of floral sex identity in oaks are more plastic than traditionally assumed, supporting a reinterpretation of oak reproductive biology based on a versatile and resilient framework responsive to different developmental contexts.

Catharanthus roseus leaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell type localizations, and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialized idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts of C. roseus leaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterized. This involved the assembly of an improved C. roseus transcriptome from short- and long-read data, IDIO+. It was observed that C. roseus mesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast to surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the key to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.

To explore the connection between chloroplast and coffee resistance factors, designated as SH1 to SH9, whole genomic DNA of 42 coffee genotypes was sequenced, and entire chloroplast genomes were de novo assembled. The chloroplast phylogenetic haplotype network clustered individuals per species instead of SH factors. However, for the first time, it allowed the molecular validation of Coffea arabica as the maternal parent of the spontaneous hybrid "Híbrido de Timor". Individual reads were also aligned on the C. arabica reference genome to relate SH factors with chloroplast metabolism, and an in-silico analysis of selected nuclear-encoded chloroplast proteins (132 proteins) was performed. The nuclear-encoded thioredoxin-like membrane protein HCF164 enabled the discrimination of individuals with and without the SH9 factor, due to specific DNA variants linked to chromosome 7c (from C. canephora-derived sub-genome). The absence of both the thioredoxin domain and redox-active disulphide center in the HCF164 protein, observed in SH9 individuals, raises the possibility of potential implications on redox regulation. For the first time, the identification of specific DNA variants of chloroplast proteins allows discriminating individuals according to the SH profile. This study introduces an unexplored strategy for identifying protein/genes associated with SH factors and candidate targets of H. vastatrix effectors, thereby creating new perspectives for coffee breeding programs.

Catharanthus roseus leaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell-type localizations and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialised idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts of C. roseus leaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterised. This involved the assembly of an improved C. roseus transcriptome from short- and long-read data, IDIO+. It was observed that C. roseus mesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast with surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the keys to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.

AbstractCatharanthus roseusleaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell-type localizations and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialised idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts ofC. roseusleaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterised. This involved the assembly of an improvedC. roseustranscriptome from short- and long-read data, IDIO+. It was observed thatC. roseusmesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast with surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the keys to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.HighlightCatharanthus mesophyll idioblasts are a hotspot of anticancer alkaloid accumulation. The idioblast transcriptome reveals commitment with stress responses and provides a roadmap towards the increase of anticancer alkaloid levels.

Iron (Fe) is a micronutrient that is essential for plant growth and development as well as for crop productivity and the quality of their derived products. Despite its high abundance in Earth’s crust, Fe is poorly available to plants in one-third of the cultivated land. This is because at neutral to alkaline pH, Fe is mostly present in the form of oxides/hydroxides that are not readily available for plants. Nevertheless, not only Fe deficiency, but also Fe excess is detrimental to the plant. This is due to the capacity of Fe to interact with oxygen in aerobic conditions, leading to the generation of reactive oxygen species via the Fenton reaction. Therefore, to maintain optimal Fe levels in plant cells, Fe homeostasis must be tightly regulated. To this end, plants have evolved several molecular mechanisms modulating Fe uptake, partitioning, and assimilation. Within this chapter, the main strategies evolved by plants to take up Fe from soil will be first described. Then, the main molecular mechanism regulating this process will be summarized. Last, an outline will be given on how abiotic (i.e., other micro- and macronutrients) and biotic factors affect Fe homeostasis in plants.

Catharanthus roseus leaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell-type localizations and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialised idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts of C. roseus leaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterised. This involved the assembly of an improved C. roseus transcriptome from short- and long-read data, IDIO+. It was observed that C. roseus mesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast with surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the keys to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs. ### Competing Interest Statement The authors have declared no competing interest.

The ubiquitin-like modifying peptide SMALL UBIQUITIN-LIKE MODIFIER (SUMO) has become a known modulator of the plant response to multiple environmental stimuli. A common feature of many of these external stresses is the production of reactive oxygen species (ROS). Taking into account that SUMO conjugates rapidly accumulate in response to an external oxidative stimulus, it is likely that ROS and sumoylation converge at the molecular and regulatory levels. In this study, we explored the SUMO-ROS relationship, using as a model the Arabidopsis (Arabidopsis thaliana) null mutant of the major SUMO-conjugation enhancer, the E3 ligase SAP AND MIZ 1 (SIZ1). We showed that SIZ1 is involved in SUMO conjugate increase when primed with both exogenous and endogenous ROS. In siz1, seedlings were sensitive to oxidative stress imposition, and mutants accumulated different ROS throughout development. We demonstrated that the deregulation in hydrogen peroxide and superoxide homeostasis, but not of singlet O-2 (O-1(2)), was partially due to SA accumulation in siz1. Furthermore, transcriptomic analysis highlighted a transcriptional signature that implicated siz1 with O-1(2) homeostasis. Subsequently, we observed that siz1 displayed chloroplast morphological defects and altered energy dissipation activity and established a link between the chlorophyll precursor protochlorophyllide and deregulation of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA), which is known to drive overproduction of O-1(2). Ultimately, network analysis uncovered known and additional associations between transcriptional control of PORA and SIZ1-dependent sumoylation. Our study connects sumoylation, and specifically SIZ1, to the control of chloroplast functions and places sumoylation as a molecular mechanism involved in ROS homeostatic and signaling events. Protein modification by sumoylation and reactive oxygen species are reciprocally regulated in Arabidopsis, with the sumoylation SIZ1 enzyme playing a critical role via control of gene expression.

