Abstract Key message We provide data on seedlot germination potential—a key trait related to regeneration—of 12 oak species. Germination was tested at the University of Granada following international protocols with 8985 acorns from 93 batches and 16 countries across Europe. Data on germination probability, acorn origin, mass, and moisture content measured on another 4544 acorns are available at https://doi.org/10.30827/Digibug.87318. Associated metadata are available at https://metadata-afs.nancy.inra.fr/geonetwork/srv/fre/catalog.search#/metadata/a742c6d8-bc37-4ca2-8b81-2447c5a8858d.

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies.

In plants, exocyst subunit isoforms exhibit significant functional diversity in that they are involved in either protein secretion or autophagy, both of which are essential for plant development and survival. Although the molecular basis of autophagy is widely reported, its contribution to plant reproduction is not very clear. Here, we have identified Exo84c, a higher plant-specific Exo84 isoform, as having a unique function in modulating exocytotic compartment degradation during stigmatic tissue senescence. This process is achieved through its interaction with the ER localised VAP27 proteins, which regulate the turnover of Exo84c through the autophagy pathway. VAP27 recruits Exo84c onto the ER membrane as well as numerous ER-derived autophagosomes that are labelled with ATG8. These Exo84c/exocyst and VAP27 positive structures are accumulated in the vacuole for degradation, and this process is partially perturbed in the exo84c knock-out mutants. Interestingly, the exo84c mutant showed a prolonged effective pollination period with higher seed sets, possibly because of the delayed stigmatic senescence when Exo84c regulated autophagy is blocked. In conclusion, our studies reveal a link between the exocyst complex and the ER network in regulating the degradation of exocytosis vesicles, a process that is essential for normal papilla cell senescence and flower receptivity.

ER-mitochondria contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a novel eukaryotic EMCS complex, comprising a family of uncharacterised mitochondrial outer-membrane proteins, TraB1 and the ER protein, VAP27-1. In Arabidopsis, there are two TraB1 isoforms and the trab1a/trab1b double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TraB1 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trab1a/b and VAP27 mutants ( vap27-1/3/4/6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed. A similar phenotype is found in both autophagy mutants ( atg5 and atg7 ), suggesting that TraB1 regulated mitophagy and ER-mitochondrial tethering are two closely related processes, necessary for normal mitochondrial function. Taken together, we ascribe a dual role to TraB1 which is a novel component of the EMCS complex in eukaryotes, regulating both tethering of the mitochondria to the ER and mitophagy.

Douglas fir is expected to play an increasingly important role in Swedish forestry under a changing climate. Thus far, seed orchards with clones of phenotypically selected trees (plus trees) have been established to supply the market with highly qualitative reproductive material. Given the high genetic variation of the species, its growth properties are significantly affected by the provenance. Here, we applied microsatellite markers to identify the origin of clones selected within the Swedish breeding programme. Moreover, we analysed the timing of bud burst in open-pollinated families of these clones. In particular, we aimed to explain the provenance effect on phenology by using molecular identification as a proxy. A Bayesian clustering analysis with microsatellite data enabled the assignment of the clones to one of the two varieties and also resolved within-variety origins. The phenological observations indicated an earlier bud burst of the interior variety. Within the coastal variety, the northern provenances exhibited a later bud burst. We found a significant effect of the identified origin on bud burst timing. The results of this study will be used to support further breeding efforts.

Extracellular ATP is a purinergic signal with important functions in regulating plant growth and stress-adaptive responses, including programmed cell death. While signalling events proximate to receptor activation at the plasma membrane have been characterised, downstream protein targets and the mechanism of cell death activation/regulation are unknown. We designed a proteomic screen to identify ATP-responsive proteins in Arabidopsis cell cultures exposed to mycotoxin stress via fumonisin B1 (FB1) application. Arabidopsis RIBONUCLEASE 1 (RNS1) was identified by the screen, and transgenic plants overexpressing native RNS1 showed greater susceptibility to FB1, while a gene knockout rns1 mutant and antisense RNS1 transgenic plants were resistant to FB1-induced cell death. Native RNS1 complemented rns1 mutants and restored the cell death response to FB1, while a catalytically inactive version of the ribonuclease could not. The FB1 resistance of salicylic acid (SA)-depleted nahG-expressing plants was abolished by transformation with native RNS1, but not the catalytically dead version. The mechanism of FB1-induced cell death is activation of RNS1-dependent RNA cleavage, which is blocked by ATP via RNS1 suppression, or enhanced by SA through induction of RNS1 expression. Our study reveals RNS1 as a previously unknown convergence point of ATP and SA signalling in the regulation of stress-induced cell death.

