A new approach for artificial pollination in walnut trees: AirPoll.

Pollination is the first step in the plant's fruit development. Therefore, fruit setting does not occur without pollination. Some problems encountered in natural pollination cause pollination not to be achieved as desired and cause significant losses in yield and fruit quality. Artificial pollination applications with drones are the best way to solve these problems. In this study, the AirPoll artificial pollination machine, which performs artificial pollination through the air using drone technology, was developed and the operating success of the machine was tested in walnut gardens. In the experiment gardens, female flowers on 18 branches of 5 trees each in the artificially pollinated area with a drone and in the control area were marked with colored strings. Control trees were selected from a distance that would not be possible to transport pollen with a drone. As a result of the study carried out in 2020 and 2021, the average fruit setting rate in trees pollinated by drone was determined as 94.61%. In control trees, 32.33% fruit setting was achieved. Thus, it was determined that the productivity increase in artificial pollination with AirPoll was 62.28%. In addition, in the study, Computational Fluid Dynamics (CFD) simulation analysis was performed using ANSYS Fluent 2024 R1 software to predict the downward air flow and pollen distribution in the walnut tree crown. The analysis was carried out in 680 iterations using drone propellers at a rotation speed of 4500 rpm, 4 m/s airflow and a k-w viscous model. In the analysis, it was observed that the pollen was distributed homogeneously with the determined height and the created artificial pollination environment. Based on the results obtained from the simulations, a convergence criterion of 5e-3 for continuity and 1e-6 for speed, k, w was determined. Considering all the results, the ease of use of the developed AirPoll artificial pollination machine and the successful results obtained in field trials reveal the effectiveness of the AirPoll artificial pollination machine.

Comparative genomics of six Juglans species reveals disease-associated gene family contractions.

Juglans (walnuts), the most speciose genus in the walnut family (Juglandaceae), represents most of the family's commercially valuable fruit and wood-producing trees. It includes several species used as rootstock for their resistance to various abiotic and biotic stressors. We present the full structural and functional genome annotations of six Juglans species and one outgroup within Juglandaceae (Juglans regia, J. cathayensis, J. hindsii, J. microcarpa, J. nigra, J. sigillata and Pterocarya stenoptera) produced using BRAKER2 semi-unsupervised gene prediction pipeline and additional tools. For each annotation, gene predictors were trained using 19 tissue-specific J. regia transcriptomes aligned to the genomes. Additional functional evidence and filters were applied to multi-exonic and mono-exonic putative genes to yield between 27 000 and 44 000 high-confidence gene models per species. Comparison of gene models to the BUSCO embryophyta dataset suggested that, on average, genome annotation completeness was 85.6%. We utilized these high-quality annotations to assess gene family evolution within Juglans, and among Juglans and selected Eurosid species. We found notable contractions in several gene families in J. hindsii, including disease resistance-related wall-associated kinase (WAK), Catharanthus roseus receptor-like kinase (CrRLK1L) and others involved in abiotic stress response. Finally, we confirmed an ancient whole-genome duplication that took place in a common ancestor of Juglandaceae using site substitution comparative analysis.

Predicting Stomatal Closure and Turgor Loss in Woody Plants Using Predawn and Midday Water Potential.

Knowledge about physiological stress thresholds provides crucial information about plant performance and survival under drought. In this study, we report on the triphasic nature of the relationship between plant water potential (Ψ) at predawn and midday and describe a method that predicts Ψ at stomatal closure and turgor loss exclusively from this water potential curve (WP curve). The method is based on a piecewise linear regression model that was developed to predict the boundaries (termed Θ1 and Θ2) separating the three phases of the curve and corresponding slope values. The method was tested for three economically important woody species. For all species, midday Ψ was much more negative than predawn Ψ during phase I (mild drought), reductions in midday Ψ were minor while predawn Ψ continued to decline during phase II (moderate drought), and midday and predawn Ψ reached similar values during phase III (severe drought). Corresponding measurement of leaf gas exchange indicated that boundary Θ1 between phases I and II coincided with Ψ at stomatal closure. Data from pressure-volume curves demonstrated that boundary Θ2 between phases II and III predicted Ψ at leaf turgor loss. The WP curve method described here is an advanced application of the Scholander-type pressure chamber to categorize plant dehydration under drought into three distinct phases and to predict Ψ thresholds of stomatal closure and turgor loss.

