Hypoxia in tomato (Solanum lycopersicum) fruit during ripening: biophysical elucidation by a 3D reaction-diffusion model.

Respiration provides energy, substrates and precursors to support physiological changes of the fruit during climacteric ripening. A key substrate of respiration is oxygen that needs to be supplied to the fruit in a passive way by gas transfer from the environment. Oxygen gradients may develop within the fruit due to its bulky size and the dense fruit tissues, potentially creating hypoxia that may have a role in the spatial development of ripening. This study presents a three-dimensional reaction-diffusion model using tomato (Solanum lycopersicum) fruit as a test subject, combining the multiscale fruit geometry generated from magnetic resonance imaging and micro-computed tomography with varying respiration kinetics and contrasting boundary resistances obtained through independent experiments. The model predicted low oxygen levels in locular tissue under atmospheric conditions and the oxygen level was markedly lower upon scar occlusion, aligning with microsensor profiling results. The locular region was in a hypoxic state, leading to its low aerobic respiration with high CO2 accumulation by fermentative respiration, while the rest of the tissues remained well oxygenated. The model further revealed that the hypoxia is caused by a combination of diffusion resistances and respiration rates of the tissue. Collectively, this study reveals the existence of the respiratory gas gradients and its biophysical causes during tomato fruit ripening, providing richer information for future studies on localized endogenous ethylene biosynthesis and fruit ripening.

Automatic 3D cell segmentation of fruit parenchyma tissue from X-ray micro CT images using deep learning.

BACKGROUND: High quality 3D information of the microscopic plant tissue morphology-the spatial organization of cells and intercellular spaces in tissues-helps in understanding physiological processes in a wide variety of plants and tissues. X-ray micro-CT is a valuable tool that is becoming increasingly available in plant research to obtain 3D microstructural information of the intercellular pore space and individual pore sizes and shapes of tissues. However, individual cell morphology is difficult to retrieve from micro-CT as cells cannot be segmented properly due to negligible density differences at cell-to-cell interfaces. To address this, deep learning-based models were trained and tested to segment individual cells using X-ray micro-CT images of parenchyma tissue samples from apple and pear fruit with different cell and porosity characteristics. RESULTS: The best segmentation model achieved an Aggregated Jaccard Index (AJI) of 0.86 and 0.73 for apple and pear tissue, respectively, which is an improvement over the current benchmark method that achieved AJIs of 0.73 and 0.67. Furthermore, the neural network was able to detect other plant tissue structures such as vascular bundles and stone cell clusters (brachysclereids), of which the latter were shown to strongly influence the spatial organization of pear cells. Based on the AJIs, apple tissue was found to be easier to segment, as the porosity and specific surface area of the pore space are higher and lower, respectively, compared to pear tissue. Moreover, samples with lower pore network connectivity, proved very difficult to segment. CONCLUSIONS: The proposed method can be used to automatically quantify 3D cell morphology of plant tissue from micro-CT instead of opting for laborious manual annotations or less accurate segmentation approaches. In case fruit tissue porosity or pore network connectivity is too low or the specific surface area of the pore space too high, native X-ray micro-CT is unable to provide proper marker points of cell outlines, and one should rely on more elaborate contrast-enhancing scan protocols.

Spatio-temporal dynamics of the metabolome of climacteric fruit during ripening and post-harvest storage.

Fruit quality traits are determined to a large extent by their metabolome. The metabolite content of climacteric fruit changes drastically during ripening and post-harvest storage, and has been investigated extensively. However, the spatial distribution of metabolites and how it changes in time has received much less attention as fruit are usually considered as homogenous plant organs. Yet, spatio-temporal changes of starch, which is hydrolyzed during ripening, has been used for a long time as a ripening index. As vascular transport of water, and hence convective transport of metabolites, slows down in mature fruit and even stalls after detachment, spatio-temporal changes in their concentration are probably affected by diffusive transport of gaseous molecules that act as substrate (O2), inhibitor (CO2), or regulator (ethylene and NO) of the metabolic pathways that are active during climacteric ripening. In this review, we discuss such spatio-temporal changes of the metabolome and how they are affected by transport of metabolic gases and gaseous hormones. As there are currently no techniques available to measure the metabolite distribution repeatedly by non-destructive means, we introduce reaction-diffusion models as an in silico tool to compute it. We show how the different components of such a model can be integrated and used to better understand the role of spatio-temporal changes of the metabolome in ripening and post-harvest storage of climacteric fruit that is detached from the plant, and discuss future research needs.

The role of chloroplast movement in C4 photosynthesis: a theoretical analysis using a three-dimensional reaction-diffusion model for maize.

