Identification, characterization, and expression of lipoxygenase genes in sweet cherry (Prunus avium L.) cv. Regina and their relationship with the development of an herbaceous off-flavor during fruit ripening.

Flavor is an essential characteristic of fruit quality and is significant for consumers. Off-flavors have been reported in several fruits, including sweet cherry. This fruit has been reported to show an herbaceous/grassy-like flavor. The herbaceous off-flavor in sweet cherries detected in cultivar Regina has been related to the differential development of aroma compounds such as short-chain aldehydes and esters. One of the main biosynthesis pathways for these compounds is the fatty acid oxidation mediated by lipoxygenases (LOX). In order to have a better understanding of the biological basis of the differences in the volatile profile, the LOX gene expression profile was characterized during fruit development with and without herbaceous off-flavor. A genome-wide analysis of LOX in sweet cherry was carried out and compared to other species such as Arabidopsis, tomato, apple, prunus and strawberry. The structural features of 9-LOX and 13-LOX genes, encoded protein domains and their synteny were examined. Moreover, we analyzed the LOX expression at four developmental stages along ripening by RT-qPCR. Thirteen LOX gene candidates (six 9-LOX and seven 13-LOX) were identified. The 13-LOXs, PaLOX10, PaLOX11, and PaLOX12 were differentially expressed in herbaceous sweet cherries. Furthermore, their expression profile positively correlated with key volatile compounds linked to the herbaceous off-flavor. Overall, this study involves the genome-wide characterization of the LOX family in Prunus avium cv. Regina and provides information that can aid in studying LOX-related fruit deterioration in sweet cherries and associated species.

Ultra-Low Oxygen and Preconditioning Storage Regulate Ethylene Synthesis to Prevent Corky Disorders in 'Fuji' Apple.

Corky disorders in apples represent a significant problem for long-term storage where controlled atmosphere (CA) is mainly used. Ultra-low oxygen (ULO) is an alternative to CA, which consists of low partial pressure of O2 to maintain a low metabolism in the apple fruit, achieving an effective decrease in the ethylene production and physiological disorders. The aim of this research was to study the effectiveness of a short hypoxia period on the development of cork physiological disorders during the storage of apple. 'Fuji' apples were prestored under ULO (0.5 kPa O2) for two periods of time (15 and 30 days) and at two temperatures (0 or 5°C). Corky physiological disorders increased at 5°C prestorage temperature; however, ULO treatments for 15 or 30 days at 0 or 5°C achieved a significant reduction in corky disorders near to 1%, compared with control treatments. In addition, a considerable reduction in ethylene production for up to 30 days was observed in ULO-treated fruit at 0 and 5°C. ULO for 30 days at 0 and 5°C increased the internal production of ethanol and acetaldehyde, causing a lower sensory quality due to the presence of fermentative flavors in fruit stored at 5°C. ULO of 15 days of conditioning decreased the relative expression of ethylene biosynthesis genes MdACS1 and MdACO1, resulting in lower ethylene production.

Low Salt Treatment Results in Plant Growth Enhancement in Tomato Seedlings.

Climate change together with excessive fertilization and poor water quality can affect soil quality and salinization. In plants, high salinity causes osmotic stress, ionic toxicity, and oxidative stress. Consequently, salt stress limits plant development, growth, productivity, and yield. Tomatoes are a very common agricultural product, and some cultivars can partially tolerate salinity. However, most studies are focused on salt excess, which does not necessarily extrapolate on how plants develop in soils with low concentrations of salts. Thus, this study characterizes plant growth and the development of different salt concentrations from 25 to 200 mM in Solanum lycopersicum cv. Moneymaker. Tomato seedlings grown in Murashige and Skoog medium supplied with different NaCl concentrations (0, 25, 50, 75, 100, 125, 150, 175, and 200 mM) showed that low salt concentrations (25 and 50 mM) have a positive impact on lateral root development. This was further observed in physiological parameters such as shoot length, primary root length, and proliferation of lateral roots versus controls. Interestingly, no significant changes in Na+ concentration were observed in 25 mM NaCl in roots or shoots versus controls. Overall, our results suggest that non-toxic salt concentrations can have a positive impact on plant development.

Wine astringency: more than just tannin-protein interactions.

Red wines are characterized by their astringency, a very important sensory attribute that affects the perceived quality of wines. Three mechanisms have been proposed to explain astringency, and two theories describe how these mechanisms work in an integrated manner to produce tactile sensations such as drying, roughening, shrinking and puckering. The factors involved include not only tannins and salivary proteins, but also anthocyanins, grape polysaccharides and mannoproteins, as well as other wine matrix components that modulate their interactions. These multifactorial interactions could be responsible for different sensory responses and therefore need to be further studied. This review presents the latest advances in astringency perception and its possible origins, with special attention on the interactions of components, their impact on oral perception and the development of astringency sub-qualities. Future research efforts should concentrate on understanding the mechanisms involved as well as on the limiting factors related to the conformation and stability of the tannin-salivary protein complexes. © 2021 Society of Chemical Industry.

