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Global warming causes heat and drought stress in plants, which affects crop production. In addition to osmotic stress and protein inactivation, reactive oxygen species (ROS) overaccumulation under heat and drought stress is a secondary stress that further impairs plant performance. Chloroplasts, mitochondria, peroxisomes, and apoplasts are the main ROS generation sites in heat- and drought-stressed plants. In this review, we summarize ROS generation and scavenging in heat- and drought-stressed plants and highlight the potential applications of plant nanobiotechnology for enhancing plant tolerance to these stresses. [ABSTRACT FROM AUTHOR]

Plants respond to pathogen exposure by activating the expression of a group of defense-related proteins known as Pathogenesis-Related (PR) proteins, initially discovered in the 1970s. These PR proteins are categorized into 17 distinct families, denoted as PR1-PR17. Predominantly secreted, most of these proteins execute their defensive roles within the apoplastic space. Several PR proteins possess well-defined enzymatic functions, such as β-glucanase (PR2), chitinases (PR3, 4, 8, 11), proteinase (PR7), or RNase (PR10). Enhanced resistance against pathogens is observed upon PR protein overexpression, while their downregulation renders plants more susceptible to pathogen infections. Many of these proteins exhibit antimicrobial activity in vitro, and due to their compact size, some are classified as antimicrobial peptides. Recent research has unveiled that phytopathogens, including nematodes, fungi, and phytophthora, employ analogous proteins to bolster their virulence and suppress plant immunity. This raises a fundamental question: how can these conserved proteins act as antimicrobial agents when produced by the host plant but simultaneously suppress plant immunity when generated by the pathogen? In this hypothesis, we investigate PR proteins produced by pathogens, which we term "PR-like proteins," and explore potential mechanisms by which this class of virulence factors operate. Preliminary data suggests that these proteins may form complexes with the host's own PR proteins, thereby interfering with their defense-related functions. This analysis sheds light on the intriguing interplay between plant and pathogen-derived PR-like proteins, providing fresh insights into the intricate mechanisms governing plant-pathogen interactions. [ABSTRACT FROM AUTHOR]

Key message: The investigation of MYMIV-infected mung bean leaf apoplast revealed viral genome presence, increased EVs secretion, and altered stress-related metabolite composition, providing comprehensive insights into plant–virus interactions. The apoplast, an extracellular space around plant cells, plays a vital role in plant–microbe interactions, influencing signaling, defense, and nutrient transport. While the involvement of apoplast and extracellular vesicles (EVs) in RNA virus infection is documented, the role of the apoplast in plant DNA viruses remains unclear. This study explores the apoplast's role in mungbean yellow mosaic India virus (MYMIV) infection. Our findings demonstrate the presence of MYMIV genomic components in apoplastic fluid, suggesting potential begomovirus cell-to-cell movement via the apoplast. Moreover, MYMIV infection induces increased EVs secretion into the apoplast. NMR-based metabolomics reveals altered metabolic profiles in both apoplast and symplast in response to MYMIV infection, highlighting key metabolites associated with stress and defense mechanisms. The data show an elevation of α- and β-glucose in both apoplast and symplast, suggesting a shift in glucose utilization. Interestingly, this increase in glucose does not contribute to the synthesis of phenolic compounds, potentially influencing the susceptibility of mung bean to MYMIV. Fructose levels increase in the symplast, while apoplastic sucrose levels rise significantly. Symplastic aspartate levels increase, while proline exhibits elevated concentration in the apoplast and reduced concentration in the cytosol, suggesting a role in triggering a hypersensitive response. These findings underscore the critical role of the apoplast in begomovirus infection, providing insights for targeted viral disease management strategies. [ABSTRACT FROM AUTHOR]

Small proteins represent a significant portion of the cargo transported through plant secretory pathways, playing crucial roles in developmental processes, fertilization, and responses to environmental stresses. Despite the importance of small secreted proteins, substantial knowledge gaps persist regarding the regulatory mechanisms governing their trafficking along the secretory pathway, and their ultimate localization or destination. To address these gaps, we conducted a comprehensive literature review, focusing particularly on trafficking and localization of Arabidopsis small secreted proteins with potential biochemical and/or signaling roles in the extracellular space, typically those within the size range of 101–200 amino acids. Our investigation reveals that while at least six members of the 21 mentioned families have a confirmed extracellular localization, eight exhibit intracellular localization, including cytoplasmic, nuclear, and chloroplastic locations, despite the presence of N-terminal signal peptides. Further investigation into the trafficking and secretion mechanisms of small protein cargo could not only deepen our understanding of plant cell biology and physiology but also provide a foundation for genetic manipulation strategies leading to more efficient plant cultivation. [ABSTRACT FROM AUTHOR]

Witches' broom disease (WBD) affects cocoa trees (Theobroma cacao L.) and is caused by the fungus Moniliophthora perniciosa that grows in the apoplast in its biotrophic phase and later progresses into the tissues, causing serious losses in the production of cocoa beans. Therefore, the apoplast of T. cacao can provide important defense responses during the interaction with M. perniciosa. In this work, the protein profile of the apoplast of the T. cacao genotypes Catongo, susceptible to WBD, and CCN-51, resistant one, was evaluated. The leaves of T. cacao were collected from asymptomatic plants grown in a greenhouse (GH) and from green witches' brooms grown under field (FD) conditions for extraction of apoplastic washing fluid (AWF). AWF was used in proteomic and enzymatic analysis. A total of 14 proteins were identified in Catongo GH and six in Catongo FD, with two proteins being common, one up-accumulated, and one down-accumulated. In CCN-51, 19 proteins were identified in the GH condition and 13 in FD, with seven proteins being common, one up-accumulated, and six down-accumulated. Most proteins are related to defense and stress in both genotypes, with emphasis on pathogenesis-related proteins (PR): PR-2 (β-1,3-glucanases), PR-3 and PR-4 (chitinases), PR-5 (thaumatine), PR-9 (peroxidases), and PR-14 (lipid transfer proteins). Furthermore, proteins from microorganisms were detected in the AWF. The enzymatic activities of PR-3 showed a significant increase (p < 0.05) in Catongo GH and PR-2 activity (p < 0.01) in CCN-51 FD. The protein profile of the T. cacao apoplastome offers insight into the defense dynamics that occur in the interaction with the fungus M. perniciosa and offers new insights in exploring future WBD control strategies. [ABSTRACT FROM AUTHOR]

