High-throughput transient transformation of Arabidopsis roots enables systematic colocalization analysis of GFP-tagged proteins

Determination of the subcellular localization of an unknown protein is a major step towards the elucidation of its function. Lately, the expression of proteins fused to fluorescent markers has been very popular and many approaches have been proposed to express these proteins. Stable transformation using Agrobacterium tumefaciens generates stable lines for downstream experiments, but is time-consuming. If only colocalization is required, transient techniques save time and effort. Several methods for transient assays have been described including protoplast transfection, biolistic bombardment, Agrobacterium tumefaciens cocultivation and infiltration. In general colocalizations are preferentially performed in intact tissues of the same species, resembling the native situation. High transformation rates were described for cotyledons of Arabidopsis, but never for roots. Here we report that it is possible to transform Arabidopsis root epidermal cells with an efficiency that is sufficient for colocalization purposes.Key words: Arabidopsis, GFP-fusions, protein localization, root, transient transformationSince the release of the Arabidopsis thaliana genome sequence plant biologists set the goal to elucidate the functions of all coded genes. Apart from the spatio-temporal expression patterns of genes, the subcellular localization of gene products can play an essential role in deciphering their function. Classical immunological approaches to localize proteins can be hindered by cross-reactivity, time-consuming generation of antibodies and the low temporal resolution. Expression of tagged proteins forms a suitable alternative. Lately, fusions with fluorescent proteins in combination with confocal (CLSM)¹ or spinning disc microscopy² allow real time protein localization and even subcellular trafficking at high resolution. An overview of fluorescent tagging approaches can be found elsewhere.³Currently several techniques to introduce the coding region for a tagged protein in a plant are available. The generation of stable lines transformed by Agrobacterium tumefaciens offers a continuous source of plant material, but it is time-consuming especially when only colocalization experiments are required. Transient assays, on the other hand, offer the advantage of being fast and amenable to high throughput strategies. Each of these techniques, however, has some limitations and drawbacks. Particle bombardment (biolistics) ^4–6 for example circumvents the host specificity of Agrobacterium strains, but requires expensive equipment. Moreover, it is rather disruptive and imposes a significant stress upon the plants, possibly influencing the results. Protoplasts lack a cell wall and protoplast transformation^7,8 is therefore not suitable for certain experiments related to cell wall proteins or when interactions between cells on tissue level might be important.⁹ Moreover, protoplasts have lost their identity which might be critical for the correct functioning of certain transgenic constructs. Agrobacterium infiltration of tobacco leaves¹⁰ is regularly used and represents an efficient, fast and relatively easy transformation technique. However, tobacco leaves easily show autofluoresence due to tissue damage as a result of experimental manipulations. As it has been reported that some protein fusions expressed in an heterologous system localize to different subcellular localizations11 it is advisable not to use tobacco when localizing Arabidopsis proteins. Leaf infiltrations have been performed in Arabidopsis,¹² but apparently their leaves are much more prone to mechanical damage and the leaf developmental stage is critical, complicating this technique. Cocultivation of Agrobacterium with seedlings offers a rapid and efficient approach applicable to many mono and dicot species. It was reported to work efficiently in Arabidopsis cotyledons, but not in roots.⁹ As an alternative method, Agrobacterium infiltration of Arabidopsis seedlings¹¹ seems an efficient technique for transient expression. However, expression in root cells could not be obtained. Colocalizations are required in the native cells or tissue for the correct localization of an unknown protein or proteins that need interaction partners. As a consequence this technique can not be reliably used when root expressed gene products are studied. Here we show evidence that it is possible to use the described technique¹¹ to induce transient expression in Arabidopsis roots.We used the Agrobacterium infiltration of Arabidopsis seedlings technique¹¹ to colocalize several C-terminal (S65T)-sGFP fusions generated in the plant binary vector pGWB6.¹³ Each construct was transformed into Agrobacterium tumefaciens (C59C1Rif^R) containing the helper plasmid pMP90. Subsequently different stable marker lines, wild type Arabidopsis (Col-0) bearing mCherry fusion constructs,¹⁴ were transiently transformed.¹¹ After 2 or 3 days seedlings were studied using CLSM. Besides being expressed in cotyledons fusion proteins were clearly observed in root epidermis and root cap cells (Fig. 1A and B). As reported¹¹ the transformation efficiency in cotyledons was considerably higher than in root cells. However, in each experiment we obtained a considerable amount of transformed root epidermal cells which was more than sufficient for colocalization studies (Fig. 2). It was remarkable that transformation was repeatedly successful in groups of cells, adjacent or close to each other.Open in a separate windowFigure 1Transient transformation of Arabidopsis root cells. Expression of the protein-GFP fusion product can be seen in the epidermal (A) and root cap cells (B) on fluorescence/transmission merged images. As seen in (A) high efficiencies of root transformation can be reached.Open in a separate windowFigure 2Colocalization of mCherry and GFP constructs. Confocal image of the mCherry fluorescence (A), the GFP signal (B) and the merged image (C).In contrast to what was reported earlier we show here that the Agrobacterium infiltration technique¹¹ is perfectly capable of transiently transforming Arabidopsis root epidermal cells. It allows the transient production and study of proteins in their native environment, considerably increasing the reliability of such experiments. Additionaly the use of RFP marker constructs in colocalisation studies in the root is free of interference by the red background autofluorescence of chlorophyll.     

pH signature for the responses of arbuscular mycorrhizal fungi to external stimuli

Environmental and developmental signals can elicit differential activation of membrane proton (H⁺) fluxes as one of the primary responses of plant and fungal cells. In recent work,¹ we could determine that during the presymbiotic growth of arbuscular mycorrhizal (AM) fungi specific domains of H⁺ flux are activated by clover root factors, namely host root exudates or whole root system. Consequently, activation on hyphal growth and branching were observed and the role of plasma membrane H⁺-ATPase was investigated. The specific inhibitors differentially abolished most of hyphal H⁺ effluxes and fungal growth. As this enzyme can act in signal transduction pathways, we believe that spatial and temporal oscillations of the hyphal H⁺ fluxes could represent a pH signature for both early events of the AM symbiosis and fungal ontogeny.Key words: H⁺-specific vibrating probe, pH signatures, arbuscular mycorrhiza, pH signalling, Gigaspora margaritaThe 450-million-year-old symbiosis between the majority of land plants and arbuscular mycorrhizal (AM) fungi is one of the most ancient, abundant and ecologically important symbiosis on Earth.^2,3The development of AM interaction starts before the physical contact between the host plant roots and the AM fungus. The hyphal growth and branching are induced by the root factors exudated by host plants, followed by the formation of appressorium leading to the hyphal penetration in the root system. These root factors seems to be specifically synthesized by host plants, since exudates from non-host plants are not able to promote neither hyphal differentiation nor appressorium formation.^4,5 Most root exudates contain several host signals or better, active compounds including flavonoids^6,19 and strigolactones,^7,8 however many of them are not yet known.Protons (H⁺) may have an important role on the fungal growth and host signal perception.¹ In plant and fungal cells, H⁺ can be pumped out through two different mechanisms: (1) the activity of the P-type plasma membrane (PM) H⁺-ATPase⁹ and (2) PM redox reactions.¹⁰ The proportional contribution from both mechanisms is not known, but in most plant cells the PM H⁺-ATPase seems to be the major responsible by the H⁺ efflux across plasma membrane. AM Fungal cells also energize their PM using P-type H⁺-pumps quite similar to the plant ones. Indeed, some genes codifying isoforms of P-type H⁺-ATPase have been isolated of AM fungi,^11–13 and AM fungal ATP hydrolysis activity was shown by cytochemistry, localized mainly in the first 70 µm from the germ tube tip.¹⁴ This structural evidence correlates with data obtained by H⁺-specific vibrating probe (Fig. 1A and B), which indicates that the H⁺ efflux in Gigaspora margarita is more intense in the subapical region of the lateral hyphae¹ (Fig. 1A). Furthermore, the correlation between the cytosolic pH profile previously obtained by Jolicoeur et al.,¹⁵ with the H⁺ efflux pattern (erythrosine-dependent), seems to clearly indicate that an active PM H⁺-ATPase takes place at the subapical hyphal region. Using orthovanadate, we could show that those H⁺ effluxes are susceptible mainly in the subapical region, but no effect in the apical was found.¹ Recently, a method to use fluorescent marker expression in an AM fungus driven by arbuscular mycorrhizal promoters was published.³¹ It could be adjusted as an alternative to measure “in vivo” PM H⁺-ATPase expression in AM fungal hyphae and their responses to root factors.³¹Open in a separate windowFigure 1(A) H⁺ flux profile along growing secondary hyphae of G. margarita in the presence (open squares) or absence (closed squares) of erythrosin B and its correlation with cytosolic pH (pHc) data described by Jolicoeur et al.,¹⁵ (dotted line). Dotted area depicts the region with higher susceptibility to erythrosin B. (B) ion-selective electrode near to AM fungal hyphae. (C) Stimulation on hyphal H⁺ efflux after incubation with root factors or whole root system. R, roots; RE, root exudates; CO₂, carbon dioxide; CWP, cell wall proteins; GR24, synthetic strigolactone. The medium pH in all treatment was monitored and remained about 5.7, including with prior CO₂ incubation. Means followed by the same letter are statistically equal by Duncan''s test at p < 5%.The H⁺ electrochemical gradient generated by PM H⁺-ATPases provides not only driving force for nutrient uptake,^9,16 but also can act as an intermediate in signal transduction pathways.¹⁸ The participation of these H⁺ pumps in cell polarity and tip growth of plant cells was recently reported,²⁷ addressing their crucial role on apical growth.²⁸ Naturally, in the absence of root factors the AM fungi have basal metabolic^8,21–23 and respiratory activity.²⁴ However when root signals are recognized and processed by AM fungal cells they might become activated.²² We thus searched for pH signatures that could reflect the alterations on fungal metabolism in response to external stimuli. In fact, preliminary analyses from our group demonstrate that AM fungal hyphae increase their H⁺ efflux in response not only to root exudates recognition, but also to other root factors (Fig. 1C). The incubation for 30 min of AM fungal hyphae with several root factors induces hyphal H⁺ efflux similar to the response to intact root system (5 days of incubation). The major increases were found with 1% CO₂ (750%) followed by root cell wall proteins (221%), root exudates (130%) and synthetic strigolactone (5%) (Fig. 1C). Those stimulations could define the transition from the state without root signals to the presymbiotic developmental stage (Fig. 1C). In the case of CO₂, the incorporation of additional carbon could represent a new source of energy, since CO₂ dark fixation takes place in Glomus intraradices germ tubes.^22,25Interestingly, after the treatment with synthetic strigolactone (10⁻⁵ M GR24), no significant stimulation was found compared to the remaining factors (Fig. 1C). It opens the question if the real effect of strigolactone is restrict to hyphal branching and does not intervene in very fast response pathways. Likewise, strigolactones need additional time to exhibit an effect, as recently discussed by Steinkellner et al.,²⁶ However, at the moment, no comprehensive electrophysiological analyses are presently available separating the effects of strigolactone and some flavonoids in AM fungal hyphae.The next target of our work is the study of ionic responses of single germ tubes or primary hyphae to root factors (Fig. 2). As reported by Ramos et al.,¹ we have been observing that the pattern of ion fluxes at the apical zone of primary hyphae is differentiated from secondary or lateral hyphae. In the primary, two interesting responses were detected in the absence of root factors: (1) a “dormant Ca²⁺ flux” and (2) Cl⁻ or anion fluxes at the same direction of H⁺ ions, suggesting a possible presence of H⁺/Cl⁻ symporters at the apex, similarly to what occurs in root hairs (Fig. 2).³⁰ In the presence of root factors such as root exudates the stimulated influxes of Cl⁻ (anion), H⁺, Na⁺ and effluxes of K⁺ and Ca²⁺ are activated. It can explain why the AM fungi hyphal tips are depolarized^20,29 during the period without root signals—“asymbiosis”—as long as K⁺ efflux and H⁺ influx occur simultaneously. Indeed, H⁺ as well as Ca²⁺ ions may act as second messengers, where extra and intracellular transient pH changes are preconditions for a number of processes, including gravity responses and possibly in plant-microbe interactions.^17,30Open in a separate windowFigure 2Ion dynamics in the apex of primary hyphae of arbuscular mycorrhizal fungi. It represents the Stage 1 described in Ramos et al.¹ After treatment with root factors, an activation of Ca²⁺ efflux is observed at the hyphal apex.Clearly, further data on the mechanism of action of signaling molecules such as strigolactones over the signal transduction and ion dynamics in AM fungi will be very important to improve our understanding of the molecular bases of the mycorrhization process. Future studies are necessary in order to provide basic knowledge of the ion signaling mechanisms and their role on the response of very important molecules playing at the early events of AM symbiosis.     