Association and linkage mapping to unravel genetic architecture of phenological traits and lateral bearing in Persian walnut (Juglans regia L.).

BACKGROUND: Unravelling the genetic architecture of agronomic traits in walnut such as budbreak date and bearing habit, is crucial for climate change adaptation and yield improvement. A Genome-Wide Association Study (GWAS) using multi-locus models was conducted in a panel of 170 walnut accessions genotyped using the Axiom™ J. regia 700 K SNP array, with phenological data from 2018, 2019 and legacy data. These accessions come from the INRAE walnut germplasm collection which is the result of important prospecting work performed in many countries around the world. In parallel, an F1 progeny of 78 individuals segregating for phenology-related traits, was genotyped with the same array and phenotyped for the same traits, to construct linkage maps and perform Quantitative Trait Loci (QTLs) detection. RESULTS: Using GWAS, we found strong associations of SNPs located at the beginning of chromosome 1 with both budbreak and female flowering dates. These findings were supported by QTLs detected in the same genomic region. Highly significant associated SNPs were also detected using GWAS for heterodichogamy and lateral bearing habit, both on chromosome 11. We developed a Kompetitive Allele Specific PCR (KASP) marker for budbreak date in walnut, and validated it using plant material from the Walnut Improvement Program of the University of California, Davis, demonstrating its effectiveness for marker-assisted selection in Persian walnut. We found several candidate genes involved in flowering events in walnut, including a gene related to heterodichogamy encoding a sugar catabolism enzyme and a cell division related gene linked to female flowering date. CONCLUSIONS: This study enhances knowledge of the genetic architecture of important agronomic traits related to male and female flowering processes and lateral bearing in walnut. The new marker available for budbreak date, one of the most important traits for good fruiting, will facilitate the selection and development of new walnut cultivars suitable for specific climates.

Precocious genotypes and homozygous tendency generated by self-pollination in walnut.

BACKGROUND: Observations of precocious (early bearing) genotypes of walnut (Juglans regia L.) under natural conditions encouraged us to study the origin and genetic control of these fascinating traits. RESULTS: In this study, the self-fertility, progeny performance, and simple sequence repeat (SSR) locus variation of iron walnut (Juglans sigillata Dode), an ecotype of J. regia, were investigated. The average self-pollinated fruit set rate of J. sigillata cv. 'Dapao' (DP) was 7.0% annually from 1979 to 1982. The average germination rate of self-pollinated seeds was 45.2% during the 4-year period. Most progeny had inbreeding depression. Nine representative self-pollinated progeny (SP1-SP9), with special or typical traits of DP, were selected. SP1-SP4 were precocious because they initiated flowers as early as 2 years after germination, compared to the 7-10-yr period that is typical of DP. SP9 had not flowered since 1980. Twelve SSR markers were used to analyze the SP and DP. The genome of SP had a tendency toward high levels of homozygosity. The high levels of homozygosity reported in 18 additional precocious walnut genotypes complemented the results of this study. CONCLUSIONS: These results provide evidence of precocious phenotypes and genomes with high levels of homozygosity that might be generated from self-pollinating walnut. This suggests that self-pollination might facilitate the generation of unique homozygous parents for subsequent use in walnut-breeding programs. The results also indicate that more attention should be focused on adequate management of precocious walnut to avoid early depression in the production of nuts.

The walnut transcription factor JrGRAS2 contributes to high temperature stress tolerance involving in Dof transcriptional regulation and HSP protein expression.

