Developing sensory modules for specific molecules of interest represents a fundamental challenge in synthetic biology and its applications. A somewhat generalizable approach for this challenge is demonstrated here by evolving a naturally occurring chemically induced heterodimer into a genetically encoded sensor for herbicides. The interaction between PYRABACTIN-RESISTANT-like receptors and type-2C protein phosphatases is induced by abscisic acid─a small-molecule hormone in plants. We considered abscisic acid receptors as a potential scaffold for the development of biosensors because of past successes in their engineering, a structurally defined ligand cavity and the availability of large-scale assays for their activation. A panel of 475 receptor variants, mutated at ligand-proximal residues, was screened for activation by 37 herbicides from several classes. Twelve compounds activated at least one member of the mutant panel. To facilitate the subsequent improvement of herbicide receptors through directed evolution, we engineered a yeast two-hybrid platform optimized for sequential positive and negative selection using fluorescence-activated cell sorting. By utilizing this system, we were able to isolate receptors with low nanomolar sensitivity and a broad dynamic range in sensing a ubiquitous group of chloroacetamide herbicides. Aside from its possible applicative value, this work lays down conceptual groundwork and provides infrastructure for the future development of biosensors through directed evolution.Developing sensory modules for specific molecules of interest represents a fundamental challenge in synthetic biology and its applications. A somewhat generalizable approach for this challenge is demonstrated here by evolving a naturally occurring chemically induced heterodimer into a genetically encoded sensor for herbicides. The interaction between PYRABACTIN-RESISTANT-like receptors and type-2C protein phosphatases is induced by abscisic acid─a small-molecule hormone in plants. We considered abscisic acid receptors as a potential scaffold for the development of biosensors because of past successes in their engineering, a structurally defined ligand cavity and the availability of large-scale assays for their activation. A panel of 475 receptor variants, mutated at ligand-proximal residues, was screened for activation by 37 herbicides from several classes. Twelve compounds activated at least one member of the mutant panel. To facilitate the subsequent improvement of herbicide receptors through directed evolution, we engineered a yeast two-hybrid platform optimized for sequential positive and negative selection using fluorescence-activated cell sorting. By utilizing this system, we were able to isolate receptors with low nanomolar sensitivity and a broad dynamic range in sensing a ubiquitous group of chloroacetamide herbicides. Aside from its possible applicative value, this work lays down conceptual groundwork and provides infrastructure for the future development of biosensors through directed evolution.

Responses of terrestrial ecosystems to climate change have been explored in many regions worldwide. While continued drying and warming may alter process rates and deteriorate the state and performance of ecosystems, it could also lead to more fundamental changes in the mechanisms governing ecosystem functioning. Here we argue that climate change will induce unprecedented shifts in these mechanisms in historically wetter climatic zones, towards mechanisms currently prevalent in dry regions, which we refer to as ‘dryland mechanisms’. We discuss 12 dryland mechanisms affecting multiple processes of ecosystem functioning, including vegetation development, water flow, energy budget, carbon and nutrient cycling, plant production and organic matter decomposition. We then examine mostly rare examples of the operation of these mechanisms in non-dryland regions where they have been considered irrelevant at present. Current and future climate trends could force microclimatic conditions across thresholds and lead to the emergence of dryland mechanisms and their increasing control over ecosystem functioning in many biomes on Earth.