ER-mitochondrial contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a EMCS complex, comprising a family of uncharacterised mitochondrial outer membrane proteins, TRB1, TRB2, and the ER protein, VAP27-1. In Arabidopsis, there are three TraB family isoforms and the trb1/trb2 double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation, and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TRB1 and TRB2 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trb1/trb2 and VAP27 mutants (vap27-1,3,4,6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed, suggesting that TRB1-regulated mitophagy and ER-mitochondrial interaction are two closely related processes. Taken together, we ascribe a dual role to TraB family proteins which are component of the EMCS complex in eukaryotes, regulating both interaction of the mitochondria to the ER and mitophagy.

Non-native forest tree species have been introduced in Europe since the 16th century, but only in the second half of the 20th century the significance of the seed source origin for their economic use was recognized, resulting in the establishment of numerous provenance trials at a national, regional, European and International level, as those led by IUFRO. Breeding programs have also been launched in the continent for the most economically important species. Aim of this work is the formulation of provenance recommendations for planting of five non-native tree species in Europe (Douglas fir, grand fir, Sitka spruce, lodgepole pine and black locust), based on the information obtained from twenty countries, in the frame of the EU FP-1403 NNEXT Cost Action. The survey revealed that official and non-official national recommendations, based on provenance research results, have been elaborated and followed at a different level and extend for the above five species, but only for Douglas fir recommendations exist in almost all the participating to the survey countries. The compilation of provenance recommendations across Europe for each species is presented in the current work. Besides the recommended introduced seed sources, European seed sources are also preferred for planting, due to ease of access and high availability of forest reproductive material. European breeding programs yielding genetic material of high productivity and quality constitute currently the seed source of choice for several species and countries. Consolidation of trial data obtained across countries will allow the joint analysis that is urgently needed to draw solid conclusions, and will facilitate the development of ‘Universal-Response-Functions’ for the species of interest, rendering possible the identification of the genetic material suitable for global change. New provenance trial series that will test seed sources from the entire climatic range of the species, established in sites falling within and outside the environmental envelopes of their natural ranges, are urgently needed to pinpoint and understand the species-specific climate constraints, as well as to correlate functional traits to the seed origin and the environmental conditions of the test sites, so that the selection of suitable forest reproductive material of non-native tree species in the face of climate change can be feasible.

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. They tether the cell’s plasma membrane (PM) to the actin network. Moreover, in a similar manner, they are also involved in the tethering of membrane bound organelles to the actin cytoskeleton; the endoplasmic reticulum (ER) and the ER to the PM. This raises the question as to whether NET proteins are involved in actin cytoskeletal remodelling. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function as downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure depends on a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune signalling events and proper actin cytoskeletal remodelling.

Competition is a concern for tree breeding because of its potential to reduce the genetic gain. Competition, if not accounted for in the analytical model, can potentially introduce a source of bias in genetic parameter estimation and breeding value prediction. This study modelled competition between trees in 20 Swedish progeny trials of Norway spruce (Picea abies (L.) Karst.), Scots pine (Pinus sylvestris L.), and lodgepole pine (Pinus contorta Douglas ex Loudon). The competition model assumed a tree has a direct additive genetic effect, which affects the tree’s own phenotype, and an indirect additive effect, which affects the phenotypes of its neighbours. Genetic parameters were estimated via a factor analytic structure in which separate indirect effects were considered for each neighbour, or via a combined indirect effect approach. We analysed diameter, as it is the trait that can be expected to be affected most by competition. Competition at the genetic level was detected in 17 of the 20 trials analysed. In most cases, the ratio of indirect to direct additive variance was less than 20% and no major changes in ranking resulted. At this stage, there is little incentive to incorporate indirect effects into program-wide genetic evaluation models. The added complexity is not commensurate with the benefit that would be gained.