Salinity stress is a major factor affecting the nutritional and metabolic profiles of crops, thus hindering optimal yield and productivity. Recent advances in nanotechnology propose an avenue for the use of nano-fertilizers as a potential solution for better nutrient management and stress mitigation. This study aimed to evaluate the benefits of conventional and nano-fertilizers (nano-Zn/nano-Si) on maize and subcellular level changes in its ionomic and metabolic profiles under salt stress conditions. Zinc and silicon were applied both in conventional and nano-fertilizer-using farms under stress (100 mM NaCl) and normal conditions. Different ions, sugars, and organic acids (OAs) were determined using ion chromatography and inductively coupled plasma mass spectroscopy (ICP-MS). The results revealed significant improvements in different ions, sugars, OAs, and other metabolic profiles of maize. Nanoparticles boosted sugar metabolism, as evidenced by increased glucose, fructose, and sucrose concentrations, and improved nutrient uptake, indicated by higher nitrate, sulfate, and phosphate levels. Particularly, nano-fertilizers effectively limited Na accumulation under saline conditions and enhanced maize's salt stress tolerance. Furthermore, nano-treatments optimized the potassium-to-sodium ratio, a critical factor in maintaining ionic homeostasis under stress conditions. With the growing threat of salinity stress on global food security, these findings highlight the urgent need for further development and implementation of effective solutions like the application of nano-fertilizers in mitigating the negative impact of salinity on plant growth and productivity. However, this controlled environment limits the direct applicability to field conditions and needs future research, particularly long-term field trials, to confirm such results of nano-fertilizers against salinity stress and their economic viability towards sustainable agriculture. [ABSTRACT FROM AUTHOR]

BACKGROUND: The phoma stem canker pathogen Leptosphaeria maculans is one of the most widespread and devastating pathogens of oilseed rape (Brassica napus) in the world. Pathogen colonization is stopped by an interaction of a pathogen Avr effector gene with the corresponding host resistance (R) gene. While molecular mechanisms of this gene‐for‐gene interaction are being elucidated, understanding of effector function remains limited. The purpose of this study was to determine the action of L. maculans effector (AvrLm) genes on incompatible interactions triggered by B. napus noncorresponding R (Rlm) genes. Specifically, effects of AvrLm4‐7 and AvrLm1 on Rlm7‐mediated resistance were studied. RESULTS: Although there was no major effect on symptom expression, induction of defence genes (e.g. PR1) and accumulation of reactive oxygen species was reduced when B. napus cv. Excel carrying Rlm7 was challenged with a L. maculans isolate containing AvrLm1 and a point mutation in AvrLm4‐7 (AvrLm1, avrLm4‐AvrLm7) compared to an isolate lacking AvrLm1 (avrLm1, AvrLm4‐AvrLm7). AvrLm7‐containing isolates, isogenic for presence or absence of AvrLm1, elicited similar symptoms on hosts with or without Rlm7, confirming results obtained with more genetically diverse isolates. CONCLUSION: Careful phenotypic examination of isogenic L. maculans isolates and B. napus introgression lines demonstrated a lack of effect of AvrLm1 on Rlm7‐mediated resistance despite an apparent alteration of the Rlm7‐dependent defence response using more diverse fungal isolates with differences in AvrLm1 and AvrLm4. As deployment of Rlm7 resistance in crop cultivars increases, other effectors need to be monitored because they may alter the predominance of AvrLm7. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

SUMMARY: The intercellular space or apoplast constitutes the main interface in plant–pathogen interactions. Apoplastic subtilisin‐like proteases—subtilases—may play an important role in defence and they have been identified as targets of pathogen‐secreted effector proteins. Here, we characterise the role of the Solanaceae‐specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post‐translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D‐deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum. Significance Statement: The Solanaceae‐specific P69 subtilase family participates in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. [ABSTRACT FROM AUTHOR]

Plant proteases are essential enzymes that play key roles during crucial phases of plant life. Some proteases are mainly involved in general protein turnover and recycle amino acids for protein synthesis. Other proteases are involved in cell signalling, cleave specific substrates and are key players during important genetically controlled molecular processes. Cathepsin B is a cysteine protease that can do both because of its exopeptidase and endopeptidase activities. Animal cathepsin B has been investigated for many years, and much is known about its mode of action and substrate preferences, but much remains to be discovered about this potent protease in plants. Cathepsin B is involved in plant development, germination, senescence, microspore embryogenesis, pathogen defence and responses to abiotic stress, including programmed cell death. This review discusses the structural features, the activity of the enzyme and the differences between the plant and animal forms. We discuss its maturation and subcellular localisation and provide a detailed overview of the involvement of cathepsin B in important plant life processes. A greater understanding of the cell signalling processes involving cathepsin B is needed for applied discoveries in plant biotechnology. [ABSTRACT FROM AUTHOR]

Abstract Pear anthracnose, caused by the fungus Colletotrichum fructicola, is a devastating disease for the pear industry. The apoplast, an extracellular compartment outside the plasma membrane, plays a crucial role in water and nutrient transport, as well as plant-microbe interactions. This study aimed to uncover the molecular mechanism of pear leaf apoplastic protein-mediated resistance to C. fructicola. Apoplast fluid was isolated using the vacuum infiltration method, and defence-related apoplastic proteins were identified through protein mass spectrometry and transcriptome sequencing. We found 213 apoplastic proteins in the leaf apoplast fluid during early C. fructicola infection, with the majority (74.64%) being enzymes, including glycosidases, proteases, and oxidoreductases. Gene Ontology analysis revealed their involvement in defence response, enzyme inhibition, carbohydrate metabolism, and phenylpropanoid biosynthesis. Transcriptome analysis showed the infection induced expression of certain apoplast proteins, potentially contributing to pear leaf resistance. Notably, the expression of PbrGlu1, an endo-β-1,3-glucanase from the glycoside hydrolase 17 family, was significantly higher in infected leaves. Silencing of the PbrGlu1 gene increased pear leaf susceptibility to C. fructicola, leading to more severe symptoms and higher reactive oxygen species content. Overall, our study provides insights into the apoplast space interaction between pear leaves and C. fructicola, identifies a key gene in infected pears, and offers a foundation and new strategy for understanding the molecular mechanisms underlying pear anthracnose and breeding disease-resistant pears.

Using an original methodological and technical approach, we studied the real-time dynamics of radial water transfer in roots and transpiration rate in intact maize plants in response to water stress. It was shown that the response of maize plants to water stress, induced by 10% PEG 6000, was accompanied by changes in the intensity and redistribution of water transfer along different pathways of radial water transport in the roots. It was shown that during the first minutes of water stress impact, the intensity of transcellular and symplastic water transport in the roots decreased with a parallel short-term increase in the transpiration rate in leaves and, presumably, in apoplastic transport in roots. Further, after a decrease in transpiration rate, the intensity of transcellular and symplastic water transport was restored to approximately the initial values and was accompanied by parallel upregulation of some PIP aquaporin genes in roots and leaves, changes in aquaporin localization in root tissues, and changes in xylem sap pH. Under water stress conditions, cell-to-cell water transport in roots becomes dominant, and aquaporins contribute to the simultaneous regulation of water transport in roots and shoots under water stress. [ABSTRACT FROM AUTHOR]