Rosemary (Salvia rosmarinus (L.) Schleid., Handb. syn. Rosmarinus officinalis L.) extracts are widely used as natural preservatives due to their antimicrobial and antioxidant properties, which are attributed to the phenolic diterpenoid carnosic acid (CA). Growers are rewarded based on CA content in their rosemary leaf harvested. Conventional methods for estimating leaf CA content are destructive and often time-consuming. This preliminary study presents a spectral non-destructive approach for in vivo estimation of CA content in different rosemary cultivars based on the reflectance spectra of their canopy. The proposed approach is based on the characteristic rosemary absorption features along the visible and shortwave infrared spectral regions at 550 nm, 1200 nm, and 1690 nm, respectively, attributed to leaf color, the oxygen-hydrogen bond bending in water molecules, and distinctive carbon-hydrogen bond features typical for terpenes and phenolic compounds. Correlations between measured CA content by high-performance liquid chromatography (HPLC) and leaf reflectance spectra, normalized spectral indices, and latent components obtained by genetic algorithm-based partial least squares regression (GA-PLSR) were assessed using data collected from 79 rosemary cultivars. The GA-PLSR model successfully predicted the CA content among the various cultivars, further providing evidence of high weightage to the above-mentioned absorption features also obtained from two best-wavelength combination selections. Randomly selected canopy spectra were used to calibrate and simultaneously cross-validate 100 iterations, using the ‘leave-k-out’ approach. The root mean squared error (RMSE) obtained for calibration and cross-validation were 0.86% and 1.15% CA content from the dry leaf matter, and the residual prediction deviation (RPD) were reported to be 2.71 and 2.05, respectively. This work will set the stage for precise planning of harvesting time to ensure increased yield and income for the farmers and improved utilization of resources.

AbstractResource competition theory predicts coexistence and exclusion patterns based on species? R*s, the minimum resource values required for a species to persist. A central assumption of the theory is that all species have equal access to resources. However, many systems are characterized by preemption exploitation, where some species deplete resources before their competitors can access them (e.g., asymmetric light competition, contest competition among animals). We hypothesized that coexistence under preemption requires an R*-preemption trade-off?that is, the species with the priority access should have a higher R* (lower ?efficiency?). Thus, we developed an extension of resource competition theory to investigate partial and total preemption (in the latter, the preemptor is unaffected by species with lower preemption rank). We found that an R*-preemption trade-off is a necessary condition for coexistence in all models. Moreover, under total preemption, the trade-off alone is sufficient for coexistence. In contrast, under partial preemption, more conditions are needed, which restricts the parameter space of coexistence. Finally, we discuss the implications of our finding for seemingly distinct trade-offs, which we view as special cases of the R*-preemption trade-off. These trade-offs include the digger-grazer trade-off, the competition-colonization trade-off, and trade-offs related to light competition between trees and understories.AbstractResource competition theory predicts coexistence and exclusion patterns based on species? R*s, the minimum resource values required for a species to persist. A central assumption of the theory is that all species have equal access to resources. However, many systems are characterized by preemption exploitation, where some species deplete resources before their competitors can access them (e.g., asymmetric light competition, contest competition among animals). We hypothesized that coexistence under preemption requires an R*-preemption trade-off?that is, the species with the priority access should have a higher R* (lower ?efficiency?). Thus, we developed an extension of resource competition theory to investigate partial and total preemption (in the latter, the preemptor is unaffected by species with lower preemption rank). We found that an R*-preemption trade-off is a necessary condition for coexistence in all models. Moreover, under total preemption, the trade-off alone is sufficient for coexistence. In contrast, under partial preemption, more conditions are needed, which restricts the parameter space of coexistence. Finally, we discuss the implications of our finding for seemingly distinct trade-offs, which we view as special cases of the R*-preemption trade-off. These trade-offs include the digger-grazer trade-off, the competition-colonization trade-off, and trade-offs related to light competition between trees and understories.

The bidirectional reflectance distribution function (BRDF) is crucial in determining the quantity of reflected light on the earth’s surface as a function of solar and view angles (i.e., azimuth and zenith angles). The Vegetation and ENvironment monitoring Micro-Satellite (VENµS) provides a unique opportunity to acquire data from the same site, with the same sensor, with almost constant solar and view zenith angles from two (or more) view azimuth angles. The present study was aimed at exploring the view angles’ effect on the stability of the values of albedo and of two vegetation indices (VIs): the normalized difference vegetation index (NDVI) and the red-edge inflection point (REIP). These products were calculated over three polygons representing urban and cultivated areas in April, June, and September 2018, under a minimal time difference of less than two minutes. Arithmetic differences of VIs and a change vector analysis (CVA) were performed. The results show that in urban areas, there was no difference between the VIs, whereas in the well-developed field crop canopy, the REIP was less affected by the view azimuth angle than the NDVI. Results suggest that REIP is a more appropriate index than NDVI for field crop studies and monitoring. This conclusion can be applied in a constellation of satellites that monitor ground features simultaneously but from different view azimuth angles.

