Pull the fuzes:Processing protein precursors to generate apoplastic danger signals for triggering plant immunity

The apoplast is one of the first cellular compartments outside the plasma membrane encountered by phytopathogenic microbes in the early stages of plant tissue invasion.Plants have developed sophisticated surveillance mechanisms to sense danger events at the cell surface and promptly activate immunity.However,a fine tuning of the activation of immune pathways is necessary to mount a robust and effective defense response.Several endogenous proteins and enzymes are synthesized as inactive precursors,and their post-translational processing has emerged as a critical mechanism for triggering alarms in the apoplast.In this review,we focus on the precursors of phytocytokines,cell wall remodeling enzymes,and proteases.The physiological events that convert inactive precursors into immunomodulatory active peptides or enzymes are described.This review also explores the functional synergies among phytocytokines,cell wall damage-associated molecular patterns,and remodeling,highlighting their roles in boosting extracellular immunity and reinforcing defenses against pests.

A G-type lectin receptor-like kinase regulates the perception of oomycete apoplastic expansin-like proteins

Phytophthora capsici is one of the most harmful pathogens in agriculture, which threatens the safe production of multiple crops and causes serious economic losses worldwide. Here, we identified a P. capsici expansin-like protein, Pc EXLX1, by liquid chromatography-tandem mass spectrometry from Nicotiana benthamiana apoplastic fluid infected with P. capsici. Clustered regularly interspaced short palindromic repeats/crispr associated protein9(CRISPR/Cas9)-mediated Pc EXLX1 knockout mutants exhibited significantly enhanced virulence,while the overexpression of Pc EXLX1 impaired the virulence. Prokaryotically expressed Pc EXLX1 activated multiple plant immune responses, which were BRI1-associated kinase 1(BAK1)-and suppressor of BIR1-1(SOBIR1)-dependent. Furthermore, overexpression of Pc EXLX1 homologs in N. benthamiana could also increase plant resistance to P. capsici. A G-type lectin receptor-like kinase from N. benthamiana, expansin-regulating kinase 1(ERK1), was shown to regulate the perception of Pc EXLX1 and positively mediate the plant resistance to P. capsici. These results reveal that the expansin-like protein, Pc EXLX1, is a novel apoplastic effector with plant immunity-inducing activity of oomycetes, perception of which is regulated by the receptor-like kinase, ERK1.

Iron toxicity resistance strategies in tropical grasses:The role of apoplastic radicular barriers

The revegetation of mined areas poses a great challenge to the iron ore mining industry.The initial recovery process in degraded areas might rely on the use of Fe-resistant grasses.Tropical grasses, such as Paspalum densum and Echinochloa crus-galli, show different resistance strategies to iron toxicity; however, these mechanisms are poorly understood.The Fe-resistance mechanisms and direct iron toxicity as a function of root apex removal were investigated. To achieve this purpose, both grass species were grown for up to 480 hr in a nutrient solution containing 0.019 or 7 mmol/L Fe-EDTA after the root apices had been removed or maintained. Cultivation in the presence of excess iron-induced leaf bronzing and the formation of iron plaque on the root surfaces of both grass species, but was more significant on those plants whose root apex had been removed. Iron accumulation was higher in the roots, but reached phytotoxic levels in the aerial parts as well. It did not hinder the biosynthesis of chloroplastidic pigments. No significant changes in gas exchange and chlorophyll a fluorescence occurred in either grass when their roots were kept intact; the contrary was true for plants with excised root apices. In both studied grasses, the root apoplastic barriers had an important function in the restriction of iron translocation from the root to the aerial plant parts, especially in E. crus-galli. Root apex removal negatively influenced the iron toxicity resistance mechanisms(tolerance in P. densum and avoidance in E. crus-galli).

Editorial: Effect of root anatomy and apoplastic barrier development on cadmium uptake in rice

Cadmium (Cd) is a toxic metal with high mobility from soil and known translocation into plants (Song et al., 2015).Because the main source of human exposure to Cd is from food consumption, there has been increased research examining Cd uptake in agricultural plants (Li et al., 2014;Rizwan et al., 2016;Song et al., 2015).