Pear anthracnose, caused by the fungus Colletotrichum fructicola, is a devastating disease for the pear industry. The apoplast, an extracellular compartment outside the plasma membrane, plays a crucial role in water and nutrient transport, as well as plant-microbe interactions. This study aimed to uncover the molecular mechanism of pear leaf apoplastic protein-mediated resistance to C. fructicola. Apoplast fluid was isolated using the vacuum infiltration method, and defence-related apoplastic proteins were identified through protein mass spectrometry and transcriptome sequencing. We found 213 apoplastic proteins in the leaf apoplast fluid during early C. fructicola infection, with the majority (74.64%) being enzymes, including glycosidases, proteases, and oxidoreductases. Gene Ontology analysis revealed their involvement in defence response, enzyme inhibition, carbohydrate metabolism, and phenylpropanoid biosynthesis. Transcriptome analysis showed the infection induced expression of certain apoplast proteins, potentially contributing to pear leaf resistance. Notably, the expression of PbrGlu1, an endo-β-1,3-glucanase from the glycoside hydrolase 17 family, was significantly higher in infected leaves. Silencing of the PbrGlu1 gene increased pear leaf susceptibility to C. fructicola, leading to more severe symptoms and higher reactive oxygen species content. Overall, our study provides insights into the apoplast space interaction between pear leaves and C. fructicola, identifies a key gene in infected pears, and offers a foundation and new strategy for understanding the molecular mechanisms underlying pear anthracnose and breeding disease-resistant pears. [ABSTRACT FROM AUTHOR]

Summary: The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae.Using activity‐based proteomics with a cocktail of biotinylated probes, we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs).The activity of 82 of these hydrolases (mostly SHs) increases during infection, while the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active β‐galactosidase‐1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis‐related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity and contains an E112Q active site substitution that is essential for antibacterial activity and is present only in Nicotiana species.This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo‐functionalised Nicotiana‐specific antibacterial NbPR3. [ABSTRACT FROM AUTHOR]

Botrytis cinerea is a necrotrophic fungal plant pathogen that causes grey mould and rot diseases in many crops. Here, we show that the B. cinerea BcCrh4 transglycosylase is secreted during plant infection and induces plant cell death and pattern‐triggered immunity (PTI), fulfilling the characteristics of a cell death‐inducing protein (CDIP). The CDIP activity of BcCrh4 is independent of the transglycosylase enzymatic activity, it takes place in the apoplast and does not involve the receptor‐like kinases BAK1 and SOBIR1. During saprophytic growth, BcCrh4 is localized in the endoplasmic reticulum and in vacuoles, but during plant infection, it accumulates in infection cushions (ICs) and is then secreted to the apoplast. Two domains within the BcCrh4 protein determine the CDIP activities: a 20aa domain at the N′ end activates intense cell death and PTI, while a stretch of 52aa in the middle of the protein induces a weaker response and suppresses the activity of the 20aa N′ domain. Deletion of bccrh4 affected fungal development and IC formation in particular, resulting in reduced virulence. Collectively, our findings demonstrate that BcCrh4 is required for fungal development and pathogenicity, and hint at a dual mechanism that balances the virulence activity of this, and potentially other CDIPs. Summary statement: 1.The Botrytis cinerea transglycosylase BcCrh4 is an apoplastic effector with weak and hyperactive cell death‐inducing domains. The protein is recognized and activates the plant defence system, but the perception and signalling do not involve the receptor‐like kinases BAK1 and SOBIR1 complex.2.Two domains within the BcCrh4 protein determine activation of cell death and pattern‐triggered immunity: a 20‐amino‐acid domain located at the N′ end activates intense cell death and plant immunity, while a stretch of 52 amino acids located in the middle of the protein has a weak cell death‐inducing activity. The two domains probably compete on the same plant target hence the full‐length protein has a milder cell death and plant immunity‐inducing activity than that of the 20 amino acids domain alone. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2), like other reactive oxygen species (ROS), plays a dual role in physiology of plant organisms. On the one hand, ROS are capable of initiating oxidative stress, thus resulting in damage and death of the cells and whole plants. On the other hand, ROS are signaling molecules that promote the formation of adaptive mechanisms and improve plant resistance. Therefore, H2O2 is now primarily considered as a signaling molecule that acts as a key component of hormonal signaling networks as well as a regulator of growth, development, stomatal guard cell movements, and adaptive responses to biotic and abiotic stressors. The majority of the methods used to measure H2O2 and other ROS are being criticized for the lack of specificity and, most important, insufficient reliability. Herein, possible causes for variability of results were analyzed, general requirements for sample pretreatment were formulated, and criteria to check if reliable data are obtained were proposed. The authors outlined in detail the benefits and shortcomings of commonly used methods for assaying H2O2 concentration in plant extracts, including those based on the oxidation of Fe2+, I–, luminol, and substrates of peroxidase reaction as well as the methods relying on the complex formation between H2O2 and Ti(IV). [ABSTRACT FROM AUTHOR]

Casparian strips are ring-like structures consisting of lignin, sealing the apoplastic space between endodermal cells. They are thought to have important functions in controlling radial transport of nutrients and toxic elements in roots. However, Arabidopsis mutants with a defective Casparian strip structure have been found to maintain nutrient homeostasis in ranges supportive of growth under standard laboratory conditions. In this study, we investigated the function of Casparian strips under excess boron (B) conditions using sgn3 and sgn4 mutants with defective Casparian strip development but which do not exhibit excessive deposition of suberin, another endodermal diffusion barrier. The growth of sgn3 and sgn4 mutants did not differ significantly from that of wild-type (WT) plants under different B conditions in plate cultures; however, they were highly sensitive to B excess in hydroponic culture, where transpiration drives the translocation of boric acid toward the shoot. In hydroponic culture with sufficient to excess boric acid, B accumulation in shoots of the sgn3 and sgn4 mutants was higher than that in the WT. A time-course tracer study using 10B-enriched boric acid at a sufficient or slightly excessive concentration showed higher translocation of B into shoots of the sgn3 and sgn4 mutants. Furthermore, a genetically encoded biosensor for boric acid expressed under a stele-specific promoter (proCIF2:NIP5;1 5′UTR : Eluc-PEST) visualized faster boric acid flux into the mutant steles. Collectively, our results demonstrate the importance of Casparian strips in preventing apoplastic diffusion of boric acid into the stele under excess supply. [ABSTRACT FROM AUTHOR]

Witches’ broom disease (WBD) affects cocoa trees (Theobroma cacao L.) and is caused by the fungus Moniliophthora perniciosa that grows in the apoplast in its biotrophic phase and later progresses into the tissues, causing serious losses in the production of cocoa beans. Therefore, the apoplast of T. cacao can provide important defense responses during the interaction with M. perniciosa. In this work, the protein profile of the apoplast of the T. cacao genotypes Catongo, susceptible to WBD, and CCN-51, resistant one, was evaluated. The leaves of T. cacao were collected from asymptomatic plants grown in a greenhouse (GH) and from green witches’ brooms grown under field (FD) conditions for extraction of apoplastic washing fluid (AWF). AWF was used in proteomic and enzymatic analysis. A total of 14 proteins were identified in Catongo GH and six in Catongo FD, with two proteins being common, one up-accumulated, and one down-accumulated. In CCN-51, 19 proteins were identified in the GH condition and 13 in FD, with seven proteins being common, one up-accumulated, and six down-accumulated. Most proteins are related to defense and stress in both genotypes, with emphasis on pathogenesis-related proteins (PR): PR-2 (β-1,3-glucanases), PR-3 and PR-4 (chitinases), PR-5 (thaumatine), PR-9 (peroxidases), and PR-14 (lipid transfer proteins). Furthermore, proteins from microorganisms were detected in the AWF. The enzymatic activities of PR-3 showed a significant increase (p < 0.05) in Catongo GH and PR-2 activity (p < 0.01) in CCN-51 FD. The protein profile of the T. cacao apoplastome offers insight into the defense dynamics that occur in the interaction with the fungus M. perniciosa and offers new insights in exploring future WBD control strategies.