To meet the ever-growing global population necessities, integrating climate-change-relevant plant traits into breeding programs is required. Developing new tools for fast and accurate estimation of chlorophyll parameters, chlorophyll a (Chl-a) content, chlorophyll b (Chl-b) content, and their ratio (Chl-a/b), can promote breeding programs of wheat with enhanced climate adaptability. Spectral reflectance of leaves is affected by changes in pigment concentration and can be used to estimate chlorophyll parameters. The current study identified and validated the top known spectral indices and developed new vegetation indices (VIs) for Chl-a and Chl-b content estimation and used them to non-destructively estimate Chl-a/b values and compare them to hyperspectral estimations. Three wild emmer introgression lines, with contrasting drought stress responsiveness dynamics, were selected. Well-watered and water-limited irrigation regimes were applied. The wheat leaves were spectrally measured with a handheld spectrometer to acquire their reflectance in the 330 to 790 nm range. Regression models based on calculated VIs as well as all hyperspectral curves were calibrated and validated against chlorophyll extracted values. The developed normalized difference spectral indices (NDSIs) resulted in high accuracy of Chl-a (NDSI415,614) and Chl-b (NDSI406,525) estimation, allowing for indirect non-destructive estimation of Chl-a/b with root mean square error (RMSE) values that could fit 6 to 10 times in the range of the measured values. They also performed similarly to the hyperspectral models. Altogether, we present here a new tool for a non-destructive estimation of Chl-a/b, which can serve as a basis for future breeding efforts of climate-resilient wheat as well as other crops.

Drought intensity as experienced by plants depends upon soil moisture status and atmospheric variables such as temperature, radiation, and air vapour pressure deficit. Although the role of shoot architecture with these edaphic and atmospheric factors is well characterized, the extent to which shoot and root dynamic interactions as a continuum are controlled by genotypic variation is less well known. Here, we targeted these interactions using a wild emmer wheat introgression line (IL20) with a distinct drought-induced shift in the shoot-to-root ratio and its drought-sensitive recurrent parent Svevo. Using a gravimetric platform, we show that IL20 maintained higher root water influx and gas exchange under drought stress, which supported a greater growth. Interestingly, the advantage of IL20 in root water influx and transpiration was expressed earlier during the daily diurnal cycle under lower vapour pressure deficit and therefore supported higher transpiration efficiency. Application of a structural equation model indicates that under drought, vapour pressure deficit and radiation are antagonistic to transpiration rate, whereas the root water influx operates as a feedback for the higher atmospheric responsiveness of leaves. Collectively, our results suggest that a drought-induced shift in root-to-shoot ratio can improve plant water uptake potential in a short preferable time window during early morning when vapour pressure deficit is low and the light intensity is not a limiting factor for assimilation.

Hydrated silica (SiO2·nH2O) aggregates in the root endodermis of grasses. Application of soluble silicates (Si) to roots is associated with variations in the balance of reactive oxygen species (ROS), increased tolerance to a broad range of stresses affecting ROS concentrations, and early lignin deposition. In sorghum (Sorghum bicolor L.), silica aggregation is patterned in an active silicification zone (ASZ) by a special type of aromatic material forming a spotted pattern. The deposition has a signature typical of lignin. Since lignin polymerization is mediated by ROS, we studied the formation of root lignin and silica controlled by ROS via modulating hydrogen peroxide (H2O2) concentrations in the growth medium. Sorghum seedlings were grown hydroponically and supplemented with Si, H2O2, and KI, an ionic compound that catalyses H2O2 decomposition. Lignin and silica deposits in the endodermis were studied by histology, scanning electron and Raman microscopies. Cell wall composition was quantified by thermal gravimetric analysis. Endodermal H2O2 concentration correlated to the extent of lignin-like deposition along the root, but did not affect its patterning in spots. Our results show that the ASZ spots were necessary for root silica aggregation, and suggest that silicification is intensified under oxidative stress as a result of increased ASZ lignin-like deposition.