BACKGROUND: GRAS transcription factor (TF) family is unique and numerous in higher plants with diverse functions that involving in plant growth and development processes, such as gibberellin (GA) signal transduction, root development, root nodule formation, and mycorrhiza formation. Walnut tree is exposed to various environmental stimulus that causing concern about its resistance mechanism. In order to understand the molecular mechanism of walnut to adversity response, a GRAS TF (JrGRAS2) was cloned and characterized from Juglans regia in this study. RESULTS: A 1500 bp promoter fragment of JrGRAS2 was identified from the genome of J. regia, in which the cis-elements were screened. This JrGRAS2 promoter displayed expression activity that was enhanced significantly by high temperature (HT) stress. Yeast one-hybrid assay, transient expression and chromatin immunoprecipitation (Chip)-PCR analysis revealed that JrDof3 could specifically bind to the DOFCOREZM motif and share similar expression patterns with JrGRAS2 under HT stress. The transcription of JrGRAS2 was induced by HT stress and up-regulated to 6.73-~11.96-fold in the leaf and 2.53-~4.50-fold in the root to control, respectively. JrGRAS2 was overexpressed in Arabidopsis, three lines with much high expression level of JrGRAS2 (S3, S7, and S8) were selected for HT stress tolerance analysis. Compared to the wild type (WT) Arabidopsis, S3, S7, and S8 exhibited enhanced seed germination rate, fresh weight accumulation, and activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione-S-transferase (GST) under HT stress. In contrast, the Evans blue staining, electrolyte leakage (EL) rates, hydrogen dioxide (H2O2) and malondialdehyde (MDA) content of transgenic seedlings were all lower than those of WT exposed to HT stress. Furthermore, the expression of heat shock proteins (HSPs) in S3, S7, and S8 was significant higher than those in WT plants. The similar results were obtained in JrGRAS2 transient overexpression walnut lines under normal and HT stress conditions. CONCLUSIONS: Our results suggested that JrDof3 TF contributes to improve the HT stress response of JrGRAS2, which could effectively control the expression of HSPs to enhance HT stress tolerance. JrGRAS2 is an useful candidate gene for heat response in plant molecular breeding.

The walnut JrVHAG1 gene is involved in cadmium stress response through ABA-signal pathway and MYB transcription regulation.

BACKGROUND: Vacuolar H+-ATPase (V-ATPase) is a vital protein complex involved in abiotic stress response in plants. The G subunit of Juglans regia (JrVHAG1) was previously identified as a drought tolerance-related gene involved in the ABA (abscisic acid)-signal pathway. Heavy metal stress is becoming a major detriment for plant growth, development, and production. In order to understand the role of JrVHAG1, the potential function mechanism of JrVHAG1 exposed to CdCl2 stress was confirmed in this study. RESULTS: Transcription of JrVHAG1 was induced by ABA and increased to 58.89-fold (roots) and 7.38-fold (leaves) and by CdCl2 to 2.65- (roots) and 11.42-fold (leaves) relative to control, respectively. Moreover, when treated simultaneously with ABA and CdCl2 (ABA+CdCl2), JrVHAG1 was up-regulated to 110.13- as well as 165.42-fold relative to control in the roots and leaves, accordingly. Compared to the wild type (WT) Arabidopsis plants, the transgenic plants with overexpression of JrVHAG1 (G2, G6, and G9) exhibited increased seed germination rate, biomass accumulation, proline content, and activities of superoxide dismutase (SOD) and peroxidase (POD) under ABA, CdCl2, and ABA+CdCl2 treatments. In contrast, the reactive oxygen species (ROS) staining, malondialdehyde (MDA) content, hydrogen dioxide (H2O2) content, as well as electrolyte leakage (EL) rates of transgenic seedlings were all lower than those of WT exposed to ABA, CdCl2 and ABA+CdCl2 stresses. Furthermore, a 1200 bp promoter fragment of JrVHAG1 was isolated by analyzing the genome of J. regia, in which the cis-elements were identified. This JrVHAG1 promoter fragment showed expression activity that was enhanced significantly when subjected to the above treatments. Yeast one-hybrid assay and transient expression analysis demonstrated that JrMYB2 specifically bound to the MYBCORE motif and shared similar expression patterns with JrVHAG1 under ABA, CdCl2 and ABA+CdCl2 stress conditions. CONCLUSIONS: Our results suggested that the JrVHAG1 gene functions as a CdCl2 stress response regulator by participating in ABA-signal pathway and MYB transcription regulation network. JrVHAG1 gene is a useful candidate gene for heavy metal stress tolerance in plant molecular breeding.

Assessing frost damages using dynamic models in walnut trees: exposure rather than vulnerability controls frost risks.