Multiple signaling pathways control nitrogen-mediated root elongation in maize

Response of root system architecture to nutrient availability is an essential way for plants to adapt to soil environments. Nitrogen can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Low soil N stimulates root elongation in maize. Recent evidence suggests that plant hormones auxin and cytokinin, as well as NO signaling pathway, are involved in the regulation of root elongation by low nitrogen nutrition.Key words: nitrogen, root growth, auxin, cytokinin, NONitrogen acquisition is determined by N demand for plant growth. At low N stress, N demand for maximum plant growth rate is not matched by plant N uptake. To acquire adequate N, plants may increase root length density to explore a larger soil volume and/or increase N uptake activity. High root density is also an important root trait for competition with soil microorganisms.¹ Since nitrate is a highly mobile, non-adsorbing ion, theoretic analysis predicts that its uptake is not limited by transport through soil, and a small root system is sufficient for nitrate acquisition.^2–4 In field conditions, however, genotypes that are efficient in N acquisition generally had a larger root system and higher root length density.^5,6 Under conditions of insufficient N supply, N mass flow to roots may not be adequate to meet the N demand for plant growth. Even in N-sufficient soils, various soil constraints (low water content, etc) may reduce the N mass flow rate. In these cases, large root size and high density will be very important for the utilization of the spatially distributed N, especially newly mineralized N, and the competition for organic N with soil microorganisms.^7,8The development of lateral roots in Arabidopsis in response to nitrate supply has been widely studied.⁹ Less attention has been paid to primary root growth in response to N, possibly because root elongtion is insensitive to increased N supply in Arabidopsis.^10,11 In maize, however, root elongation was sigificantly promoted by suboptimal N supply, and inhibited by overdose supply of N (Fig. 1).^12,13 Until recently less is known about the underlying physiological mechansms. It is well documented that cytokinin is a root-to-shoot signal communicating N availability in addition to nitrate itself.¹⁴ Exogenous cytokinin application suppresses the elongation of primary roots.¹⁵ Recent work in Arabidopsis overexpressing cytokinin synthase (IPT) demonstrate that long-term CK overproduction inhibited primary root elongation by reducing quantitative parameters of primary root meristem.¹⁶ By comparing two maize inbred lines whose root elongation had a differential response to low N stress, it was found that the change of cytokinin content in roots was closely related to low-N induced root elongation.¹³ In the N-sensitive genotype 478, cytokinin (Zeatin + Zeatin riboside) content was significantly lower at low N condition. While in N-insensitive genotype Wu312, cytokinin content was hardly affected at various N supplies. Higher N supply shortened the distance from root apex to the first visible lateral roots, a phenomenen similar to that caused by exogenous cytokinins. Furthermore, exogenous cytokinin 6-benzylaminopurine (6-BA) completely reversed the stimulatory effect of low nitrate on root elongation. All the data suggests that the inhibitory effect of high concentration of nitrate on root elongation is, at least in part, mediated by increased cytokinin level in roots.Open in a separate windowFigure 1Root elongation is inhibited at high nitrate supply.Auxin regulates many cellular responses crucial for plant development. Auxin plays a key role in establishing and elaborating patterns in root meristems.^17,18 Root elongation of Arabidopsis is enhanced by exogenous auxin at low concentrations, but is inhibited at high concentrations.¹⁹ In an earlier report, a high external nitrate supply (8 mM) did cause a 70% decrease in the auxin concentration of the root in soybean.²⁰ In maize, inhibition of root growth by high nitrate was found closely related to the reduction of IAA levels in roots and exogenous NAA and IAA restored primary root growth in high nitrate concentrations.²¹ Interesting, it was found that auxin concentrations in phloem exudates were reduced by a greater nitrate supply, suggesting that shoot-to-root auxin transport may be inhibited by high N supply. Considering the antagonism between auxin and cytokinin.²² it was possible that, by increasing the cytokinin level and decreasing the auxin level, high nitrate supply may have negative influences on root apex activity so that root apical dominance is weakened and, therefore, root elongation is suppressed and lateral roots grow closer to the root apex.Nitric oxide (NO) is emerging as an important messenger molecule associated with many biochemical and physiological processes in plants. The involvement of NO in IAA-induced adventitious root development has also been reported.²³ Given that nitrate is a substrate for NR-catalysed NO production, and root development and growth are closely related to NO, it is expected that NO may play a role in nitrate-dependent root growth. Surprisingly, endogenous levels of NO in the root apices of maize seedlings grown in high nitrate solution were much lower than those in apices grown in low nitrate. The nitrate-induced inhibition of root elongation in maize was markedly reversed by treatments of the roots with a NO donor (SNP) and IAA.²⁴ These data suggest that the arrest of root elongation by high levels of external nitrate concentrations may result from an alteration of endogenous NO levels in root apical cells. NR mediated NO production is unlikely to be involved in the nitrate-dependent NO production and root elongation because NR activity is lower at low N supply. A NO synthase (NOS) inhibitor reduced root elongation in maize plants grown in the low-nitrate medium, suggest that NOS activity may be inhibited in plants grown in high-nitrate solution, thus leading to a reduction of the endogenous NO levels.Taken together, high nitrogen supply increases cytokinin level, but decreases auxin and NO levels in roots of maize. Besides, it was well documented ethylene has a negative effect on root elongation of various plants.^25–27 Exogenous supply of cytokinin increase ethylene production (Stenlid 1982; Bertell et al., 1990). Recently, it was demonstrated in Arabidopsis that auxin transport from the root apex via the lateral root cap is required for ethylene-mediated inhibition of root growth.²⁸ Therefore, a complex multiple siganlling pathways may be involved in N-mediated root elongation (Fig. 2). Further study is required to understand how these pathways interact with each other to reduce root elongation in response to high nitrate supply.Open in a separate windowFigure 2A simplified model explaining nitogen-mediated root elongation in maize.     

ARF-GTPase as a Molecular Switch for Polar Auxin Transport Mediated by Vesicle Trafficking in Root Development

