The influence of the pollination compatibility type on the pistil S-RNase expression in European pear (Pyrus communis).

The S-RNase gene plays an essential role in the gametophytic self-incompatibility (GSI) system of Pyrus. It codes for the stylar-expressed S-RNase protein which inhibits the growth of incompatible pollen tubes through cytotoxicity and the induction of programmed cell death in the pollen tube. While research on the Pyrus GSI system has primarily focused on the S-RNase gene, there is still a lack of insight into its spatiotemporal expression profile and the factors that regulate it. Previous studies have suggested that S-RNase expression in the style is influenced by pollination and is dependent on the compatibility type. We here continue on this basic hypothesis by analyzing the spatiotemporal expression of the S-RNase alleles in Pyrus communis "Conference" styles in response to different types of pollination; namely, upon full- and semi-compatible pollination and upon incompatible selfing. The results revealed that temporal dynamics of S-RNase expression are influenced by the pollen's compatibility type, indicating the presence of a signaling mechanism between pollen and style to control S-RNase production during pollen tube growth. In our experiment, S-RNase expression continuously decreased after cross-pollination and in the unpollinated control. However, after a fully incompatible pollination, S-RNase expression remained constant. Finally, semi-compatible pollination showed a initially constant S-RNase expression for both alleles followed by a strong decrease in expression. Based on these results and previous findings, we propose a regulatory mechanism to explain the effect of pollination and the associated compatibility type on S-RNase expression in the style. This proposed mechanism could be used as a starting point for future research.

Leaf ontogeny modulates epinasty through shifts in hormone dynamics during waterlogging in tomato.

Waterlogging leads to hypoxic conditions in the root zone that subsequently cause systemic adaptive responses in the shoot, including leaf epinasty. Waterlogging-induced epinasty in tomato has long been ascribed to the coordinated action of ethylene and auxins. However, other hormonal signals have largely been neglected, despite evidence of their importance in leaf posture control. To cover a large group of growth regulators, we performed a tissue-specific and time-dependent hormonomics analysis. This revealed that multiple hormones are differentially affected throughout a 48 h waterlogging treatment, and that leaf age determines hormone homeostasis and modulates their changes during waterlogging. In addition, we distinguished early hormonal signals that contribute to fast responses to oxygen deprivation from those that potentially sustain the waterlogging response. We found that abscisic acid (ABA) levels peak in petioles within the first 12 h of the treatment, while its precursors only increase much later, suggesting that ABA transport is altered. At the same time, cytokinins (CKs) and their derivatives drastically decline during waterlogging in leaves of all ages. This drop in CKs possibly releases the inhibition of ethylene- and auxin-mediated cell elongation to establish epinastic bending. Auxins themselves rise substantially in the petiole of mature leaves, but mostly after 48 h of root hypoxia. Based on our hormone profiling, we propose that ethylene and ABA might act synergistically as an early signal to induce epinasty, while the balance of indole-3-acetic acid and CKs in the petiole ultimately regulates differential growth.

Ethylene inhibits photosynthesis via temporally distinct responses in tomato plants.

Ethylene is a volatile plant hormone that regulates many developmental processes and responses toward (a)biotic stress. Studies have shown that high levels of ethylene repress vegetative growth in many important crops, including tomato (Solanum lycopersicum), possibly by inhibiting photosynthesis. We investigated the temporal effects of ethylene on young tomato plants using an automated ethylene gassing system to monitor the physiological, biochemical, and molecular responses through time course RNA-seq of a photosynthetically active source leaf. We found that ethylene evokes a dose-dependent inhibition of photosynthesis, which can be characterized by 3 temporally distinct phases. The earliest ethylene responses that marked the first phase and occurred a few hours after the start of the treatment were leaf epinasty and a decline in stomatal conductance, which led to lower light perception and CO2 uptake, respectively, resulting in a rapid decline of soluble sugar levels (glucose, fructose). The second phase of the ethylene effect was marked by low carbohydrate availability, which modulated plant energy metabolism to adapt by using alternative substrates (lipids and proteins) to fuel the TCA cycle. Long-term continuous exposure to ethylene led to the third phase, characterized by starch and chlorophyll breakdown, which further inhibited photosynthesis, leading to premature leaf senescence. To reveal early (3 h) ethylene-dependent regulators of photosynthesis, we performed a ChIP-seq experiment using anti-ETHYLENE INSENSITIVE 3-like 1 (EIL1) antibodies and found several candidate transcriptional regulators. Collectively, our study revealed a temporal sequence of events that led to the inhibition of photosynthesis by ethylene and identified potential transcriptional regulators responsible for this regulation.