Chloroplasts movement within mesophyll cells in C4 plants is hypothesized to enhance the CO2 concentrating mechanism, but this is difficult to verify experimentally. A three-dimensional (3D) leaf model can help analyse how chloroplast movement influences the operation of the CO2 concentrating mechanism. The first volumetric reaction-diffusion model of C4 photosynthesis that incorporates detailed 3D leaf anatomy, light propagation, ATP and NADPH production, and CO2, O2 and bicarbonate concentration driven by diffusional and assimilation/emission processes was developed. It was implemented for maize leaves to simulate various chloroplast movement scenarios within mesophyll cells: the movement of all mesophyll chloroplasts towards bundle sheath cells (aggregative movement) and movement of only those of interveinal mesophyll cells towards bundle sheath cells (avoidance movement). Light absorbed by bundle sheath chloroplasts relative to mesophyll chloroplasts increased in both cases. Avoidance movement decreased light absorption by mesophyll chloroplasts considerably. Consequently, total ATP and NADPH production and net photosynthetic rate increased for aggregative movement and decreased for avoidance movement compared with the default case of no chloroplast movement at high light intensities. Leakiness increased in both chloroplast movement scenarios due to the imbalance in energy production and demand in mesophyll and bundle sheath cells. These results suggest the need to design strategies for coordinated increases in electron transport and Rubisco activities for an efficient CO2 concentrating mechanism at very high light intensities.

Innovative Fabrication of Hollow Microneedle Arrays Enabling Blood Sampling with a Self-Powered Microfluidic Patch.

Microneedles are gaining a lot of attention in the context of sampling cutaneous biofluids such as capillary blood. Their minimal invasiveness and user-friendliness make them a prominent substitute for venous puncture or finger-pricking. Although the latter is suitable for self-sampling, the impracticality of manual handling and the difficulty of obtaining enough qualitative sample is driving the search for better solutions. In this context, hollow microneedle arrays (HMNAs) are particularly interesting for completely integrating sample-to-answer solutions as they create a duct between the skin and the sampling device. However, the fabrication of sharp-tipped HMNAs with a high aspect ratio (AR) is challenging, especially since a length of ≥1500 µm is desired to reach the blood capillaries. In this paper, we first described a novel two-step fabrication protocol for HMNAs in stainless steel by percussion laser drilling and subsequent micro-milling. The HMNAs were then integrated into a self-powered microfluidic sampling patch, containing a capillary pump which was optimized to generate negative pressure differences up to 40.9 ± 1.8 kPa. The sampling patch was validated in vitro, showing the feasibility of sampling 40 µL of liquid. It is anticipated that our proof-of-concept is a starting point for more sophisticated all-in-one biofluid sampling and point-of-care testing systems.

Dust drift during mechanical and pneumatic wheat sowing and insights into the physicochemical characteristics of the abraded dust.

BACKGROUND: During sowing, plant protection products (PPP)-laden dust particles can be abraded from coated seeds and emitted into the atmosphere. Drift of these particles is a very complex phenomenon and depends on many factors, including the physicochemical characteristics of the dust. Currently, the available data needed to obtain a better understanding of the phenomenon and to build a risk assessment tool remain very limited. In this study, new data on dust drift and on the physochemical characteristics of dust abraded from wheat seeds generated using a pneumatic and a mechanical seeder were obtained. These data will serve as input to optimize a much-needed computational fluid dynamics (CFD) model. RESULTS: The dust generated by the pneumatic seeder contained a greater volume of smaller particles (<150 µm) than the mechanical seeder dust, which contained a greater volume of larger particles (>1000 µm) than pneumatic seeder dust. Compared to the pneumatic seeder, the mechanical seeder showed lower drift values. With both seeders, the drift depositions decreased with increasing distance from the sowing area but no clear relationship between dust drift and wind speed could be found. CONCLUSION: The gathered physicochemical and drift data for wheat seed drilling extend the current dust drift database, and help to better understand the complex dust drift phenomenon. These data will serve as input to refine and validate a CFD dust drift model. Such a model will allow a better and quicker assessment of different scenarios (e.g. varying wind speeds and direction, treatment, drilling technique) at a lower cost than conducting more field trials. © 2023 Society of Chemical Industry.

Causes and consequences of endogenous hypoxia on growth and metabolism of developing maize kernels.