Polar auxin transport (PAT), which is controlled precisely by both auxin efflux and influx facilitators and mediated by the cell trafficking system, modulates organogenesis, development and root gravitropism. ADP-ribosylation factor (ARF)-GTPase protein is catalyzed to switch to the GTP-bound type by a guanine nucleotide exchange factor (GEF) and promoted for hybridization to the GDP-bound type by a GTPase-activating protein (GAP). Previous studies showed that auxin efflux facilitators such as PIN1 are regulated by GNOM, an ARF-GEF, in Arabidopsis. In the November issue of The Plant Journal, we reported that the auxin influx facilitator AUX1 was regulated by ARF-GAP via the vesicle trafficking system.¹ In this addendum, we report that overexpression of OsAGAP leads to enhanced root gravitropism and propose a new model of PAT regulation: a loop mechanism between ARF-GAP and GEF mediated by vesicle trafficking to regulate PAT at influx and efflux facilitators, thus controlling root development in plants.Key Words: ADP-ribosylation factor (ARF), ARF-GAP, ARF-GEF, auxin, GNOM, polar transport of auxinPolar auxin transport (PAT) is a unique process in plants. It results in alteration of auxin level, which controls organogenesis and development and a series of physiological processes, such as vascular differentiation, apical dominance, and tropic growth.² Genetic and physiological studies identified that PAT depends on efflux facilitators such as PIN family proteins and influx facilitators such as AUX1 in Arabidopsis.Eight PIN family proteins, AtPIN1 to AtPIN8, exist in Arabidopsis. AtPIN1 is located at the basal side of the plasma membrane in vascular tissues but is weak in cortical tissues, which supports the hypothesis of chemical pervasion.³ AtPIN2 is localized at the apical side of epidermal cells and basally in cortical cells.^1,4 GNOM, an ARF GEF, modulates the localization of PIN1 and vesicle trafficking and affects root development.^5,6 The PIN auxin-efflux facilitator network controls root growth and patterning in Arabidopsis.⁴ As well, asymmetric localization of AUX1 occurs in the root cells of Arabidopsis plants,⁷ and overexpression of OsAGAP interferes with localization of AUX1.¹ Our data support that ARF-GAP mediates auxin influx and auxin-dependent root growth and patterning, which involves vesicle trafficking.¹ Here we show that OsAGAP overexpression leads to enhanced gravitropic response in transgenic rice plants. We propose a model whereby ARF GTPase is a molecular switch to control PAT and root growth and development.Overexpression of OsAGAP led to reduced growth in primary or adventitious roots of rice as compared with wild-type rice.¹ Gravitropism assay revealed transgenic rice overxpressing OsAGAP with a faster response to gravity than the wild type during 24-h treatment. However, 1-naphthyl acetic acid (NAA) treatment promoted the gravitropic response of the wild type, with no difference in response between the OsAGAP transgenic plants and the wild type plants (Fig. 1). The phenotype of enhanced gravitropic response in the transgenic plants was similar to that in the mutants atmdr1-100 and atmdr1-100/atpgp1-100 related to Arabidopsis ABC (ATP-binding cassette) transporter and defective in PAT.⁸ The physiological data, as well as data on localization of auxin transport facilitators, support ARF-GAP modulating PAT via regulating the location of the auxin influx facilitator AUX1.¹ So the alteration in gravitropic response in the OsAGAP transgenic plants was explained by a defect in PAT.Open in a separate windowFigure 1Gravitropism of OsAGAP overexpressing transgenic rice roots and response to 1-naphthyl acetic acid (NAA). (A) Gravitropism phenotype of wild type (WT) and OsAGAP overexpressing roots at 6 hr gravi-stimulation (top panel) and 0 hr as a treatment control (bottom panel). (B) Time course of gravitropic response in transgenic roots. (C and D) results correspond to those in (A and B), except for treatment with NAA (5 × 10⁻⁷ M).The polarity of auxin transport is controlled by the asymmetric distribution of auxin transport proteins, efflux facilitators and influx carriers. ARF GTPase is a key member in vesicle trafficking system and modulates cell polarity and PAT in plants. Thus, ARF-GDP or GTP bound with GEF or GAP determines the ARF function on auxin efflux facilitators (such as PIN1) or influx ones (such as AUX1).ARF1, targeting ROP2 and PIN2, affects epidermal cell polarity.⁹ GNOM is involved in the regulation of PIN1 asymmetric localization in cells and its related function in organogenesis and development.⁶ Although VAN3, an ARF-GAP in Arabidopsis, is located in a subpopulation of the trans-Golgi transport network (TGN), which is involved in leaf vascular network formation, it does not affect PAT.¹⁰ OsAGAP possesses an ARF GTPase-activating function in rice.¹¹ Specifically, our evidence supports that ARF-GAP bound with ARF-GTP modulates PAT and gravitropism via AUX1, mediated by vesicle trafficking, including the Golgi stack.¹Therefore, we propose a loop mechanism between ARF-GAP and GEF mediated by the vascular trafficking system in regulating PAT at influx and efflux facilitators, which controls root development and gravitropism in plants (Fig. 2). Here we emphasize that ARF-GEF catalyzes a conversion of ARF-bound GDP to GTP, which is necessary for the efficient delivery of the vesicle to the target membrane.¹² An opposite process of ARF-bound GDP to GTP is promoted by ARF-GTPase-activating protein via binding. A loop status of ARF-GTP and ARF-GDP bound with their appurtenances controls different auxin facilitators and regulates root development and gravitropism.Open in a separate windowFigure 2Model for ARF GTPase as a molecular switch for the polar auxin transport mediated by the vesicle traffic system.     

Dynamic protein trafficking to the cell wall

Recently we have studied the secretion pattern of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2) in tobacco protoplast using the protein fusions, secGFP-PMEI1 and PGIP2-GFP. Both chimeras reach the cell wall by passing through the endomembrane system but using distinct mechanisms and through a pathway distinguishable from the default sorting of a secreted GFP. After reaching the apoplast, sec-GFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP undergoes endocytic trafficking. Here we describe the final localization of PGIP2-GFP in the vacuole, evidenced by co-localization with the marker Aleu-RFP, and show a graphic elaboration of its sorting pattern. A working model taking into consideration the presence of a regulated apoplast-targeted secretion pathway is proposed.Key words: cell wall trafficking, endocytosis, GPI-anchor, PGIP2, PMEI1, secretion pathway, vacuole fluorescent markerCell wall biogenesis, growth, differentiation and remodeling, as well as wall-related signaling and defense responses depend on the functionality of the secretory pathway. Matrix polysaccharides, synthesized in the Golgi stacks, and cell wall proteins, synthesized in the ER, are packaged into secretory vesicles that fuse with the plasma membrane (PM) releasing their cargo into the cell wall. Also the synthesis and deposition of cellulose itself are driven by the endomembrane system which controls the assembly, within the Golgi, and the export to the plasma membrane of rosette complexes of cellulose synthase.¹ Secretion to the cell wall has always been considered a default pathway² but recent studies have evidenced a complex regulation of wall component trafficking that does not seem to follow the default secretion model. Recent evidence that several cell wall proteins are retained in the Golgi stacks until specific signals at the N-terminal domain are proteolitically removed is a case in point.^3–5 Moreover, it has previously been reported that secretion of exogenous marker proteins (secGFP and secRGUS) and cell wall polysaccharides reach the PM through different pathways.⁶ More recently, we have reported that cell wall protein trafficking also occurs through mechanisms distinguishable from that of a secreted GFP suggesting that more complex events than the mechanisms of bulk flow control cell wall growth and differentiation.⁷ To follow cell wall protein trafficking we used a Phaseolus vulgaris polygalacturonase inhibitor protein (PGIP2) and an Arabidopsis pectin methylesterase inhibitor protein (PMEI1) fused to GFP (PGIP2-GFP and secGFP-PMEI1). Both apoplastic proteins are involved in the remodeling of pectin network with different mechanisms. PGIP2 specifically inhibits exogenous fungal polygalacturonases (PGs) and is involved in the plant defense mechanisms against pathogenic fungi.^8,9 PMEI1 counteracts endogenous PME and takes part in the physiological synthesis and remodeling of the cell wall during growth and differentiation.^10,11 The specific functions of the two apoplastic proteins seem to be strictly related to the distinct mechanisms that control their secretion and stability in the cell wall. In fact, while secGFP-PMEI1 moves through ER and Golgi stacks linked to a glycosyl phosphatidylinositol (GPI)-anchor, PGIP2-GFP moves as a cargo soluble protein. Furthermore, secGFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP, over the time, is internalized into endosomes and targeted to vacuole, likely for degradation. After reaching the cell wall, the different fate of the two proteins seems to be strictly related to the presence/absence of their physiological counteractors. PMEI regulates the demethylesterification of homogalacturonan by inhibiting pectin methyl esterase (PME) activity through the formation of a reversible 1:1 complex which is stable in the acidic cell wall environment.¹² Stable wall localization of PMEI1 is likely related to its interaction with endogenous PME, always present in the wall. Unlike PMEs, fungal polygalacturonases (PGs), the physiological interactors of PGIP2, are present in the cell wall only during a pathogen attack. The absence of PGs may determine PGIP2 internalization. Internalization events have been already reported for PM proteins,^13–16 while cell wall protein internalization is surely a less well-known event. To date, only internalization of an Arabidopsis pollen-specific PME^4,5,17 and PGIP2 ⁷ has been reported.To further confirm the internalization of PGIP2-GFP and its final localization into the vacuole, we constructed a red fluorescent variant (RFP) of the green fluorescent marker protein that accumulates in lytic or acidic vacuole because of the barley aleurain sorting determinants (Aleu-RFP).¹⁸ The localization of PGIP2-GFP was compared to that of Aleu-RFP by confocal microscopy in tobacco protoplasts transiently expressing both fusions. Sixty hours after transformation, PGIP2-GFP labeled the central vacuole as indicated by complete co-localization with the vacuolar marker (Fig. 1A–D). Instead, at the same time point, secGFP-PMEI1 still labeled the cell wall (Fig. 1E–H) and never reached the vacuolar compartment. To summarize PGIP2-GFP secretion pattern, a graphic elaboration of confocal images is reported describing the sorting of PGIP2GFP in tobacco protoplast (Fig. 1I). The protein transits through the endomembrane system (green) and reaches the cell wall which is rapidly regenerating as evidenced by immunostaining with the red monoclonal antibody JIM7 that binds to methylesterified pectins.¹⁹ PGIP2-GFP is then internalized in endosomes, labeled in yellow because of the co-localization with the styryl dye FM4-64, a red marker of the endocytic pathway.Open in a separate windowFigure 1PGIP2-GFP, but not secGFP-PMEI1, is internalized and reaches the vacuole in tobacco leaf protoplasts. (A) Approximately 60 h after transformation, PGIP2-GFP labeled the central vacuole as indicated by co-localization with the vacuole marker Aleu-RFP (B). (C) Merged image of (A and B). (D) Differential interference contrast (DIC) image of (A–C). On the contrary, secGFP-PMEI1 still labeled cell wall (E). (F) No co-localization is present in the vacuole labeled by Aleu-RFP. (G) Merged image of (E and F). (H) DIC image of (E–G). (I) Graphic elaboration of confocal images describing the sorting of PGIP2. The protein is sorted by the endomembrane system (green) to the cell wall (red) that is regenerated by the protoplast. Lacking the specific ligand, it is then internalized in endosome (yellow). Details are reported in the text.In Figure 2 we propose a model of the mechanism of secGFP-PMEI1 and PGIP2-GFP secretion derived from the different lines of evidence previously reported in reference ⁷. SecGFPPMEI1 (Fig. 2-1), but not PGIP2-GFP (Fig. 2-2), carries a GPI-anchor, required for its secretion to the cell wall. When the anchorage of GPI is inhibited by mannosamine (Fig. 2-a) or by the fusion of GFP to the C-terminus of PMEI1 (Fig. 2-b), the two non-anchored proteins accumulate in the Golgi stacks. Evidence of retention in Golgi stacks has already been reported for other two cell wall proteins.^3–5 Unlike secGFP-PMEI1, PGIP2-GFP is not stably accumulated in the cell wall and undergoes endocytic trafficking (Fig. 2-3). PGIP2-GFP internalization, likely due to the absence of PGs, might also be related with its ability to interact with homogalacturonan and oligogalacturonides,²⁰ which have been reported to internalize^21,22 (Fig. 2-4). Since SYP 121, a Qa-SNARE, is involved in the default secretion of secGFP,²³ but not in secretion of PGIP2-GFP and secGFP-PMEI1, trafficking mechanisms underlying secretion into the apoplast are likely different from those underlying the default route (Figs. 2-5). Taken as a whole, evidence suggests the existence of currently undefined signals that control apoplast-targeted secretion.Open in a separate windowFigure 2Schematic illustration for secGFP-PMEI1 and PGIP2-GFP trafficking. See text for details.     