Reactive oxygen species are, at high levels, harmful to plant cells, whereas, at a balanced and controlled level, they are involved in transduction of oxidative signaling. The apoplast, also known as the extracellular matrix plays a vital role in transferring the extracellular signals to internal organelles and the signal from cell to cell. Moreover, having a lower level of antioxidants renders it a favorable compartment for ROS production and maintenance. Multiple routes for H2O2 production exist in the apoplast; however, a prominent source is the NADPH oxidases, also named respiratory burst oxidase homologs (RBOHs). Many stimuli, such as pathogens, extracellular ATP, hormones, and abiotic stresses can trigger the RBOH-dependent ROS production in the apoplast. RBOHD and RBOHF are extensively studied and proposed typical roles in stomatal conductance and pathogen responses. In this review, we provide an update on RBOH enzymes and the regulatory mechanisms controlling the key enzyme of the family, RBOHD. Furthermore, we performed a phylogenetic analysis by including the RBOH protein family of Arabidopsis and some important crop species which have not been studied before, such as Brasssica rapa, Brassica oleracea, Oriza sativa, Glycine max, and Zea mays to characterize the evolutionary relationship of these isoforms in order to better classify them. We further describe how ROS are perceived and transduced inside the cell and how this interlinks with other systemic signals, including hormones, and abiotic stress responses such as those toward light and salt stress.

In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

Background: Plant-pathogen interactions occur in the apoplast comprising the cell wall matrix and the fluid in the extracellular space outside the plasma membrane. However, little is known regarding the contribution of the apoplastic proteome to systemic acquired resistance (SAR). Methods: Specifically, SAR was induced by inoculating plants with Pst DC3000 avrRps4. The apoplast washing fluid (AWF) was collected from the systemic leaves of the SAR-induced or mock-treated plants. A label free quantitative proteomic analysis was performed to identified the proteins related to SAR in AWF. Results: A total of 117 proteins were designated as differentially accumulated proteins (DAPs), including numerous pathogenesis-related proteins, kinases, glycosyl hydrolases, and redox-related proteins. Functional enrichment analyses shown that these DAPs were mainly enriched in carbohydrate metabolic process, cell wall organization, hydrogen peroxide catabolic process, and positive regulation of catalytic activity. Comparative analysis of proteome data indicated that these DAPs were selectively enriched in the apoplast during the induction of SAR. Conclusions: The findings of this study indicate the apoplastic proteome is involved in SAR. The data presented herein may be useful for future investigations on the molecular mechanism mediating the establishment of SAR. [ABSTRACT FROM AUTHOR]

In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by phosphoribohydrolase, an enzyme encoded by a gene named LONELY GUY (LOG). This reaction accounts for most of the cytokinin supply needed for regulating plant growth and development. In contrast, the LOG-independent pathway, in which dephosphorylation and deribosylation sequentially occur, is also thought to play a role in cytokinin biosynthesis, but the gene entity and physiological contribution have been elusive. In this study, we profiled the phytohormone content of chromosome segment substitution lines of Oryza sativa and searched for genes affecting the endogenous levels of cytokinin ribosides by quantitative trait loci analysis. Our approach identified a gene encoding an enzyme that catalyzes the deribosylation of cytokinin nucleoside precursors and other purine nucleosides. The cytokinin/purine riboside nucleosidase 1 (CPN1) we identified is a cell wall–localized protein. Loss-of-function mutations (cpn1) were created by inserting a Tos17-retrotransposon that altered the cytokinin composition in seedling shoots and leaf apoplastic fluid. The cpn1 mutation also abolished cytokinin riboside nucleosidase activity in leaf extracts and attenuated the trans-zeatin riboside-responsive expression of cytokinin marker genes. Grain yield of the mutants declined due to altered panicle morphology under field-grown conditions. These results suggest that the cell wall–localized LOG-independent cytokinin activating pathway catalyzed by CPN1 plays a role in cytokinin control of rice growth. Our finding broadens our spatial perspective of the cytokinin metabolic system. [ABSTRACT FROM AUTHOR]

Plant non-specific lipid transfer proteins (nsLTPs) are small proteins capable of transferring phospholipids between membranes and binding non-specifically fatty acids in vitro. They constitute large gene families in plants, e.g., 83 in potato (Solanum tuberosum). Despite their recognition decades ago, very few have been functionally characterized. Here, we set out to better understand the function of one of the potato members, StnsLTPI.33. Using quantitative polymerase chain reaction, we show that StnsLTPI.33 is expressed throughout the potato plant, but at relatively higher levels in roots and leaves compared to petals, anthers, and the ovary. We also show that ectopically-expressed StnsLTPI.33 fused to green fluorescent protein colocalized with an apoplastic marker in Nicotiana benthamiana leaves, indicating that StnsLTPI.33 is targeted to the apoplast. Constitutive overexpression of the StnsLTPI.33 gene in potato led to increased levels of superoxide anions and reduced plant growth, particularly under salt stress conditions, and enhanced susceptibility to Alternaria solani. In addition, StnsLTPI.33-overexpressing plants had a depleted leaf pool of pipecolic acid, threonic acid, and glycine, while they accumulated putrescine. To our knowledge, this is the first report of an nsLTP that is associated with enhanced susceptibility to a pathogen in potato. [ABSTRACT FROM AUTHOR]

Purinergic signaling activated by extracellular nucleotides and their derivative nucleosides trigger sophisticated signaling networks. The outcome of these pathways determine the capacity of the organism to survive under challenging conditions. Both extracellular ATP (eATP) and Adenosine (eAdo) act as primary messengers in mammals, essential for immunosuppressive responses. Despite the clear role of eATP as a plant damage-associated molecular pattern, the function of its nucleoside, eAdo, and of the eAdo/eATP balance in plant stress response remain to be fully elucidated. This is particularly relevant in the context of plant-microbe interaction, where the intruder manipulates the extracellular matrix. Here, we identify Ado as a main molecule secreted by the vascular fungus Fusarium oxysporum. We show that eAdo modulates the plant’s susceptibility to fungal colonization by altering the eATP-mediated apoplastic pH homeostasis, an essential physiological player during the infection of this pathogen. Our work indicates that plant pathogens actively imbalance the apoplastic eAdo/eATP levels as a virulence mechanism.

