Search

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]

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]

Iron (Fe) is an essential metal ion that plays a major role as a cofactor in many biological processes. The balance between the Fe2+ and Fe3+ forms is central for cellular Fe homeostasis because it regulates its transport, utilization, and storage. Contrary to Fe3+ reduction that is crucial for Fe uptake by roots in deficiency conditions, ferroxidation has been much less studied. In this work, we have focused on the molecular characterization of two members of the MultiCopper Oxidase family (MCO1 and MCO3) that share high identity with the Saccharomyces cerevisiae ferroxidase Fet3. The heterologous expression of MCO1 and MCO3 restored the growth of the yeast fet3fet4 mutant, impaired in high and low affinity Fe uptake and otherwise unable to grow in Fe deficient media, suggesting that MCO1 and MCO3 were functional ferroxidases. The ferroxidase enzymatic activity of MCO3 was further confirmed by the measurement of Fe2+‐dependent oxygen consumption, because ferroxidases use oxygen as electron acceptor to generate water molecules. In planta, the expression of MCO1 and MCO3 was induced by increasing Fe concentrations in the medium. Promoter‐GUS reporter lines showed that MCO1 and MCO3 were mostly expressed in shoots and histochemical analyses further showed that both promoters were highly active in mesophyll cells. Transient expression of MCO1‐RFP and MCO3‐RFP in tobacco leaves revealed that both proteins were localized in the apoplast. Moreover, cell plasmolysis experiments showed that MCO1 remained closely associated to the plasma membrane whereas MCO3 filled the entire apoplast compartment. Although the four knock out mutant lines isolated (mco1‐1, mco1‐2, mco3‐1, and mco3‐2) did not display any macroscopic phenotype, histochemical staining of Fe with the Perls/DAB procedure revealed that mesophyll cells of all four mutants overaccumulated Fe inside the cells in Fe‐rich structures in the chloroplasts, compared with wild‐type. These results suggested that the regulation of Fe transport in mesophyll cells had been disturbed in the mutants, in both standard condition and Fe excess. Taken together, our findings strongly suggest that MCO1 and MCO3 participate in the control of Fe transport in the mesophyll cells, most likely by displacing the Fe2+/Fe3+ balance toward Fe3+ in the apoplast and therefore limiting the accumulation of Fe2+, which is more mobile and prone to be transported across the plasma membrane. [ABSTRACT FROM AUTHOR]

Plant root absorbs water and nutrients from the soil, and the root apoplastic fluid (AF) is an important intermediate between cells and the surrounding environment. The acid growth theory suggests that an acidic AF is needed for cell wall expansion during root growth. However, technical limitations have precluded the quantification of root apoplastic fluid pH (AF‐pH). Here, we used Green‐enhanced Nano‐lantern (GeNL), a chimeric protein of the luciferase NanoLuc (Nluc) and the green fluorescent protein mNeonGreen (mNG), as a ratiometric pH indicator based on the pH dependency of bioluminescence resonance energy transfer efficiency from Nluc to mNG. Luminescence spectrum of GeNL changed reciprocally from pH 4.5 to 7.5, with a pKa of 5.5. By fusing GeNL to a novel signal peptide from Arabidopsis thaliana Cellulase 1, we localised GeNL in A. thaliana AF. We visualised AF dynamics at subcellular resolution over 30 min and determined flow velocity in the maturation zone to be 0.97± 0.06 μm/s. We confirmed that the developing root AF is acidic in the pH range of 5.1−5.7, suggesting that the AF‐pH is tightly regulated during root elongation. These results support the acid growth theory and provide evidence for AF‐pH maintenance despite changes in ambient pH. Summary statement: We visualised and quantified the pH dynamics by localising a bioluminescent ratiometric indicator to the root apoplastic fluid (AF) of Arabidopsis thaliana. The AF‐pH was dynamically acidified as the root developed regardless of different ambient pH, suggesting a strict regulation of AF‐pH. [ABSTRACT FROM AUTHOR]

SUMMARY: Photosynthetic efficiency and sink demand are tightly correlated with rates of phloem loading, where maintaining low cytosolic sugar concentrations is paramount to prevent the downregulation of photosynthesis. Sugars Will Eventually be Exported Transporters (SWEETs) are thought to have a pivotal role in the apoplastic phloem loading of C4 grasses. SWEETs have not been well studied in C4 species, and their investigation is complicated by photosynthesis taking place across two cell types and, therefore, photoassimilate export can occur from either one. SWEET13 homologues in C4 grasses have been proposed to facilitate apoplastic phloem loading. Here, we provide evidence for this hypothesis using the C4 grass Setaria viridis. Expression analyses on the leaf gradient of C4 species Setaria and Sorghum bicolor show abundant transcript levels for SWEET13 homologues. Carbohydrate profiling along the Setaria leaf shows total sugar content to be significantly higher in the mature leaf tip compared with the younger tissue at the base. We present the first known immunolocalization results for SvSWEET13a and SvSWEET13b using novel isoform‐specific antisera. These results show localization to the bundle sheath and phloem parenchyma cells of both minor and major veins. We further present the first transport kinetics study of C4 monocot SWEETs by using a Xenopus laevis oocyte heterologous expression system. We demonstrate that SvSWEET13a and SvSWEET13b are high‐capacity transporters of glucose and sucrose, with a higher apparent Vmax for sucrose, compared with glucose, typical of clade III SWEETs. Collectively, these results provide evidence for an apoplastic phloem loading pathway in Setaria and possibly other C4 species. [ABSTRACT FROM AUTHOR]

The apoplast is the extracellular space for signalling, nutrient transport, and plant–microbe interactions, but little is known about how plant viruses use the foliar apoplast. Proteomic analysis of the apoplasts isolated from potato virus X (PVX)‐infected Nicotiana benthamiana plants showed that the coat protein (CP) is the dominant viral component. The presence of the CP in the apoplast was confirmed by western blot, viral nucleic acid was detected by reverse transcription‐PCR and northern blot, and viral particles were observed by transmission electron microscopy (TEM). The apoplast from infected leaves was infectious if rubbed onto healthy leaves but not when infiltrated into them. The exosomes were separated from the apoplast fluid by high‐speed centrifugation and TEM showed that PVX particles were not associated with the exosomes. These results suggest that PVX virions are released to the N. benthamiana apoplast in a one‐way manner and do not share the bidirectional transport of exosomes. [ABSTRACT FROM AUTHOR]