The zinc deficiency response in Arabidopsis thaliana is regulated by F-group basic region leucine-zipper (F-bZIP) transcription factors, and there is evidence of evolutionary conservation of this regulatory network in land plants. Fundamental knowledge on the zinc homeostasis regulation in crop species will contribute to improving their zinc nutritional value. Legumes are protein-rich crops, used worldwide as part of traditional diets and as animal forage, being therefore a good target for micronutrient biofortification. Here, we identified F-bZIP transcription factors in representative legume species and functionally characterized the two F-bZIPs from Medicago truncatula. Results indicate that MtFbZIP1 is the functional homolog of A. thaliana bZIP19 and bZIP23, while MtFbZIP2 does not play a role in the zinc deficiency response. Additionally, analysis of M. truncatula genes from the Zrt/Irt-like protein (ZIP) family of zinc transporters or encoding nicotianamine synthase enzymes that produce the zinc ligand nicotianamine, support the conservation of the F-bZIP-regulated zinc deficiency response in M. truncatula. Phylogenetic analysis of F-bZIP homologs enriched in legume species reinforces the branching into two groups, with MtFbZIP1 and MtFbZIP2 mapping in Groups 1 and 2, respectively. This phylogeny combined with the functional characterization of MtFbZIPs supports the suggested conservation of the zinc deficiency response associated with Group 1 F-bZIPs, and the more variable evolutionary paths associated with Group 2. Overall, we provide novel insight on the mechanisms of response to zinc deficiency in M. truncatula, which contributes to developing strategies for improving zinc content in legume crops.

Educational gardens can be a significant resource in the promotion of environmental education, engaging both the school population and the general public. The main goal of the present study was to implement and assess a hands-on interventional program to promote knowledge and awareness of plant-related topics at a basic school level. We report on a hands-on educational project implemented with 8th-grade Portuguese students (mostly 13–14 years of age), associated with the establishment, on school grounds, of three educational gardens representing distinct Portuguese ecosystems. This was a collaborative project and encompassed several activities and subjects, including garden creation, plant propagation and plant care, plant identification, the study of form–function relationships, and lectures by plant researchers. A survey instrument with pre- and post-test assessments demonstrated the effectiveness of the program in raising student knowledge and awareness on topics centered around the native flora. Specifically, we noted that scores increased in all questions addressing different plant biology-related topics in the post-test assessment. This study supports the benefits of incorporating field/laboratory work and educational gardens in educational programs geared toward plant-oriented environmental education.

ABSTRACTGrapevine (Vitis viniferaL.) is one of the most significant crops in the world. Today’s richness in grapevine diversity results from a complex domestication history over multiple historical periods. Here, we employed whole genome resequencing to elucidate different aspects of the recent evolutionary history of this crop. Our results support a model in which a central domestication event in grapevine was followed by post-domestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties, which show large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by geneflow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.

Background and Aims The characterisation of plant microbiomes using metabarcoding strategies is expected to be progressively replaced by shotgun metagenomic (SMg) approaches. In the present report we explore the potential of applying SMg to grapevine leaf, rhizosphere and soil samples. Methods and Results Our strategy involved combining a single method for column-based DNA extraction of multiple tissues, with sequencing using the BGISEQ short-read next-generation sequencing platform. This study details aspects of DNA isolation, library construction, sequencing and early bioinformatics treatment of read data, including the use of subsampling as a proxy for detection of sample microbial proportions. The combination of an innovative sequencing platform with recent mapping tools allowed for the characterisation of the microbiome associated with the grapevine leaf, rhizosphere and adjacent soil. Conclusions We coupled a robust single extraction/library preparation protocol suitable for contrasting samples, with cost-effective BGISEQ short-read next-generation sequencing technology, to generate a landscape of grapevine-associated microbial diversity. Significance of the Study Shotgun metagenomics is emerging as a state-of-the-art approach to grapevine microbiome characterisation. The present report provides a detailed and technical roadmap targeting multiple aspects of this important approach.

Grapevine (Vitis vinifera L.) diversity richness results from a complex domestication history over multiple historical periods. Here, we used whole-genome resequencing to elucidate different aspects of its recent evolutionary history. Our results support a model in which a central domestication event in grapevine was followed by postdomestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties showing large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by gene flow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.