Anatomical and Chemical Alterations but not Photosynthetic Dynamics and Apoplastic Transport Changes are Involved in the Brittleness Culm Mutation of Rice

Brittleness culm is an important agronomic trait that has a potential usefulness in agricultural activity as animal forage although the developmental mechanism is not clear yet. In the present study, the anatomical and chemical characteristics as well as some ecophysiological features in the brittleness culm mutation of rice （Oryza sativa L.） were investigated. Compared with the wild type （WT）, the brittleness culm mutant （bcm） exhibited higher culm vascular bundle distance and lower culm wall thickness, leaf interveinal distance and leaf thickness. Ratio of bundle sheath cell/whole bundle and areas of whole vascular bundles and bundle sheath of leaves were reduced while ratios of xylem and phloem to whole bundles were elevated in bcm. The Fourier transform infrared （FTIR） microspectroscopy analysis and further histochemical and physiological measurements revealed that the different contents and depositions of cell wall components such as pectins, lignin, suberin and cellulose all participated in the mutation of brittleness. However, the mutant presented no significant changes in leaf photosynthetic dynamics and apoplastic transport ability. These results strongly indicate that the alterations in anatomical and chemical characteristics, rather than changes in major ecophysiological features such as photosynthesis and apoplastic transport were involved in the brittleness mutation of rice.

Apoplastic Proteases: Powerful Weapons against Pathogen Infection in Plants

Plants associate with diverse microbes that exert beneficial,neutral,or pathogenic effects inside the host.During the initial stages of invasion,the plant apoplast constitutes a hospitable environment for invading microbes,providing both water and nutrients.In response to microbial infection,a number of secreted proteins from host cells accumulate in the apoplastic space,which is related to microbial association or colonization processes.However,the molecular mechanisms underlying plant modulation of the apoplast environment and how plant-secreted proteases are involved in pathogen resistance are still poorly understood.Recently,several studies have reported the roles of apoplastic proteases in plant resistance against bacteria,fungi,and oomycetes.On the other hand,microbe-secreted proteins directly and/or indirectly inhibit host-derived apoplastic proteases to promote infection.These findings illustrate the importance of apoplastic proteases in plant–microbe interactions.Therefore,understanding the protease-mediated apoplastic battle between hosts and pathogens is of fundamental importance for understanding plant–pathogen interactions.Here,we provide an overviewof plant–microbe interactions in the apoplastic space.We define the apoplast,summarize the physical and chemical properties of these structures,and discuss the roles of plant apoplastic proteases and pathogen protease inhibitors in host–microbe interactions.Challenges and future perspectives for research into protease-mediated apoplastic interactions are discussed,which may facilitate the engineering of resistant crops.

Effect of Soil Moisture on Release of Low-Molecular Weight Organic Acids in Root Exudates and the Accu-mulation of Iron in Root Apoplasm of Peanut

A three-compartments rhizobox was designed and used to study the low-molecular-weight organic acids in root exudates and the root apoplastic iron of "lime-induced chlorosis" peanut grown on a calcareous soil in relation to different soil moisture conditions. Results showed that chlorosis of peanuts developed under condition of high soil moisture level (250 g kg-1), while peanuts grew well and chlorosis did not develop when soil moisture was managed to a normal level (150 g kg-1). The malic acid, maleic acid and succinic acid contents of chlorotic peanut increased by 108.723, 0.029 and 22.446 ig cm-2, respectively, compared with healthy peanuts. The content of citric acid and fumaric acid also increased in root exudates of chlorotic peanuts. On Days 28 and 42 of peanut growth, the accumulation of root apoplastic iron in chlorotic peanuts was higher than that of healthy peanuts. From Day 28 to Day 42, the mobilization percentages of chlorotic peanuts and healthy peanuts to root apoplastic iron were almost the same, being 52.4% and 52.8%, respectively, indicating that the chlorosis might be caused by the inactivation of iron within peanut plant grown on a calcareous soil under high soil moisture conditions.