Author summary Plants form organs continuously throughout their lives, and the number and shape of their organs is determined in a flexible manner according to the internal and external circumstances. Alongside this flexibility, plants maintain basic developmental programs to ensure proper functioning. Among the ways by which plants achieve flexible development is by tuning the pace of their maturation and differentiation, at both the plant and organ levels. One of the ways plants regulate the rate of maturation and differentiation is by changing gene expression. Here, we identified a gene that promotes plant and organ maturation and differentiation. This gene, CRAWLING ELEPHANT (CREL) acts by bringing a repressing complex to target genes. We show the importance of CREL in multiple developmental processes and in the expression of multiple genes throughout the tomato genome.

A major gene controls flowering pattern in peanut, possibly encoding a TFL1-like. It was subjected to gain/loss events of a deletion and changes in mRNA expression levels, partly explaining the evolution of flowering pattern in Arachis.

Grapevines are susceptible and responsive to their surrounding environment. Factors such as climate region and terroir are known to affect polyphenolic compounds in wine and therefore, its quality. The uniqueness of the terroir in Israel is the variety of soil types and the climatic conditions, ranging from Mediterranean to arid climates. Thus, understanding the effects of climate on grapevine performance in Israel may be a test case for the effect of climate change on grapevine at other areas in the future. First, we present a preliminary survey (2012–2014) in different climate zones and terroirs, which showed that trans-resveratrol concentrations in Merlot and Shiraz were high, while those of Cabernet Sauvignon were significantly lower. A further comprehensive countrywide survey (2016) of Merlot wines from 62 vineyards (53 wineries) compared several phenolic compounds’ concentrations between five areas of different climate and terroir. Results show a connection between trans-resveratrol concentrations, variety, and terroir properties. Furthermore, we show that trans-resveratrol concentrations are strongly correlated to humidity levels at springtime, precipitation, and soil permeability. This work can be considered a glimpse into the possible alterations of wine composition in currently moderate-climate wine-growing areas.

The Arabidopsis (Arabidopsis thaliana) leaf veins bundle-sheath cells (BSCs)—a selective barrier to water and solutes entering the mesophyll—increase the leaf radial hydraulic conductance (Kleaf) by acidifying the xylem sap by their plasma membrane H+-ATPase, AHA2. Based on this and on the BSCs’ expression of phototropins PHOT1 and PHOT2, and the known blue light (BL)-induced Kleaf increase, we hypothesized that, resembling the guard cells, BL perception by the BSCs’ phots activates its H+-ATPase, which, consequently, upregulates Kleaf. Indeed, under BL, the Kleaf of the knockout mutant lines phot1-5, phot2-1, phot1-5 phot2-1, and aha2-4 was lower than that of the wild-type (WT). BSC-only-directed complementation of phot1-5 or aha2-4 by PHOT1 or AHA2, respectively, restored the BL-induced Kleaf increase. BSC-specific silencing of PHOT1 or PHOT2 prevented such Kleaf increase. A xylem-fed kinase inhibitor (tyrphostin 9) replicated this also in WT plants. White light—ineffective in the phot1-5 mutant—acidified the xylem sap (relative to darkness) in WT and in the PHOT1-complemented phot1-5. These results, supported by BL increase of BSC protoplasts’ water permeability and cytosolic pH and their hyperpolarization by BL, identify the BSCs as a second phot-controlled water conductance element in leaves, in series with stomatal conductance. Through both, BL regulates the leaf water balance.