The F-bZIP transcription factors bZIP19 and bZIP23 are the central regulators of the zinc deficiency response in Arabidopsis, and phylogenetic analysis of F-bZIP homologs across land plants indicates that the regulatory mechanism of the zinc deficiency response may be conserved. Here, we identified the rice F-bZIP homologs and investigated their function. OsbZIP48 and OsbZIP50, but not OsbZIP49, complement the zinc deficiency-hypersensitive Arabidopsis bzip19bzip23 double mutant. Ectopic expression of OsbZIP50 in Arabidopsis significantly increases plant zinc accumulation under control zinc supply, suggesting an altered Zn sensing in OsbZIP50. In addition, we performed a phylogenetic analysis of F-bZIP homologs from representative monocot species that supports the branching of plant F-bZIPs into Group 1 and Group 2. Our results suggest that regulation of the zinc deficiency response in rice is conserved, with OsbZIP48 being a functional homolog of AtbZIP19 and AtbZIP23. A better understanding of the mechanisms behind the Zn deficiency response in rice and other important crops will contribute to develop plant-based strategies to address the problems of Zn deficiency in soils, crops, and cereal-based human diets.

Figure 1: Hexokinase SUMO site and SUMO-interacting motif (SIM) prediction and evolutionary conservation in plants. (A) Predicted sumoylation and SIM sites are indicated in red and blue, respectively. Light yellow and brown correspond to hexokinase small and large subdomains, respectively. The orange colour indicates the residues that were previously determined to have a role in AtHXK1 functioning (Moore et al., 2003). Scale bar indicates 10 amino acids (aa). (B) The plant Hexokinase phylogenetic tree was generated using RaxML with 1000 bootstrap values (numbers represent the bootstrap percentage). Lysines with high or medium probability of sumoylation, inferred bioinformatically using GPSSUMO 1.0, are indicated between brackets. The prefix for each sequence corresponds to prefixes for GeneIDs as implemented in PLAZA (Van Bel et al., 2018), and represent the following species: At (Arabidopsis thaliana, ARATH), Solyc (Solanum lycopersicum, SOLYC), GSVIV (Vitis vinifera, VITVI), LOC_Os and Os (Oryza sativa ssp. Japonica, ORYSJ), ATR (Amborella trichopoda, AMBTC), PAB (Picea abies, PICAB), SMO (Selaginella moellendorffii, SELML), Pp (Physcomitrella patens, PHYPA), and Cre (Chlamydomonas reinhardtii, CHLRE). (C) Multiple sequence alignment of SUMO and SIM sites in Arabidopsis thaliana, Amborella trichopoda, and group 6/7 HXKs. Amino acid consistency is classified from 0 (unconserved) to 10 (conserved). Arrows indicate motifs with predicted SUMO-targeted lysine. Asterisk indicates a SUMO site predicted by GSP-SUMO to be low probability rather than high/medium probability. The first set of alignments corresponds to Arabidopsis thaliana HXKs (excluding the truncated AtHXK7). The second set of alignments was produced with Amborella trichopoda sequences (excluding the truncated ATR0481G001). The last panel of alignment was produced using members from HXK group 6 and 7, as classified by Karve et al. (2010). 6/29/2020 Open Access

Figure 1: Characterization of the double T-DNA insertion mutant siz1-2 kxk1-1 (siz1 hxk1). (A) Summarized information regarding HXK1 T-DNA insertion mutants in the Arabidopsis thaliana Columbia-0 (Col-0) ecotype background, with updated designations; KD and KO stand for knockdown and knockout, respectively. (B) Representation of the HXK1 gene displaying exons (white boxes), introns (thin lines), and UTRs (grey boxes). The site and orientation of the T-DNA insertion (triangle with insertion mutant line code) and location of primers used for genotyping are represented; scale bar indicates 100 base pairs (bp). (C) Genotyping PCR, confirming the presence or absence of the T-DNA insertion for the hxk1-1 and siz1-2 mutant lines. (D) Morphology of 1-month-old plants grown under long days. Scale bar indicates 1 cm. (E) Morphological measurement of the maximum rosette radius for each genotype. Error bars represent SEM (n≥5). (F) Seed germination percentages scored by green cotyledon appearance 10 days after sowing onto MS media; error bars represent SEM (n≥4). Asterisks represent statistically significant differences between genotypes (unpaired t test; NS, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Description 6/29/2020 Open Access

Functional insight on the post-translational modifier SUMO and its biochemical pathway in plants has steadily increased over the past decade. In contrast to the low number of core components that catalytically control SUMO attachment to targets, the enzymes that control deconjugation and SUMO maturation seem to have diversified in terms of both gene number and biological function. However, studies on these deSUMOylating proteases have been accompanied by diversity in nomenclature and unclear evolutionary categorization. We provide a state-of-the-art assessment of the evolutionary subclades within the ULP gene family of plant deSUMOylating proteases, and propose a nomenclature for this protease subgroup for consistent annotation of ULP-encoding genes in plant genomes.