Maize (Zea mays) kernels are the largest cereal grains, and their endosperm is severely oxygen deficient during grain fill. The causes, dynamics, and mechanisms of acclimation to hypoxia are minimally understood. Here, we demonstrate that hypoxia develops in the small, growing endosperm, but not the nucellus, and becomes the standard state, regardless of diverse structural and genetic perturbations in modern maize (B73, popcorn, sweet corn), mutants (sweet4c, glossy6, waxy), and non-domesticated wild relatives (teosintes and Tripsacum species). We also uncovered an interconnected void space at the chalazal pericarp, providing superior oxygen supply to the placental tissues and basal endosperm transfer layer. Modeling indicated a very high diffusion resistance inside the endosperm, which, together with internal oxygen consumption, could generate steep oxygen gradients at the endosperm surface. Manipulation of oxygen supply induced reciprocal shifts in gene expression implicated in controlling mitochondrial functions (23.6 kDa Heat-Shock Protein, Voltage-Dependent Anion Channel 2) and multiple signaling pathways (core hypoxia genes, cyclic nucleotide metabolism, ethylene synthesis). Metabolite profiling revealed oxygen-dependent shifts in mitochondrial pathways, ascorbate metabolism, starch synthesis, and auxin degradation. Long-term elevated oxygen supply enhanced the rate of kernel development. Altogether, evidence here supports a mechanistic framework for the establishment of and acclimation to hypoxia in the maize endosperm.

Drying banana seeds for ex situ conservation.

The ability of seeds to withstand drying is fundamental to ex situ seed conservation but drying responses are not well known for most wild species including crop wild relatives. We look at drying responses of seeds of Musa acuminata and Musa balbisiana, the two primary wild relatives of bananas and plantains, using the following four experimental approaches: (i) We equilibrated seeds to a range of relative humidity (RH) levels using non-saturated lithium chloride solutions and subsequently measured moisture content (MC) and viability. At each humidity level we tested viability using embryo rescue (ER), tetrazolium chloride staining and germination in an incubator. We found that seed viability was not reduced when seeds were dried to 4% equilibrium relative humidity (eRH; equating to 2.5% MC). (ii) We assessed viability of mature and less mature seeds using ER and germination in the soil and tested responses to drying. Findings showed that seeds must be fully mature to germinate and immature seeds had negligible viability. (iii) We dried seeds extracted from ripe/unripe fruit to 35-40% eRH at different rates and tested viability with germination tests in the soil. Seeds from unripe fruit lost viability when dried and especially when dried faster; seeds from ripe fruit only lost viability when fast dried. (iv) Finally, we dried and re-imbibed mature and less mature seeds and measured embryo shrinkage and volume change using X-ray computer tomography. Embryos of less mature seeds shrank significantly when dried to 15% eRH from 0.468 to 0.262 mm3, but embryos of mature seeds did not. Based on our results, mature seeds from ripe fruit are desiccation tolerant to moisture levels required for seed genebanking but embryos from immature seeds are mechanistically less able to withstand desiccation, especially when water potential gradients are high.

Time Is of the Essence-Early Activation of the Mevalonate Pathway in Apple Challenged With Gray Mold Correlates With Reduced Susceptibility During Postharvest Storage.

Apple is typically stored under low temperature and controlled atmospheric conditions to ensure a year round supply of high quality fruit for the consumer. During storage, losses in quality and quantity occur due to spoilage by postharvest pathogens. One important postharvest pathogen of apple is Botrytis cinerea. The fungus is a broad host necrotroph with a large arsenal of infection strategies able to infect over 1,400 different plant species. We studied the apple-B. cinerea interaction to get a better understanding of the defense response in apple. We conducted an RNAseq experiment in which the transcriptome of inoculated and non-inoculated (control and mock) apples was analyzed at 0, 1, 12, and 28 h post inoculation. Our results show extensive reprogramming of the apple's transcriptome with about 28.9% of expressed genes exhibiting significant differential regulation in the inoculated samples. We demonstrate the transcriptional activation of pathogen-triggered immunity and a reprogramming of the fruit's metabolism. We demonstrate a clear transcriptional activation of secondary metabolism and a correlation between the early transcriptional activation of the mevalonate pathway and reduced susceptibility, expressed as a reduction in resulting lesion diameters. This pathway produces the building blocks for terpenoids, a large class of compounds with diverging functions including defense. 1-MCP and hot water dip treatment are used to further evidence the key role of terpenoids in the defense and demonstrate that ethylene modulates this response.

Corrigendum to: Drying banana seeds for ex situ conservation.

[This corrects the article DOI: 10.1093/conphys/coab099.].

Computer-aided food engineering.