Cellulose nanocrystals (CNCs) are composed of chiral cellulose units, which form chiral nematic liquid crystals in water that, upon drying, self-assemble to more complex spiral chiral sheets. This secondary structure arrangement is found to change with an external magnetic or electric field. Here, we show that one of the basic organization driving forces is electron spin, which is produced as the charge redistributes in the organization process of the chiral building blocks. It is important to stress that the electron spin-exchange interactions supply the original driving force and not the magnetic field per se. The results present the first utilization of the chiral-induced spin selectivity (CISS) effect in sugars, enabling one to regulate the CNC bottom-up fabrication process. Control is demonstrated on the organization order of the CNC by utilizing different magnetization directions of the ferromagnetic surface. The produced spin is probed using a simple Hall device. The measured Hall resistance shows that the CNC sheets’ arrangement is affected during the first four hours as long as the CNC is in its wet phase. On introducing the 1,2,3,4-butanetetracarboxylic acid cross-linker into the CNC sheet, the packing density of the CNC helical structure is enhanced, presenting an increase in the Hall resistance and the chiral state.Cellulose nanocrystals (CNCs) are composed of chiral cellulose units, which form chiral nematic liquid crystals in water that, upon drying, self-assemble to more complex spiral chiral sheets. This secondary structure arrangement is found to change with an external magnetic or electric field. Here, we show that one of the basic organization driving forces is electron spin, which is produced as the charge redistributes in the organization process of the chiral building blocks. It is important to stress that the electron spin-exchange interactions supply the original driving force and not the magnetic field per se. The results present the first utilization of the chiral-induced spin selectivity (CISS) effect in sugars, enabling one to regulate the CNC bottom-up fabrication process. Control is demonstrated on the organization order of the CNC by utilizing different magnetization directions of the ferromagnetic surface. The produced spin is probed using a simple Hall device. The measured Hall resistance shows that the CNC sheets’ arrangement is affected during the first four hours as long as the CNC is in its wet phase. On introducing the 1,2,3,4-butanetetracarboxylic acid cross-linker into the CNC sheet, the packing density of the CNC helical structure is enhanced, presenting an increase in the Hall resistance and the chiral state.

Litchi is a highly perishable fruit. Ripe litchi fruit loses quality quickly as they hang on tree, giving a very short hanging life and thus harvest period. This study attempted to explore the roles of cytokinin in regulating fruit ripening and senescence of litchi and examine the possibility of applying cytokinin in “on-tree storage” of the fruit. Exogenous cytokinin, forchlorfenuron (CPPU), was applied at 20 mg L⁻¹ 7 weeks after full bloom on litchi (Litchi chinensis cv. Feizixiao) fruit clusters. Color parameters, chlorophylls, anthocyanins, fruit and fruit part weights, total soluble solutes (TSSs), soluble sugars, organic acids, non-anthocyanin flavonoids, ethanol, and also CPPU residue in fruit were traced. CPPU residue was higher but decreased faster in the pericarp than in the aril, where it maintained < 10 μg kg⁻¹. CPPU had no significant effect on fruit weight but tended to increase pericarp weight. The treatment suppressed chlorophyll loss and anthocyanin accumulation in the pericarp, increased non-anthocyanin flavonoids in the aril, but had no significant effects on non-anthocyanin flavonoids in the pericarp and total sugar and organic acids in the aril. As the commercially ripe fruit hanged on tree, TSSs, total sugar, and sucrose decreased with ethanol and acetic acid accumulation in the aril. CPPU significantly suppressed the loss of sucrose and total sugar and the accumulation of ethanol and acetic acid in the aril and inhibited malondialdehyde accumulation in the pericarp of the overripe fruit. Soluble invertase, alcohol dehydrogenase, and pyruvate decarboxylase (PDC) activity and gene expression in the aril were downregulated by CPPU. The results suggest that cytokinin partially suppresses the ripening process in litchi and is effective to slow quality loss in the overripe fruit on tree.