Summary: The plant apoplast is a harsh environment in which hydrolytic enzymes, especially proteases, accumulate during pathogen infection. However, the defense functions of most apoplastic proteases remain largely elusive.We show here that a newly identified small cysteine‐rich secreted protein PC2 from the potato late blight pathogen Phytophthora infestans induces immunity in Solanum plants only after cleavage by plant apoplastic subtilisin‐like proteases, such as tomato P69B.A minimal 61 amino acid core peptide carrying two key cysteines, conserved widely in most oomycete species, is sufficient for PC2‐induced cell death. Furthermore, we showed that Kazal‐like protease inhibitors, such as EPI1, produced by P. infestans prevent PC2 cleavage and dampen PC2 elicited host immunity.This study reveals that cleavage of pathogen proteins to release immunogenic peptides is an important function of plant apoplastic proteases. [ABSTRACT FROM AUTHOR]

SUMMARY: Lipids have been observed attached to lumen‐facing surfaces of mature xylem conduits of several plant species, but there has been little research on their functions or effects on water transport, and only one lipidomic study of the xylem apoplast. Therefore, we conducted lipidomic analyses of xylem sap from woody stems of seven plants representing six major angiosperm clades, including basal magnoliids, monocots and eudicots, to characterize and quantify phospholipids, galactolipids and sulfolipids in sap using mass spectrometry. Locations of lipids in vessels of Laurus nobilis were imaged using transmission electron microscopy and confocal microscopy. Xylem sap contained the galactolipids di‐ and monogalactosyldiacylglycerol, as well as all common plant phospholipids, but only traces of sulfolipids, with total lipid concentrations in extracted sap ranging from 0.18 to 0.63 nmol ml−1 across all seven species. Contamination of extracted sap from lipids in cut living cells was found to be negligible. Lipid composition of sap was compared with wood in two species and was largely similar, suggesting that sap lipids, including galactolipids, originate from cell content of living vessels. Seasonal changes in lipid composition of sap were observed for one species. Lipid layers coated all lumen‐facing vessel surfaces of L. nobilis, and lipids were highly concentrated in inter‐vessel pits. The findings suggest that apoplastic, amphiphilic xylem lipids are a universal feature of angiosperms. The findings require a reinterpretation of the cohesion‐tension theory of water transport to account for the effects of apoplastic lipids on dynamic surface tension and hydraulic conductance in xylem. [ABSTRACT FROM AUTHOR]

Stomatal movements are enabled by changes in guard cell turgor facilitated via transient accumulation of inorganic and organic ions imported from the apoplast or biosynthesized within guard cells. Under salinity, excess salt ions accumulate within plant tissues resulting in osmotic and ionic stress. To elucidate whether (a) Na+ and Cl− concentrations increase in guard cells in response to long‐term NaCl exposure and how (b) guard cell metabolism acclimates to the anticipated stress, we profiled the ions and primary metabolites of leaves, the apoplast and isolated guard cells at darkness and during light, that is, closed and fully opened stomata. In contrast to leaves, the primary metabolism of guard cell preparations remained predominantly unaffected by increased salt ion concentrations. Orchestrated reductions of stomatal aperture and guard cell osmolyte synthesis were found, but unlike in leaves, no increases of stress responsive metabolites or compatible solutes occurred. Diverging regulation of guard cell metabolism might be a prerequisite to facilitate the constant adjustment of turgor that affects aperture. Moreover, the photoperiod‐dependent sucrose accumulation in the apoplast and guard cells changed to a permanently replete condition under NaCl, indicating that stress‐related photosynthate accumulation in leaves contributes to the permanent closing response of stomata under stress. Guard cell metabolism differentially responds to altered ion composition resulting from salt stress (NaCl) compared with whole leaf tissue in Vicia faba L. [ABSTRACT FROM AUTHOR]

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze–thaw cycle. Many plant species increase their freezing tolerance during exposure to low, non‐freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub‐zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodelling may be particularly important for sub‐zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub‐zero acclimation, compared to the Col‐0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centres of rosette indicated that epitopes of the xyloglucan‐specific antibody LM25 were highly abundant in the vasculature of Col‐0 plants after sub‐zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodelling for the increased freezing tolerance observed after low temperature acclimation. xth19 mutant had reduced freezing tolerance after cold or sub‐zero acclimation. Microscopic and biochemical characterization of the cell wall indicated altered xyloglucan deposition in xth19 after sub‐zero acclimation showing the importance of cell wall remodelling for increased freezing tolerance. [ABSTRACT FROM AUTHOR]

Summary: Plants utilising crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reducing transpirational water loss associated with photosynthesis. Unlike stomata of C3 and C4 species, CAM stomata open at night for the mesophyll to fix CO2 into malate (Mal) and store it in the vacuole. CAM plants decarboxylate Mal in the light, generating high CO2 concentrations within the leaf behind closed stomata for refixation by RuBisCO.CO2 may contribute to stomatal closure but additional mechanisms, plausibly including Mal activation of anion channels, ensure closure in the light.In the CAM species Kalanchoë fedtschenkoi, we found that guard cell anion channel activity, recorded under voltage clamp, follows KfSLAC1 and KfALMT12 transcript abundance, declining to near zero by the end of the light period. Unexpectedly, however, we found that extracellular Mal inhibited the anion current of Kalanchoë guard cells, both in wild‐type and RNAi mutants with impaired Mal metabolism.We conclude that the diurnal cycle of anion channel gene transcription, rather than the physiological signal of Mal release, is a key factor in the inverted CAM stomatal cycle. [ABSTRACT FROM AUTHOR]