A network of stress-related genes regulates hypocotyl elongation downstream of selective auxin perception.

The plant hormone auxin, a master coordinator of development, regulates hypocotyl elongation during seedling growth. We previously identified the synthetic molecule RubNeddin 1 (RN1), which induces degradation of the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors INDOLE-3-ACETIC ACID-INDUCIBLE3 (IAA3) and IAA7 in planta and strongly promotes hypocotyl elongation. In the present study, we show that despite the structural similarity of RN1 to the synthetic auxin 2,4-dichlorophenoxyacetic-acid (2,4-D), direct treatments with these compounds in Arabidopsis (Arabidopsis thaliana) result in distinct effects, possibly due to enhanced uptake of RN1 and low-level, chronic release of 2,4-D from RN1 in planta. We confirm RN1-induced hypocotyl elongation occurs via specific TRANSPORT INHIBITOR RESISTANT1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) receptor-mediated auxin signaling involving TIR1, AFB2, and AFB5. Using a transcriptome profiling strategy and candidate gene approach, we identify the genes ZINC FINGER OF ARABIDOPSIS THALIANA10 (ZAT10), ARABIDOPSIS TOXICOS EN LEVADURA31 (ATL31), and WRKY DNA-BINDING PROTEIN33 (WRKY33) as being rapidly upregulated by RN1, despite being downregulated by 2,4-D treatment. RN1-induced expression of these genes also occurs via TIR1/AFB-mediated auxin signaling. Our results suggest both hypocotyl elongation and transcription of these genes are induced by RN1 via the promoted degradation of the AUX/IAA transcriptional repressor IAA7. Moreover, these three genes, which are known to be stress-related, act in an inter-dependent transcriptional regulatory network controlling hypocotyl elongation. Together, our results suggest ZAT10, ATL31, and WRKY33 take part in a common gene network regulating hypocotyl elongation in Arabidopsis downstream of a selective auxin perception module likely involving TIR1, AFB2, and AFB5 and inducing the degradation of IAA7.

Nitrate triggered phosphoproteome changes and a PIN2 phosphosite modulating root system architecture.

Nitrate commands genome-wide gene expression changes that impact metabolism, physiology, plant growth, and development. In an effort to identify new components involved in nitrate responses in plants, we analyze the Arabidopsis thaliana root phosphoproteome in response to nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. 176 phosphoproteins show significant changes at 5 or 20 min after nitrate treatments. Proteins identified by 5 min include signaling components such as kinases or transcription factors. In contrast, by 20 min, proteins identified were associated with transporter activity or hormone metabolism functions, among others. The phosphorylation profile of NITRATE TRANSPORTER 1.1 (NRT1.1) mutant plants was significantly altered as compared to wild-type plants, confirming its key role in nitrate signaling pathways that involves phosphorylation changes. Integrative bioinformatics analysis highlights auxin transport as an important mechanism modulated by nitrate signaling at the post-translational level. We validated a new phosphorylation site in PIN2 and provide evidence that it functions in primary and lateral root growth responses to nitrate.

Stable Transformation of Arabidopsis thaliana Cell Suspension Cultures: A Case Study for The Overexpression of the COI1 Receptor.

Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value chemicals reproducibly and in elevated quantities. In this protocol, a method to stably transform and maintain Arabidopsis cell suspension cultures is devised and presented in detail. Arabidopsis cell cultures were directly transformed with A. tumefaciens for the overexpression of the CORONATINE INSENSITIVE 1 (COI1) jasmonate receptor. Cell cultures were established after transformation and continuously maintained and tested for the overexpression of COI1. The protocol was also previously used to silence Arabidopsis peroxidases and allows for long term maintenance of transformed cells. Details on culture maintenance, both in liquid and solid media are provided, alongside with evidence of protein expression to confirm transformation. The system described provides a powerful tool for synthetic biology to study signaling independent of developmental control and to obtain metabolites of interest for the biotechnological and medical sectors.

Genome-wide analysis in response to nitrogen and carbon identifies regulators for root AtNRT2 transporters.

In Arabidopsis (Arabidopsis thaliana), the High-Affinity Transport System (HATS) for root nitrate (NO3-) uptake depends mainly on four NRT2 NO3- transporters, namely NRT2.1, NRT2.2, NRT2.4, and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 gene expression to the interaction of these signals has never been specifically investigated, and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in response to N/C signals. Our systems analysis of the data identified three transcription factors, TGA3, MYC1, and bHLH093. Functional analysis of mutants combined with yeast one-hybrid experiments confirmed that all three transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals. These results reveal a role for TGA3, MYC1, and bHLH093 in controlling the expression of root NRT2 transporter genes.

Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport.

Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate-dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments.

Overexpression of the Auxin Receptor AFB3 in Arabidopsis Results in Salt Stress Resistance and the Modulation of NAC4 and SZF1.

Soil salinity is a key problem for crop production worldwide. High salt concentration in soil negatively modulates plant growth and development. In roots, salinity affects the growth and development of both primary and lateral roots. The phytohormone auxin regulates various developmental processes during the plant's life cycle, including several aspects of root architecture. Auxin signaling involves the perception by specialized receptors which module several regulatory pathways. Despite their redundancy, previous studies have shown that their functions can also be context-specific depending on tissue, developmental or environmental cues. Here we show that the over-expression of Auxin Signaling F-Box 3 receptor results in an increased resistance to salinity in terms of root architecture and germination. We also studied possible downstream signaling components to further characterize the role of auxin in response to salt stress. We identify the transcription factor SZF1 as a key component in auxin-dependent salt stress response through the regulation of NAC4. These results give lights of an auxin-dependent mechanism that leads to the modulation of root system architecture in response to salt identifying a hormonal cascade important for stress response.

Auxin-mediated responses under salt stress: from developmental regulation to biotechnological applications.

As sessile organisms, plants are exposed to multiple abiotic stresses commonly found in nature. To survive, plants have developed complex responses that involve genetic, epigenetic, cellular, and morphological modifications. Among different environmental cues, salt stress has emerged as a critical problem contributing to yield losses and marked reductions in crop production. Moreover, as the climate changes, it is expected that salt stress will have a significant impact on crop production in the agroindustry. On a mechanistic level, salt stress is known to be regulated by the crosstalk of many signaling molecules such as phytohormones, with auxin having been described as a key mediator of the process. Auxin plays an important role in plant developmental responses and stress, modulating a complex balance of biosynthesis, transport, and signaling that among other things, finely tune physiological changes in plant architecture and Na+ accumulation. In this review, we describe current knowledge on auxin's role in modulating the salt stress response. We also discuss recent and potential biotechnological approaches to tackling salt stress.

Nitrate and hormonal signaling crosstalk for plant growth and development.

Nitrate is an essential macronutrient for plants, a primary nitrogen source in natural and human-made ecosystems. Nitrate can also act as a signaling molecule that directs genome-wide gene expression changes with an impact on plant metabolism, physiology, growth and development. Nitrate and phytohormone signaling pathways crosstalk to modulate growth and developmental programs in a multifactorial manner. Nitrate-signaling controls plant growth and development using molecular mechanisms that involve phytohormone-signaling pathways. In contrast, many phytohormones modulate or impact nitrate signaling in interconnected pathways. In this review, we explore recent progress in our understanding of well-documented connections between nitrate and phytohormones such as auxin, cytokinin and abscisic acid. We also discuss recent studies connecting nitrate to other phytohormones such as ethylene, salicylic acid, gibberellins and brassinosteroids. While many molecular details remain to be elucidated, a number of core signaling components at the intersection between nitrate and the major hormonal pathways have been described. We focus on established interactions of nitrate and different hormonal pathways to bring about cellular, growth and developmental processes in Arabidopsis thaliana.

Local Changes in Chromatin Accessibility and Transcriptional Networks Underlying the Nitrate Response in Arabidopsis Roots.

Transcriptional regulation, determined by the chromatin structure and regulatory elements interacting at promoter regions, is a key step in plant responses to environmental cues. Nitrate (NO3-) is a nutrient signal that regulates the expression of hundreds of genes in Arabidopsis thaliana. Here, we integrate mRNA sequencing, genome-wide RNA polymerase II (RNPII), chromatin immunoprecipitation sequencing, and DNase sequencing datasets to establish the relationship between RNPII occupancy and chromatin accessibility in response to NO3- treatments in Arabidopsis roots. Genomic footprinting allowed us to identify in vivo regulatory elements controlling gene expression in response to NO3- treatments. NO3--modulated transcription factor (TF) footprints are important for a rapid increase in RNPII occupancy and transcript accumulation over time. We mapped key TF regulatory interactions and functionally validated the role of NAP, an NAC-domain containing TF, as a new regulatory factor in NO3- transport. Taken together, our study provides a comprehensive view of transcriptional networks in response to a nutrient signal in Arabidopsis roots.

COI1-dependent jasmonate signalling affects growth, metabolite production and cell wall protein composition in arabidopsis.

Background and Aims: Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described. Methods: Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels. Key Results: Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as ß-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses. Conclusions: This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.

Nitrate Transport, Sensing, and Responses in Plants.