Effect of ethylene pretreatment on tomato plant responses to salt, drought, and waterlogging stress.

Salinity, drought, and waterlogging are common environmental stresses that negatively impact plant growth, development, and productivity. One of the responses to abiotic stresses is the production of the phytohormone ethylene, which induces different coping mechanisms that help plants resist or tolerate stress. In this study, we investigated if an ethylene pretreatment can aid plants in activating stress-coping responses prior to the onset of salt, drought, and waterlogging stress. Therefore, we measured real-time transpiration and CO2 assimilation rates and the impact on biomass during and after 3 days of abiotic stress. Our results showed that an ethylene pretreatment of 1 ppm for 4 h did not significantly influence the negative effects of waterlogging stress, while plants were more sensitive to salt stress as reflected by enhanced water losses due to a higher transpiration rate. However, when exposed to drought stress, an ethylene pretreatment resulted in reduced transpiration rates, reducing water loss during drought stress. Overall, our findings indicate that pretreating tomato plants with ethylene can potentially regulate their responses during the forthcoming stress period, but optimization of the ethylene pre-treatment duration, timing, and dose is needed. Furthermore, it remains tested if the effect is related to the stress duration and severity and whether an ethylene pretreatment has a net positive or negative effect on plant vigor during stress recovery. Further investigations are needed to elucidate the mode of action of how ethylene priming impacts subsequent stress responses.

Evolution of ethylene as an abiotic stress hormone in streptophytes.

All land plants modulate their growth and physiology through intricate signaling cascades. The majority of these are at least modulated-and often triggered-by phytohormones. Over the past decade, it has become apparent that some phytohormones have an evolutionary origin that runs deeper than plant terrestrialization-many emerged in the streptophyte algal progenitors of land plants. Ethylene is such a case. Here we synthesize the current knowledge on the evolution of the phytohormone ethylene and speculate about its deeply conserved role in adjusting stress responses of streptophytes for more than half a billion years of evolution.

Targeted system approach to ethylene biosynthesis and signaling of a heat tolerant tomato cultivar; the impact of growing season on fruit ripening.

Growing tomato in hot weather conditions is challenging for fruit production and yield. Tomato cv. Savior is a heat-tolerant cultivar which can be grown during both the Vietnamese winter (mild condition) and summer (hot condition) season. Understanding the mechanisms of ethylene biosynthesis and signaling are important for agriculture, as manipulation of these pathways can lead to improvements in crop yield, stress tolerance, and fruit ripening. The objective of this study was to investigate an overview of ethylene biosynthesis and signaling from target genes to proteins and metabolites and the impact of growing season on a heat tolerant tomato cultivar throughout fruit ripening and postharvest storage. This work also showed the feasibility of absolute protein quantification of ethylene biosynthesis enzymes. Summer fruit showed the delayed peak of ethylene production until the red ripe stage. The difference in postharvest ethylene production between winter and summer fruit appears to be regulated by the difference in accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) which depends on the putative up-regulation of SAM levels. The lack of differences in protein concentrations between winter and summer fruit indicate that heat stress did not alter the ethylene biosynthesis-related protein abundance in heat tolerant cultivar. The analysis results of enzymatic activity and proteomics showed that in both winter and summer fruit, the majority of ACO activity could be mainly contributed to the abundance of ACO5 and ACO6 isoforms, rather than ACO1. Likewise, ethylene signal transduction was largely controlled by the abundance of ethylene receptors ETR1, ETR3, ETR6, and ETR7 together with the constitute triple response regulator CTR1 for both winter and summer grown tomatoes. Altogether our results indicate that in the heat tolerant tomato cv. Savior, growing season mainly affects the ethylene biosynthesis pathway and leaves the signaling pathway relatively unaffected.

From a different angle: genetic diversity underlies differentiation of waterlogging-induced epinasty in tomato.