Summary: Epichloë festucae is an endophytic fungus that forms a symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae.A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from uninfected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem MS was used to identify a novel product of a secondary metabolite gene cluster.Metabolites likely to be present in the apoplast were identified using MarVis in combination with the BioCyc and KEGG databases, and an in‐house Epichloë metabolite database. We were able to identify the known endophyte‐specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers.To determine whether these methods can be applied to the identification of novel Epichloë‐derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative MS analysis of apoplastic wash fluid from wild‐type‐ vs mutant‐infected plants identified a novel Leu/Ile glycoside metabolite present in the former. [ABSTRACT FROM AUTHOR]

Ascorbate in leaf apoplast (ASCapo) reacts with ozone (O3) and thereby reduces O3 flux reaching plasmalemma (Fpl). Some studies have shown significant protection of cells from O3 by ASCapo, while others have questioned its efficacy. Hypothesizing that the protection by ASCapo depends on other variables, we quantified determinants of O3 detoxification with a model of O3 transport and reaction in apoplast. The model determines ascorbic acid concentration in apoplast (AAapo) using measured values of O3 concentration (co), leaf tissue ascorbic acid concentration (AAleaf), cell wall thickness (L3), apoplastic pH (pHapo), and stomatal conductance (Gsw). We compared the measured and model‐estimated AAapo in leaves of peach (Prunus persica) grown in open‐top chambers under non‐filtered air (NF) and elevated (EO3: NF + 80 ppb) O3 concentrations. The estimated AAapo in individual leaves agreed well with the measured values (R2 =.91). Analyses of the simulation results yielded the following findings: (a) The efficacy of O3 reduction with ASCapo as quantified by fractional reduction (ϕ3) of O3 flux at the surface of plasmalemma (Fpl) was lowered from 70% in NF to 40% in EO3 due to the reduction of L3. The EO3 reduced AAapo, but the lower Gsw and L3 in EO3 increased AAapo resulting in no significant change in AAapo due to EO3. ϕ3 can be calculated with measured values of AAapo and L3, and Fpl can be estimated with the measurement‐based ϕ3. (b) When c0 is increased, Fpl increased curvilinearly with the increase of Fst: nominal O3 flux via stomatal diffusion, exhibiting apparent threshold on Fst. The deviation of Fpl from Fst became greater when L3, pHapo, and AAleaf were increased. The quantification of ϕ3 and Fpl using leaf traits shall facilitate the understanding of the mechanisms of differential plant sensitivity to O3 and improve quantification of the O3 impacts on plants. [ABSTRACT FROM AUTHOR]

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5‐fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA‐sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit. Wild barley is using different strategies to cope with osmotic stress especially in terms of root suberization compared with cultivated barley. Wild barley is also better adapted to osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit. [ABSTRACT FROM AUTHOR]

Summary: Anion transport by aluminium‐activated malate transporter (ALMT) proteins is negatively regulated by gamma‐aminobutyric acid (GABA), which increases in concentration during stress.Here, the interaction between GABA and wheat (Triticum aestivum, Ta) TaALMT1 heterologously‐expressed in Xenopus laevis oocytes was investigated.GABA inhibited anion transport by TaALMT1 in membrane patches from the cytosolic, not extracellular membrane face, via a reduction in open probability (NPopen), not an inhibition of channel current magnitude. TaALMT1 currents in patches frequently exhibited rundown with complete removal of cytosolic factors, but were partially sustained by protein kinase C dependent phosphorylation. When applied to whole oocytes a GABA‐analogue‐BODIPY conjugate inhibited TaALMT1 anion currents from the cytoplasmic face only, whereas free GABA inhibited from both the inside and outside consistent with GABA traversing the TaALMT1 pore then acting from the inside.We propose GABA does not competitively inhibit ALMT conductance through the same pore but rather leads to an allosteric effect, reducing anion channel opening frequency. Across plants GABA is a conserved regulator of anion transport via ALMTs – a family with numerous physiological roles beyond Al3+ tolerance. Our data suggests that a GABA–ALMT interaction from the cytosolic face has the potential to form part of a novel plant signalling pathway. [ABSTRACT FROM AUTHOR]

Summary: Mesophyll conductance (gm), the diffusion of CO2 from substomatal cavities to the carboxylation sites in the chloroplasts, is a highly complex trait driving photosynthesis (net CO2 assimilation, AN). However, little is known concerning the mechanisms by which it is dynamically regulated. The apoplast is considered as a 'key information bridge' between the environment and cells. Interestingly, most of the environmental constraints affecting gm also cause apoplastic responses, cell wall (CW) alterations and metabolic rearrangements. Since CW thickness is a key determinant of gm, we hypothesize that other changes in this cellular compartiment should also influence gm. We study the relationship between the antioxidant apoplastic system and CW metabolism and the gm responses in tobacco plants (Nicotiana sylvestris L.) under two abiotic stresses (drought and salinity), combining in vivo gas‐exchange measurements with analyses of antioxidant activities, CW composition and primary metabolism. Stress treatments imposed substantial reductions in AN (58–54%) and gm (59%), accompanied by a strong antioxidant enzymatic response at the apoplastic and symplastic levels. Interestingly, apoplastic but not symplastic peroxidases were positively related to gm. Leaf anatomy remained mostly stable; however, the stress treatments significantly affected the CW composition, specifically pectins, which showed significant relationships with AN and gm. The treatments additionally promoted a differential primary metabolic response, and specific CW‐related metabolites including galactose, glucosamine and hydroxycinnamate showed exclusive relationships with gm independent of the stress. These results suggest that gm responses can be attributed to specific changes in the apoplastic antioxidant system and CW metabolism, opening up more possibilities for improving photosynthesis using breeding/biotechnological strategies. Significance Statement: Mesophyll conductance is a complex trait which imposes considerable limitations on photosynthesis. However, beyond the current knowledge from gas‐exchange measurements and anatomical characterization, the mechanisms driving its dynamic response to the environment are still poorly understood. Here, we relate changes in apoplastic antioxidant metabolism, cell wall composition and primary metabolism with responses of mesophyll conductance to different abiotic stresses. This information improves our understanding about mesophyll conductance responses and opens up more possibilities to improve photosynthetic performance. [ABSTRACT FROM AUTHOR]