We have recently characterised NET2A as a pollen-specific actin-binding protein that binds F-actin at the plasma membrane of growing pollen tubes. However, the role of NET2 proteins in pollen development and fertilisation have yet to be elucidated. To further characterise the role of Arabidopsis NET2 proteins in pollen development and fertilisation, we analysed the subcellular localisation of NET2A over the course of pollen grain development and investigated the role of the NET2 family using net2 loss-of-function mutants. We observed NET2A to localise to the F-actin cytoskeleton in developing pollen grains as it underwent striking structural reorganisations at specific stages of development and during germination and pollen tube growth. Furthermore, net2 loss-of-function mutants exhibited striking morphological defects in the early stages of pollen tube growth, arising from frequent changes to pollen tube growth trajectory. We observed defects in the cortical actin cytoskeleton and actin-driven subcellular processes in net2 mutant pollen tubes. We demonstrate that NET2 proteins are essential for normal actin-driven pollen development highlighting an important role for the NET2 family members in regulating pollen tube growth during fertilisation.

ATP is secreted to the extracellular matrix, where it activates plasma membrane receptors for controlling plant growth and stress-adaptive processes. DOES NOT RESPOND TO NUCLEOTIDES 1 (DORN1), was the first plant ATP receptor to be identified but key downstream proteins remain sought after. Here, we identified 120 proteins secreted by Arabidopsis cell cultures and screened them for putative stress-responsive proteins using ATP-affinity purification. We report three Arabidopsis proteins isolated by ATP-affinity: PEROXIDASE 52, SUBTILASE-LIKE SERINE PROTEASE 1.7 and PHOSPHOLIPASE C-LIKE 1. In wild-type Arabidopsis, the expression of genes encoding all three proteins responded to fumonisin B1, a cell death-activating mycotoxin. The expression of PEROXIDASE 52 and PHOSPHOLIPASE C-LIKE 1 was altered in fumonisin B1-resistant salicylic acid induction-deficient (sid2) mutants. Exposure to fumonisin B1 suppressed PHOSPHOLIPASE C-LIKE 1 expression in sid2 mutants, suggesting that the inactivation of this gene might provide mycotoxin tolerance. Accordingly, gene knockout mutants of PHOSPHOLIPASE C-LIKE 1 were resistant to fumonisin B1-induced death. The activation of PHOSPHOLIPASE C-LIKE 1 gene expression by exogenous ATP was not blocked in dorn1 loss-of-function mutants, indicating that DORN1 is not required. Furthermore, exogenous ATP rescued both the wild type and the dorn1 mutants from fumonisin-B1 toxicity, suggesting that different ATP receptor(s) are operational in this process. Our results point to the existence of additional plant ATP receptor(s) and provide crucial downstream targets for use in designing screens to identify these receptors. Finally, PHOSPHOLIPASE C-LIKE 1 serves as a convergence point for fumonisin B1 and extracellular ATP signalling, and functions in the Arabidopsis stress response to fumonisin B1.

In boreal forests, autumn frost tolerance in seedlings is a critical fitness component because it determines survival rates during regeneration. To understand the forces that drive local adaptation in this trait, we conducted freezing tests in a common garden setting for 54 Pinus sylvestris (Scots pine) populations (>5000 seedlings) collected across Scandinavia into western Russia, and genotyped 24 of these populations (>900 seedlings) at >10 000 SNPs. Variation in cold hardiness among populations, as measured by QST , was above 80% and followed a distinct cline along latitude and longitude, demonstrating significant adaptation to climate at origin. In contrast, the genetic differentiation was very weak (mean FST 0.37%). Despite even allele frequency distribution in the vast majority of SNPs among all populations, a few rare alleles appeared at very high or at fixation in marginal populations restricted to northwestern Fennoscandia. Genotype-environment associations showed that climate variables explained 2.9% of the genetic differentiation, while genotype-phenotype associations revealed a high marker-estimated heritability of frost hardiness of 0.56, but identified no major loci. Very extensive gene flow, strong local adaptation, and signals of complex demographic history across markers are interesting topics of forthcoming studies on this species to better clarify signatures of selection and demography.

The cambium and procambium generate the majority of biomass in vascular plants. These meristems constitute a bifacial stem cell population from which xylem and phloem are specified on opposing sides by positional signals. The PHLOEM INTERCALATED WITH XYLEM (PXY) receptor kinase promotes vascular cell division and organization. However, how these functions are specified and integrated is unknown. Here, we mapped a putative PXY-mediated transcriptional regulatory network comprising 690 transcription factor-promoter interactions in Arabidopsis (Arabidopsis thaliana). Among these interactions was a feedforward loop containing transcription factors WUSCHEL HOMEOBOX RELATED14 (WOX14) and TARGET OF MONOPTEROS6 (TMO6), each of which regulates the expression of the gene encoding a third transcription factor, LATERAL ORGAN BOUNDARIES DOMAIN4 (LBD4). PXY signaling in turn regulates the WOX14, TMO6, and LBD4 feedforward loop to control vascular proliferation. Genetic interaction between LBD4 and PXY suggests that LBD4 marks the phloem-procambium boundary, thus defining the shape of the vascular bundle. These data collectively support a mechanism that influences the recruitment of cells into the phloem lineage, and they define the role of PXY signaling in this context in determining the arrangement of vascular tissue.

