Diversity in spindle morphology in Arabidopsis root tip

A primary function of the spindle apparatus is to segregate chromosomes into two equal sets in a dividing cell. It is unclear whether spindles in different cell types play additional roles in cellular regulation. As a first step in revealing new functions of spindles, we investigated spindle morphology in different cell types in Arabidopsis roots in the wild-type and the cytokinesis defective1 (cyd1) mutant backgrounds. cyd1 provides cells larger than those of the wild type for testing the cell size effect on spindle morphology. Our observations indicate that cell type (shape), not cell size, is likely a factor affecting spindle morphology. At least three spindle types were observed, including small spindles with pointed poles in narrow cells, large barrel-shaped spindles (without pointed poles) in wide cells, and spindles intermediate in pole focus and size in other cells. We hypothesize that the cell-type-associated spindle diversity may be an integral part of the cell differentiation processes.Key words: spindle pole, microtubule, morphogenesis, cell type, metaphaseThe cellular apparatus for chromosome segregation during mitosis is typically described as a spindle composed of microtubules and microtubule-associated proteins. Research on the structure and function of the spindle is usually conducted under the assumption that spindles are structurally the same or alike in different cell types in an organism. If the assumption is true, it would indicate that either the intracellular conditions in different dividing cells are very similar or the assembly and maintenance of the spindle are insensitive to otherwise variable intracellular conditions. But experimental evidence related to this assumption is relatively sparse.The root tip in Arabidopsis, as in other higher plants, contains dividing cells of different shapes and sizes. These cells include both meristem initial and derivative cells, with the former and latter being proximal and distal to the quiescent center, respectively.¹ The diversity in dividing cells in the root tip provides an opportunity for testing whether the spindles also exhibit diversity in morphology. To visualize the spindles at the metaphase stage in the root tip cells, we conducted indirect immunofluorescence labeling of the β-tubulin in single cells prepared from wild-type Arabidopsis (in Col-0 background) root tips as previously described in references ² and ³. The spindles in cells of different morphologies were then observed under a confocal laser scanning microscope.³ Three types of spindle were detected. The first type (Fig. 1A) was the smallest in width and length and had the most-pointed poles among the three types. The second type (Fig. 1B) was wider and longer than the first type but with less-pointed poles than the first type. The third type (Fig. 1C) was similar in height to the second type but lacked the pointed poles. In fact, the third type is shaped more like a barrel than a spindle. The first type was found in cells narrow in the direction parallel to the equatorial plane of the spindle, a situation opposite to that of the third type whose cells were wide in the equatorial direction. The wide cells containing the barrel-shaped spindles likely belonged to the epidermal layer in the root tip.¹ The second type was found in cells intermediate in width. Examples of metaphase spindles morphologically resembling the three types of spindles in Arabidopsis root can also be found in a previous report by Xu et al. even although spindle diversity was not the subject of the report.⁴ In Xu et al.''s report, type 1- or 2-like metaphase spindles can be identified in Figures 2B and 3A, and type 3-like metaphase spindles can be identified in Figures 1A and 3B. These observations indicate that at least three types of spindles exist in the root cells.Open in a separate windowFigure 1Spindles in wild-type root cells. (A) Type-1 spindle. (B) Type-2 spindle. (C) Type-3 spindle. The spots without fluorescence signals in the middle of the spindles are where the chromosomes were located. Scale bar for all the figures = 20 µm.Open in a separate windowFigure 2Spindles in cyd1 root cells. (A) Type-1 spindle. Arrows indicate the upper and lower boundaries of the cell. (B and C) Two type-2 spindles. (D and E) Two type-3 spindles. (F) DAPI-staining image corresponding to (E), showing chromosomes at the equatorial plane. Scale bar for the images = 20 µm.The above observations suggest that either the cell size or the cell type (shape) might be a factor in the type of spindle found in a specific cell. To further investigate the relationship between cell morphology and spindle morphology, we studied metaphase spindles in root cells of the cytokinesis defective1 (cyd1) mutant.⁵ Because the root cells in cyd1 were larger than corresponding cells in the wild type, presumably due to abnormal polyploidization prior to the collection of the root cells,^5,6 this investigation might reveal a relationship between increasing cell size and altered spindle morphology. A pattern of different spindle types in different cell types similar to that in the wild type was observed in cyd1 (Fig. 2). Figures 2A–C show narrow cells that contained spindles with pointed poles even though the spindles differed in size and focus. Figure 2D shows a barrel-shaped spindle in a wide cell, resembling Figure 1C in overall appearance. The large number of chromosomes at metaphase (more than the diploid number of 10) in Figure 2F indicates that the cells in Figure 2 were polyploid. These figures thus demonstrate that the enlargement in cell size did not alter the pattern of types 1 and 2 spindles in narrow cells, as well as type 3 spindles in wide cells. Moreover, the edges of the spindles in Figure 2B and E were similarly distanced to the cell walls in the equatorial plane, and yet they differ greatly in shape with the former being type 2 and the latter being type 3. This finding argues against that the cell width in the equatorial direction dictates the spindle shape. On the other hand, the cells in Figure 2B and E are obviously of different types. Taken together, these observations suggest that the spindle diversity in both wild type and cyd1 is associated with cell-type diversity.It is unclear whether the different spindle types have different functions in their respective cell types, in addition to the usual role for chromosome segregation. One possibility is that, at the ensuing telophase, the pointed spindles result in compact chromosomal congregation at the poles whereas the barrel-shaped spindles result in loose chromosomal congregation at the poles, which in turn may differentially affect the shape of the subsequently formed daughter nuclei and their organization. Different nuclear shape and organization are likely to be integrated into the processes that confer cell differentiation.     