Although excess cadmium (Cd) accumulation is harmful to plants, the molecular mechanisms underlying Cd detoxification and accumulation in Arabidopsis thaliana remain largely undetermined. In this study, we demonstrated that the A. thaliana PLANT DEFENSIN 2 gene AtPDF2.5 is involved in Cd tolerance and accumulation. In vitro Cd‐binding assays revealed that AtPDF2.5 has Cd‐chelating activity. Site‐directed mutagenesis of AtPDF2.5 identified eight cysteine residues that were essential for mediating Cd tolerance and chelation. Histochemical analysis demonstrated that AtPDF2.5 was mainly expressed in root xylem vascular bundles, and that AtPDF2.5 was significantly induced by Cd. Subcellular localization analysis revealed that AtPDF2.5 was localized to the cell wall. The overexpression of AtPDF2.5 significantly enhanced Cd tolerance and accumulation in A. thaliana and its heterologous overexpression in rice increased Cd accumulation; however, the functional disruption of AtPDF2.5 decreased Cd tolerance and accumulation. Physiological analysis suggested that AtPDF2.5 promoted Cd efflux from the protoplast and its subsequent accumulation in the cell wall. These data suggest that AtPDF2.5 promotes cytoplasmic Cd efflux via chelation, thereby enhancing Cd detoxification and apoplastic accumulation. Plants need to accumulate and detoxify cadmium for adapting to and remediating heavy metal polluted soils. The PLANT DEFENSIN 2 protein AtPDF2.5 mediates cadmium tolerance and accumulation in Arabidopsis thaliana by chelating this metal and facilitating the efflux of the AtPDF2.5‐Cd complex from protoplasts, indicating this might be a molecular tool for phytoremediation. [ABSTRACT FROM AUTHOR]

Summary: C‐type lectins (CTLs), a class of multifunctional proteins, are numerous in nematodes. One CTL gene, Mg01965, shown to be expressed in the subventral glands, especially in the second‐stage juveniles of the root‐knot nematode Meloidogyne graminicola, was further analysed in this study. In vitro RNA interference targeting Mg01965 in the preparasitic juveniles significantly reduced their ability to infect host plant roots. Immunolocalizations showed that Mg01965 is secreted by M. graminicola into the roots during the early parasitic stages and accumulates in the apoplast. Transient expression of Mg01965 in Nicotiana benthamiana and targeting it to the apoplast suppressed the burst of reactive oxygen species triggered by flg22. The CTL Mg01965 suppresses plant innate immunity in the host apoplast, promoting nematode parasitism in the early infection stages. [ABSTRACT FROM AUTHOR]

Summary: Barley (Hordeum vulgare) is more drought tolerant than other cereals, thus making it an excellent model for the study of the chemical, transcriptomic and physiological effects of water deficit. Roots are the first organ to sense soil water deficit. Therefore, we studied the response of barley seminal roots to different water potentials induced by polyethylene glycol (PEG) 8000.We investigated changes in anatomical parameters by histochemistry and microscopy, quantitative and qualitative changes in suberin composition by analytical chemistry, transcript changes by RNA‐sequencing (RNA‐Seq), and the radial water and solute movement of roots using a root pressure probe.In response to osmotic stress, genes in the suberin biosynthesis pathway were upregulated that correlated with increased suberin amounts in the endodermis and an overall reduction in hydraulic conductivity (Lpr). In parallel, transcriptomic data indicated no or only weak effects of osmotic stress on aquaporin expression.These results indicate that osmotic stress enhances cell wall suberization and markedly reduces Lpr of the apoplastic pathway, whereas Lpr of the cell‐to‐cell pathway is not altered. Thus, the sealed apoplast markedly reduces the uncontrolled backflow of water from the root to the medium, whilst keeping constant water flow through the highly regulated cell‐to‐cell path. [ABSTRACT FROM AUTHOR]

ContentsSummary67I.Introduction68II.Silicon transport in plants: to absorb or not to absorb69III.The role of silicon in plants: not just a matter of semantics71IV.Silicon and biotic stress: beyond mechanical barriers and defense priming76V.Silicon and abiotic stress: a proliferation of proposed mechanisms78VI.The apoplastic obstruction hypothesis: a working model79VII.Perspectives and conclusions80Acknowledgements81References81 Summary: Silicon (Si) is not classified as an essential plant nutrient, and yet numerous reports have shown its beneficial effects in a variety of species and environmental circumstances. This has created much confusion in the scientific community with respect to its biological roles. Here, we link molecular and phenotypic data to better classify Si transport, and critically summarize the current state of understanding of the roles of Si in higher plants. We argue that much of the empirical evidence, in particular that derived from recent functional genomics, is at odds with many of the mechanistic assertions surrounding Si's role. In essence, these data do not support reports that Si affects a wide range of molecular‐genetic, biochemical and physiological processes. A major reinterpretation of Si's role is therefore needed, which is critical to guide future studies and inform agricultural practice. We propose a working model, which we term the 'apoplastic obstruction hypothesis', which attempts to unify the various observations on Si's beneficial influences on plant growth and yield. This model argues for a fundamental role of Si as an extracellular prophylactic agent against biotic and abiotic stresses (as opposed to an active cellular agent), with important cascading effects on plant form and function. [ABSTRACT FROM AUTHOR]

Plant pathogens have developed different strategies during their evolution to infect and colonize their hosts. In the same way, plants have evolved different mechanisms acting against potential pathogens trying to infect and colonize their tissues. Regulation of a wide variety of proteins is required in order to perceive the pathogen and to activate the plant defence mechanisms. The apoplast is the first compartment where these recognition phenomena occur in most plant–pathogen interactions, allowing the exchange of different molecules and facilitating inter‐ and intracellular communication in plant cells. Proteomic analysis of the apoplast in recent years has found the initial biochemical responses involved in pathogen recognition and early defence responses. However, this proteomic approach requires some specific experimental conditions to obtain an extract free of cytoplasmic proteins and nonprotein contaminants that affect the subsequent stages of separation and quantification. Obtaining the highest proportion of proteins from the apoplastic space in infected tissues requires different steps such as extraction of apoplastic washing fluids and preparation of total secreted proteins (protein precipitation, solubilization, separation and digestion). Protein identification using mass spectrometry techniques and bioinformatics tools identifying peptides for the extracellular exportation is required to confirm the apoplastic location. This review compiles the most commonly used techniques for proteomic studies, focusing on the early biochemical changes occurring in the apoplast of plants infected by a wide range of pathogens. The scope of this approach to discover the molecular mechanisms involved in the plant–pathogen interaction is discussed. [ABSTRACT FROM AUTHOR]