Frost damages develop when exposure overtakes frost vulnerability. Frost risk assessment therefore needs dynamic simulation of frost hardiness using temperature and photoperiod in interaction with developmental stage. Two models, including or not the effect of photoperiod, were calibrated using five years of frost hardiness monitoring (2007-2012), in two locations (low and high elevation) for three walnut genotypes with contrasted phenology and maximum hardiness (Juglans regia cv Franquette, J. regia × nigra 'Early' and 'Late'). The photothermal model predicted more accurate values for all genotypes (efficiency = 0.879; Root Mean Standard Error Predicted (RMSEP) = 2.55 °C) than the thermal model (efficiency = 0.801; RMSEP = 3.24 °C). Predicted frost damages were strongly correlated to minimum temperature of the freezing events (ρ = -0.983) rather than actual frost hardiness (ρ = -0.515), or ratio of phenological stage completion (ρ = 0.336). Higher frost risks are consequently predicted during winter, at high elevation, whereas spring is only risky at low elevation in early genotypes exhibiting faster dehardening rate. However, early frost damages, although of lower value, may negatively affect fruit production the subsequent year (R2  = 0.381, P = 0.057). These results highlight the interacting pattern between frost exposure and vulnerability at different scales and the necessity of intra-organ studies to understand the time course of frost vulnerability in flower buds along the winter.

Phenological calendar in some walnut genotypes grown in Romania and its correlations with air temperature.

Phenology is the study of periodic biological events in the plant world that are influenced by the environment. Temperature increase in spring season can advance the spring phases, but warming in autumn and winter may slow the fulfillment of chilling requirements and lead to later onset of spring events. This study examined different genotypes of walnut trees under environmental conditions, and measured the cold demand, heat requirements, and average time from budburst till flowering for 28 walnut genotypes. The information obtained provides a better understanding of the phenological temperature response of walnuts that will be useful for walnut production and will also contribute to the development of adaptation measures in the light of the expected climate change.

Spring bud growth depends on sugar delivery by xylem and water recirculation by phloem Münch flow in Juglans regia.

MAIN CONCLUSION: During spring, bud growth relies on long-distance transport of remotely stored carbohydrates. A new hypothesis suggests this transport is achieved by the interplay of xylem and phloem. During the spring, carbohydrate demand of developing buds often exceeds locally available storage, thus requiring the translocation of sugars from distant locations like limbs, stems and roots. Both the phloem and xylem have the capacity for such long-distance transport, but their functional contribution is unclear. To address this ambiguity, the spatial and temporal dynamics of carbohydrate availability in extension shoots of Juglans regia L. were analyzed. A significant loss of extension shoot carbohydrates in remote locations was observed while carbohydrate availability near the buds remained unaffected. This pattern of depletion of carbohydrate reserves supports the notion of long-distance translocation. Girdling and dye perfusion experiments were performed to assess the role of phloem and xylem in the transport of carbohydrate and water towards the buds. Girdling caused a decrease in non-structural carbohydrate concentration above the point of girdling and an unexpected concurrent increase in water content associated with impeded xylem transport. Based on experimental observations and modeling, we propose a novel mechanism for maintenance of spring carbohydrate translocation in trees where xylem transports carbohydrates and this transport is maintained with the recirculation of water by phloem Münch flow. Phloem Münch flow acts as a pump for generating water flux in xylem and allows for transport and mobilization of sugars from distal locations prior to leaves photosynthetic independence and in the absence of transpiration.

Characterization of a vacuolar H+-ATPase G subunit gene from Juglans regia (JrVHAG1) involved in mannitol-induced osmotic stress tolerance.

KEY MESSAGE: JrVHAG1 is an important candidate gene for plant osmotic tolerance regulation. Vacuolar H+-ATPase (V-ATPase) is important for plant responses to abiotic stress; the G subunit is a vital part of V-ATPase. In this study, a G subunit of V-ATPase was cloned from Juglans regia (JrVHAG1) and functionally characterized. JrVHAG1 transcription was induced by mannitol that increasing 17.88-fold in the root at 12 h and 19.16-fold in the leaf at 96 h compared to that under control conditions. JrVHAG1 was overexpressed in Arabidopsis and three lines (G2, G6, and G9) with highest expression levels were selected for analysis. The results showed that under normal conditions, the transgenic and wild-type (WT) plants displayed similar germination, biomass accumulation, reactive oxygen species (ROS) level, and physiological index. However, when treated with mannitol, the fresh weight, root length, water-holding ability, and V-ATPase, superoxide dismutase, and peroxidase activity of G2, G6, and G9 were significantly higher than those of WT. In contrast, the ROS and cell damage levels of the transgenic seedlings were lower than those of WT. Furthermore, the transcription levels of V-ATPase subunits, ABF, DREB, and NAC transcription factors (TFs), all of which are factors of ABA signaling pathway, were much higher in JrVHAG1 transgenic plants than those in WT. The positive induction of JrVHAG1 gene under abscisic acid (ABA) treatments in root and leaf tissues indicates that overexpression of JrVHAG1 improves plant tolerance to osmotic stress relating to the ABA signaling pathway, which is transcriptionally activated by ABF, DREB, and NAC TFs, and correlated to ROS scavenging and V-ATPase activity.