Thigmomorphogenesis in Solanum lycopersicum: Morphological and biochemical responses in stem after mechanical stimulation

The activation of the phenylpropanoid pathway in plants by environmental stimuli is one of the most universal biochemical stress responses known. In tomato plant, rubbing applied to a young internode inhibit elongation of the rubbed internode and his neighboring one. These morphological changes were correlated with an increase in lignification enzyme activities, phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidases (POD), 24 hours after rubbing of the forth internode. Furthermore, a decrease in indole-3-acetic acid (IAA) content was detected in the rubbed internode and the upper one. Taken together, our results suggest that decrease in rubbed internode length is a consequence of IAA oxidation, increases in enzyme activities (PAL, CAD and POD), and cell wall rigidification associated with induction of lignification process.Key words: Mechanical stimulation, PAL, CAD, POD, IAAIn their environment, plants are constantly submitted to several stimuli such as wind, rain and wounding. The growth response of plants to such stimuli was termed thigmomorphogenesis and was observed in a wide range of plants.^1–3 The most common thigmomorphogenetic response is a retardation of tissue elongation accompanied by an increase in thickness.⁴ The plant response to mechanical perturbation is mainly restricted to the young developing internode, since no influence can be detected when the internode has reached its final length.^5,6 These plant growth modifications, which characterize thigmomorphogenesis, are related to biochemical events associated with lignification process⁷ and ethylene production.^8,9In tomato plant the length of internodes 4 (N4) and 5 (N5) was measured 14 days after rubbing of the fourth internode. Results reported in Figure 1 show that rubbing led to a significant reduction of elongation of the stressed internode (N4) (decrease of N4 length from 4.3 cm in the control plant to 2.9 in the rubbed one). This effect was not limited to the rubbed area but affected also the elongation of the neighboring internodes (N5) that were shorter in rubbed plants than in control ones.Open in a separate windowFigure 1Internode lengths of control and rubbed plants measured 14 day after mechanical stress applied to the fourth internode. Standard errors are indicated by vertical bars.Results reported in Figure 2 show an increase in PAL activity in both internodes N4 and N5, 24 hours after mechanical stress application as compared with corresponding controls. CAD activity was also investigated in N4 and N5, 24 h after rubbing of the fourth internode. Results presented in Figure 3 show that mechanical stress application induces a strong increase of CAD activity in the rubbed internode N4 (5.3 nkatal μg-1 protein) with an approximately two-fold increase when compared to control tomato internodes (2.3 nkatal μg-1 protein). Further, CAD activity in N5 was also increased in the rubbed internode (5.538 nkatal μg-1 protein) as compared with the control one (3.256 nkatal μg-1 protein).Open in a separate windowFigure 2PAL activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Open in a separate windowFigure 3CAD activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Syringaldazine (S-POD) and gaïacol (G-POD) peroxidase activities were measured in tomato N4 and N5. Results reported in Figure 4 show an increase in soluble peroxidase activity with both substrates in the rubbed internode N4 as compared with control plant. Enhancement in peroxidase activities in N4 was more pronounced with gaïacol (80.7 U) as an electron donor than syringaldazine (33.8 U). Similar results were observed in internode 5 as compared with control one (Fig. 4).Open in a separate windowFigure 4(A) Syringaldazine-POD (Syr-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars. (B) Gaiacol-POD (G-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.IAA was quantified in control and rubbed plant internodes 24 h after rubbing of the fourth internode. Results reported in figure 5 show that in control sample and as expected, the content of IAA was found to be higher in the younger internode (N5) as compared to the older one (N4). Rubbing led to a significant decrease in IAA levels in N4 (5.06 nmol g⁻¹ MF⁻¹) as compared with corresponding controls (7.27 nmol g⁻¹ MF⁻¹). Similar results were observed in internode 5, where IAA content was reduced from 16.52 nmol g⁻¹ MF⁻¹ in control internode to 12.35 nmol g⁻¹ MF⁻¹ in the rubbed internode (Fig. 5).Open in a separate windowFigure 5IAA Level of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.The results reported here establish an evident correlation between growth limitation of the rubbed internode and their degree of lignification, the increase in lignification enzymes activities and auxin degradation after mechanical stress application.Auxin seems to be involved in thigmomorphogenesis.¹⁰ It was proposed that MIS (Mechanically-induced stress) has opposite effects on auxin levels in the two species studied to date, Phaseolus vulgaris¹⁰ and Bryonia dioica.^11,12 Auxin level as measured by bioassay, increased in Phaseolus vulgaris following rubbing of the stem.¹⁰ It was proposed that a build up of auxin may result from the reduced polar transport of IAA at the rubbed internode, causing a build up of IAA in the stem tissue. Exogenous IAA did not reverse the MIS inhibition of growth in Phaseolus vulgaris and high levels of IAA retarded growth in non-stressed plants.¹⁰ Thus, retardation of extension growth in Phaseolus vulgaris may have been caused by high levels of endogenous auxin and the increase in stem diameter by increased ethylene production.⁴ However, ethylene increases radial growth only if auxin is present.¹³Boyer¹¹ reported a decrease in auxinlike activity in Bryonia dioica following MIS and this was confirmed in the same species by Hofinger et al.¹² who reported a decrease in IAA using gas chromatography-mass spectrometry. Auxin catabolism was accompanied with changes in both soluble and ionically bound cell wall basic peroxidases¹⁴ and the appearance of an additional peroxidase. This can suggest that in Bryonia, auxin catabolism is hastened by mechanical stimulated peroxidase. In addition, Boyer et al.¹⁵ reported that lithium pre-treatment prevents both thigmomorphogenesis and appearance of specific cathodic isoperoxidase in Bryonia plants subjected to MIS. This is give further credence to the possibility that the peroxidase-auxin system is involved in Bryonia thigmomorphogenesis. In addition, ethylene increases peroxidase activity which reduces the auxin content in the tissue to a level low enough not to support normal growth. We have evidence that decrease of auxin level contribute to mechanism leading to tomato internode inhibition subjected to mechanical stress.Growth inhibition has been suggested to be the result of tissues lignification.⁶ As the initial enzyme in the monolignol biosynthesis pathway, PAL has a direct influence on lignin accumulation.¹⁶ The characteristics of lignin differ among cell wall tissues and plant organs.¹⁷ It comprises polyphenolic polymers derived from the oxidative polymerization of different monolignols, including p-coumaryl, coniferyl and sinapyl alcohols via a side pathway of phenylalanine metabolism leading to lignin synthesis.¹⁸ The increase in lignin content in the rubbed tomato internode could be a response mechanism to mechanical damage caused by rubbing.³ It is known that plants create a natural barrier that includes lignin and suberin synthesis, components directly linked to support systems.^19,20The increase in lignin content of rubbed tomato internode³ is paralleled by a rise in CAD activity and whilst such direct proportionality between CAD activity and lignin accumulation does not always agree with the results in the literature, it clearly is responding in ways similar to those of the other enzymes in the pathway.²¹Mechanical stress-induced membrane depolarization would generate different species of free radicals and peroxides, which in turn initiate lipid peroxidation.²² The degradation of cell membranes is suggested to bring about rapid changes in ionic flux, especially release of K⁺ which would result in an enhanced endogenous Ca/K ratio and in leakage of solutes, among them electron donors such as ascorbic acid and phenolic substances. The increased intracellular relative calcium level activated secretion of basic peroxidases²³ into the free space where, in association with the electron donors and may be with the circulating IAA, they eliminate the peroxides, and facilitated binding of basic peroxidases to membrane structures allowing a role as 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidases. The resulting IAA and ACC oxidase-mediated changes in ethylene production²⁴ would further induce (this time through the protein synthesis machinery) an increase in activity of phenylalanine ammonia-lyase and peroxidases. The resulting lignification and cell wall rigidification determines the growth response of tomato internode to the mechanical stress.     