Root Morphology and Anatomy Affect Cadmium Translocation and Accumulation in Rice

Paddy fields contaminated with cadmium(Cd)present decreased grain yield and produce Cckcontaminated grains.Screening for low-Cd-accumulating cultivars is a useful method to reduce the amount of Cd in the grains.The present study aimed to examine the roles of the root morphology and an atomy in Cd tran slocati on and accumulati on in rice plants.Twenty・two rice cultivars were used in the first experiment,after which two cultivars[Zixiangnuo(ZXN)and Jinyou T36(JYT36)]were selected and used in subsequent experiments under hydroponic conditions.The results showed that there were significant differences in Cd concentrations in the shoots(ranging from 4 to 100 mg/kg)and the Cd translocation rates(shoot/root)(from 7%to 102%)among the 22 cultivars,and the shoot Cd concentration was significantly correlated with the Cd translocation rate of the 22 cultivars under 0.1 mg/L Cd treatment.Compared with cultivar ZXN,JYT36 had greater root Cd uptake and accumulation but lower shoot Cd accumulation and Cd translocation rate.The number of root tips per surface area of cultivar ZXN was greater than that of JYT36,while the average root diameter was lower than that of JYT36.Compared with ZXN,JYT36 had stronger apoplastic barriers,and the Casparian bands and suberin lamellae in the root endodermis and exodermis were closer to the root apex in both the control and Cd treatments,especially for suberin lamellae in the root exodermis with Cd treatments,with a differenee of 25 mm.The results also showed that,compared with ZXN,JYT36 had greater percentages of Cd bound in cell walls and intracellular Cd but lower Cd concentrations in the apoplastic fluid under the Cd treatment.The results suggested that Cd translocation,rather than root Cd uptake,is a key process that determi nes Cd accumulati on in the rice shoots.The root morphological and an atomical characteristics evidently affect Cd accumulation in the shoots by inhibiting Cd translocation,especially via the apoplastic pathway.It was possible to pre-screen low・Cd・accumulating rice cultivars on the basis of their root morphology,an atomical characteristics and Cd tran slocati on rate at the seedling stage.

Anatomical and Chemical Characteristics of a Rolling Leaf Mutant of Rice and Its Ecophysiological Properties

The anatomical and chemical characteristics of a rolling leaf mutant （rlm） of rice （Oryza sativa L.） and its ecophysiological properties in photosynthesis and apoplastic transport were investigated. Compared with the wild type （WT）, the areas of whole vascular bundles and xylem as well as the ratios of xylem area/whole vascular bundles area and xylem area/phloem area were higher in rim, whereas the area and the width of foliar bulliform cell were lower. The Fourier transform infrared （FTIR） microspectroscopy spectra of foliar cell walls differed greatly between rim and WT. The rim exhibited lower protein and polysaccharide contents of foliar cell walls. An obvious reduction of pectin content was also found in rim by biochemical measurements. Moreover, the rate of photosynthesis was depressed while the conductance of stoma and the intercellular CO2 concentration were enhanced in rim. The PTS fluorescence, which represents the ability of apoplastic transport, was 11% higher in rim than in WT. These results suggest that the changes in anatomical and chemical characteristics of foliar vascular bundles, such as the reduction of proteins, pectins, and other polysaccharides of foliar cell walls, participate in the leaf rolling mutation, and consequently lead to the reduced photosynthetic dynamics and apoplastic transport ability in the mutant.

Observation on the modifying activity of the protein from Elytrzgia repens rhizome for ice crystal

In winter, spring and summer, the rhizome of wild Elytrzgia repens of Heilongjiang Province was selected to extract the soluble which whole protein and the apoplastic protein, and analyzed by SDS-PAGE. The result indicated that there were two specific polypeptides in two types protein from winter; their relative molecular weight were identified as 52 ku and 26 ku by analyzing software; the apoplastic protein from winter had the ability of modifing the growth of ice crystal which appeared hexagonal in shape observed with the phase-contrast photomicroscope. So the apoplastic protein from winter has the antifreeze characters and the 52 ku protein is more likely the antifreeze protein

Proteomic analysis of pathogen-responsive proteins from maize stem apoplast triggered by Fusarium verticillioides