Cavitation and water fluxes driven by ice water potential in Juglans regia during freeze-thaw cycles.

Freeze-thaw cycles induce major hydraulic changes due to liquid-to-ice transition within tree stems. The very low water potential at the ice-liquid interface is crucial as it may cause lysis of living cells as well as water fluxes and embolism in sap conduits, which impacts whole tree-water relations. We investigated water fluxes induced by ice formation during freeze-thaw cycles in Juglans regia L. stems using four non-invasive and complementary approaches: a microdendrometer, magnetic resonance imaging, X-ray microtomography, and ultrasonic acoustic emissions analysis. When the temperature dropped, ice nucleation occurred, probably in the cambium or pith areas, inducing high water potential gradients within the stem. The water was therefore redistributed within the stem toward the ice front. We could thus observe dehydration of the bark's living cells leading to drastic shrinkage of this tissue, as well as high tension within wood conduits reaching the cavitation threshold in sap vessels. Ultrasonic emissions, which were strictly emitted only during freezing, indicated cavitation events (i.e. bubble formation) following ice formation in the xylem sap. However, embolism formation (i.e. bubble expansion) in stems was observed only on thawing via X-ray microtomography for the first time on the same sample. Ultrasonic emissions were detected during freezing and were not directly related to embolism formation. These results provide new insights into the complex process and dynamics of water movements and ice formation during freeze-thaw cycles in tree stems.

Characteristics of ultrasonic acoustic emissions from walnut branches during freeze-thaw-induced embolism formation.

Ultrasonic acoustic emission (UAE) methods have been applied for the detection of freeze-thaw-induced embolism formation in water conduits of tree species. Until now, however, the exact source(s) of UAE has not been identified especially in angiosperm species, in which xylem tissues are composed of diverse types of cells. In this study, UAE was recorded from excised branches of walnut (Juglans regia cv. Franquette) during freeze-thaw cycles, and attempts were made to characterize UAEs generated by cavitation events leading to embolism formation according to their properties. During freeze-thaw cycles, a large number of UAEs were generated from the sample segments. However, the cumulative numbers of total UAE during freeze-thawing were not correlated with the percentage loss of hydraulic conductivity after thawing, suggesting that the sources of UAE were not only cavitation leading to embolism formation in vessels. Among the UAEs, cumulative numbers of UAEs with absolute energy >10.0 fJ strongly correlated with the increase in percentage loss of hydraulic conductivity. The high absolute energy of the UAEs might reflect the formation of large bubbles in the large lumen of vessels. Therefore, UAEs generated by cavitation events in vessels during freeze-thawing might be distinguished from other signals according to their magnitudes of absolute energy. On the other hand, the freezing of xylem parenchyma cells was followed by a certain number of UAEs. These results indicate the possibility that UAE methods can be applied to the detection of both freeze-thaw-induced embolism and supercooling breakdown in parenchyma cells in xylem.

Dynamic modeling of stem water content during the dormant period in walnut trees.

Water content (WC) is a key variable in plant physiology even during the winter period. To simulate stem WC during the dormant season, a series of experiments were carried out on walnut trees under controlled conditions. In the field, WC was significantly correlated with soil temperature at 50 cm depth (R2 = 0.526). In the greenhouse, WC remained low as long as soil temperature was kept cold (<+5 °C) and increased after the soil temperature was warmed to +15 °C regardless of the date. Stem dehydration rate was significantly influenced by the WC and evaporative demand. A parsimonious model with functions describing the main experimental results was calibrated and validated with field data from 13 independent winter dynamics in Juglans regia L. orchards. Three functions of water uptake were tested, and these gave equivalent accuracies (root-mean-square error (RMSE) = 0.127-8; predictive root-mean-square error = 0.116). However, only a sigmoid function describing the relationship between the root water uptake and soil temperature gave values in agreement with the experimental results. Finally, the simulated WC provided a similar accuracy in predicting frost hardiness compared with the measured WC (RMSE ca 3 °C) and was excellent in spring (RMSE ca 2 °C). This model may be a relevant tool for predicting the risk of spring frost in walnut trees. Its genericity should be tested in other fruit and forest tree species.