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.     

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.     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

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.     

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.     

Biphasic ethylene production during the hypersensitive response in Arabidopsis: A window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H₂O₂, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.¹ Much work on the regulation of the HR has indicated the importance of H₂O₂,² and NO.³ A feature of H₂O₂ generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)⁴ and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.⁵Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H₂O₂ (●, Mur¹⁸); (B) nitric oxide (◇; Mur¹² (C) salicylic acid (SA, ■¹⁹) and (D) ethylene (○ Mur⁹) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H₂O₂—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.⁶ Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H₂O₂ act to initiate SA biosynthesis, where SA and NO act to initiate a “H₂O₂ biphasic switch”. This could initially suppress both SA and the H₂O₂ generation but subsequently acts to potentiate a second phase of H₂O₂ generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C₂H₄ biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.⁶ Such secondary stimuli include SA³ or PAMPs⁷ and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H₂O₂ generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.^6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.⁸We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.⁹ As with H₂O₂ generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H₂O₂ (Fig. 1A) which initiated SA biosynthesis^10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models⁵ this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H₂O₂ and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.⁹ Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H₂O₂ generation. Significantly, the second phases in both H₂O₂ and ethylene production occur exactly where SA and NO production coincides; in the case of H₂O₂ generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco^9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with ​with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.¹³ who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.¹⁴ Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.⁶Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m².sec⁻¹) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 10⁶ colony forming units.ml⁻¹) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.⁹ The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 10⁶ c.f.u.mL⁻¹ with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H₂O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.⁹A further point requires consideration; the role of ethylene as a direct contributor to plant defense.¹⁵ The contribution of ethylene to the HR has been disputed,¹⁶ but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.⁹ In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD.     

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.     