During the attack of a pathogen, a variety of defense-associated proteins are released by the host plant in the apoplast to impede the perceived attack. This study utilized the mass spectrometry(LC-MS/MS) and label-free quantification method to analyze the apoplastic fluid(APF) from maize stalk and identified the proteins responsive to the Fusarium verticillioides infection. We have identified 742 proteins, and among these, 119 proteins were differentially accumulated(DAPs), i.e., 35 up-regulated, 18 down-regulated, and 66 proteins were only induced by the pathogen infection. The differentially accumulated proteins were analyzed for their Gene Ontology(GO) and Kyoto Encyclopedia of Gene and Genomes(KEGG) pathway enrichment. The highly enriched Biological Process(BP) term was the L-serine biosynthesis process, whereas the most enriched Molecular Function(MF) term was the cysteine-type endopeptidase inhibitor activity. It was also found that the pathways related to the biosynthesis of amino acid, biosynthesis of secondary metabolites, protein processing in the endoplasmic reticulum, and carbohydrate metabolic pathways were significantly enriched. Moreover, 61 out of 119 differentially accumulated proteins were predicted as secretory proteins. The secretory pathways analysis showed that a greater number of proteins were secreted through the conventional secretion system compared to the unconventional secretion system. The identified secreted proteins were related to a variety of pathways in defense responses including cell redox homeostasis, cell wall modification, signal transduction, carbohydrate metabolism, binding proteins(metal ion binding, RNA binding and heme-binding), maintenance and stabilization of other proteins, indicating a complex response from the plant to the fungal infection. Our data suggested that a number of host proteins belonging to various pathways have been modulated in the apoplastic region.

Photorespiration and Its Role in the Regulation of Photosynthesis and Plant Productivity

The results of long-term studies of photorespiration are summarized and the unsuccessful attempts to increase productivity by suppressing this process are shown. It has been shown that photorespiration and glycolate metabolism are involved in the regulation of the relationship between light processes in chloroplasts and the dark reactions of carbon dioxide assimilation. The studies were conducted on plants in vivo and were associated with the activity of the apoplastic invertase enzyme, affecting assimilate transport. In violation of donor-acceptor relations between photosynthetic and plant-assimilating organs (removal of part of organs-consumers of assimilates or leaves, increase in nitrate nutrition), the kinetics of inclusion of 14C in glycolate was changed. This is due to the strengthening of the role of the transketolase mechanism of its formation. The study of genetically transformed plants, in which either an additional apoplastic invertase gene was introduced, or the existing gene was blocked and did not act, showed a different change in the ratio of 14C-labeled sucrose/hexose and the transpiration response to reduced light. In this connection, the concept of the mechanism of photorespiration interaction with apoplastic invertase and stomatal apparatus of the leaf is proposed when the ratio of light and dark reactions of photosynthesis or assimilate transport is changed. The essence of the concept is that when the ratio of light and dark processes is disturbed, the concentration of organic acids changes first in mesophilic cells (mainly by photorespiration), and then in the extracellular space. It changes the activity of apoplastic invertase, which hydrolyzes sucrose and prevents it from being exported from the leaf. Hydrolysis of sucrose increases the osmoticity of the aquatic environment of the apoplast, which increases with movement to the stomata. The changed osmoticity of the environment around the stomatal guard cells changes the resistance of CO2 diffusion into the leaf. This normalizes the ratio of light and dark processes in the sheet. Therefore, when illumination decreases, nitrate nutrition increases or difficulties arise with the use of photosynthesis products in acceptor organs, the ratio of 14C-labeled sucrose/hexose decreases, and the stomata close. With increasing illumination, reverse events occur.

Ecological Significance of the Interaction of Photosynthesis Light and Dark Processes

The kinetics of 14C incorporation into glycolate was studied after changing the export of photosynthetic products from the leaf. It has been shown that the ribulose-bisphosphate-oxygenase pathway of glycolate formation works in the stationary state of the plant. An excess of photosyntates or a decrease in the amount of light primary products, as well as nitrates in the leaves, immediately turns on the transketolase pathway of glycolate formation. In this case, part of the oxygen formed in the photochemical reactions of chloroplasts ceases to be released from the leaf. After oxygen receives an electron from ferredoxin in the electron transport chain of chloroplasts, it starts (through photorespiration) the formation of non-carbohydrate photosyntates and metabolic processes in the cytoplasm. It was concluded that the main function of photorespiration in the regulation of photosynthesis is maintaining a balance between light and dark processes of photosynthesis on change of living conditions.