In tomato, downward leaf bending is a morphological adaptation towards waterlogging, which has been shown to induce a range of metabolic and hormonal changes. This kind of functional trait is often the result of a complex interplay of regulatory processes starting at the gene level, gated through a plethora of signaling cascades and modulated by environmental cues. Through phenotypical screening of a population of 54 tomato accessions in a Genome Wide Association Study (GWAS), we have identified target genes potentially involved in plant growth and survival during waterlogging and subsequent recovery. Changes in both plant growth rate and epinastic descriptors revealed several associations to genes possibly supporting metabolic activity in low oxygen conditions in the root zone. In addition to this general reprogramming, some of the targets were specifically associated to leaf angle dynamics, indicating these genes might play a role in the induction, maintenance or recovery of differential petiole elongation in tomato during waterlogging.

Plant microbiota controls an alternative root branching regulatory mechanism in plants.

The establishment of beneficial interactions with microbes has helped plants to modulate root branching plasticity in response to environmental cues. However, how the plant microbiota harmonizes with plant roots to control their branching is unknown. Here, we show that the plant microbiota influences root branching in the model plant Arabidopsis thaliana. We define that the microbiota's ability to control some stages in root branching can be independent of the phytohormone auxin that directs lateral root development under axenic conditions. In addition, we revealed a microbiota-driven mechanism controlling lateral root development that requires the induction of ethylene response pathways. We show that the microbial effects on root branching can be relevant for plant responses to environmental stresses. Thus, we discovered a microbiota-driven regulatory pathway controlling root branching plasticity that could contribute to plant adaptation to different ecosystems.

The plant hormone ethylene promotes abiotic stress tolerance in the liverwort Marchantia polymorpha.

Plants are often faced with an array of adverse environmental conditions and must respond appropriately to grow and develop. In angiosperms, the plant hormone ethylene is known to play a protective role in responses to abiotic stress. Here we investigated whether ethylene mediates resistance to abiotic stress in the liverwort Marchantia polymorpha, one of the most distant land plant relatives of angiosperms. Using existing M. polymorpha knockout mutants of Mpein3, and Mpctr1, two genes in the ethylene signaling pathway, we examined responses to heat, salinity, nutrient deficiency, and continuous far-red light. The Mpein3 and Mpctr1 mutants were previously shown to confer ethylene insensitivity and constitutive ethylene responses, respectively. Using mild or sub-lethal doses of each stress treatment, we found that Mpctr1 mutants displayed stress resilience similar to or greater than the wild type. In contrast, Mpein3 mutants showed less resilience than the wild type. Consistent with ethylene being a stress hormone, we demonstrated that ethylene production is enhanced by each stress treatment. These results suggest that ethylene plays a role in protecting against abiotic stress in M. polymorpha, and that ethylene has likely been conserved as a stress hormone since before the evolutionary divergence of bryophytes from the land plant lineage approximately 450 Ma.

Mild and severe salt stress responses are age-dependently regulated by abscisic acid in tomato.

Salt stress hampers plant growth and development through both osmotic and ionic imbalances. One of the key players in modulating physiological responses towards salinity is the plant hormone abscisic acid (ABA). How plants cope with salinity largely depends on the magnitude of the soil salt content (stress severity), but also on age-related developmental processes (ontogeny). Here we studied how ABA directs salt stress responses in tomato plants for both mild and severe salt stress in leaves of different ages. We used the ABA-deficient mutant notabilis, which contains a null-mutation in the gene of a rate-limiting ABA biosynthesis enzyme 9-cis-epoxycarotenoid dioxygenase (NCED1), leading to impaired stomatal closure. We showed that both old and young leaves of notabilis plants keep a steady-state transpiration and photosynthesis rate during salt stress, probably due to their dysfunctional stomatal closure. At the whole plant level, transpiration declined similar to the wild-type, impacting final growth. Notabilis leaves were able to produce osmolytes and accumulate ions in a similar way as wild-type plants, but accumulated more proline, indicating that osmotic responses were not impaired by the NCED1 mutation. Besides NCED1, also NCED2 and NCED6 are strongly upregulated under salt stress, which could explain why the notabilis mutant did not show a lower ABA content upon salt stress, except in young leaves. This might be indicative of a salt-mediated feedback mechanism on NCED2/6 in notabilis and might explain why notabilis plants seem to perform better under salt stress compared to wild-type plants with respect to biomass and water content accumulation.