Light is a crucial factor affecting plant growth and development. Besides providing the energy for photosynthesis, light serves as a sensory cue to control the adaptation of plants to environmental changes. We used the etiolated maize (Zea mays) seedlings as a model system to study the red light-regulated growth. Exposure of the maize seedlings to red light resulted in growth inhibition of mesocotyls. We demonstrate for the first time (to the best our knowledge) that red light affected the patterns of apoplastic fluid (AF) metabolites extracted from the mesocotyl segments. By means of the untargeted gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach, we identified 44 metabolites in the AF of maize mesocotyls and characterised the dynamics of their relative tissue abundances. The characteristic metabolite patterns of mesocotyls dominated with mono- and disaccharides, organic acids, amino acids, and other nitrogen-containing compounds. Upon red light irradiation, the contents of β -alanine, putrescine and trans -aconitate significantly increased (P -value < 0.05). In contrast, there was a significant decrease in the total ascorbate content in the AF of maize mesocotyls. The regulatory role of apoplastic metabolites in the red light-induced inhibition of maize mesocotyl elongation is discussed. Light is a crucial factor affecting plant growth and development. Besides providing the energy for photosynthesis, light serves as a sensory cue to control the adaptation of plants to environmental changes. Exposure of the maize (Zea mays) seedlings to red light resulted in growth inhibition of growing mesocotyls and decreased the total ascorbate contents. Red light also affected the primary metabolite patterns in mesocotyl apoplastic fluid. Among metabolites, the contents of β-alanine, putrescine and trans -aconitate significantly increased. Our results get a new insight into the mechanisms of red light-induced growth inhibition in plants. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria, P. syringae relies on the secretion of toxins, which manipulate host-pathogen interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic P. syringae pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates, whereas a distinct subgroup of four effectors was exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins was exclusively secreted in medium and high virulence strains. Taken together, our data show that P. syringae pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicate distinct strategies for establishing virulence of P. syringae pv. aptata in plants. [ABSTRACT FROM AUTHOR]

In the developing seeds of all higher plants, filial cells are symplastically isolated from the maternal tissue supplying photosynthate to the reproductive structure. Photoassimilates must be transported apoplastically, crossing several membrane barriers, a process facilitated by sugar transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been proposed to play a crucial role in apoplastic sugar transport during phloem unloading and the post-phloem pathway in sink tissues. Evidence for this is presented here for developing seeds of the C4 model grass Setaria viridis. Using immunolocalization, SvSWEET4 was detected in various maternal and filial tissues within the seed along the sugar transport pathway, in the vascular parenchyma of the pedicel, and in the xylem parenchyma of the stem. Expression of SvSWEET4a in Xenopus laevis oocytes indicated that it functions as a high-capacity glucose and sucrose transporter. Carbohydrate and transcriptional profiling of Setaria seed heads showed that there were some developmental shifts in hexose and sucrose content and consistent expression of SvSWEET4 homologues. Collectively, these results provide evidence for the involvement of SWEETs in the apoplastic transport pathway of sink tissues and allow a pathway for post-phloem sugar transport into the seed to be proposed. [ABSTRACT FROM AUTHOR]

Monocots and dicots differ in their boron (B) requirement, but also in their capacity to accumulate silicon (Si). Although an ameliorative effect of Si on B toxicity has been reported in various crops, differences among monocots and dicots are not clear, in particular in light of their ability to retain B in the leaf apoplast. In hydroponic experiments under controlled conditions, we studied the role of Si in the compartmentation of B within the leaves of wheat (Triticum vulgare L.) as a model of a high-Si monocot and sunflower (Helianthus annuus L.) as a model of a low-Si dicot, with the focus on the leaf apoplast. The stable isotopes 10B and 11B were used to investigate the dynamics of cell wall B binding capacity. In both crops, the application of Si did not affect B concentration in the root, but significantly decreased the B concentration in the leaves. However, the application of Si differently influenced the binding capacity of the leaf apoplast for excess B in wheat and sunflower. In wheat, whose capacity to retain B in the leaf cell walls is lower than in sunflower, the continuous supply of Si is crucial for an enhancement of high B tolerance in the shoot. On the other hand, the supply of Si did not contribute significantly in the extension of the B binding sites in sunflower leaves. [ABSTRACT FROM AUTHOR]

Extracellular RNA (exRNA) has long been considered as cellular waste that plants can degrade and utilize to recycle nutrients. However, recent findings highlight the need to reconsider the biological significance of RNAs found outside of plant cells. A handful of studies suggest that the exRNA repertoire, which turns out to be an extremely heterogenous group of non-coding RNAs, comprises species as small as a dozen nucleotides to hundreds of nucleotides long. They are found mostly in free form or associated with RNA-binding proteins, while very few are found inside extracellular vesicles (EVs). Despite their low abundance, small RNAs associated with EVs have been a focus of exRNA research due to their putative role in mediating trans-kingdom RNAi. Therefore, non-vesicular exRNAs have remained completely under the radar until very recently. Here we summarize our current knowledge of the RNA species that constitute the extracellular RNAome and discuss mechanisms that could explain the diversity of exRNAs, focusing not only on the potential mechanisms involved in RNA secretion but also on post-release processing of exRNAs. We will also share our thoughts on the putative roles of vesicular and extravesicular exRNAs in plant–pathogen interactions, intercellular communication, and other physiological processes in plants. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is one of the most toxic compounds released into our environment and is harmful to human health, urging the need to remediate Cd-polluted soils. To this end, it is important to increase our insight into the molecular mechanisms underlying Cd stress responses in plants, ultimately leading to acclimation, and to develop novel strategies for economic validation of these soils. Albeit its non-redox-active nature, Cd causes a cellular oxidative challenge, which is a crucial determinant in the onset of diverse signalling cascades required for long-term acclimation and survival of Cd-exposed plants. Although it is well known that Cd affects reactive oxygen species (ROS) production and scavenging, the contribution of individual organelles to Cd-induced oxidative stress responses is less well studied. Here, we provide an overview of the current information on Cd-induced organellar responses with special attention to redox biology. We propose that an integration of organellar ROS signals with other signalling pathways is essential to finetune plant acclimation to Cd stress. [Display omitted] • Cadmium exposure rapidly induces an oxidative challenge in plants. • Cadmium increases reactive oxygen species levels in different compartments. • Redox signals are integrated with pathways mediating acclimation to cadmium stress. [ABSTRACT FROM AUTHOR]

Microplastic (MP) Pollution has become a global environmental concern now. Aquatic, and terrestrial ecosystems and agricultural land have become a major sink for MPs (100nm-5mm) and poses risk to the ecosystem and human health. As per the reports, MPs are capable of penetrating plant parts, if it is happening in edible plants, the safety of crop plants and human health need urgent attention. Hence, this pilot-scale study is aimed to study the effects of MPs (glitters) in the seed germination of Vigna radiata (Mung bean). Since, much attention is not being paid to glitters (50 to > 5000 μm), which are primary microplastics and an emerging source of pollutants, V. radiata seeds were grown in Murashige-Skoog (MS) medium with glitters of varying concentrations (0.2, 0.4 and 2g) and were examined under a fluorescence microscope (10X) after 14 days. The cross-section of the root confirmed the entry of glitters and plant parts were found to be stained by the dye from the glitter. Though, the results shed light on the entry of MPs into the food chain, a detailed field study needs to be conducted to confirm the penetration and accumulation of MPs in the various parts of the plants and their impact on the growth of the plants. [ABSTRACT FROM AUTHOR]