Accumulation of Sugars and Liquid in Apoplast of Fruit Flesh Result in Pineapple Translucency

Translucency is a recurring problem for pineapple industry. Translucent fruit contained more sucrose, glucose and fructose in apoplast than those in apoplast of normal fruit. There were more liquid in intercellular space of translucent fruit than that of normal flesh. The contents of alcohol and ethylene in translucent fruit were higher than those in normal fruit. Translucent fruit contained less calcium than normal fruit. Electrolyte leakage of translucent flesh was more than that of normal flesh. There were 205 proteins of which the expressions in translucent flesh were higher than those in normal flesh. Calcium-ions-binding protein EF-hand domain-containing protein, ethylene-synthesizing enzyme 1-aminpcyclopropane-1-carboxylate oxidase, ROS-producing protein universal stress protein A-like protein were the top three proteins of which the expressions in translucent flesh were higher than those in normal fruit. When much sugar was transferred into fruit pulp and accumulated in intercellular space, water will be absorbed from cells around and translucence formed. The accumulation of sugar and liquid in apoplast were due to that cell wall and membrane were degraded, which was from being attacked by ROS. There might be more and larger pores in cell wall and membranes of translucent flesh. These data played foundations for researching methods for controlling pineapple translucency.

Small holes,big impact:Stomata in plant–pathogen–climate epic trifecta

The regulation of stomatal aperture opening and closure represents an evolutionary battle between plants and pathogens,characterized by adaptive strategies that influence both plant resistance and pathogen virulence.The ongoing climate change introduces further complexity,affecting pathogen invasion and host immunity.This review delves into recent advances on our understanding of the mechanisms governing immunity-related stomatal movement and patterning with an emphasis on the regulation of stomatal opening and closure dynamics by pathogen patterns and host phytocytokines.In addition,the review explores how climate changes impact plant–pathogen interactions by modulating stomatal behavior.In light of the pressing challenges associated with food security and the unpredictable nature of climate changes,future research in this field,which includes the investigation of spatiotemporal regulation and engineering of stomatal immunity,emerges as a promising avenue for enhancing crop resilience and contributing to climate control strategies.

Recognition of the inducible,secretory small protein OsSSP1 by the membrane receptor OsSSR1 and the co-receptor OsBAK1 confers rice resistance to the blast fungus

The plant apoplast,which serves as the frontline battleground for long-term host–pathogen interactions,harbors a wealth of disease resistance resources.However,the identification of the disease resistance proteins in the apoplast is relatively lacking.In this study,we identified and characterized the rice secretory protein OsSSP1(Oryza sativa secretory small protein 1).OsSSP1 can be secreted into the plant apoplast,and either in vitro treatment of recombinant OsSSP1 or overexpression of OsSSP1 in rice could trigger plant immune response.The expression of OsSSP1 is suppressed significantly during Magnaporthe oryzae infection in the susceptible rice variety Taibei 309,and OsSSP1-overexpressing lines all show strong resistance to M.oryzae.Combining the knockout and overexpression results,we found that OsSSP1 positively regulates plant immunity in response to fungal infection.Moreover,the recognition and immune response triggered by OsSSP1 depend on an uncharacterized transmembrane OsSSR1(secretory small protein receptor 1)and the key co-receptor OsBAK1,since most of the induced immune response and resistance are lost in the absence of OsSSR1 or OsBAK1.Intriguingly,the OsSSP1 protein is relatively stable and can still induce plant resistance after 1 week of storage in the open environment,and exogenous OsSSP1 treatment for a 2-week period did not affect rice yield.Collectively,our study reveals that OsSSP1 can be secreted into the apoplast and percepted by OsSSR1 and OsBAK1 during fungal infection,thereby triggering the immune response to enhance plant resistance to M.oryzae.These findings provide novel resources and potential strategies for crop breeding and disease control.

Effects of root morphology and anatomy on cadmium uptake and translocation in rice(Oryza sativa L.)

A clear description of the certain mechanisms of cadmium(Cd) uptake and translocation in rice(Oryza sativa L.) may help to reduce Cd accumulation in rice grain. Hydroponic experiments were carried out to determine the effects of cultivation conditions(aerated and stagnant) on the uptake, translocation and subcellular distribution of Cd in relation to the morphology and anatomy of roots in two rice genotypes with different Cd accumulations in grains. Marked differences in morphology and anatomy were observed between these two genotypes under different cultivation conditions. Genotypes with low Cd accumulation in grains tended to develop fewer root tips per root surface area, larger root porosity and more mature apoplastic barriers. The stagnant cultivation condition decreased the number of root tips per root surface area but increased root porosity and accelerated apoplastic barrier formation in root tissues.Correlative Cd uptake studies revealed that rice plants with fewer number of root tips per root surface area reduced root Cd uptake ability, while mature apoplastic barriers increased root Cd retention in cell walls and the symplast. Thus, the fewer number of root tips per root surface area and the earlier formation of mature apoplastic barriers led to lower Cd uptake and translocation. The results indicated that the morphology and anatomy of roots could play important roles in Cd uptake and translocation in rice, and could be influenced by both genotype and cultivation conditions. The present results would be useful in screening and planting rice plants with low Cd accumulation.