Linking protein kinase CK2 and auxin transport

Studies performed in different organisms have highlighted the importance of protein kinase CK2 in cell growth and cell viability. However, the plant signaling pathways in which CK2 is involved are largely unknown. We have reported that a dominant-negative mutant of CK2 in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. We demonstrated that auxin transport is, indeed, impaired in these mutant plants, and that this correlates with misexpression and mislocalization of PIN efflux transporters and of PINOID. Our data establishes a link between CK2 activity and the regulation of auxin homeostasis in plants, strongly suggesting that CK2 might be required at multiple points of the pathways regulating auxin fluxes.Key words: protein kinase CK2, root development, auxin, PIN, PINOIDThe plant hormone auxin plays critical roles in plant growth and development.¹ The most abundant natural auxin is the indol-3-acetic acid (IAA), which is synthesized in young apical tissues and then transported to the growing zones of the stem and root. The major route for long distance IAA movement is via the vascular tissue, but, additionally, a slower transport via cell-to-cell (called polar transport) is critical to generate auxin gradients within tissues. Formation of correct auxin gradients is thought to be essential for many plant developmental processes.² In recent years, the IAA transporters have been identified, establishing the molecular basis to understand how auxin transport is regulated. In particular, the identification of the family of plasma-resident PIN proteins, the members of which function as IAA efflux carriers, and the knowledge of their polar localization in the plasma membrane (PM), contributed to generate models predicting the direction of IAA fluxes.^3,4The factors that govern PIN targeting to a particular membrane domain are still not understood. It is known that PIN proteins constitutively undergo cycles of exocytosis and endocytosis to and from the PM, using distinct sorting and recycling endosome trafficking pathways.^5–7 Phosphorylation/dephosphorylation by the Ser/Thr kinase PINOID (PID) and the protein phosphatase 2A, respectively, controls PIN proteins apical/basal localization at the PM, via the GNOM-mediated vesicle trafficking system.⁸ Interestingly, PID is a member of the plant AGC kinases, and, as it happens with its mammals AGC counterparts, is activated by a membrane-associated 3-phosphoinositide-dependent kinase (PDK1).⁹ Moreover, a functional similarity between PIN polar localization in response to auxin and glucose receptor (GLUT4) asymmetrical distribution in response to insulin, has been pointed out.¹⁰ In both cases, cargo proteins (GLUT4 and PIN, respectively) are transported from endosomal vesicles to PM and the process is mediated by PDK1-activated AGC kinases.Protein kinase CK2 is a Ser/Thr kinase evolutionary conserved in eukaryotes, which plays key roles in cell survival, cell division and other cellular processes. A loss-of-function mutant of CK2 in Arabidopsis, obtained by overexpression of a CK2α-inactive subunit, confirmed the essential role of this protein kinase for plant viability.¹¹ Moreover, CK2mut plants showed a dramatic decrease of lateral root formation, inhibition of root growth and overproliferation of root hairs. We have further demonstrated that auxin transport is impaired in this plants, which is concomitant with missexpression of most of the PM-resident PIN proteins, and of PID.¹² In addition, PIN proteins accumulated in endosomal vesicles and auxin gradients were disturbed, both in roots and shoots of CK2mut plants. In particular, root columella cells were depleted of auxin, although the maximum at the quiescent center was unchanged. Starch granule staining with lugol revealed that columella cells retained their fate, although their organization and/or cell shape were clearly affected (Fig. 1).Open in a separate windowFigure 1Lugol-stained starch granules in uninduced (−Dex) and Dex-induced (+Dex) CK2mut roots. In the central part of the figure, a sketch of the main morphogenetic characteristics of mutant roots (right plantlet) as compared to wild-type roots (left plantlet) is shown. Note the shorter roots, wavy phenotype, absence of lateral roots and overproliferation of root hairs in mutant plants.Our results strongly suggest that CK2 is a regulator of auxin-dependent responses, most likely by participating in the regulation of auxin transport. Strikingly, depletion of CK2 activity inhibits some auxin-dependent physiological responses whereas it enhances others. For instance, whereas shoot phototropism was completely absent, root gravitropism was enhanced.¹² Figure 2 shows a time-course of DR5rev::GFP-derived signal after changing the gravity vector, in mutant and control Arabidopsis roots. The progressive auxin translocation to the lower side of the root after gravistimulation is more rapid and sustained in mutant than in control roots, which is likely responsible for the enhanced response to gravity found in mutant roots. Based on these results, we postulate that CK2 might act at different points of the auxin-induced regulatory pathway. As far as is known, the core module that regulates auxin transport is constituted by the protein kinase PID and a protein of the NPH3-domain family. NPH3-containing proteins play important roles in phototropic and gravitropic responses, and regulate polarity and endocytosis of PIN proteins.¹³ As has been proposed by other authors, the participation of one AGC kinase and one NPH3-like protein upstream of an ARF factor might be a common theme in response to different stimulus that are signaled by auxin.¹⁴ We propose that one of the functions of CK2 is the regulation of the activity of core proteins (Fig. 3). Mammalian AGC kinases are well known substrates of CK2 and CK2-dependent phosphorylation is critical for a full display of their activity. The PID and the NPH3-containing protein sequences contain numerous acidic-based motifs that are predicted CK2 phosphorylation sites. Moreover, according to Arabidopsis phosphoproteome databases, several members of the NPH3-containing protein family are predicted to be phosphorylated.¹⁵ In addition, we do not discard the possibility that other proteins involved in PIN transport might also be regulated by CK2-dependent phosphorylation. Experiments are in progress in our laboratory to assess the regulatory role of CK2 in auxin transport.Open in a separate windowFigure 2Time course of auxin relocation during root gravitropic response, as visualized by DR5rev::GFP fluorescence. Root pictures were taken at the indicated times after changing the direction of the gravity vector. Translocation of auxin to the lower part of the root is more rapid in Dex-induced CK2mut plants. Arrows indicate asymmetrical DR5::GFP fluorescence.Open in a separate windowFigure 3Proposed model for the role of CK2 in regulating auxin transport. The core module that regulates auxin transport (shown here as a black box) is constituted by the protein kinase PID and a protein of the NPH3-domain family. PID regulates apical-basal targeting of PIN proteins, by phosphorylating conserved Ser residues present in PIN hydrophilic loops.¹⁶ On the other hand, the family of NPH3-containing proteins regulates polarity and endocytosis of PIN proteins.¹³ There is also a functional similarity between the intracellular transport of PIN proteins and that of the glucose receptor (GLUT4),¹⁰ two processes that are signaled by AGC kinases. We propose that CK2 might be a regulator of the activity of the core proteins, by phosphorylating either the AGC kinase and/or the NPH3-containing protein. Mammalian CK2 is a known regulator of the activity of AGC kinases and other proteins participating in signaling pathways, such as in the Wnt/β-catenin signaling pathway.¹⁷     

Brassinosteroid negatively regulates jasmonate inhibition of root growth in Arabidopsis

Jasmonate (JA) inhibits root growth of Arabidopsis thaliana seedlings. The mutation in COI1, that plays a central role in JA signaling, displays insensitivity to JA inhibition of root growth. To dissect JA signaling pathway, we recently isolated one mutant named psc1, which partially suppresses coi1 insensitivity to JA inhibition of root growth. As we identified the PSC1 gene as an allele of DWF4 that encodes a key enzyme in brassinosteroid (BR) biosynthesis, we hypothesized and demonstrated that BR is involved in JA signaling and negatively regulates JA inhibition of root growth. In our Plant Physiology paper, we analyzed effects of psc1 or exogenous BR on the inhibition of root growth by JA. Here we show that treatment with brassinazole (Brz), a BR biosynthesis inhibitor, increased JA sensitivity in both coi1-2 and wild type, which further confirms that BR negatively regulates JA inhibition of root growth. Since effects of psc1, Brz and exogenous BR on JA inhibition of root growth were mild, we suggests that BR negatively finely regulates JA inhibition of root growth in Arabidopsis.Key words: jasmonate signaling, root growth, brassinosteroid, brassinazole, arabidopsisJasmonate (JA) regulates many plant developmental processes and stress responses.^1,2 COI1 plays a central role in JA signaling and is required for all JA responses in Arabidopsis.^3,4 coi1-1, a strong mutation in COI1, is male sterile and exhibits loss of all JA responses tested to date, such as JA inhibition of root growth, the expression of JA-induced genes, and susceptibility to insect attack and pathogen infection, and coi1-2, a weak mutant of COI1, shows similar JA responses to coi1-1 except for partially fertile that makes it able to produce a small quantity of seeds.⁵To investigate COI1-mediated JA responses and dissect JA signaling pathway, we conducted genetic screens for suppressors of coi1-2. Previously, we identified cos1 that completely suppresses coil-2 insensitive to JA.⁶ Recently, we isolated the psc1 mutant that partially suppresses coi1-2 insensitivity to JA, and found that PSC1 is an allele of DWF4.⁷Since the DWF4 gene encodes a key enzyme in brassinosteroid (BR) biosynthesis,⁸ we hypothesized that BR is involved in JA signaling. By physiological analysis, we showed that psc1 partially restored JA inhibition of root growth in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background, the effects of psc1 were eliminated by exogenous BR, and that exogenous BR could attenuated JA inhibition of root growth in wild type. These findings demonstrated that BR is involved in JA signaling and indicated that BR negatively regulates JA inhibition of root growth.BR is a family of polyhydroxylated steroid hormones involved in many aspects of plant growth and development. The BR-deficient mutants exhibited severely retarded growth that was able to be rescued by exogenous BR.⁹ Brassinazole (Brz) is a BR biosynthesis inhibitor. The Arabidopsis seedlings treated with Brz displayed a BR deficient-mutant-like phenotype, which could be elimilated by exogenous BR.¹⁰To determine wether treatment with Brz affects JA inhibition of root growth, the seedlings of wild type and coi1-2 were grown in MS medium supplemented with MeJA and/or Brz. As shown in Figure 1, the relative root length was obviously reduced in both coi1-2 and wild type when treated with Brz relative to without Brz, indicating that the repression of BR biosynthesis by Brz could increase JA sensitivity. These results further confirm BR negatively regulates JA inhibition of root growth.Open in a separate windowFigure 1Effect of Brz on JA inhibition of root growth. Brz increased JA inhibition of root growth in both coi1-2 and wild type (WT). Root length of 7-day-old seedlings grown in MS medium containing 0, 5 and 10 μM MeJA without (−) or with (+) 0.5 μM Brz was expressed as a percentage of root length in MS without (−) or with (+) 0.5 µM Brz. Error bars represent SE (n > 30).It has been demonstrated that JA connects with other plant hormones including auxin, ethylene, abscisic acid, salicylic acid and gibberellin to form complex regulatory networks modulating plant developmental and stress responses.^11–15 We found that BR negatively regulates JA inhibition of root growth, suggesting that a cross talk between JA and BR exists in planta, which extends our understandings on the JA signal transduction.COI1 is a JA receptor¹⁶ and DWF4 catalyzes the rate-limiting step in BR-biosynthesis pathway.⁸ We found that JA inhibits DWF4 expression, this inhibition was dependent on COI1,⁷ indicating that DWF4 is downregulated by JA and is located downstream of COI1 in the JA signaling pathway.Since the effects of psc1, Brz, and exogenous BR on JA inhibition of root growth were mild, and the DWF4 expression was partially repressed by JA (Ren et al. 2009, Fig. 1), we suggest that BR negatively finely regulates JA inhibition of root growth, and propose a model for these regulations. As shown in Figure 2A, JA signal passes COI1 repressing substrates, such as JAZs,^17,18 i.e., JA activates degradation of substrates via SCF^COI1-26S proteasome,^16–18 whereas substrates positively regulate root growth through other regulators. JA also partially inhibits DWF4 expression through COI1, reducing BR that is required for root growth.^7,9 Mutation in COI1 interrupts JA signaling for failing in degradation of substrates and repression of DWF4 as well, resulting in JA-insensitivity (Fig. 2B). However, mutation in DWF4 or treatment with Brz causes a reduction in BR, which affects root growth, leading to JA-hypersensitivity in wild-type COI1 background (Fig. 2C and E) and partial restoration of JA sensitivity in coi1-2 background (Fig. 2D and F). Whereas, an application of exogenous BR could eliminate the effect of BR reduction resulted from repression of DWF4 by JA on root growth, attenuating JA sensitivity in wild type (Fig. 2G). Because the inhibition of DWF4 expression by JA is dependent on COI1, the coi1 mutant treated with exogenous BR do not show alteration in JA sensitivity (Fig. 2H).Open in a separate windowFigure 2A model for that BR negatively finely regulates JA inhibition of root growth in Arabidopsis. (A–D) Treatment with JA in wild type (A), coi1-2 (B), psc1 (C) and psc1coi1 (D). (E and F) Treatments with JA and Brz in wild type (E) and coi1-2 (F). (G and H) Treatments with JA and exogenous BR in wild type (G) and coi1-2 (H). Arrows indicate positive regulation or enhancement, whereas blunted lines indicate repression or negative regulation. Crosses indicate interruption or impairment. The letter “S” indicates substrates of SCF^COI1. Thicker arrows and blunted lines represent the central JA signaling pathway regulating JA inhibition of root growth. Broken arrows represent JA signaling pathway in which other regulators are involved. The intensity of gray boxes represents the degree of JA inhibition on root growth.     