A 1-aminocyclopropane-1-carboxylic-acid (ACC) dipeptide elicits ethylene responses through ACC-oxidase mediated substrate promiscuity.

Plants produce the volatile hormone ethylene to regulate many developmental processes and to deal with (a)biotic stressors. In seed plants, ethylene is synthesized from 1-aminocyclopropane-1-carboxylic acid (ACC) by the dedicated enzyme ACC oxidase (ACO). Ethylene biosynthesis is tightly regulated at the level of ACC through ACC synthesis, conjugation and transport. ACC is a non-proteinogenic amino acid, which also has signaling roles independent from ethylene. In this work, we investigated the biological function of an uncharacterized ACC dipeptide. The custom-synthesized di-ACC molecule can be taken up by Arabidopsis in a similar way as ACC, in part via Lysine Histidine Transporters (e.g., LHT1). Using Nano-Particle Assisted Laser Desoprtion/Ionization (Nano-PALDI) mass-spectrometry imaging, we revealed that externally fed di-ACC predominantly localizes to the vasculature tissue, despite it not being detectable in control hypocotyl segments. Once taken up, the ACC dimer can evoke a triple response phenotype in dark-grown seedlings, reminiscent of ethylene responses induced by ACC itself, albeit less efficiently compared to ACC. Di-ACC does not act via ACC-signaling, but operates via the known ethylene signaling pathway. In vitro ACO activity and molecular docking showed that di-ACC can be used as an alternative substrate by ACO to form ethylene. The promiscuous nature of ACO for the ACC dimer also explains the higher ethylene production rates observed in planta, although this reaction occurred less efficiently compared to ACC. Overall, the ACC dipeptide seems to be transported and converted into ethylene in a similar way as ACC, and is able to augment ethylene production levels and induce subsequent ethylene responses in Arabidopsis.

A transporter of 1-aminocyclopropane-1-carboxylic acid affects thallus growth and fertility in Marchantia polymorpha.

In seed plants, 1-aminocyclopropane-1-carboxylic acid (ACC) is the precursor of the plant hormone ethylene but also has ethylene-independent signaling roles. Nonseed plants produce ACC but do not efficiently convert it to ethylene. In Arabidopsis thaliana, ACC is transported by amino acid transporters, LYSINE HISTIDINE TRANSPORTER 1 (LHT1) and LHT2. In nonseed plants, LHT homologs have been uncharacterized. Here, we isolated an ACC-insensitive mutant (Mpain) that is defective in ACC uptake in the liverwort Marchantia polymorpha. Mpain contained a frameshift mutation (1 bp deletion) in the MpLHT1 coding sequence, and was complemented by expression of a wild-type MpLHT1 transgene. Additionally, ACC insensitivity was re-created in CRISPR/Cas9-Mplht1 knockout mutants. We found that MpLHT1 can also transport l-hydroxyproline and l-histidine. We examined the physiological functions of MpLHT1 in vegetative growth and reproduction based on mutant phenotypes. Mpain and Mplht1 plants were smaller and developed fewer gemmae cups compared to wild-type plants. Mplht1 mutants also had reduced fertility, and archegoniophores displayed early senescence. These findings reveal that MpLHT1 serves as an ACC and amino acid transporter in M. polymorpha and has diverse physiological functions. We propose that MpLHT1 contributes to homeostasis of ACC and other amino acids in M. polymorpha growth and reproduction.

Ethylene Insensitive 3-Like 2 is a Brassicaceae-specific transcriptional regulator involved in fine-tuning ethylene responses in Arabidopsis thaliana.