To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete Aphanomyces euteiches, which causes root rot diseases on legumes. Genome mining and expression analysis highlighted an overrepresentation of microbial tandemly repeated proteases, which are upregulated during host infection. Activity Based Protein Profiling and mass spectrometry (ABPP-MS) on apoplastic fluids isolated from pea roots infected by the pathogen led to the identification of 35 active extracellular microbial proteases, which represents around 30% of the genes expressed encoding serine and cysteine proteases during infection. Notably, eight of the detected active secreted proteases carry an additional Cterminal domain. This study reveals novel active modular extracellular eukaryotic proteases as potential pathogenicity factors in Aphanomyces genus. [ABSTRACT FROM AUTHOR]

This study was aimed to investigate the effects of boron on Ca movement, yield, and quality of tomatoes. A soilless culture experiment with three levels of B (0, 25, and 50 µM) and three concentrations of Ca (2, 4, and 8 mM) was carried out to grow tomatoes. The results showed that although the application of boron to the sample was more effective in the vegetative growth and the fruit induction, resulting in higher numbers of fruit, the addition of Ca could more effectively enhance the weight and quality of the fruit. With B application, Ca-symplast increased, while pectin methylesterase activity decreased, resulting in less Blossom End Rot indices and more fruit firmness. The Ca-transfer index from apoplast to symplast slightly increased with the B application. Overall, in terms of fruit quality and quantity, the best result was seen in higher Ca application as well as higher B concentrations. Although exogenous boron could help Ca absorption in the root and the shoot, it lowered the Ca transfer from xylem to apoplast and symplast, from shoot to fruit, and from the proximal to distal end of the fruit. Nevertheless, loading 4 and 8 mM of Ca seems to provide enough calcium reserves in fruits to have better quality and yield. [ABSTRACT FROM AUTHOR]

Aluminium (Al) toxicity is one of the major constraints for crop growth and productivity in most of the acid soils worldwide. The primary lesion of Al toxicity is the rapid inhibition of root elongation. The root apex, especially the transition zone (TZ), has been identified as the major site of Al accumulation and injury. The signalling, in particular through phytohormones in the root apex TZ in response to Al stress, has been reported to play crucial roles in the regulation of Al-induced root growth inhibition. The binding of Al in the root apoplast is the initial event leading to inhibition of root elongation. Much progress has been made during recent years in understanding the molecular functions of cell wall modification and Al resistance-related genes in Al resistance or toxicity, and several signals including phytohormones, Ca2+, etc. have been reported to be involved in these processes. Here we summarize the recent advances in the understanding of Al-induced signalling and regulatory networks in the root apex involved in the regulation of Al-induced inhibition of root growth and Al toxicity/resistance. This knowledge provides novel insights into how Al-induced signals are recognized by root apical cells, transmitted from the apoplast to symplast, and finally initiate the defence system against Al. We conclude that the apoplast plays a decisive role in sensing and transmitting the Al-induced signals into the symplast, further stimulating a series of cellular responses (e.g. exudation of organic acid anions from roots) to adapt to the stress. We expect to stimulate new research by focusing on the signalling events in the root apex in response to Al stress, particularly taking into consideration the signal transduction between the meristem zone, TZ, and elongation zone and the apoplast and symplast. [ABSTRACT FROM AUTHOR]

Aluminum (Al) toxicity is the primary factor limiting agricultural productivity in acid soils. The cell wall is mainly composed of saccharides and the first barrier for aluminum (Al) to enter plant root cells, but it is unknown whether and how root saccharide components are involved in regulating the Al tolerance of Lespedeza that is well adapted to acid soils. Here, we used Al-tolerant Lespedeza bicolor and Al-sensitive Lespedeza cuneata to examine the association of root saccharide components with Lespedeza Al tolerance through analyzing the saccharide changes of roots exposed to Al toxicity. Al-sensitive Lespedeza accumulated more Al and pectin but less hemicellulose in the root cell walls than Al-tolerant Lespedeza. Al treatment decreased the amounts of total sugar secreted from and within the roots of only Al-tolerant Lespedeza. Al treatment decreased the content of root monosaccharides including glucose and mannose in both Lespedeza species, but increased the xylose contents in only Al-sensitive Lespedeza. Taken together, less cell wall pectin rather than hemicellulose is responsible for less root Al accumulation, and Al-decreased root saccharide contents may enhance root organic-acid secretion to chelate toxic Al, both of which contribute to Lespedeza Al tolerance. [ABSTRACT FROM AUTHOR]

Using an original methodological and technical approach, we studied the real-time dynamics of radial water transfer in roots and transpiration rate in intact maize plants in response to water stress. It was shown that the response of maize plants to water stress, induced by 10% PEG 6000, was accompanied by changes in the intensity and redistribution of water transfer along different pathways of radial water transport in the roots. It was shown that during the first minutes of water stress impact, the intensity of transcellular and symplastic water transport in the roots decreased with a parallel short-term increase in the transpiration rate in leaves and, presumably, in apoplastic transport in roots. Further, after a decrease in transpiration rate, the intensity of transcellular and symplastic water transport was restored to approximately the initial values and was accompanied by parallel upregulation of some PIP aquaporin genes in roots and leaves, changes in aquaporin localization in root tissues, and changes in xylem sap pH. Under water stress conditions, cell-to-cell water transport in roots becomes dominant, and aquaporins contribute to the simultaneous regulation of water transport in roots and shoots under water stress.