Splenogonadal Fusion: An Unusual Case of an Acute Scrotum

We highlight a case on a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophilic infiltrate and thrombus consistent with chronic infarction and torsion. Splenogonadal fusion (SGF) is a rather rare entity, with approximately 184 cases reported in the literature. The most comprehensive review was that of 123 cases completed by Carragher in 1990. Since then, an additional 61 cases have been reported in the scientific literature. We have studied these 61 cases in detail and have included a summary of that information here.Key words: Splenogonadal fusion, Acute scrotumA 10-year-old boy presented with worsening left-sided scrotal pain of 12 hours’ duration. The patient reported similar previous episodes occurring intermittently over the past several months. His past medical history was significant for left hip dysplasia, requiring multiple hip surgeries. On examination, he was found to have an edematous left hemiscrotum with a left testicle that was rigid, tender, and noted to be in a transverse lie. The ultrasound revealed possible polyorchism, with two testicles on the left and one on the right (Figure 1), and left epididymitis. One of the left testicles demonstrated a loss of blood flow consistent with testicular torsion (Figure 2).Open in a separate windowFigure 1Ultrasound of the left hemiscrotum reveals two spherical structures; the one on the left is heterogeneous and hyperdense in comparison to the right.Open in a separate windowFigure 2Doppler ultrasound of left hemiscrotum. No evidence of blood flow to left spherical structure.The patient was taken to the operating room for immediate scrotal exploration. A normalappearing left testicle with a normal epididymis was noted. However, two accessory structures were noted, one of which was torsed 720°; (Figure 3). An inguinal incision was then made and a third accessory structure was noted. All three structures were connected with fibrous tissue, giving a “rosary bead” appearance. The left accessory structures were removed, a left testicular biopsy was taken, and bilateral scrotal orchipexies were performed.Open in a separate windowFigure 3Torsed accessory spleen with splenogonadal fusion.Pathology revealed a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophillic infiltrate and thrombus consistent with chronic infarction and torsion (Figure 4).Open in a separate windowFigure 4Splenogonadal fusion, continuous type with three accessory structures.     

Evolutionary origin of rhizobium Nod factor signaling

For over two decades now, it is known that the nodule symbiosis between legume plants and nitrogen fixing rhizobium bacteria is set in motion by the bacterial signal molecule named nodulation (Nod) factor.¹ Upon Nod factor perception a signaling cascade is activated that is also essential for endomycorrhizal symbiosis (Fig. 1). This suggests that rhizobium co-opted the evolutionary far more ancient mycorrhizal signaling pathway in order to establish an endosymbiotic interaction with legumes.² As arbuscular mycorrhizal fungi of the Glomeromycota phylum can establish a symbiosis with the vast majority of land plants, it is most probable that this signaling cascade is wide spread in the plant kingdom.³ However, Nod factor perception generally is considered to be unique to legumes. Two recent breakthroughs on the evolutionary origin of rhizobium Nod factor signaling demonstrate that this is not the case.^4,5 The purification of Nod factor-like molecules excreted by the mycorrhizal fungus Glomus intraradices and the role of the LysM-type Nod factor receptor PaNFP in the non-legume Parasponia andersonii provide novel understanding on the evolution of rhizobial Nod factor signaling.Open in a separate windowFigure 1Schematic representation of the genetically dissected symbiosis signaling pathway. In legumes rhizobium Nod factors and mycorrhizal Myc factors are perceived by distinct receptor complexes. In case of Nod factors these are the LysM-RK type receptors MtLYK3/LjNFR1 and MtNFP/LjNFR5, whereas Myc factors remain to be elucidated. In Parasponia PaNFP fulfils a dual function and acts in both symbioses. The subsequent common signaling pathway consists of several components including a plasma membrane localized LRR-type receptor (MtDMI2/LjSymRK), a cation channel in the nuclear envelope (MtDMI1/LjCASTOR/LjPOLLUX) and subunits of the nuclear pore (NUP85, NUP133), and a nuclear localized complex of calcium calmodulin dependent kinase (CCaMK) and interactor protein MtIPD3/LjCYCLOPS. Downstream of CCaMK the rhizobium and mycorrhiza induced responses bifurcate.Key words: parasponia, legumes, rhizobium, mycorrhizae, Nod factor     

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.     