Freeze dehydration vs supercooling in tree stems: physical and physiological modelling.

Frost resistance is the major factor affecting the distribution of plant species at high latitude and elevation. The main effects of freeze-thaw cycles are damage to living cells and formation of gas embolism in tree xylem vessels. Lethal intracellular freezing can be prevented in living cells by two mechanisms, such as dehydration and deep supercooling. We developed a multiphysics numerical model coupling water flow, heat transfer and phase change, considering different cell types in plant tissues, to study the dynamics and extent of cell dehydration, xylem pressure changes and stem diameter changes in response to freezing and thawing. Results were validated using experimental data for stem diameter changes of walnut trees (Juglans regia). The effect of cell mechanical properties was found to be negligible as long as the intracellular tension developed during dehydration was sufficiently low compared with the ice-induced cryostatic suction. The model was finally used to explore the coupled effects of relevant physiological parameters (initial water and sugar content) and environmental conditions (air temperature variations) on the dynamics and extent of dehydration. It revealed configurations where cell dehydration could be sufficient to protect cells from intracellular freezing, and situations where supercooling was necessary. This model, freely available with this paper, could easily be extended to explore different anatomical structures, different species and more complex physical processes.

On the mechanism for winter stem pressure build-up in walnut trees.

Xylem embolism is a significant factor in tree mortality. Restoration of hydraulic conductivity after massive embolization of the vascular system requires the application of positive pressure to the vessels and/or the creation of new conductive elements. Some species generate positive pressure from the root system to propagate pressure in distal, aboveground organs in spring, whereas other species generate positive pressure locally at the stem level during winter. We provide a mechanistic explanation for winter stem pressure build-up in the walnut tree. We have developed a physical model that accounts for temperature fluctuations and phase transitions. This model is based on the exchange of water and sugars between living cells and vessels. Our computations demonstrate that vessel pressurization can be attributed to the transfer of water between vessels across the parenchyma rays, which is facilitated by a radial imbalance in sugar concentration. The ability to dispose of soluble sugars in living cells, and to transport them between living cells and up to the vessels, is identified as the main drivers of stem pressure build-up in the walnut tree.

The regulating mechanism of salt tolerance of black walnut seedlings was revealed by the physiological and biochemical integration analysis.

Salt stress is an important abiotic stress that seriously affects plant growth. In order to research the salt tolerance of walnut rootstocks so as to provide scientific basis for screening salt-tolerant walnut rootstocks, two kinds of black walnut seedlings, Juglans microcarpa L. (JM) and Juglans nigra L. (JN), were treated under salt stress with different concentrations of NaCl (0, 50, 100, and 200 mM) and the growth situation of seedlings were observed. The physiological indexes of JM and JN seedlings were also measured in different days after treatment. Our study showed salt stress inhibited seedlings growth and limited biomass accumulation. Walnut mainly increased osmotic adjustment ability by accumulation Pro and SS. Furthermore, with the duration of treatment time increased, SOD and APX activities decreased, TPC and TFC contents increased. Walnut accumulated Na mostly in roots and transported more K and Ca to aboveground parts. The growth and physiological response performance differed between JM and JN, specifically, the differences occurred in the ability to absorb minerals, regulate osmotic stress, and scavenge ROS. Salt tolerance of JM and JN was assessed by principal component analysis (PCA) and resulted in JN > JM. In conclusion, our results indicated that JN has higher salt tolerance than JM, and JN might be used as a potential germplasm resource for the genetic breeding of walnuts.

Balance between carbon gain and loss in warmer environments: impacts on photosynthesis and leaf respiration in four temperate tree species.