Computer-aided food engineering (CAFE) can reduce resource use in product, process and equipment development, improve time-to-market performance, and drive high-level innovation in food safety and quality. Yet, CAFE is challenged by the complexity and variability of food composition and structure, by the transformations food undergoes during processing and the limited availability of comprehensive mechanistic frameworks describing those transformations. Here we introduce frameworks to model food processes and predict physiochemical properties that will accelerate CAFE. We review how investments in open access, such as code sharing, and capacity-building through specialized courses could facilitate the use of CAFE in the transformation already underway in digital food systems.

Microstructural changes enhance oxygen transport in tomato (Solanum lycopersicum) fruit during maturation and ripening.

Climacteric ripening of tomato fruit is initiated by a characteristic surge of the production rate of ethylene, accompanied by an increase in respiration rate. As both activities consume O2 and produce CO2 , gas concentration gradients develop in the fruit that cause diffusive transport. This may, in turn, affect respiration and ethylene biosynthesis. Gas diffusion in fruit depends on the amount and connectivity of cells and intercellular spaces in 3D. We investigated micromorphological changes in different tomato tissues during development and ripening by visualizing cells and pores based on high-resolution micro-computed tomography, and computed effective O2 diffusivity coefficients based on microstructural features of the tissues. We demonstrated that mesocarp and septa tissues have larger cells but small and more disconnected pores than the placenta and columella, resulting in relatively lower effective O2 diffusivity coefficients. Cell disintegration occurred in the mesocarp and septa during ripening, indicating lysigenous air pore formation and resulting in a gradual increase of the effective O2 diffusivity. The results suggest that hypoxic conditions caused by the increasing size and, hence, diffusion resistance of the growing fruit may induce an increase of tissue porosity that results in a greatly enhanced O2 diffusivity and, thus, helps to alleviate them.

X-ray computed tomography for 3D plant imaging.

X-ray computed tomography (CT) is a valuable tool for 3D imaging of plant tissues and organs. Applications include the study of plant development and organ morphogenesis, as well as modeling of transport processes in plants. Some challenges remain, however, including attaining higher contrast for easier quantification, increasing the resolution for imaging subcellular features, and decreasing image acquisition and processing time for high-throughput phenotyping. In addition, phase contrast, multispectral, dark-field, soft X-ray, and time-resolved imaging are emerging. At the same time, a large amount of 3D image data are becoming available, posing challenges for data management. We review recent advances in the area of X-ray CT for plant imaging, and describe opportunities for using such images for studying transport processes in plants.

3D Printing of Monolithic Capillarity-Driven Microfluidic Devices for Diagnostics.

Rapid diagnostic testing at the site of the patient is essential when a fully equipped laboratory is not accessible. To maximize the impact of this approach, low-cost, disposable tests that require minimal user-interference and external equipment are desired. Fluid transport by capillary wicking removes the need for bulky ancillary equipment to actuate and control fluid flow. Nevertheless, current microfluidic paper-based analytical devices based on this principle struggle with the implementation of multistep diagnostic protocols because of fabrication-related issues. Here, 3D-printed microfluidic devices are demonstrated in a proof-of-concept enzyme-linked immunosorbent assay in which a multistep assay timeline is completed by precisely engineering capillary wetting within printed porous bodies. 3D printing provides a scalable route to low-cost microfluidic devices and obviates the assembly of discrete components. The resulting rapid and seamless transition between digital data and physical objects allows for rapid design iterations, and opens up perspectives on distributed manufacturing.

Designing Mechanical Properties of 3D Printed Cookies through Computer Aided Engineering.

Additive manufacturing or 3D printing can be applied in the food sector to create food products with personalized properties such as shape, texture, and composition. In this article, we introduce a computer aided engineering (CAE) methodology to design 3D printed food products with tunable mechanical properties. The focus was on the Young modulus as a proxy of texture. Finite element modelling was used to establish the relationship between the Young modulus of 3D printed cookies with a honeycomb structure and their structure parameters. Wall thickness, cell size, and overall porosity were found to influence the Young modulus of the cookies and were, therefore, identified as tunable design parameters. Next, in experimental tests, it was observed that geometry deformations arose during and after 3D printing, affecting cookie structure and texture. The 3D printed cookie porosity was found to be lower than the designed one, strongly influencing the Young modulus. After identifying the changes in porosity through X-ray micro-computed tomography, a good match was observed between computational and experimental Young's modulus values. These results showed that changes in the geometry have to be quantified and considered to obtain a reliable prediction of the Young modulus of the 3D printed cookies.

Correction: 4D synchrotron microtomography and pore-network modelling for direct in situ capillary flow visualization in 3D printed microfluidic channels.

Correction for '4D synchrotron microtomography and pore-network modelling for direct in situ capillary flow visualization in 3D printed microfluidic channels' by Agnese Piovesan et al., Lab Chip, 2020, 20, 2403-2411, DOI: .