Ethylene signaling directs a pleiotropy of developmental processes in plants. In Arabidopsis, ethylene signaling converges at the master transcription factor Ethylene Insensitive 3 (EIN3), which has five homologs, EIN3-like 1-5 (EIL1-EIL5). EIL1 is most fully characterized and operates similarly to EIN3, while EIL3-5 are not involved in ethylene signaling. EIL2 remains less investigated. Our phylogenetic analysis revealed that EIL2 homologs have only been retrieved in the Brassicaceae family, suggesting that EIL2 diverged to have specific functions in the mustard family. By characterizing eil2 mutants, we found that EIL2 is involved in regulating ethylene-specific developmental processes in Arabidopsis thaliana, albeit in a more subtle way compared with EIN3/EIL1. EIL2 steers ethylene-triggered hypocotyl elongation in light-grown seedlings and is involved in lateral root formation. Furthermore, EIL2 takes part in regulating flowering time as eil2 mutants flower on average 1 d earlier and have fewer leaves. A pEIL2:EIL2:GFP translational reporter line revealed that EIL2 protein abundance is restricted to the stele of young developing roots. EIL2 expression, and not EIL2 protein stability, is regulated by ethylene in an EIN3/EIL1-dependent way. Despite EIL2 taking part in several developmental processes, the precise upstream and downstream regulation of this ethylene- and Brassicaceae-specific transcription factor remains to be elucidated.

Overview of Witloof Chicory (Cichorium intybus L.) Discolorations and Their Underlying Physiological and Biochemical Causes.

Many fruits and vegetables suffer from unwanted discolorations that reduce product quality, leading to substantial losses along the supply chain. Witloof chicory (Cichorium intybus L. var. foliosum), a specialty crop characterized by its unique bitter taste and crunchiness, is particularly sensitive to various types of red and brown discolorations. The etiolated vegetable suffers from three predominant color disorders, i.e., core browning, internal leaf reddening, and leaf edge browning. Additionally, several less frequently observed color disorders such as hollow pith, external red, and point noir can also negatively affect crop quality. In this article, we bring together fragmented literature and present a comprehensive overview of the different discoloration types in chicory, and discuss their potential underlying physiological causes, including laticifer rupture, calcium deficiency, and a disturbed water distribution. We also describe the role of environmental cues that influence discoloration incidence, including cultivation and postharvest storage conditions such as forcing and storage temperature, root ripeness and the duration of the forcing process. Finally, we zoom in on the underlying biochemical pathways that govern color disorders in witloof chicory, with a strong emphasis on polyphenol oxidase.

A semi in vivo pollination technique to assess the level of gametophytic self-incompatibility and pollen tube growth in pear (Pyrus communis L.).

KEY MESSAGE: We describe a semi in vivo pollination technique to determine the compatibility relation between different pear cultivars. This assay provides a valuable addition to existing tools in GSI research. The gametophytic self-incompatibility (GSI) system in Pyrus inhibits fertilization by pollen that shares one of the two S-alleles of the style. Depending on their S-locus genotype, two pear cultivars therefore either show a cross-compatible, semi-compatible or incompatible interaction. Because GSI greatly influences seed and fruit set, accurate knowledge of the compatibility type of a cultivar is key for both pear fruit production and breeding. Currently, compatibility relations between different pear cultivars are generally assessed via S-genotyping. However, this approach is restricted to the currently known S-alleles in pear, and does not provide functional assessment of the level of (self-)incompatibility. We here present an optimized semi in vivo pollination assay, that enables quantitative analysis of (self-)incompatibility in pear, and that can also serve useful for more fundamental studies on pollen tube development and pollen-style interactions. This assay involves in vitro incubation of cut pollinated styles followed by microscopic counting of emerging pollen tubes at a specific time interval. The validity and selectivity of this method to determine compatibility interactions in pear is demonstrated in the cultivars "Celina" and "Packham's Triumph." Overall, this technique constitutes a valuable tool for quantitatively determining in vivo pollen tube growth and (cross-)compatibility in pear.

Something old, something new: Conservation of the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid as a signaling molecule.

In seed plants, 1-amino-cyclopropane-1-carboxylic acid (ACC) is the well-known precursor of the plant hormone ethylene. In nonseed plants, the current view is that ACC is produced but is inefficiently converted to ethylene. Distinct responses to ACC that are uncoupled from ethylene biosynthesis have been discovered in diverse aspects of growth and development in liverworts and angiosperms, indicating that ACC itself can function as a signal. Evolutionarily, ACC may have served as a signal before acquiring its role as the ethylene precursor in seed plants. These findings pave the way for unraveling a potentially conserved ACC signaling pathway in plants and have ramifications for the use of ACC as a substitute for ethylene treatment in seed plants.

A digital sensor to measure real-time leaf movements and detect abiotic stress in plants.