Phytophthora sojae Effector PsAvh240 Inhibits Host Aspartic Protease Secretion to Promote Infection

Plants secrete defense molecules into the extracellular space (the apoplast) to combat attacking microbes. However, the mechanisms by which successful pathogens subvert plant apoplastic immunity remain poorly understood. In this study, we show that PsAvh240, a membrane-localized effector of the soybean pathogen Phytophthora sojae, promotes P. sojae infection in soybean hairy roots. We found that PsAvh240 interacts with the soybean-resistant aspartic protease GmAP1 in planta and suppresses the secretion of GmAP1 into the apoplast. By solving its crystal structure we revealed that PsAvh240 contain six a helices and two WY motifs. The first two a helices of PsAvh240 are responsible for its plasma membrane-localization and are required for PsAvh240's interaction with GmAP1. The second WY motifs of two PsAvh240 molecules form a handshake arrangement resulting in a handshake-like dimer. This dimerization is required for the effector's repression of GmAP1 secretion. Taken together, these data reveal that PsAvh240 localizes at the plasma membrane to interfere with GmAP1 secretion, which represents an effective mechanism by which effector proteins suppress plant apoplastic immunity.

The pH of the Apoplast： Dynamic Factor with =unctional Impact Under Stress

The apoplast is an interconnected compartment with a thin water-film that alkalinizes under stress. This systemic pH increase may be a secondary effect without functional implications, arising from ion movements or proton-pump regulations. On the other hand, there are increasing indications that it is part of a mechanism to withstand stress. Regardless of this controversy, alkalinization of the apoplast has received little attention. The apoplastic pH （PHapo） increases not only during plant-pathogen interactions but also in response to salinity or drought. Not much is known about the mechanisms that cause the leaf apoplast to alkalinize, nor whether, and if so, how functional impact is conveyed. Controversial explanations have been given, and the unusual complexity of pHapo regulation is considered as the primary reason behind this lack of knowledge. A gathering of scattered information revealed that changes in PHapo convey functionality by regulating stomatal aperture via the effects exerted on abscisic acid. Moreover, apoplastic alkalinization may regulate growth under stress, whereas this needs to be verified. In this review, a comprehensive survey about several physiological mechanisms that alkalize the apoplast under stress is given, and the suitability of apoplastic alkalinization as transducing element for the transmission of sensory information is discussed.

PDX1.1-dependent biosynthesis of vitamin B6 protects roots from ammonium-induced oxidative stress

Despite serving as a major inorganic nitrogen source for plants,ammonium causes toxicity at elevated con-centrations,inhibiting root elongation early on.While previous studies have shown that ammonium-inhibited root development relates to ammonium uptake and formation of reactive oxygen species(ROS)in roots,it remains unclear about the mechanisms underlying the repression of root growth and how plants cope with this inhibitory effect of ammonium.In this study,we demonstrate that ammonium-induced apo-plastic acidification co-localizes with Fe precipitation and hydrogen peroxide(H_(2)O_(2))accumulation along the stele of the elongation and differentiation zone in root tips,indicating Fe-dependent ROS formation.By screening ammonium sensitivity in T-DNA insertion lines of ammonium-responsive genes,we identified PDX1.1,which is upregulated by ammonium in the root stele and whose product catalyzes de novo biosyn-thesis of vitamin B6.Root growth of pdx1.1 mutants is hypersensitive to ammonium,while chemical complementation or overexpression of PDX1.1 restores root elongation.This salvage strategy requires non-phosphorylated forms of vitamin B6 that are able to quench ROS and rescue root growth from ammo-nium inhibition.Collectively,these results suggest that PDX1.1-mediated synthesis of non-phosphorylated B6 vitamers acts as a primary strategy to protect roots from ammonium-dependent ROS formation.