4D synchrotron microtomography and pore-network modelling for direct in situ capillary flow visualization in 3D printed microfluidic channels.

Powder-based 3D printing was employed to produce porous, capillarity-based devices suitable for passive microfluidics. Capillary imbibition in such devices was visualized in situ through dynamic synchrotron X-ray microtomography performed at the European Synchrotron Radiation Facility (ESRF) with sub-second time resolution. The obtained reconstructed images were segmented to observe imbibition dynamics, as well as to compute the system effective contact angle and to generate a pore-network to model capillary imbibition. A contact angle gradient was observed resulting in a preferential wicking direction, with the central portion of the microfluidic channel filling faster than the edge areas. The contact angle analysis and the pore-network model results suggest that this is due to spatial variations in the material surface properties arising from both the 3D printing and the subsequent drying processes.

3-D microstructural changes in relation to the evolution of quality during ripening of mango (Mangifera indica L. cv. Carabao).

BACKGROUND: The ripening of mango involves changes in texture, flavor, and color, affecting the quality of the fruit. Previous studies have investigated the physiology on the evolution of quality during ripening but only a few have looked at microstructural changes during ripening. None of them has provided an insight into the relationhip between 3-D microstructure and the evolution of quality during ripening. As the 3-D microstructure of fruit tissue determines its mechanical and gas-transport properties, it is likely to affect fruit texture, respiratory metabolism, and other ripening processes. RESULTS: The present study focuses on the role of 3-D microstructural changes in relation to quality changes during mango ripening. Microstructural imaging using X-ray micro-computed tomography suggested the incidence of cell leakage, which was confirmed by the measurement of electrolyte leakage from the fruit peel. Due to cell leakage, porosity, pore connectivity, and pore local diameter were decreased whereas the tissue local diameter and pore specific area were increased. The decline in respiration and respiratory quotient during ripening followed the microstructural changes observed. Meanwhile, changes in aroma were observed such as a decrease in monoterpenes and an increase in esters and other fermentative metabolites. CONCLUSION: Overall, the results provide a complete, integrated picture of microstructural changes during ripening accompanying the evolution of fruit quality, suggesting functional relationships between the two. © 2020 Society of Chemical Industry.

The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size.

The physiological constraints on bud burst in woody perennials, including vascular development and oxygenation, remain unresolved. Both light and tissue oxygen status have emerged as important cues for vascular development in other systems; however, grapevine buds have only a facultative light requirement, and data on the tissue oxygen status have been confounded by the spatial variability within the bud. Here, we analysed apoplastic development at early stages of grapevine bud burst and combined molecular modelling with histochemical techniques to determine the pore size of cell walls in grapevine buds. The data demonstrate that quiescent grapevine buds were impermeable to apoplastic dyes (acid fuchsin and eosin Y) until after bud burst was established. The molecular exclusion size was calculated to be 2.1 nm, which would exclude most macromolecules except simple sugars and phytohormones until after bud burst. We used micro-computed tomography to demonstrate that tissue oxygen partial pressure data correlated well with structural heterogeneity of the bud and differences in tissue density, confirming that the primary bud complex becomes rapidly and preferentially oxygenated during bud burst. Taken together, our results reveal that the apoplastic porosity is highly regulated during the early stages of bud burst, suggesting a role for vascular development in the initial, rapid oxygenation of the primary bud complex.

In silico study of the role of cell growth factors in photosynthesis using a virtual leaf tissue generator coupled to a microscale photosynthesis gas exchange model.

Computational tools that allow in silico analysis of the role of cell growth and division on photosynthesis are scarce. We present a freely available tool that combines a virtual leaf tissue generator and a two-dimensional microscale model of gas transport during C3 photosynthesis. A total of 270 mesophyll geometries were generated with varying degrees of growth anisotropy, growth extent, and extent of schizogenous airspace formation in the palisade mesophyll. The anatomical properties of the virtual leaf tissue and microscopic cross-sections of actual leaf tissue of tomato (Solanum lycopersicum L.) were statistically compared. Model equations for transport of CO2 in the liquid phase of the leaf tissue were discretized over the geometries. The virtual leaf tissue generator produced a leaf anatomy of tomato that was statistically similar to real tomato leaf tissue. The response of photosynthesis to intercellular CO2 predicted by a model that used the virtual leaf tissue geometry compared well with measured values. The results indicate that the light-saturated rate of photosynthesis was influenced by interactive effects of extent and directionality of cell growth and degree of airspace formation through the exposed surface of mesophyll per leaf area. The tool could be used further in investigations of improving photosynthesis and gas exchange in relation to cell growth and leaf anatomy.