Summary: In the plant apoplast, ascorbate is oxidised, via dehydroascorbic acid, to O‐oxalyl esters [oxalyl‐ l‐threonate (OxT) and cyclic oxalyl‐ l‐threonate (cOxT)]. We tested whether OxT and cOxT can donate the oxalyl group in transacylation reactions to form oxalyl‐polysaccharides, potentially modifying the cell wall. [oxalyl‐14C]OxT was incubated with living spinach (Spinacia oleracea) and Arabidopsis cell‐suspension cultures in the presence or absence of proposed acceptor substrates (carbohydrates). In addition, [14C]OxT and [14C]cOxT were incubated in vitro with cell‐wall enzyme preparations plus proposed acceptor substrates. Radioactive products were monitored electrophoretically. Oxalyltransferase activity was detected. Living cells incorporated oxalate groups from OxT into cell‐wall polymers via ester bonds. When sugars were added, [14C]oxalyl‐sugars were formed, in competition with OxT hydrolysis. Preferred acceptor substrates were carbohydrates possessing primary alcohols e.g. glucose. A model transacylation product, [14C]oxalyl‐glucose, was relatively stable in vivo (half‐life >24 h), whereas [14C]OxT underwent rapid turnover (half‐life ~6 h). Ionically wall‐bound enzymes catalysed similar transacylation reactions in vitro with OxT or cOxT as oxalyl donor substrates and any of a range of sugars or hemicelluloses as acceptor substrates. Glucosamine was O‐oxalylated, not N‐oxalylated. We conclude that plants possess apoplastic acyltransferase (oxalyltransferase) activity that transfers oxalyl groups from ascorbate catabolites to carbohydrates, forming relatively long‐lived O‐oxalyl‐carbohydrates. The findings increase the range of known metabolites whose accumulation in vivo indicates vitamin C catabolism. Possible signalling roles of the resulting oxalyl‐sugars can now be investigated, as can the potential ability of polysaccharide oxalylation to modify the wall's physical properties. [ABSTRACT FROM AUTHOR]

Summary: The plant apoplast is integral to intercellular signalling, transport and plant–pathogen interactions. Plant pathogens deliver effectors both into the apoplast and inside host cells, but no computational method currently exists to discriminate between these localizations. We present ApoplastP, the first method for predicting whether an effector or plant protein localizes to the apoplast. ApoplastP uncovers features of apoplastic localization common to both effectors and plant proteins, namely depletion in glutamic acid, acidic amino acids and charged amino acids and enrichment in small amino acids. ApoplastP predicts apoplastic localization in effectors with a sensitivity of 75% and a false positive rate of 5%, improving the accuracy of cysteine‐rich classifiers by > 13%. ApoplastP does not depend on the presence of a signal peptide and correctly predicts the localization of unconventionally secreted proteins. The secretomes of fungal saprophytes as well as necrotrophic, hemibiotrophic and extracellular fungal pathogens are enriched for predicted apoplastic proteins. Rust pathogens have low proportions of predicted apoplastic proteins, but these are highly enriched for predicted effectors. ApoplastP pioneers apoplastic localization prediction using machine learning. It will facilitate functional studies and will be valuable for predicting if an effector localizes to the apoplast or if it enters plant cells. [ABSTRACT FROM AUTHOR]

Contents521I.521II.522III.523IV.524525References525 Summary: Plant iron (Fe) uptake relies to a large extent on the capacity of cells to control and extract Fe pools safely conserved in extracytoplasmic environments such as the apoplast and vacuoles, at least as much as on the transport machinery nested in plasma membranes. Recent studies on root and embryo Fe nutrition support this assertion and show that the root Fe‐deficiency response also includes the dynamic use of a large Fe reservoir bound to cell wall components in the root apoplast, secretion in the apoplast of phenolic compounds of the coumarin family, which solubilize Fe in calcareous soils, and inhibition of suberization of endodermal cells in order to allow apoplastic and transcellular radial transport of Fe. All of these responses are regulated by the stress hormones ethylene and abscisic acid (ABA), suggesting an integrated strategy within the root to adapt to Fe shortage. For its nutrition, the embryo has developed both an original uptake mechanism, in which ascorbate is effluxed to chemically reduce Fe3+ to the transport‐competent Fe2+ form, and an efficient strategy to control utilization of a large Fe pool in vacuoles. This review will attempt to summarize exciting new insights into the diverse routes that Fe takes to feed plant tissues. [ABSTRACT FROM AUTHOR]

One of the most fundamental questions in plant pathology is what determines whether a pathogen grows within a plant? This question is frequently studied in terms of the role of elicitors and pathogenicity factors in the triggering or overcoming of host defences. However, this focus fails to address the basic question of how the environment in host tissues acts to support or restrict pathogen growth. Efforts to understand this aspect of host-pathogen interactions are commonly confounded by several issues, including the complexity of the plant environment, the artificial nature of many experimental infection systems and the fact that the physiological properties of a pathogen growing in association with a plant can be very different from the properties of the pathogen in culture. It is also important to recognize that the phenotype and evolution of pathogen and host are inextricably linked through their interactions, such that the environment experienced by a pathogen within a host, and its phenotype within the host, is a product of both its interaction with its host and its evolutionary history, including its co-evolution with host plants. As the phenotypic properties of a pathogen within a host cannot be defined in isolation from the host, it may be appropriate to think of pathogens as having an 'extended phenotype' that is the product of their genotype, host interactions and population structure within the host environment. This article reflects on the challenge of defining and studying this extended phenotype, in relation to the questions posed below, and considers how knowledge of the phenotype of pathogens in the host environment could be used to improve disease control. What determines whether a pathogen grows within a plant?, What aspects of pathogen biology should be considered in describing the extended phenotype of a pathogen within a host?, How can we study the extended phenotype in ways that provide insights into the phenotypic properties of pathogens during natural infections? [ABSTRACT FROM AUTHOR]