Creating drought- and salt-tolerant cotton by overexpressing a vacuolar pyrophosphatase gene

Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proton pump activity of the vacuolar pyrophosphatase, which generates a higher proton electrochemical gradient across the vacuolar membrane, leading to lower water potential in the plant vacuole and higher secondary transporter activities that prevent ion accumulation to toxic levels in the cytoplasm. Additionally, overexpression of AVP1 appears to stimulate auxin polar transport, which in turn stimulates root development. The larger root system allows AVP1-overexpressing plants to absorb water more efficiently under drought and saline conditions, resulting in stress tolerance and increased yields. Multi-year field-trial data indicate that overexpression of AVP1 in cotton leads to at least 20% more fiber yield than wild-type control plants in dry-land conditions, which highlights the potential use of AVP1 in improving drought tolerance in crops in arid and semiarid areas of the world.Key words: drought tolerance, proton pump, salt tolerance, transgenic cotton, vacuolar membraneDrought and salinity are major environmental factors that limit agricultural productivity in most parts of the world.¹ Climate change will likely make many places worse in terms of water availability and soil salinization,² which will have negative impacts on food production in world agriculture. Yet, the demand for more food will continue to rise because of the growing world population that may reach 9 billon people by 2050.³ Therefore, the primary challenge we face during this century is the production of more food under the constraints of limited water and fertilizer on marginal soils.Many genes that respond to abiotic stresses have been identified in the model plant Arabidopsis,⁴ and some of them were shown to play important roles in protecting plants under abiotic stress conditions.⁵ The Arabidopsis vacuolar pyrophosphatase gene AVP1 appears to be one of the most promising genes that may be used to improve drought- and salt-tolerance in crops.⁶ Roberto Gaxiola''s group first demonstrated that overexpression of AVP1 could lead to significantly improved drought- and salt-tolerance in transgenic Arabidopsis plants.⁷ Later when this gene was introduced into tomato⁸ and rice,⁹ similar tolerance phenotypes were observed. Overexpression of AVP1 in cotton, not only improved drought- and salt-tolerance in greenhouse conditions, but also increased fiber yield in dryland field conditions.⁶ AVP1-expressing cotton plants produced larger root systems and bigger shoot biomass than controls when grown under hydroponic conditions in the presence of up to 200 mM NaCl.⁶ In the greenhouse, AVP1-expressing cotton plants also produced more root and shoot biomass than controls when grown under saline conditions or reduced irrigation.⁶ The increased yield by AVP1-expressing cotton plants is due to more bolls produced, which in turn is due to larger shoot system that AVP1-expressing cotton plants develop under saline or drought conditions.⁶The larger root systems of AVP1-expressing cotton plants under saline and water-deficit conditions allow transgenic plants access to more of the soil profile and available soil water resulting in increased biomass production and yield. Li et al. showed that the larger root systems of AVP1-overexpressing Arabidopsis is caused by increased auxin polar transport in the root, which stimulates root development in AVP1-overexpressing Arabidopsis plants.¹⁰ Furthermore, a recent comparative study of transgenic Arabidopsis lines that produce enlarged leaves showed that auxin levels were increased by 50% in AVP1-overexpressing plants.¹¹ To test if altered auxin level is responsible for the observed larger root systems in AVP1-expressing cotton plants, we germinated wild-type and AVP1-expressing cotton plants in the absence or presence of the auxin polar transport inhibitor Naphthylphthalamic acid (NPA). Both wild-type and AVP1-expressing cotton plants developed robust lateral root systems in the absence of NPA (Fig. 1A). The presence of 50 µM NPA resulted in nearly complete inhibition of lateral root development in wild-type plants, while lateral root development in AVP1-expressing plants was reduced, it was significantly greater than wild-type (Fig. 1B). These data indicate that AVP1-overexpression could overcome the inhibitory effects of NPA on root development in AVP1-expressing cotton plants, suggesting that either increased auxin transport or higher auxin concentration in the root systems of AVP1-expressing cotton plants is responsible for the observed larger root systems, and eventually for the increased boll numbers and fiber yields under dryland field conditions.Open in a separate windowFigure 1Root development of wild-type and AVP1-expressing cotton plants in the absence and presence of auxin transport inhibitor NPA. (A) Phenotype of cotton roots after 10 days of growth in the absence of NPA. WT, Wild-type; 1, 5, 9, three independent AVP1-overexpressing cotton lines. (B) Phenotype of cotton roots after 10 days of growth in the presence of 50 µm NPA.Many genes that may play important roles under water-deficit conditions have been tested in laboratory conditions,^4,5 but very few have been tested vigorously in field conditions. A bacterial cold shock protein gene was shown to improve drought tolerance in maize based on multi-year and multi-place field trial experiments,¹² and it appears that this gene will likely gain approval for commercial release and become the first genetically engineered product that demonstrates improved drought tolerance in a major crop in the U.S. Another example of increased drought tolerance supported by multiple field trial experiments is through downregulation of farnesylation in transgenic canola plants.¹³ Downregulation of farnesyltransferase by antisense or RNAi techniques in transgenic canola leads to increased sensitivity to abscisic acid, consequently resulting in smaller guard cell aperture under drought conditions. These transgenic canola plants lose less water through transpiration and are more drought resistant. Data from more than 5 years of field studies in Canada consistently proved that this approach can indeed increase drought tolerance in transgenic canola. Our study with AVP1-expressing cotton over the last several years in field conditions is another example that genetic engineering approach can be an efficient tool in generating drought-tolerant crops. AVP1-expressing cotton plants can establish a larger shoot mass in dryland conditions (Fig. 2), which results in increased boll numbers and fiber production. Our approach is likely applicable to other major crops as well.Open in a separate windowFigure 2Wild-type and AVP1-expressing cotton plants grown in the dryland field condition. Plants were planted in the middle of may 2009 and the picture was taken in the middle of July 2009 at the USDA experimental Farm in Lubbock, Texas.     

Conserved residues in the ankyrin domain of VAPYRIN indicate potential protein-protein interaction surfaces