A comprehensive collection of 1254 tomato accessions, corresponding to European traditional and modern varieties, early domesticated varieties, and wild relatives, was analyzed by genotyping by sequencing. A continuous genetic gradient between the traditional and modern varieties was observed. European traditional tomatoes displayed very low genetic diversity, with only 298 polymorphic loci (95% threshold) out of 64 943 total variants. European traditional tomatoes could be classified into several genetic groups. Two main clusters consisting of Spanish and Italian accessions showed higher genetic diversity than the remaining varieties, suggesting that these regions might be independent secondary centers of diversity with a different history. Other varieties seem to be the result of a more recent complex pattern of migrations and hybridizations among the European regions. Several polymorphic loci were associated in a genome-wide association study with fruit morphological traits in the European traditional collection. The corresponding alleles were found to contribute to the distinctive phenotypic characteristic of the genetic varietal groups. The few highly polymorphic loci associated with morphological traits in an otherwise a low-diversity population suggests a history of balancing selection, in which tomato farmers likely maintained the morphological variation by inadvertently applying a high selective pressure within different varietal types.

Nutrient enrichment of natural ecosystems is a primary characteristic of the Anthropocene and a known cause of biodiversity loss, particularly in grasslands. In a global meta-analysis of 630 resource addition experiments, we conduct a simultaneous test of the three most prominent explanations of this phenomenon. Our results conclusively indicate that nitrogen is the leading cause of species loss. This result is important because of the increase in nitrogen deposition and the frequent use of nitrogen-based fertilizers worldwide. Our findings provide global-scale, experimental evidence that minimizing nitrogen inputs to ecological systems may help to conserve the diversity of grassland ecosystems. Eutrophication is a major driver of species loss in plant communities worldwide. However, the underlying mechanisms of this phenomenon are controversial. Previous studies have raised three main explanations: 1) High levels of soil resources increase standing biomass, thereby intensifying competitive interactions (the “biomass-driven competition hypothesis”). 2) High levels of soil resources reduce the potential for resource-based niche partitioning (the “niche dimension hypothesis”). 3) Increasing soil nitrogen causes stress by changing the abiotic or biotic conditions (the “nitrogen detriment hypothesis”). Despite several syntheses of resource addition experiments, so far, no study has tested all of the hypotheses together. This is a major shortcoming, since the mechanisms underlying the three hypotheses are not independent. Here, we conduct a simultaneous test of the three hypotheses by integrating data from 630 resource addition experiments located in 99 sites worldwide. Our results provide strong support for the nitrogen detriment hypothesis, weaker support for the biomass-driven competition hypothesis, and negligible support for the niche dimension hypothesis. The results further show that the indirect effect of nitrogen through its effect on biomass is minor compared to its direct effect and is much larger than that of all other resources (phosphorus, potassium, and water). Thus, we conclude that nitrogen-specific mechanisms are more important than biomass or niche dimensionality as drivers of species loss under high levels of soil resources. This conclusion is highly relevant for future attempts to reduce biodiversity loss caused by global eutrophication.