See also the Commentary on this article by Ökmen & Doehlemann, 212: 799–801. [ABSTRACT FROM AUTHOR]

902I.902II.903III.903IV.903V.905VI.905VII.905906References906 Summary: Plants deploy a sophisticated immune system to cope with different microbial pathogens and other invaders. Recent research provides an increasing body of evidence for papain‐like cysteine proteases (PLCPs) being central hubs in plant immunity. PLCPs are required for full resistance of plants to various pathogens. At the same time, PLCPs are targeted by secreted pathogen effectors to suppress immune responses. Consequently, they are subject to a co‐evolutionary host–pathogen arms race. When activated, PLCPs induce a broad spectrum of defense responses including plant cell death. While the important role of PLCPs in plant immunity has become more evident, it remains largely elusive how these enzymes are activated and which signaling pathways are triggered to orchestrate different downstream responses. See also the Commentary on this article by Ökmen & Doehlemann, 212: 799–801. [ABSTRACT FROM AUTHOR]

Plants have evolved a series of tolerance mechanisms to saline stress, which perturbs physiological processes throughout the plant. To identify genetic mechanisms associated with salinity tolerance, we performed linkage analysis and genome-wide association study (GWAS) on maintenance of root growth of Arabidopsis thaliana in hydroponic culture with weak and severe NaCl toxicity. The top 200 single-nucleotide polymorphisms (SNPs) determined by GWAS could cumulatively explain approximately 70% of the variation observed at each stress level. The most significant SNPs were linked to the genes of ATP-binding cassette B10 and vacuolar proton ATPase A2. Several known salinity tolerance genes such as potassium channel KAT1 and calcium sensor SOS3 were also linked to SNPs in the top 200. In parallel, we constructed a gene co-expression network to independently verify that particular groups of genes work together to a common purpose. We identify molecular mechanisms to confer salt tolerance from both predictable and novel physiological sources and validate the utility of combined genetic and network analysis. Additionally, our study indicates that the genetic architecture of salt tolerance is responsive to the severity of stress. These gene datasets are a significant information resource for a following exploration of gene function. [ABSTRACT FROM AUTHOR]

The ribosome-inactivating protein BE27 from sugar beet ( Beta vulgaris L.) leaves is an apoplastic protein induced by signalling compounds, such as hydrogen peroxide and salicylic acid, which has been reported to be involved in defence against viruses. Here, we report that, at a concentration much lower than that present in the apoplast, BE27 displays antifungal activity against the green mould Penicillium digitatum, a necrotrophic fungus that colonizes wounds and grows in the inter- and intracellular spaces of the tissues of several edible plants. BE27 is able to enter into the cytosol and kill fungal cells, thus arresting the growth of the fungus. The mechanism of action seems to involve ribosomal RNA ( rRNA) N-glycosylase activity on the sarcin-ricin loop of the major r RNA which inactivates irreversibly the fungal ribosomes, thus inhibiting protein synthesis. We compared the C-terminus of the BE27 structure with antifungal plant defensins and hypothesize that a structural motif composed of an α-helix and a β-hairpin, similar to the γ-core motif of defensins, might contribute to the specific interaction with the fungal plasma membranes, allowing the protein to enter into the cell. [ABSTRACT FROM AUTHOR]

Information about the 12th New Phytologist Workshop held in Castle Rauischholzhausen, Germany, in July 2015 is presented. Topics include recent advances in the field of apoplastic immunity, role of regulation of secreted cysteine proteases in apoplastic immunity, plant immune system, and recent advances in studying plant receptors, apoplastic effectors, downstream signaling, perception and mechanisms.

We tested the common assumption that fleshy fruits become dependent on phloem water supply because xylem inflow declines at the onset of ripening. Using two distinct grape genotypes exposed to drought stress, we found that a sink-driven rise in phloem inflow at the beginning of ripening was sufficient to reverse drought-induced berry shrinkage. Rewatering accelerated berry growth and sugar accumulation concurrently with leaf photosynthetic recovery. Interrupting phloem flow through the peduncle prevented the increase in berry growth after rewatering, but interrupting xylem flow did not. Nevertheless, xylem flow in ripening berries, but not berry size, remained responsive to root or shoot pressurization. A mass balance analysis on ripening berries sampled in the field suggested that phloem water inflow may exceed growth and transpiration water demands. Collecting apoplastic sap from ripening berries showed that osmotic pressure increased at distinct rates in berry vacuoles and apoplast. Our results indicate that the decrease in xylem inflow at the onset of ripening may be a consequence of the sink-driven increase in phloem inflow. We propose a conceptual model in which surplus phloem water bypasses the fruit cells and partly evaporates from the berry surface and partly moves apoplastically to the xylem for outflow. [ABSTRACT FROM AUTHOR]

Ammonium nutrition inhibits the growth of many plant species, including A rabidopsis thaliana. The toxicity of ammonium is associated with changes in the cellular redox state. The cellular oxidant/antioxidant balance is controlled by mitochondrial electron transport chain. In this study, we analysed the redox metabolism of frostbite1 ( fro1) plants, which lack mitochondrial respiratory chain complex I. Surprisingly, the growth of fro1 plants increased under ammonium nutrition. Ammonium nutrition increased the reduction level of pyridine nucleotides in the leaves of wild-type plants, but not in the leaves of fro1 mutant plants. The observed higher activities of type II NADH dehydrogenases and cytochrome c oxidase in the mitochondrial electron transport chain may improve the energy metabolism of fro1 plants grown on ammonium. Additionally, the observed changes in reactive oxygen species ( ROS) metabolism in the apoplast may be important for determining the growth of fro1 under ammonium nutrition. Moreover, bioinformatic analyses showed that the gene expression changes in fro1 plants significantly overlap with the changes previously observed in plants with a modified apoplastic pH. Overall, the results suggest a pronounced connection between the mitochondrial redox system and the apoplastic pH and ROS levels, which may modify cell wall plasticity and influence growth. [ABSTRACT FROM AUTHOR]