Plant VAPYRINs are required for the establishment of arbuscular mycorrhiza (AM) and root nodule symbiosis (RNS). In vapyrin mutants, the intracellular accommodation of AM fungi and rhizobia is blocked, and in the case of AM, the fungal endosymbiont cannot develop arbuscules which serve for nutrient exchange. VAPYRINs are plant-specific proteins that consists of a major sperm protein (MSP) domain and an ankyrin domain. Comparison of VAPYRINs of dicots, monocots and the moss Physcomitrella patens reveals a highly conserved domain structure. We focused our attention on the ankyrin domain, which closely resembles the D34 domain of human ankyrin R. Conserved residues within the petunia VAPYRIN cluster to a surface patch on the concave side of the crescent-shaped ankyrin domain, suggesting that this region may represent a conserved binding site involved in the formation of a protein complex with an essential function in intracellular accommodation of microbial endosymbionts.Key words: VAPYRIN, arbuscular mycorrhiza, petunia, symbiosis, glomus, ankyrin, major sperm protein, VAPPlants engage in mutualistic interactions such as root nodule symbiosis (RNS) with rhizobia and arbuscular mycorrhiza (AM) with Glomeromycotan fungi. These associations are referred to as endosymbioses because they involve transcellular passage through the epidermis and intracellular accommodation of the microbial partner within root cortical cells of the host.^1,2 Infection by AM fungi and rhizobia is actively promoted by the plant and requires the establishment of infection structures namely the prepenetration apparatus (PPA) in AM and a preinfection thread in RNS, respectively.^3–5 In both symbioses the intracellular microbial accommodation in epidermal and root cortical cells involves rebuilding of the cytoskeleton and of the entire membrane system.^6–8 Recently, intracellular accommodation of rhizobia and AM fungi, and in particular morphogenesis of the AM fungal feeding structures, the arbuscules, was shown to depend on the novel VAPYRIN protein.^9–11VAPYRINs are plant-specific proteins consisting of two protein-protein interaction domains, an N-terminal major sperm protein (MSP) domain and a C-terminal ankyrin (ANK) domain. MSP of C. elegans forms a cytoskeletal network required for the motility of the ameboidal sperm.¹² MSP domains also occur in VAP proteins that are involved in membrane fusion processes in various eukaryotes.¹³ The ANK domain, on the other hand, closely resembles animal ankyrins which serve to connect integral membrane proteins to elements of the spectrin cytoskeleton,¹⁴ thereby facilitating the assembly of functional membrane microdomains in diverse animal cells.¹⁵ Ankyrin repeats exhibit features of nano-springs, opening the possibility that ankyrin domains may be involved in mechanosensing.¹⁶ Based on these structural similarities, VAPYRIN may promote intracellular accommodation of endosymbionts by interacting with membranes and/or with the cytoskeleton. Indeed, VAPYRIN protein associates with small subcellular compartments in petunia and in Medicago truncatula.^9,10Ankyrin repeats typically consist of 33 amino acids, of which 30–40% are highly conserved across most taxa. These residues confer to the repeats their basic helix-turn-helix structure.¹⁷ Ankyrin domains often consist of arrays of several repeats that form a solenoid with a characteristic crescent shape.¹⁷ Besides the ankyrin-specific motiv-associated amino acids there is little conservation between the ankyrin domains of different proteins, or between the individual repeats of a given ankyrin domain,¹⁷ a feature that was also observed in petunia VAPYRIN (Fig. 1A).⁹ However, sequence comparison of VAPYRINs from eight dicots, three monocots and the moss Physcomitrella patens revealed a high degree of sequence conservation beyond the ankyrin-specific residues (Fig. 1B and Sup. Fig. S1). When the degree of conservation was determined for the individual ankyrin repeats among all the 12 species, it appeared that repeats 7, 9 and 10 exhibited particularly high conservation (Fig. 1C).Open in a separate windowFigure 1Sequence analysis and phylogeny of VAPYRIN from diverse plants. (A) Predicted amino acid sequence of the petunia VAPYRIN protein PAM1. The 11 repeats of the ankyrin domain are aligned, and the ankyrin consensus sequence is shown below the eleventh ankyrin repeat (line c). Conserved residues that are characteristic for ankyrin repeats (Mosavi et al. 2004)¹⁷ are depicted in bold face. (B) Unrooted phylogenetic tree representing the VAPYRINs of eight dicot species (Petunia hybrida, Solanum lycopersicon, Solanum tuberosum, Vitis vinifera, Populus trichocarpa, Ricinus communis, Medicago truncatula and Glycine max) three monocot species (Sorghum bicolor, Zea mays and Oryza sativa), and the moss Physcomitrella patens. (C) Degree of conservation of the individual ankyrin repeats of VAPYRIN. Schematic representation of the MSP domain as N-terminal barrel-shaped structure, and of the individual ankyrin repeats as pairs of alpha-helices. An additional loop occurring only in monocots (grass-loop) is inserted above repeat 4, and the deletion between repeat 7 and 8 is indicated (gap). This latter feature is common to all VAPYRIN proteins. The percentage of amino acid residues that are identical in at least 11 of the 12 VAPYRINS is given below the MSP domain and the eleven ankyrin repeats. The box highlights repeats 7–10 which contribute to the predicted binding site (compare with Figs. 3 and ​and44).Sequence comparison of the eleven repeats of all the twelve plant species revealed that the individual repeats clustered according to their position in the domain, rather than according to their origin (plant species) (Fig. 2). This shows that the repeats each are well conserved across species, but show little similarity among each other within a given VAPYRIN protein. The higher conservation of repeats 9 and 10 was reflected by the compact appearance of the respective branches, in which the monocot and moss sequences were nested closely with the dicot sequences, compared to other repeats, where the branches appeared fragmented between monocots and dicots, and where the P. patens sequence fell out of the branch as in the case of repeats 4–6 (Fig. 2). Taken together, this points to an old evolutionary origin of the entire ankyrin domain in lower land plants, with no subsequent rearrangement of ankyrin repeats.Open in a separate windowFigure 2Phylogenetic analysis of the individual ankyrin repeats of VAPYRIN. Phylogenetic representation of an alignment of all the 11 repeats of the 12 VAPYRINs compared in Figure 1B and C. The repeats cluster according to their position within the domain, rather than to their origin (plant species). Numbers indicate the position of the repeats within the domain (compare with Fig. 1C). P. patens repeats are highlighted (small circles) for clarity. The monocot repeat 4 sequences (boxed) are remote from the remaining repeat 4 sequences because of the grass loop (compare with Fig. 1C).Ankyrin domains function as protein-protein interaction domains,¹⁷ in which the residues on the surface are involved in the binding of their protein partners.¹⁴ The fact that repeats 9 and 10 exhibited particularly high levels of conservation across species from moss to angiosperms indicated that this region may contain functionally important residues. Within repeat 10, sixteen amino acid positions were identical in >90% of the analyzed species (Fig. 3A and grey bars). Nine of those represent residues that are characteristic for ankyrin repeats (red letters) and determine their typical 3D shape.¹⁷ These residues are considered ankyrin-specific, and are unlikely to be involved in a VAPYRIN-specific function. The remaining seven highly conserved residues in repeat 10, however, are VAPYRIN-specific, since they have been under positive selection, without being essential for the basic structure of the ankyrin repeat. Ankyrin-specific and VAPYRIN-specific residues where identified throughout the entire ankyrin domain (Sup. Fig. 1), and subsequently mapped on a 3-dimensional model of petunia VAPYRIN to reveal their position in the protein (Fig. 3B–G). The ankyrin-specific residues were found to be localized primarily to the interior of the ankyrin domain, with the characteristic glycines (brown) marking the turns between helices and loops (Fig. 3B, D and F, compare with A). In contrast, the VAPYRIN-specific residues were localized primarily on the surface of the ankyrin domain (Fig. 3C, E and G). A prominent clustering of VAPYRIN-specific residues was identified on the concave side of the crescent-shaped ankyrin domain comprising repeats 7–10 close to the gap (Figs. 3G and ​and44). This highly conserved VAPYRIN-specific region contains several negatively and positively charged residues (D, E and K, R, respectively) and aromatic residues (W, Y, F), which may together form a conserved binding site for an interacting protein.Open in a separate windowFigure 33D-Mapping of conserved positions within the ankyrin domain of VAPYRIN. (A) Conserved amino acid residues were evaluated for ankyrin repeat 10 of petunia VAPYRIN as an example. The degree of conservation between the 12 VAPYRINs analyzed in Figures 1B and ​and22 is depicted with grey bars. Average conservation between all the 132 ankyrin repeats of the 12 VAPYRIN sequences is shown with black bars. Residues that are conserved in all 132 repeats (red letters) define the ankyrin consensus sequence, which confers to the repeats their characteristic basic structure.¹⁷ Residues that are >90% conserved but are not part of the basic ankyrin sequence (highlighted with asterisks) are VAPYRIN-specific and may therefore have been conserved because of their specific function in VAPYRIN. Arrows indicate the characteristic antiparallel helices, the turns are marked by conserved glycine residues (underlined; compare with B, D and F). (B–G) 3D-models of the petunia VAPYRIN PAM1. Conserved amino acid residues were color-coded according to their physico-chemical properties (http://life.nthu.edu.tw/∼fmhsu/rasframe/SHAPELY.HTM) with minor modification (see below). In (B, D and F) the ankyrin-specific residues are highlighted (corresponding to the bold letters in Fig. 1A). In (C, E and G), the VAPYRIN-specific residues are highlighted. Note the patch of high conservation on the concave side of the crescent-shaped ankyrin domain between repeats 7–10 next to the gap. (B–E) represent respective side views of the ankyrin domain, (F and G) exhibit the concave inner side of the domain. Color code: Bright red: aspartic acid (D), glutamic acid (E); Yellow: cysteine (C); Blue: lysine (K), arginine (R); Orange: serine (S), threonine (T); Dark blue: phenylalanine (F), tyrosin (Y); Brown: glycine (G); Green: leucin (L), valine (V), isoleucin (I), alanine (A); Lilac: tryptophane (W); Purple: histidine (H); Pink: proline (P).Open in a separate windowFigure 4The highly conserved surface area in domain 8–10 of the ankyrin domain of petunia VAPYRIN. Close-up of the highly conserved region of petunia PAM1 as shown in Figure 3G. Amino acids were color-coded as in Figure 3 and their position in the amino acid sequence is indicated (compare with Sup. Fig. 1).In this context, it is interesting to note that human ankyrin R also contains a binding surface on the concave side of the D34 domain for the interaction with the CBD3 protein.¹⁴ Consistent with an essential function of the C-terminal third of the ankyrin domain, mutations that abolish this relatively short portion of VAPYRIN, have a strong phenotype, indicating that they may represent null alleles.⁹ Based on this collective evidence, we hypothesize that repeats 7–10 are involved in the formation of a protein complex that is essential for intracellular accommodation of rhizobia and AM fungi. Biochemical and genetic studies are now required to identify the binding partners of VAPYRINs, and to elucidate their role in plant endosymbioses.     

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H₂O₂ generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H₂O₂ signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H₂O₂ generation, inhibition of inward K⁺ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H₂O₂ signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H₂O₂, is essential to plant growth and development in response to stresses,^1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.^1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,^4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H₂O₂ generation.^6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H₂O₂ signaling regardless of SA mimicking ABA in regulating stomatal closure.^2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H₂O₂ generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.^11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,^14–16 Pisum sativum,¹⁷ Medicago sativa¹⁸ and tobacco.¹⁹ The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,²⁰ tobacco tissue²¹ and suspension-cultured Oryza sativa cells.²² Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H₂O₂ signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H₂O₂ generation and decreasing K⁺ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.^23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H₂O₂ signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H₂O₂ signaling,^23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase^23,24 is not always identical to that of MEK1/2^8,25 in ABA-induced H₂O₂ signaling of Vicia guard cells. For example, H₂O₂^- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H₂O₂, the effects of both ABA- and H₂O₂-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H₂O₂-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H₂O₂-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.^8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.²⁶ Similar to this, SB203580 improves H₂O₂-inhibited inward K⁺ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H₂O₂ signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,^1,2,8,23,24 and the ABA and H₂O₂ pathways diverge further downstream in their actions on the K⁺ channels and, thus, on stomatal control. Other differences in action between ABA and H₂O₂ are known. For example, Köhler et al. (2001) reported that H₂O₂ inhibited the K⁺ outward rectifier in guard cells shows that H₂O₂ does not mimic ABA action on guard cell ion channels as it acts on the K⁺ outward rectifier in a manner entirely contrary to that of ABA.²⁷Open in a separate windowFigure 2Effect of SB203580 on ABA- and H₂O₂-inhibited inward K⁺ currents. The experimental procedure and data analysis are according to the previous publication.²⁴ SB203580 directs ABA- and H₂O₂-inactivated inward K⁺ currents across plasma membrane of Vicia guard cells. Here the inward K⁺ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H₂O₂ signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,^1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H₂O₂ signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.     