The distribution and stability of biomes are critical for understanding, modeling, and managing the land biosphere. While studies have emphasized abiotic factors such as climate, geology, or fire regimes, we here identify a biological mechanism—plant–plant competition for belowground resources—as critical for maintaining the boundary between the Fynbos and Afrotemperate Forest biomes in South Africa. We demonstrate an apparent general mechanism in which local competition triggers a biome-scale feedback between plant traits and soil resources, which, in turn, stabilizes the biome boundary by allowing plants to maintain their own preferred soil conditions. Our findings are of general importance for understanding the organization of biodiversity across landscapes, for managing alien plant invasions, and for modeling the future of biome boundaries. Recent findings point to plant root traits as potentially important for shaping the boundaries of biomes and for maintaining the plant communities within. We examined two hypotheses: 1) Thin-rooted plant strategies might be favored in biomes with low soil resources; and 2) these strategies may act, along with fire, to maintain the sharp boundary between the Fynbos and Afrotemperate Forest biomes in South Africa. These biomes differ in biodiversity, plant traits, and physiognomy, yet exist as alternative stable states on the same geological substrate and in the same climate conditions. We conducted a 4-y field experiment to examine the ability of Forest species to invade the Fynbos as a function of growth-limiting nutrients and belowground plant–plant competition. Our results support both hypotheses: First, we found marked biome differences in root traits, with Fynbos species exhibiting the thinnest roots reported from any biome worldwide. Second, our field manipulation demonstrated that intense belowground competition inhibits the ability of Forest species to invade Fynbos. Nitrogen was unexpectedly the resource that determined competitive outcome, despite the long-standing expectation that Fynbos is severely phosphorus constrained. These findings identify a trait-by-resource feedback mechanism, in which most species possess adaptive traits that modify soil resources in favor of their own survival while deterring invading species. Our findings challenge the long-held notion that biome boundaries depend primarily on external abiotic constraints and, instead, identify an internal biotic mechanism—a selective feedback among traits, plant–plant competition, and ecosystem conditions—that, along with contrasting fire regime, can act to maintain biome boundaries.

Wood warping is a phenomenon known as a deformation in wood that occurs when changes in moisture content cause an unevenly volumetric change due to fiber orientation. Here we present an investigation of wood warped objects that were fabricated by 3D printing. Similar to natural wood warping, water evaporation causes volume decrease of the printed object, but in contrast, the printing pathway pattern and flow rate dictate the direction of the alignment and its intensity, all of which can be predesigned and affect the resulting structure after drying. The fabrication of the objects was performed by an extrusion-based 3D printing technique that enables the deposition of water-based inks into 3D objects. The printing ink was composed of 100% wood-based materials, wood flour, and plant-extracted natural binders cellulose nanocrystals, and xyloglucan, without the need for any additional synthetic resins. Two archetypal structures were printed: cylindrical structure and helices. In the former, we identified a new length scale that gauges the effect of gravity on the shape. In the latter, the structure exhibited a shape transition analogous to the opening of a seedpod, quantitatively reproducing theoretical predictions. Together, by carefully tuning the flow rate and printing pathway, the morphology of the fully dried wooden objects can be controlled. Hence, it is possible to design the printing of wet objects that will form different final 3D structures.

Earliness and ripening behavior are important attributes of fruits on and off the vine, and affect quality and preference of both growers and consumers. Fruit ripening is a complex physiological process that involves metabolic shifts affecting fruit color, firmness, and aroma production. Melon is a promising model crop for the study of fruit ripening, as the full spectrum of climacteric behavior is represented across the natural variation. Using Recombinant Inbred Lines (RILs) population derived from the parental lines “Dulce” (reticulatus, climacteric) and “Tam Dew” (inodorus, non-climacteric) that vary in earliness and ripening traits, we mapped QTLs for ethylene emission, fruit firmness and days to flowering and maturity. To further annotate the main QTL intervals and identify candidate genes, we used Oxford Nanopore long-read sequencing in combination with Illumina short-read resequencing, to assemble the parental genomes de-novo. In addition to 2.5 million genome-wide SNPs and short InDels detected between the parents, we also highlight here the structural variation between these lines and the reference melon genome. Through systematic multi-layered prioritization process, we identified 18 potential polymorphisms in candidate genes within multi-trait QTLs. The associations of selected SNPs with earliness and ripening traits were further validated across a panel of 177 diverse melon accessions and across a diallel population of 190 F1 hybrids derived from a core subset of 20 diverse parents. The combination of advanced genomic tools with diverse germplasm and targeted mapping populations is demonstrated as a way to leverage forward genetics strategies to dissect complex horticulturally important traits.