Arabidopsis glutathione reductase 1 is dually targeted to peroxisomes and the cytosol

We recently established a proteome methodology for Arabidopsis leaf peroxisomes and identified more than 90 putative novel proteins of the organelle. These proteins included glutathione reductase isoform 1 (GR1), a major enzyme of the antioxidative defense system that was previously reported to be cytosolic. In this follow-up study, we validated the proteome data by analyzing the in vivo subcellular targeting of GR1 and the function of its C-terminal tripeptide, TNL>, as a putative novel peroxisome targeting signal type 1 (PTS1). The full-length protein was targeted to peroxisomes in onion epidermal cells when fused N-terminally with the reporter protein. The efficiency of peroxisome targeting, however, was weak upon expression from a strong promoter, consistent with the idea that the enzyme is dually targeted to peroxisomes and the cytosol in vivo. The reporter protein that was extended C-terminally by 10 amino acid residues of GR1 was directed to peroxisomes, characterizing TNL> as a novel PTS1. The data thus identify plant peroxisomal GR at the molecular level in the first plant species and complete the plant peroxisomal ascorbate-glutathione cycle. Moreover, GR1 is the first plant protein that is dually targeted to peroxisomes and the cytosol. The evolutionary origin and regulatory mechanisms of dual targeting are discussed.Key words: ascorbate-glutathione cycle, dual targeting, proteome analyses, reactive oxygen species, targeting signalsMassive amounts of hydrogen peroxide (H₂O₂) are produced during photosynthesis in peroxisomes by glycolate oxidase activity as part of the photorespiratory cycle.¹ Next to catalase, the ascorbate-glutathione cycle is the secondary scavenging system for H₂O₂ detoxification.^2–4 The cycle comprises four enzymes, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and NADPH-dependent glutathione reductase (GR). GR plays a major physiological role in maintaining and regenerating reduced glutathione in response to biotic and abiotic stresses in plants.⁵ Jiminez et al. (1997) provided biochemical evidence for the presence of the antioxidants ascorbate and glutathione and the enzymes of the ascorbate-glutathione cycle in pea peroxisomes.^6–8 While Arabidopsis APX3, MDAR1 and MDAR4 have been characterized as peroxisomal isoforms,^9–11 the molecular identity of plant peroxisomal GR and DHAR have not been determined in any plant species to date.⁵ Arabidopsis encodes two GR and five DHAR isoforms that are either shown to be or predicted to be cytosolic, mitochondrial or plastidic.¹² We recently identified specific isoforms of GR (GR1, At3g24170) and DHAR (DHAR1, At1g19570) as being peroxisome-associated by proteome analysis of Arabidopsis leaf peroxisomes.^13,14 Both isoforms were previously reported to be or predicted to be cytosolic.¹⁵Arabidopsis GR1 terminates with TNL>, which is related to functional plant PTS1 tripeptides such as SNL> and ANL>.^16,17 Threonine (T), however, has not yet been described as an allowed residue at position −3 of PTS1s in any plant peroxisomal protein.¹⁶ Analysis of homologous plant proteins and expressed sequence tags (ESTs) shows that TNL> is generally highly conserved in putative plant GR1 orthologs (Fig. 1). A few other sequences terminate with related tripeptides, such TSL>, TTL>, NNL> and TKL>. Only a single EST (Picrorhiza kurrooa) carries the canonical PTS1, SKI> (Fig. 1). The data provide only weak additional support for peroxisome targeting of plant GR1 orthologs. However, GR homologs from green algae (chlorophyta) carry canonical PTS1 tripeptides, such as SKL> (Chlamydomonas, Volvox) and AKM> (Micromonas, Fig. 1, Suppl. Fig. 1).Open in a separate windowFigure 1Analysis of PTS1 conservation in plant GR1 homologs. Sequences of full-length protein (FLP) plant GR1 homologs or ESTs (“EST”) were identified by BLAST and phylogenetic analysis, aligned by ClustalX, and conserved residues were shaded by Genedoc. In addition to spermatophyta, homologs from bryophyta and chlorophyta were analyzed for PTS1 conservation. For a phylogenetic analysis of the full-length proteins, see also Supplementary Figure 1. The species abbreviations are as follows: Aa, Artemisia annua; At, Arabidopsis thaliana; Bn, Brassica napus; Br, Brassica rapa; Ci, Cichorium intybus; Cr, Chlamydomonas reinhardtii; Cs, Cynara scolymus; Fv, Fragaria vesca; Ha, Helianthus annuus; Msp, Micromonas sp. RCC 299; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, Oryza sativa; Pk, Picrorhiza kurrooa; Ppat, Physcomitrella patens subsp. patens; Ps, Pisum sativum; Ptri, Populus trichocarpa; Rc, Ricinus communis; Rs, Raphanus sativus; Tp, Trifolium pratense; Tpus, Triphysaria pusilla; Vc, Volvox carteri f. nagariensis; Vv, Vitis vinifera; Zm, Zea mays.     