Nitric oxide functions in both methyl jasmonate signaling and abscisic acid signaling in Arabidopsis guard cells

Intracellular components in methyl jasmonate (MeJA) signaling remain largely unknown, to compare those in well-understood abscisic acid (ABA) signaling. We have reported that nitric oxide (NO) is a signaling component in MeJA-induced stomatal closure, as well as ABA-induced stomatal closure in the previous study. To gain further information about the role of NO in the guard cell signaling, NO production was examined in an ABA- and MeJA-insensitive Arabidopsis mutant, rcn1. Neither MeJA nor ABA induced NO production in rcn1 guard cells. Our data suggest that NO functions downstream of the branch point of MeJA and ABA signaling in Arabidopsis guard cells.Key words: abscisic acid, Arabidopsis thaliana, guard cells, methyl jasmonate, nitric oxideStomatal pores that are formed by pairs of guard cells respond to various environmental stimuli including plant hormones. Some signal components commonly function in MeJA- and ABA-induced stomatal closing signals,¹ such as cytosolic alkalization, ROS generation and cytosolic free calcium ion elevation. Recently, we demonstrated that NO functions in MeJA signaling, as well as ABA signaling in guard cells.²NO production by nitric oxide synthase (NOS) and nitrate reductase (NR) plays important roles in physiological processes in plants.^3,4 It has been shown that NO functions downstream of ROS production in ABA signaling in guard cells.⁵ NO mediates elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt), inactivation of inward-rectifying K⁺ channels and activation of S-type anion channels,⁶ which are known to be key factors in MeJA- and ABA-induced stomatal closure.^2,7–9It has been reported that ROS was not induced by MeJA and ABA in the MeJA- and ABA-insensitive mutant, rcn1 in which the regulatory subunit A of protein phosphatase 2A, RCN1, is impaired.^7,10 We examined NO production induced by MeJA and ABA in rcn1 guard cells (Fig. 1). NO production by MeJA and ABA was impaired in rcn1 mutant (p = 0.87 and 0.25 for MeJA and ABA, respectively) in contrast to wild type. On the other hand, the NO donor, SNP induced stomatal closure both in wild type and rcn1 mutant (data not shown). These results are consistent with our previous results, i.e., NO is involved in both MeJA- and ABA-induced stomatal closure and functions downstream of the branching point of MeJA and ABA signaling in Arabidopsis guard cells.⁷ Our finding implies that protein phosphatase 2A might positively regulate NO levels in guard cells (Fig. 2).Open in a separate windowFigure 1Impairment of MeJA- and ABA-induced NO production in rcn1 guard cells. (A) Effects of MeJA (n = 10) and ABA (n = 9) on NO production in wild-type guard cells. (B) Effects of MeJA (n = 7) and ABA (n = 7) on NO production in rcn1 guard cells. The vertical scale represents the percentage of diaminofluorescein-2 diacetate (DAF-2 DA) fluorescent levels when fluorescent intensities of MeJA- or ABA-treated cells are normalized to control value taken as 100% for each experiment. Each datum was obtained from at least 30 guard cells. Error bars represent standard errors. Significance of differences between data sets was assessed by Student''s t-test analysis in this paper. We regarded differences at the level of p < 0.05 as significant.Open in a separate windowFigure 2A model of signal interaction in MeJA-induced and ABA-induced stomatal closure. Neither MeJA nor ABA induces ROS production, NO production, I_Kin and stomatal closure in rcn1 mutant. These results suggest that NO functions downstream of the branch point of MeJA signaling and ABA signaling in Arabidopsis guard cells.     

A new function for odorant receptors: MOR23 is necessary for normal tissue repair in skeletal muscle

Myofibers with an abnormal branching cytoarchitecture are commonly found in various neuromuscular diseases as well as after severe muscle injury. These aberrant myofibers are fragile and muscles containing a high percentage of these myofibers are weaker and more prone to injury. To date the mechanisms and molecules regulating myofiber branching have been obscure. Recent work analyzing the role of mouse odorant receptor 23 (MOR23) in muscle regeneration revealed that MOR23 is necessary for proper skeletal muscle regeneration in mice as loss of MOR23 leads to increased myofiber branching. Further studies demonstrated that MOR23 expression is induced when muscle cells were extensively fusing and plays an important role in controlling cell migration and adhesion. These data demonstrate a novel role for an odorant receptor in tissue repair and identify the first molecule with a functional role in myofiber branching.Key words: muscle regeneration, odorant receptor, olfactory receptor, MOR23, myofiber splitting, myofiber branching, myoblast, fusion, myotube, olfr16Skeletal muscle is characterized by an extensive ability to regenerate after injury due to trauma or disease. Muscle regeneration results from a finely orchestrated series of steps that are spatially and temporally regulated, many of which are not understood. Elucidation of the mechanisms that regulate muscle regeneration may be beneficial for enhancing the rate or extent of muscle regeneration in injury, disease or age.Skeletal muscle is composed of myofibers, which are long cylindrical cells containing hundreds of myonuclei in a common cytoplasm (Fig. 1). Each myofiber is surrounded by a basal lamina sheath; between the myofiber and the basal lamina lie myogenic stem cells called satellite cells. When muscle is injured, myofibers degenerate and satellite cells proliferate to give rise to progeny myoblasts. Myoblasts differentiate and undergo migration, adhesion and fusion to form regenerated myofibers and normal tissue architecture is restored. In many neuromuscular diseases muscle regeneration is aberrant and various abnormalities, such as variation in myofiber size, decreased myofiber number, fibrosis and branched myofibers, are observed. In the clinical literature, branched myofibers are more commonly referred to as “split myofibers.”Open in a separate windowFigure 1Myofiber growth during normal muscle regeneration. (A) Myofibers contain many myonuclei within a common cytoplasm and are surrounded by a basal lamina sheath. Underneath the basal lamina lie satellite cells, myogenic stem cells responsible for muscle regeneration. (B) Myofiber degeneration leads to activation of quiescent satellite cells and their reentry into the cell cycle. Their progeny myoblasts proliferate to yield a pool of progenitor cells. (C) Myoblasts differentiate and undergo migration, adhesion and fusion to form nascent myofibers within the original basal lamina sheath. Additional myoblasts fuse with these newly formed myofibers and the myofiber will continue to grow in size. (D) At later time points regenerated myofibers are similar in size to undamaged myofibers but contain centrally located myonuclei, a hallmark of a regenerated myofiber.Branched myofibers are malformed cells which, instead of having a normal cylindrical shape, contain one or more offshoots of small daughter myotubes contiguous with the parent myofiber (Fig. 2). Branched myofibers can be simple with only one branch (Fig. 2A) or complex with many anastomosing branches resembling a gnarled tree root (Fig. 2B).¹ In myofibers with complex cytoarchitecture, individual branches can persist up to hundreds of microns and then eventually recombine with the parent myofiber. Each daughter branch is enclosed in its own basal lamina, which is contiguous with the basal lamina of the parent myofiber.² The frequency of branched myofibers in rodent muscle under normal conditions is low, on the order of 0.003%.³ However, the frequency in both rodent and human muscle is increased in response to hypertrophy^4,5 as well as regeneration due to induced injury,^6–8 muscle transplantation,^9,10 or muscular dystrophy.^1,11–16 In mdx mice, a model of Duchenne Muscular Dystrophy, up to 65–90% of myofibers by 7 months and older display this abnormal branched morphology compared to 6–17% of myofibers at 1–3 months of age.^12–14 Branched myofibers display functional abnormalities such as alterations in myofiber calcium signaling.¹³ Additionally, isolated branched myofibers are more prone to rupture at branches during stimulation¹⁷ and mdx muscles containing a high percentage of branched myofibers are more vulnerable to contraction-induced injury.^12,14 Thus, decreasing the number of branched myofibers would likely be beneficial to muscle function.Open in a separate windowFigure 2Myofiber branching during aberrant muscle regeneration. (A) Phase contrast microscopy of a normal (left) and a branched (right) myofiber. The branched myofiber contains one branch at the end of the myofiber. (B) Schematic diagrams of myofibers with more complex patterns of branching than depicted in (A).Although branched myofibers have been reported in literature for over 100 years, the mechanism by which they arise is unknown and no causative molecules have been identified. The aberrant cytoarchitecture of branched myofibers likely arises from incomplete fusion of small myotubes during muscle regeneration⁸ though direct proof is lacking. Evidence in favor of these malformed myofibers arising from abortive regenerative processes includes the expression of neonatal myosin, a marker of early muscle regeneration, in the small branches.¹³ That these branches are newly formed is further suggested by the presence of centrally located nuclei,^13,18 a hallmark of regenerated myofibers. The observation that during muscle regeneration multiple small myotubes can form within the old basal lamina sheath^8,16,19 leads credence to the idea that aberrant fusion of such small myotubes underlies generation of branched myofibers. Indeed, electron microscopic studies support the ability of myotubes to fuse with one another in vivo;²⁰ myotubes readily fuse with one another in vitro.²¹ Recent studies of odorant receptor function during muscle regeneration in mice¹⁸ have identified the first molecule with a functional role in controlling myofiber branching and suggest that defects in muscle cell migration and/or adhesion may underlie the genesis of these branches.     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.