Europe has a history rich in examples of successful and problematic introductions of trees with a native origin outside of Europe (non-native trees, NNT). Many international legal frameworks such as treaties and conventions and also the European Union have responded to the global concern about potential negative impacts of NNT that may become invasive in natural ecosystems. It is, however, national and regional legislation in particular that affects current and future management decisions in the forest sector and shapes the landscapes of Europe. We identified all relevant legal instruments regulating NNT, the different legal approaches and the regulatory intensity in 40 European countries (no microstates). Information on hard and effective soft law instruments were collected by means of a targeted questionnaire and consultation of international and national legislation information systems and databases. In total, 335 relevant legal instruments were in place in June/July 2019 to regulate the use of NNT in the investigated 116 geopolitical legal units (countries as well as sub-national regions with their own legislation). Countries and regions were empirically categorized according to ad hoc-defined legislation indicators. These indicators pay respect to the general bans on the introduction of non-native species, the generally allowed and prohibited NNT, approval mechanisms and specific areas or cases where NNT are restricted or prohibited. Our study revealed a very diverse landscape of legal frameworks across Europe, with a large variety of approaches to regulating NNT being pursued and the intensity of restriction ranging from very few restrictions on species choice and plantation surface area to the complete banning of NNT from forests. The main conclusion is that there is a clear need for more co-ordinated, science-based policies both at the local and international levels to enhance the advantages of NNT and mitigate potential negative effects.

38 The cambium and procambium generate the majority of biomass in vascular plants. These 39 meristems constitute a bifacial stem cell population from which xylem and phloem are 40 specified on opposing sides by positional signals. The PHLOEM INTERCALATED WITH 41 XYLEM (PXY) receptor kinase promotes vascular cell division and organisation. However, how these functions are specified and integrated is unknown. Here, we mapped a putative PXY-mediated transcriptional regulatory network comprising 690 transcription factor- promoter interactions in Arabidopsis thaliana (Arabidopsis). Among these interactions was a feed-forward loop containing transcription factors WUSCHEL HOMEOBOX RELATED14 (WOX14) and TARGET OF MONOPTEROS6 (TMO6), which each regulate the expression 47 of the gene encoding a third transcription factor, LATERAL ORGAN BOUNDARIES 48 DOMAIN4 (LBD4). PXY signalling in turn regulates the WOX14, TMO6, LBD4 loop to 49 control vascular proliferation. Genetic interaction between LBD4 and PXY suggests that LBD4 marks the phloem-procambium boundary, thus defining the shape of the vascular 51 bundle. These data collectively support a mechanism that influences recruitment of cells into 52 the phloem lineage, and they define the role of PXY signalling in this context in determining 53 the arrangement of vascular tissue.

In plants, cells do not migrate. Tissues are frequently arranged in concentric rings; thus, expansion of inner layers is coordinated with cell division and/or expansion of cells in outer layers. In Arabidopsis stems, receptor kinases, PXY and ER, genetically interact to coordinate vascular proliferation and organisation via inter-tissue signalling. The contribution of PXY and ER paralogues to stem patterning is not known, nor is their function understood in hypocotyls, which undergo considerable radial expansion. Here, we show that removal of all PXY and ER gene-family members results in profound cell division and organisation defects. In hypocotyls, these plants failed to transition to true radial growth. Gene expression analysis suggested that PXY and ER cross- and inter-family transcriptional regulation occurs, but it differs between stem and hypocotyl. Thus, PXY and ER signalling interact to coordinate development in a distinct manner in different organs. We anticipate that such specialised local regulatory relationships, where tissue growth is controlled via signals moving across tissue layers, may coordinate tissue layer expansion throughout the plant body.