Water movement from the xylem to stomata is poorly understood. There is still no consensus about whether apoplastic or symplastic pathways are more important, and recent work suggests vapour diffusion may also play a role. The objective of this study was to estimate the proportions of hydraulic conductance outside the bundle sheath contributed by apoplastic, symplastic and gas phase pathways, using a novel analytical framework based on measurable anatomical and biophysical parameters. The calculations presented here suggest that apoplastic pathways provide the majority of conductance outside the bundle sheath under most conditions, whereas symplastic pathways contribute only a small proportion. The contributions of apoplastic and gas phase pathways vary depending on several critical but poorly known or highly variable parameters namely, the effective Poiseuille radius for apoplastic bulk flow, the thickness of cell walls and vertical temperature gradients within the leaf. The gas phase conductance should increase strongly as the leaf centre becomes warmer than the epidermis - providing up to 44% of vertical water transport for a temperature gradient of 0.2 K. These results may help to explain how leaf water transport is influenced by light absorption, temperature and differences in leaf anatomy among species. [ABSTRACT FROM AUTHOR]

Adenosine deaminase ( ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol ( PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16 mg L−1, a 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct. [ABSTRACT FROM AUTHOR]

This article is a Commentary on De Wit, 212: 805–813; Derevnina et al., 212: 888–895; Rovenich et al., 212: 896–901 and Misas‐Villamil et al., 212: 902–907. [ABSTRACT FROM AUTHOR]

Abscisic acid ( ABA) has been demonstrated to be involved in iron ( Fe) homeostasis, but the underlying mechanism is largely unknown. Here, we found that Fe deficiency induced ABA accumulation rapidly (within 6 h) in the roots of Arabidopsis. Exogenous ABA at 0.5 μM decreased the amount of root apoplastic Fe bound to pectin and hemicellulose, and increased the shoot Fe content significantly, thus alleviating Fe deficiency-induced chlorosis. Exogenous ABA promoted the secretion of phenolics to release apoplastic Fe and up-regulated the expression of AtNRAMP3 to enhance reutilization of Fe stored in the vacuoles, leading to a higher level of soluble Fe and lower ferric-chelate reductase ( FCR) activity in roots. Treatment with ABA also led to increased Fe concentrations in the xylem sap, partially because of the up-regulation of AtFRD3, AtYSL2 and AtNAS1, genes related to long-distance transport of Fe. Exogenous ABA could not alleviate the chlorosis of abi5 mutant resulting from the significantly low expression of AtYSL2 and low transport of Fe from root to shoot. Taken together, our data support the conclusion that ABA is involved in the reutilization and transport of Fe from root to shoot under Fe deficiency conditions in Arabidopsis. [ABSTRACT FROM AUTHOR]

The cell-wall pectic domain rhamnogalacturonan- II ( RG- II) is cross-linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross-linking. Using polyacrylamide gel electrophoresis ( PAGE) to separate monomeric from dimeric (boron-bridged) RG- II, we confirmed that Pb2+ promotes H3 BO3-dependent dimerisation in vitro. H3 BO3 concentrations as high as 50 m m did not prevent cross-linking. For in-vivo experiments, we successfully cultured 'Paul's Scarlet' rose ( Rosa sp.) cells in boron-free medium: their wall-bound pectin contained monomeric RG- II domains but no detectable dimers. Thus pectins containing RG- II domains can be held in the wall other than via boron bridges. Re-addition of H3 BO3 to 3.3 μ m triggered a gradual appearance of RG- II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG- II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG- II dimers. We conclude that RG- II normally becomes boron-bridged during synthesis or secretion but not post-secretion. Supporting this conclusion, exogenous [3H] RG- II was neither dimerised in the medium nor cross-linked to existing wall-associated RG- II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG- II domains have a brief window of opportunity for boron-bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron-bridging does not readily occur. [ABSTRACT FROM AUTHOR]

Previous studies have suggested that the mesophyll contributes to stomatal CO2 responses., The effects of changes in CO2 concentration (100 or 700 ppm) on stomatal responses in red or white light were examined microscopically in a leaf segment, an epidermal strip and an epidermal strip placed on a mesophyll segment of Commelina communis, all mounted on a buffer-containing gel., In both red and white light, stomata of the leaf segment opened/closed rapidly at low/high CO2. In red light, epidermal strip stomata barely responded to CO2. In white light, they opened at low CO2, but hardly closed at high CO2. Stomata of the epidermal strip placed on the mesophyll responded in the same manner as those on the leaf segment. Insertion of a doughnut-shaped cellophane spacer (but not polyethylene spacer) between the epidermal strip and the mesophyll hardly altered these responses. Stomata in leaf segments treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea ( DCMU), a photosynthesis inhibitor, did not open in red light, but opened/closed at low/high CO2 in white light., These results indicate that the apoplast transfer of 'mesophyll signals' and the stomatal opening at low CO2 are dependent on photosynthesis, whereas the stomatal closure at high CO2 is independent of photosynthesis. [ABSTRACT FROM AUTHOR]

Highlights: [•] A taste-modifier protein named miraculin is secreted outside the plasma membrane. [•] This secretion is dependent on a signal peptide conserved in Kunitz-type STIs. [•] Transgenic seedlings of miraculin were cultured in liquid medium. [•] Miraculin was present in the supernatant after cellulase treatment. [•] Miraculin may have lost trypsin inhibitory activity during its evolution. [ABSTRACT FROM AUTHOR]

Salinity primarily affects plants by inhibiting shoot growth. Salt-sensitive plants have been suggested to accumulate Na+ within their leaf apoplast under salinity, leading to a reduced water status. Evidence related to apoplastic Na+ accumulation is still enigmatic. We have focused on the effect of a short-term salt treatment by using the salt-sensitive V icia faba. Moreover, we have examined the role of silicon in alleviating sodium accumulation in the apoplast. Salt-sensitive field beans have been subjected to increasing levels of salinity, with and without the addition of silicon under hydroponic conditions. We have demonstrated that the dicot V icia faba exhibits a rise in Na+ concentration in the leaf apoplast at higher salinity levels; this is significantly ameliorated by the addition of silicon. Further, enhanced shoot growth under high salt treatment in the presence of added silicon is correlated with a significant decrease in Na+ concentration in the leaves. The novelty of the current study is the detection of a high Na+ concentration in the leaf apoplast of the salt-sensitive dicot field bean. Our results support Oertli's hypothesis that extracellular salt accumulation can lead to wilting leaves, plant growth reduction and cell death. [ABSTRACT FROM AUTHOR]