Efflux transporters such as MexAB-OprM contribute to bacterial resistance to diverse antimicrobial compounds. Here, we show that MexB contributes to epiphytic and late-stage apoplastic growth of Pseudomonas syringae strain B728a, as well as lesion formation in common bean (Phaseolus vulgaris). Although a ∆mexB mutant formed fewer lesions after topical application to common bean, these lesions contain the same number of cells (105 to 107 cells) as those caused by the wild-type strain. The internalized population size of both the wild-type and the ∆mexB mutant within small samples of surface-sterilized asymptomatic portions of leaves varied from undetectably low to as high as 105 cells/cm2. Localized bacterial populations within individual lesions consistently exceeded 105 cells/cm2. Strain B728a was capable of moderate to extensive apoplastic growth in diverse host plants, including lima bean (P. lunatus), fava bean (Vicia faba), pepper (Capsicum annuum), Nicotiana benthamiana, sunflower (Helianthus annuus), and tomato (Solanum lycopersicum), but MexB was not required for growth in a subset of these plant species. A model is proposed that MexB provides resistance to as-yet-unidentified antimicrobials that differ between plant species. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete Aphanomyces euteiches, which causes root rot diseases on legumes. Genome mining and expression analysis highlighted an overrepresentation of microbial tandemly repeated proteases, which are upregulated during host infection. Activity Based Protein Profiling and mass spectrometry (ABPP-MS) on apoplastic fluids isolated from pea roots infected by the pathogen led to the identification of 35 active extracellular microbial proteases, which represents around 30% of the genes expressed encoding serine and cysteine proteases during infection. Notably, eight of the detected active secreted proteases carry an additional C-terminal domain. This study reveals novel active modular extracellular eukaryotic proteases as potential pathogenicity factors in Aphanomyces genus.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a devastating soilborne disease in tomatoes. Magnesium oxide nanoparticles (MgO NPs) induce strong immunity against Fusarium wilt in tomatoes. However, the mechanisms underlying this immunity remain poorly understood. Comparative transcriptome analysis and microscopy of tomato roots were performed to determine the mechanism of MgO NP-induced immunity against FOL. Eight transcriptomes were prepared from tomato roots treated under eight different conditions. Differentially expressed genes were compared among the transcriptomes. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that in tomato roots pretreated with MgO NPs, Rcr3 encoding apoplastic protease and RbohD encoding NADPH oxidase were upregulated when challenge-inoculated with FOL. The gene encoding glycine-rich protein 4 (SlGRP4) was chosen for further analysis. SlGRP4 was rapidly transcribed in roots pretreated with MgO NPs and inoculated with FOL. Immunomicroscopy analysis showed that SlGRP4 accumulated in the cell walls of epidermal and vascular vessel cells of roots pretreated with MgO NPs, but upon FOL inoculation, SlGRP4 further accumulated in the cell walls of cortical tissues within 48 h. The results provide new insights into the probable mechanisms of MgO NP-induced tomato immunity against Fusarium wilt. [ABSTRACT FROM AUTHOR]

Contrary to the biosynthetic pathways of many terpenoids, which are well characterized and elucidated, their transport inside subcellular compartments and the secretion of reaction intermediates and final products at the short- (cell-to-cell), medium- (tissue-to-tissue), and long-distance (organ-to-organ) levels are still poorly understood, with some limited exceptions. In this review, we aim to describe the state of the art of the transport of several terpene classes that have important physiological and ecological roles or that represent high-value bioactive molecules. Among the tens of thousands of terpenoids identified in the plant kingdom, only less than 20 have been characterized from the point of view of their transport and localization. Most terpenoids are secreted in the apoplast or stored in the vacuoles by the action of ATP-binding cassette (ABC) transporters. However, little information is available regarding the movement of terpenoid biosynthetic intermediates from plastids and the endoplasmic reticulum to the cytosol. Through a description of the transport mechanisms of cytosol- or plastid-synthesized terpenes, we attempt to provide some hypotheses, suggestions, and general schemes about the trafficking of different substrates, intermediates, and final products, which might help develop novel strategies and approaches to allow for the future identification of terpenoid transporters that are still uncharacterized. [ABSTRACT FROM AUTHOR]

Since the available pH biosensor repertoire has delivered evidence for pH gradients, microdomains, and transients in the alkaline, neutral, and mildly acidic pH range ([3]; [7], Preprint; [18]; [20]; [21]; [22]), a similarly heterogeneous and dynamic situation is likely to apply also for the more strongly acidic cellular milieus. Keywords: Apoplast; biosensor; fluorescence; live imaging; pH EN Apoplast biosensor fluorescence live imaging pH 7199 7203 5 12/12/22 20221208 NES 221208 This article comments on: B Moreau H, Gaillard I, Paris N. b 2022. A characteristic apoplastic pH landscape was apparent starting from acidic in the root cap (less than pH 4), to more alkaline in the meristem (pH 5.4), to more acidic in the transition zone (pH 5.0) and the elongation zone (pH 4.6). The three Acidins taken together cover a pH range from pH 6.5 down to pH 3.0, while each of the sensor variants is responsive within a range of ~2.0 pH units. [Extracted from the article]

As in metazoans, life in oxygenic photosynthetic organisms relies on the accurate regulation of cell death. During development and in response to the environment, photosynthetic cells activate and execute cell death pathways that culminate in the death of a specific group of cells, a process known as regulated cell death (RCD). RCD control is instrumental, as its misregulation can lead to growth penalties and even the death of the entire organism. Intracellular molecules released during cell demise may act as 'survival' or 'death' signals and control the propagation of cell death to surrounding cells, even in unicellular organisms. This review explores different signals involved in cell-cell communication and systemic signalling in photosynthetic organisms, in particular Ca2+, reactive oxygen species, lipid derivates, nitric oxide, and eATP. We discuss their possible mode-of-action as either 'survival' or 'death' molecules and their potential role in determining cell fate in neighbouring cells. By comparing the knowledge available across the taxonomic spectrum of this coherent phylogenetic group, from cyanobacteria to vascular plants, we aim at contributing to the identification of conserved mechanisms that control cell death propagation in oxygenic photosynthetic organisms. [ABSTRACT FROM AUTHOR]

Background: Leaf hydration is controlled by feedback mechanisms, e.g. stomatal responses, adjustments of osmotic potential and hydraulic conductivity. Leaf water content thus is an input into related feedback-loops controlling the balance of water uptake and loss. Apoplastic alkalisation upon leaf dehydration is hypothesized to be involved together and in interaction with abscisic acid (ABA) in water stress related signaling on tissue level. However, important questions are still unresolved, e.g. the mechanisms leading to pH changes and the exact nature of its interaction with ABA. When studying these mechanisms and their intermediate signaling steps, an experimenter has only poor means to actually control the central experimental variable, leaf water content (LWC), because it is not only dependent on external variables (e.g. air humidity), which are under experimental control, but is also governed by the biological influences controlling transpiration and water uptake. Those are often unknown in their magnitude, unpredictable and fluctuating throughout an experiment and will prevent true repetitions of an experiment. The goal of the method presented here is to experimentally control and manipulate leaf water content (LWC) of attached intact leaves enclosed in a cuvette while simultaneously measuring physiological parameters like, in this case, apoplastic pH. Results: An experimental setup was developed where LWC is measured by a sensor based on IR-transmission and its signal processed to control a pump which circulates air from the cuvette through a cold trap. Hereby a feedback-loop is formed, which by adjusting vapour pressure deficit (VPD) and consequently leaf transpiration can precisely control LWC. This technique is demonstrated here in a combination with microscopic fluorescence imaging of apoplastic pH (pHapo) as indicated by the excitation ratio of the pH sensitive dye OregonGreen. Initial results indicate that pHapo of the adaxial epidermis of Vicia faba is linearly related to reductions in LWC. Conclusions: Using this setup, constant LWC levels, step changes or ramps can be experimentally applied while simultaneously measuring physiological responses. The example experiments demonstrate that bringing LWC under experimental control in this way allows better controlled and more repeatable experiments to probe quantitative relationships between LWC and signaling and regulatory processes. [ABSTRACT FROM AUTHOR]