The temperature sensitivities of photosynthesis and respiration remain a key uncertainty in predicting how forests will respond to climate warming. We grew seedlings of four temperate tree species, including Betula platyphylla, Fraxinus mandshurica, Juglans mandshurica and Tilia amurensis, at three temperature regimes (ambient, +2 °C, and +4 °C in daytime air temperature). We investigated net photosynthesis (Anet25), maximum rate of RuBP-carboxylation (Vcmax25) and RuBP-regeneration (Jmax25), stomatal conductance (gs25), mesophyll conductance (gm25), and leaf respiration (Rleaf) in dark (Rdark25) and in light (Rlight25) at 25 °C in all species. Additionally, we examined the temperature sensitivities of Anet, Vcmax, Jmax, Rdark and Rlight in F. mandshurica. Our findings showed that the warming-induced decreases in Anet25, Vcmax25 and Jmax25 were more prevalent in the late-successional species T. amurensis. Warming had negative impacts on gs25 in all species. Overall, Anet25 was positively correlated with Vcmax25 and Jmax25 across all growth temperatures. However, a positive correlation between Anet25 and gs25 was observed only under warming conditions, and gs25 was negatively associated with vapor pressure deficit. This implies that the vapor pressure deficit-induced decrease in gs25 was responsible for the decline in Anet25 at higher temperatures. The optimum temperature of Anet in F. mandshurica increased by 0.59 °C per 1.0 °C rise in growth temperature. While +2 °C elevated the thermal optima of Jmax, it did not affect the other temperature sensitivity parameters of Vcmax and Jmax. Rdark25 was not affected by warming in any species, and Rlight25 was stimulated in T. amurensis. The temperature response curves of Rdark and Rlight in F. mandshurica were not altered by warming, implying a lack of thermal acclimation. The ratios of Rdark25 and Rlight25 to Anet25 and Vcmax25 in T. amurensis increased with warming. These results suggest that Anet and Rleaf did not acclimate to warming synchronously in these temperate tree species.

Plant establishment and invasions: an increase in a seed disperser combined with land abandonment causes an invasion of the non-native walnut in Europe.

Successful invasive species often are established for a long time period before increasing exponentially in abundance. This lag phase is one of the least understood phenomena of biological invasions. Plant invasions depend on three factors: a seed source, suitable habitat and a seed disperser. The non-native walnut, Juglans regia, has been planted for centuries in Central Europe but, until recently, has not spread beyond planted areas. However, in the past 20 years, we have observed a rapid increase in walnut abundance, specifically in abandoned agricultural fields. The dominant walnut disperser is the rook, Corvus frugilegus. During the past 50 years, rooks have increased in abundance and now commonly inhabit human settlements, where walnut trees are planted. Central Europe has, in the past few decades, experienced large-scale land abandonment. Walnut seeds dispersed into ploughed fields do not survive, but when cached into ploughed and then abandoned fields, they successfully establish. Rooks preferentially cache seeds in ploughed fields. Thus, land-use change combined with disperser changes can cause rapid increase of a non-native species, allowing it to become invasive. This may have cascading effects on the entire ecosystem. Thus, species that are non-native and not invasive can become invasive as habitats and dispersers change.

Walnuts improve semen quality in men consuming a Western-style diet: randomized control dietary intervention trial.

We tested the hypothesis that 75 g of whole-shelled walnuts/day added to the Western-style diet of healthy young men would beneficially affect semen quality. A randomized, parallel two-group dietary intervention trial with single-blind masking of outcome assessors was conducted with 117 healthy men, age 21-35 yr old, who routinely consumed a Western-style diet. The primary outcome was improvement in conventional semen parameters and sperm aneuploidy from baseline to 12 wk. Secondary endpoints included blood serum and sperm fatty acid (FA) profiles, sex hormones, and serum folate. The group consuming walnuts (n = 59) experienced improvement in sperm vitality, motility, and morphology, but no change was seen in the group continuing their usual diet but avoiding tree nuts (n = 58). Comparing differences between the groups from baseline, significance was found for vitality (P = 0.003), motility (P = 0.009), and morphology (normal forms; P = 0.04). Serum FA profiles improved in the walnut group with increases in omega-6 (P = 0.0004) and omega-3 (P = 0.0007) but not in the control group. The plant source of omega-3, alpha-linolenic acid (ALA) increased (P = 0.0001). Sperm aneuploidy was inversely correlated with sperm ALA, particularly sex chromosome nullisomy (Spearman correlation, -0.41, P = 0.002). Findings demonstrated that walnuts added to a Western-style diet improved sperm vitality, motility, and morphology.