A PXY-Mediated Transcriptional Network Integrates Signaling 2 Mechanisms to Control Vascular Development in Arabidopsis 3 4 Margot E. SmitŸ, Shauni McGregorŸ, Heng Sun, Catherine Gough, Anne-Maarit 5 Bågman, Cara L. Soyars, Johannes T. Kroon, Allison Gaudinier, Clara J. 6 Williams, Xiyan Yang, Zachary L. Nimchuk, Dolf Weijers, Simon R. Turner, 7 Siobhán M. Brady* and J. Peter Etchells* 8 9 1 University of California Davis, Plant Biology & Genome Center, Davis, California, 95616, 10 USA 11 2 Laboratory of Biochemistry, Wageningen University, the Netherlands. 12 3 Stanford University, Stanford, CA, USA, 94305-5020 13 4 Durham University, Department of Biosciences, Durham, DH1 3LE, UK 14 5 Current address: University of Sheffield, APS, Western Bank 15 6 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 16 Wuhan, Hubei, 430070, PR China 17 7 Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North 18 Carolina, USA. 19 8 Thermo Fisher Scientific 20 9 Department of Plant and Microbial Biology, University of California Berkeley, CA 94720, 21 USA 22 10 Current address: UGhent, VIB Department of Plant Systems Biology, Belgium. 23 11 University of Manchester, Faculty of Life Sciences, Manchester, M13 9PT, UK. 24 Ÿ Equal contribution 25 *Corresponding authors: Peter.Etchells@durham.ac.uk; sbrady@ucdavis.edu 26 27 Short title: An Arabidopsis vascular development network 28 29 One sentence summary: A feed-forward loop that controls vascular development was 30 uncovered by identifying a transcriptional network mediated by the receptor kinase 31 PHLOEM INTERCALATED WITH XYLEM. 32 33 The authors responsible for distribution of materials integral to the findings in this article in 34 accordance with the policy described in the instructions for authors (www.plantcell.org) are J. 35 Peter Etchells (Peter.Etchells@durham.ac.uk) and Siobhán M. Brady (sbrady@ucdavis.edu) 36 37 ABSTRACT 38 The cambium and procambium generate the majority of biomass in vascular plants. These 39 meristems constitute a bifacial stem cell population from which xylem and phloem are 40 specified on opposing sides by positional signals. The PHLOEM INTERCALATED WITH 41 XYLEM (PXY) receptor kinase promotes vascular cell division and organisation. However, 42 how these functions are specified and integrated is unknown. Here, we mapped a putative 43 Plant Cell Advance Publication. Published on December 5, 2019, doi:10.1105/tpc.19.00562

Quantitative genetic variation of fiber-dimension traits and their relationship with diameter at breast height (DBH) and solid-wood traits (i.e., density and modulus of elasticity (MOE)) was investigated in lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.). A total of 823 increment cores were sampled from 207 half-sib families at two independent progeny trials, aged 34–35 years, located in northern Sweden. High-resolution pith-to-bark profiles were obtained for radial fiber width (RFW), tangential fiber width (TFW), fiber wall thickness (FWT), and fiber coarseness (FC) using SilviScan. Heritabilities ranged from 0.29 to 0.74, and inheritance increased with cambial maturity. Estimated age–age genetic correlations indicate that early selection between ages 5 and 8 years is highly efficient. Our results indicate that selection for a 1% increase in DBH or MOE incurs a negligible effect on fiber-dimension traits and maximum genetic gains are reached when DBH and MOE are considered jointly. Moreover, simultaneous improvement of growth and stiffness is achievable when a selection index with 7 to 10 economical weights for MOE relative to 1 for DBH is incorporated. However, the unfavorable relationship between solid-wood traits and pulp and paper related traits suggests that breeding strategies must be implemented to improve wood quality of lodgepole pine for multiple uses.

Mixing tree species could be a silviculture model that allows early harvest of short-rotation trees, while longer-rotation crop trees remain in the stand. We examined the effects on growth and tree characteristics in a planted experiment with lodgepole pine (LP) and elite-bred Scots pine (SP) in mixed (50/50) and monospecific plots in three different spacings (at 28 years of age after planting). The future development under different thinning regimes, including net present value for one rotation, was analyzed using the Heureka simulation software. As expected, LP had higher survival and initially more rapid growth than SP, with highest stand productivity and biomass production in LP monoculture during a rotation period as a result. However, intimate mixtures of SP and LP at the two widest spacings could give greater production and economic benefits, compared to SP in monoculture. It seems that elite-bred SP will differ in competitiveness against LP, depending on spacing for growth and some quality traits (branch and bark thickness, height of green crown). The findings support developing management systems for combining sparsely planted, and expensive, elite-bred SP in mixture with other trees that maintains high stem volume production and secures certain properties of trees and stands.