Manganese (Mn) and aluminum (Al) toxicities are serious edaphic limitations to crop production in acidic soils. Excess Mn can be countered using a stress-adapted soil microbiota that establish symbiotic relationships with native plants. The arbuscular mycorrhizal fungi (AMF) associated with Lolium rigidum L. develop extraradical mycelia (ERM) that quickly colonize wheat and lead to greater shoot growth by promoting stress-evading mechanisms that are not yet completely explained. In the present study, wheat growth was assessed after 3 weeks on disturbed and undisturbed (intact ERM) acidic soil where the native non-mycotrophic Silene gallica L. or strongly mycotrophic L. rigidum were previously developed. The physiological and biochemical mechanisms responsible for increased growth were analyzed by assessing wheat leaf chlorophyll content, photosystem II quantum yield and performance index, enzymatic activity of ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX), superoxide dismutase (SOD) and contents and subcellular localization of Mn, Mg, Si and K. The soil from native plants had a beneficial effect on shoot weight and chlorophyll levels. The highest benefits were obtained for wheat grown in soil with intact ERM associated with L. rigidum. In this condition, where earlier mycorrhization was favored, the Mn content decreased, alongside the content of Si, while the Mg/Mn ratio increased. Mn was redirected to the apoplast, while Si was redirected to the symplast. The activity of APX, GPX and SOD increased, probably due to increased metabolic growth (higher shoot weight and chlorophyll content). Understanding the mechanisms induced by native AMF responsible for increasing wheat performance can contribute to the establishment of sustainable approaches for crop production in acidic soils with Mn toxicity. The use of native plant AMF developers can improve the sustainable use of natural resources in the scope of greener agricultural practices.

Abstract Background Leaf hydration is controlled by feedback mechanisms, e.g. stomatal responses, adjustments of osmotic potential and hydraulic conductivity. Leaf water content thus is an input into related feedback-loops controlling the balance of water uptake and loss. Apoplastic alkalisation upon leaf dehydration is hypothesized to be involved together and in interaction with abscisic acid (ABA) in water stress related signaling on tissue level. However, important questions are still unresolved, e.g. the mechanisms leading to pH changes and the exact nature of its interaction with ABA. When studying these mechanisms and their intermediate signaling steps, an experimenter has only poor means to actually control the central experimental variable, leaf water content (LWC), because it is not only dependent on external variables (e.g. air humidity), which are under experimental control, but is also governed by the biological influences controlling transpiration and water uptake. Those are often unknown in their magnitude, unpredictable and fluctuating throughout an experiment and will prevent true repetitions of an experiment. The goal of the method presented here is to experimentally control and manipulate leaf water content (LWC) of attached intact leaves enclosed in a cuvette while simultaneously measuring physiological parameters like, in this case, apoplastic pH. Results An experimental setup was developed where LWC is measured by a sensor based on IR-transmission and its signal processed to control a pump which circulates air from the cuvette through a cold trap. Hereby a feedback-loop is formed, which by adjusting vapour pressure deficit (VPD) and consequently leaf transpiration can precisely control LWC. This technique is demonstrated here in a combination with microscopic fluorescence imaging of apoplastic pH (pHapo) as indicated by the excitation ratio of the pH sensitive dye OregonGreen. Initial results indicate that pHapo of the adaxial epidermis of Vicia faba is linearly related to reductions in LWC. Conclusions Using this setup, constant LWC levels, step changes or ramps can be experimentally applied while simultaneously measuring physiological responses. The example experiments demonstrate that bringing LWC under experimental control in this way allows better controlled and more repeatable experiments to probe quantitative relationships between LWC and signaling and regulatory processes.

Abstract After the Fukushima Daiichi Nuclear Power Plant accident, radiocesium, one of the main radioactive materials, has been accumulated inside konara oak trunks. Radiocesium has been thought to move radially through the trunk, but it has not been scientifically vindicated because the mechanism of the radial movement of minerals has not yet been experimentally determined. In this study, mineral radial movement was investigated in konara oak trunks of standing trees. A stable isotope cesium (Cs) solution was injected as a tracer into the outer sapwood of standing konara oak tree trunks. A trunk part was subsequently freeze-fixed with liquid nitrogen and subjected to Cs distribution analysis using cryo-scanning electron microscopy/energy-dispersive X-ray spectroscopy. By comparing normal samples that included living cells and freeze–thaw treated samples that contained no living cells, it was concluded that the injected Cs has been moving through the sapwood by the combination of rapid symplasmic movement by virtue of the living xylem parenchyma cells and slow apoplastic diffusion. Conversely, the Cs solution did not diffuse from the sapwood to the heartwood, implying that Cs is exuded from the living parenchyma cells to the apoplast, i.e., cell walls or adjoined dead parenchyma cells in the layer at the sapwood–heartwood boundary, and then diffused into the heartwood. By integrating the results of this study and our previous results obtained on Japanese cedar, we conclude that the mechanism of the radial movement of minerals through the sapwood seems to be a universal characteristic of tree species. In contrast, since mineral concentrations varied among tree species, the movement mechanism across the sapwood–heartwood boundary can differ among tree species.

In recent years, the presence of antibiotics in soil is major concern due to its hazardous effect on human health, soil microbiota, and food crops. The phytoremediation is considered an effective method for removing antibiotics from contaminated sites. However, the effectiveness and efficiency of many plants in the removal of antibiotics are still unknown. In this study, the potential of Pelargonium graveolens L. in the removal of tetracycline (TC) from soil was investigated at three different concentrations (100 mg kg−1, 200 mg kg−1 TC, and 400 mg kg−1). The mechanism of antibiotic sequestration in the plant was also examined in a hydroponic system at three different concentrations (5 mg L−1, 10 mg L−1, and 15 mg L−1). The results demonstrated that the concentrations of TC in leaves, stems, and roots ranged from 13.57 to 21.57 mg kg−1, 0.27–0.48 mg kg−1, and 5.19–7.09 mg kg−1 in different soil treatments. The TC treatments led to a significant reduction in biomass (23.3–54.8 %), root length (42.3–69.2 %), as well as chlorophyll a (24.48–52.18 %), chlorophyll b (16.35–31.70 %), and protein content (23–42.4 %) compared to the control. Moreover, there was a notable increase in proline and SOD activity (19.54–45.72 %) in plant leaves. Hydroponic study demonstrated that more uptake of TC by symplast (1.75–10.51 mg kg−1) than in the apoplast (0.09–9.63 mg kg−1). The Michaelis-Menten kinetic suggested that a higher upward translocation capacity and greater affinity of TC in the aboveground parts of P. graveolens. The higher sequestration of TC was observed in soluble fraction of all tissues. Tetracycline accumulation and distribution suggested that P. graveolens can withstand tetracycline stress and can be used for remediation purpose for antibiotic contaminated areas. [Display omitted] • P. graveolens demonstrated a good tolerance index for tetracycline. • The symplast was the dominant pathway for uptake. • Michaelis-Menten kinetic suggested favourable upward translocation in plant. • P. graveolens displayed good potential for remediation for tetracycline. [ABSTRACT FROM AUTHOR]