Nitrogen (N) is an essential macronutrient that affects plant growth and development. N is an important component of chlorophyll, amino acids, nucleic acids, and secondary metabolites. Nitrate is one of the most abundant N sources in the soil. Because nitrate and other N nutrients are often limiting, plants have developed sophisticated mechanisms to ensure adequate supply of nutrients in a variable environment. Nitrate is absorbed in the root and mobilized to other organs by nitrate transporters. Nitrate sensing activates signaling pathways that impinge upon molecular, metabolic, physiological, and developmental responses locally and at the whole plant level. With the advent of genomics technologies and genetic tools, important advances in our understanding of nitrate and other N nutrient responses have been achieved in the past decade. Furthermore, techniques that take advantage of natural polymorphisms present in divergent individuals from a single species have been essential in uncovering new components. However, there are still gaps in our understanding of how nitrate signaling affects biological processes in plants. Moreover, we still lack an integrated view of how all the regulatory factors identified interact or crosstalk to orchestrate the myriad N responses plants typically exhibit. In this review, we provide an updated overview of mechanisms by which nitrate is sensed and transported throughout the plant. We discuss signaling components and how nitrate sensing crosstalks with hormonal pathways for developmental responses locally and globally in the plant. Understanding how nitrate impacts on plant metabolism, physiology, and growth and development in plants is key to improving crops for sustainable agriculture.

Cytokinin response factors regulate PIN-FORMED auxin transporters.

Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development.

Cytokinin cross-talking during biotic and abiotic stress responses.

As sessile organisms, plants have to be able to adapt to a continuously changing environment. Plants that perceive some of these changes as stress signals activate signaling pathways to modulate their development and to enable them to survive. The complex responses to environmental cues are to a large extent mediated by plant hormones that together orchestrate the final plant response. The phytohormone cytokinin is involved in many plant developmental processes. Recently, it has been established that cytokinin plays an important role in stress responses, but does not act alone. Indeed, the hormonal control of plant development and stress adaptation is the outcome of a complex network of multiple synergistic and antagonistic interactions between various hormones. Here, we review the recent findings on the cytokinin function as part of this hormonal network. We focus on the importance of the crosstalk between cytokinin and other hormones, such as abscisic acid, jasmonate, salicylic acid, ethylene, and auxin in the modulation of plant development and stress adaptation. Finally, the impact of the current research in the biotechnological industry will be discussed.

Reactive oxygen species and their role in plant defence and cell wall metabolism.

Harnessing the toxic properties of reactive oxygen species (ROS) to fight off invading pathogens can be considered a major evolutionary success story. All aerobic organisms have evolved the ability to regulate the levels of these toxic intermediates, whereas some have evolved elaborate signalling pathways to dramatically increase the levels of ROS and use them as weapons in mounting a defence response, a process commonly referred to as the oxidative burst. The balance between steady state levels of ROS and the exponential increase in these levels during the oxidative burst has begun to shed light on complex signalling networks mediated by these molecules. Here, we discuss the different sources of ROS that are present in plant cells and review their role in the oxidative burst. We further describe two well-studied ROS generating systems, the NADPH oxidase and apoplastic peroxidase proteins, and their role as the primary producers of ROS during pathogen invasion. We then discuss what is known about the metabolic and proteomic fluxes that occur in plant cells during the oxidative burst and after pathogen recognition, and try to highlight underlying biochemical processes that may provide more insight on the complex regulation of ROS in plants.

A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense.

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity.

In plants, reactive oxygen species (ROS) associated with the response to pathogen attack are generated by NADPH oxidases or apoplastic peroxidases. Antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase (FBP1) cDNA in Arabidopsis thaliana was previously shown to diminish the expression of two Arabidopsis peroxidases (peroxidase 33 [PRX33] and PRX34), block the oxidative burst in response to a fungal elicitor, and cause enhanced susceptibility to a broad range of fungal and bacterial pathogens. Here we show that mature leaves of T-DNA insertion lines with diminished expression of PRX33 and PRX34 exhibit reduced ROS and callose deposition in response to microbe-associated molecular patterns (MAMPs), including the synthetic peptides Flg22 and Elf26 corresponding to bacterial flagellin and elongation factor Tu, respectively. PRX33 and PRX34 knockdown lines also exhibited diminished activation of Flg22-activated genes after Flg22 treatment. These MAMP-activated genes were also downregulated in unchallenged leaves of the peroxidase knockdown lines, suggesting that a low level of apoplastic ROS production may be required to preprime basal resistance. Finally, the PRX33 knockdown line is more susceptible to Pseudomonas syringae than wild-type plants. In aggregate, these data demonstrate that the peroxidase-dependent oxidative burst plays an important role in Arabidopsis basal resistance mediated by the recognition of MAMPs.