Plant and plant organ movements are the result of a complex integration of endogenous growth and developmental responses, partially controlled by the circadian clock, and external environmental cues. Monitoring of plant motion is typically done by image-based phenotyping techniques with the aid of computer vision algorithms. Here we present a method to measure leaf movements using a digital inertial measurement unit (IMU) sensor. The lightweight sensor is easily attachable to a leaf or plant organ and records angular traits in real-time for two dimensions (pitch and roll) with high resolution (measured sensor oscillations of 0.36 ± 0.53° for pitch and 0.50 ± 0.65° for roll). We were able to record simple movements such as petiole bending, as well as complex lamina motions, in several crops, ranging from tomato to banana. We also assessed growth responses in terms of lettuce rosette expansion and maize seedling stem movements. The IMU sensors are capable of detecting small changes of nutations (i.e. bending movements) in leaves of different ages and in different plant species. In addition, the sensor system can also monitor stress-induced leaf movements. We observed that unfavorable environmental conditions evoke certain leaf movements, such as drastic epinastic responses, as well as subtle fading of the amplitude of nutations. In summary, the presented digital sensor system enables continuous detection of a variety of leaf motions with high precision, and is a low-cost tool in the field of plant phenotyping, with potential applications in early stress detection.

Comparative Transcriptomics and Metabolomics Reveal an Intricate Priming Mechanism Involved in PGPR-Mediated Salt Tolerance in Tomato.

Plant-associated beneficial strains inhabiting plants grown under harsh ecosystems can help them cope with abiotic stress factors by positively influencing plant physiology, development, and environmental adaptation. Previously, we isolated a potential plant growth promoting strain (AXSa06) identified as Pseudomonas oryzihabitans, possessing 1-aminocyclopropane-1-carboxylate deaminase activity, producing indole-3-acetic acid and siderophores, as well as solubilizing inorganic phosphorus. In this study, we aimed to further evaluate the effects of AXSa06 seed inoculation on the growth of tomato seedlings under excess salt (200 mM NaCl) by deciphering their transcriptomic and metabolomic profiles. Differences in transcript levels and metabolites following AXSa06 inoculation seem likely to have contributed to the observed difference in salt adaptation of inoculated plants. In particular, inoculations exerted a positive effect on plant growth and photosynthetic parameters, imposing plants to a primed state, at which they were able to respond more robustly to salt stress probably by efficiently activating antioxidant metabolism, by dampening stress signals, by detoxifying Na+, as well as by effectively assimilating carbon and nitrogen. The primed state of AXSa06-inoculated plants is supported by the increased leaf lipid peroxidation, ascorbate content, as well as the enhanced activities of antioxidant enzymes, prior to stress treatment. The identified signatory molecules of AXSa06-mediated salt tolerance included the amino acids aspartate, threonine, serine, and glutamate, as well as key genes related to ethylene or abscisic acid homeostasis and perception, and ion antiporters. Our findings represent a promising sustainable solution to improve agricultural production under the forthcoming climate change conditions.

UV-B light and its application potential to reduce disease and pest incidence in crops.

Ultraviolet-B radiation (280-315 nm), perceived by the plant photoreceptor UVR8, is a key environmental signal that influences plant growth and development and can reduce disease and pest incidence. The positive effect of UV-B on disease resistance and incidence in various plant species supports the implementation of supplemental UV-B radiation in sustainable crop production. However, despite many studies focusing on UV-B light, there is no consensus on the best mode of application. This review aims to analyze, evaluate, and organize the different application strategies of UV-B radiation in crop production with a focus on disease resistance. We summarize the physiological effects of UV-B light on plants and discuss how plants perceive and transduce UV-B light by the UVR8 photoreceptor as well as how this perception alters plant specialized metabolite production. Next, we bring together conclusions of various studies with respect to different UV-B application methods to improve plant resistance. In general, supplemental UV-B light has a positive effect on disease resistance in many plant-pathogen combinations, mainly through the induction of the production of specialized metabolites. However, many variables (UV-B light source, plant species, dose and intensity, timing during the day, duration, background light, etc.) make it difficult to compare and draw general conclusions. We compiled the information of recent studies on UV-B light applications, including e.g., details on the UV-B light source, experimental set-up, calculated UV-B light dose, intensity, and duration. This review provides practical insights and facilitates future research on UV-B radiation as a promising tool to reduce disease and pest incidence.