ABSTRACT We examined the role of aquaporins (AQPs) in regulating leaf hydraulic conductance ( Kleaf) in Vitis vinifera L. (cv Chardonnay) by studying effects of AQP inhibitors, and AQP gene expression during water stress (WS) and recovery (REC). Kleaf was measured after 3 h of petiole perfusion with different solutions and to introduce inhibitors. The addition of 0.1 m m HgCl2 to 15 m m KCl reduced Kleaf compared with perfusion in 15 mM KNO3 or KCl, and these solutions were used for leaves from control, WS and REC plants. Perfusion for 3 h did not significantly alter stomatal conductance ( gs) though expression of VvTIP1;1 was increased. WS decreased Kleaf by about 30% and was correlated with gs. The expression of VvTIP2;1 and VvPIP2;1 correlated with Kleaf, and VvTIP2;1 was highly correlated with gs. There was no association between the expression of particular AQPs during WS and REC and inhibition of Kleaf by HgCl2; however, HgCl2 treatment itself increased expression of VvPIP2;3 and decreased expression of VvPIP2;1 . Inhibition by HgCl2 of Kleaf only at early stages of WS and then after REC suggested that apoplasmic pathways become more important during WS. This was confirmed using fluorescent dyes confined to apoplasm or preferentially accumulated in symplasm. [ABSTRACT FROM AUTHOR]

Transient apoplastic alkalinization has been discussed as a general stress factor, and is thought to represent a root-to-shoot signal that transmits information regarding an ongoing Na Cl stress event from the site of the trigger to the distant plant tissue. Surprisingly, despite this importance, a number of gaps exist in our knowledge of Na Cl-induced apoplastic p H alkalinization., This study was designed in order to shed light onto the mechanisms responsible for the initiation and transiency of leaf apoplastic alkalinization under conditions of Na Cl stress as supplied to roots., An H+-sensitive fluorescence probe, in combination with ratiometric microscopy imaging, was used for in planta live recording of leaf apoplastic p H., The use of a nonionic solute demonstrated that the alkalinization is induced in response to ionic, and not osmotic, components of Na Cl stress. Tests with Cl−- or Na+-accompanying counter-ions strengthened the idea that the stress factor itself, namely Cl−, is transferred from root to shoot and elicits the p H alterations. Investigations with a plasma membrane ATPase inhibitor suggest that ATPase activity influences the course of the alkalinization by having a shaping re-acidifying effect on the alkalinization. [ABSTRACT FROM AUTHOR]

ABSTRACT Boron (B) is known to accumulate in the leaf margins of different plant species, arguably a passive consequence of enhanced transpiration at the ends of the vascular system. However, transpiration rate is not the only factor affecting ion distribution. We examine an alternative hypothesis, suggesting the participation of the leaf bundle sheath in controlling radial water and solute transport from the xylem to the mesophyll in analogy to the root endodermis. In banana, excess B that remains confined to the vascular system is effectively disposed of via dissolution in the guttation fluid; therefore, impairing guttation should aggravate B damage to the leaf margins. Banana plants were subjected to increasing B concentrations. Guttation rates were manipulated by imposing a moderate osmotic stress. Guttation fluid was collected and analysed continuously. The distribution of ions across the lamina was determined. Impairing guttation indeed led to increased B damage to the leaf margins. The kinetics of ion concentration in guttation samples revealed major differences between ion species, corresponding to their distribution in the lamina dry matter. We provide evidence that the distribution pattern of B and other ions across banana leaves depends on active filtration of the transpiration stream and on guttation. [ABSTRACT FROM AUTHOR]

ABSTRACT The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato ( Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild-type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose : sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species. [ABSTRACT FROM AUTHOR]

ABSTRACT The BURP-domain protein family comprises a diverse group of plant-specific proteins that share a conserved BURP domain at the C terminus. However, there have been only limited studies on the functions and subcellular localization of these proteins. Members of the RD22-like subfamily are postulated to associate with stress responses due to the stress-inducible nature of some RD22-like genes. In this report, we used different transgenic systems (cells and in planta) to show that the expression of a stress-inducible RD22-like protein from soybean (GmRD22) can alleviate salinity and osmotic stress. We also performed detailed microscopic studies using both fusion proteins and immuno-electron microscopic techniques to demonstrate the apoplast localization of GmRD22, for which the BURP domain is a critical determinant of the subcellular localization. The apoplastic GmRD22 interacts with a cell wall peroxidase and the ectopic expression of GmRD22 in both transgenic Arabidopsis thaliana and transgenic rice resulted in increased lignin production when subjected to salinity stress. It is possible that GmRD22 regulates cell wall peroxidases and hence strengthens cell wall integrity under such stress conditions. [ABSTRACT FROM AUTHOR]

Salinity and boron (B) toxicity often occur simultaneously and may have interactive effects on plant responses. This study aims at further investigating long-term effects of salinity, B toxicity and their combination on wheat plants. Plants grown in hydroponics were treated with 2.5 μ m H3BO3 (control), 75 m m NaCl, 200 μ m H3BO3 or 75 m m NaCl + 200 μ m H3BO3 and were analysed on a subcellular level 6 weeks after germination. Shoot fresh and dry weight (DW), water content, transpiration rate and osmolality were reduced, while Na+ and Cl− concentrations increased under salinity stress. However, Cl− was reduced in all compartments by an additional application of B toxicity. At adequate B supply, NaCl increased apoplastic and symplastic soluble B concentrations, whereas the total B content remained unchanged. At high B level, however, soluble and total B was reduced by additional salt stress. Despite this antagonistic effect of a combined salt and B toxicity stress on Cl− and B concentrations, an additive effect was observed regarding shoot fresh weight reduction. Our results confirm an alleviating effect of the combined stresses on toxic ion concentrations, which did not prevent additive growth reductions. [ABSTRACT FROM AUTHOR]