ERECTA controls low light intensity-induced differential petiole growth independent of phytochrome B and cryptochrome 2 action in Arabidopsis thaliana

Plants can respond quickly and profoundly to changes in their environment. Several species, including Arabidopsis thaliana, are capable of differential petiole growth driven upward leaf movement (hyponastic growth) to escape from detrimental environmental conditions. Recently, we demonstrated that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA, explains a major effect Quantitative Trait Locus (QTL) for ethylene-induced hyponastic growth in Arabidopsis. Here, we demonstrate that ERECTA controls the hyponastic growth response to low light intensity treatment in a genetic background dependent manner. Moreover, we show that ERECTA affects low light-induced hyponastic growth independent of Phytochrome B and Cryptochrome 2 signaling, despite that these photoreceptors are positive regulators of low light-induced hyponastic growth.Key words: hyponastic growth, petiole, Arabidopsis, low light, ERECTA, differential growth, phytochrome B, cryptochrome 2Plants must adjust growth and reproduction to adverse environmental conditions. Among the strategies that plants employ to escape from unfavorable conditions is differential petiole growth-driven upward leaf movement, called hyponastic growth. Arabidopsis thaliana is able to exhibit a marked hyponastic response upon flooding, which is triggered by endogenous accumulation of the gaseous phytohormone ethylene.¹ Moreover, a similar response is triggered upon low light intensity perception and in response to supra-optimal temperatures.^2–5 By tilting the leaves to a more vertical position during submergence and shading, the plants restore contact with the atmosphere and light, respectively. The kinetics of the hyponastic growth response induced by the various stimuli is remarkably similar. This led to the hypothesis that shared functional genetic components may be employed to control hyponastic growth. Yet, at least part of the signaling cascades is parallel, as the hormonal control of the response differs between the stimuli. Low light-induced hyponastic growth for example does not require ethylene action.² Whereas the response to heat is antagonized by this hormone.⁵ The abiotic stress hormone abscisic acid (ABA) antagonizes ethylene-induced hyponastic growth and stimulates heat-induced hyponastic growth.^5,6 Moreover, ethylene-induced hyponasty does not involve auxin action⁷ whereas both heat- and low light-induced hyponasty require functional auxin signaling and transport components.^2,5In our recent paper, published in The Plant Journal,⁸ we employed Quantitative Trait Locus (QTL) analysis to identify loci involved in the control of ethylene-induced hyponastic petiole growth. By analyzing induced mutants and by complementation analysis of naturally occurring mutant accessions, we found that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA (ER) is a positive regulator of ethylene-induced hyponastic growth and most likely is causal to one of the identified QTLs. In addition, we demonstrated that the ER dependency is not via ER mediated control of ethylene production or sensitivity.Since low light-induced hyponasty does not require ethylene action,² ER may be part of the proposed shared signaling cascade leading to hyponastic growth where ethylene and low light signals meet. Therefore, we studied low light intensity-induced hyponasty in various erecta mutants. Moreover, natural occurring er mutant accessions complemented with a functional, Col-0 derived, ER allele were tested. The response of Lan-0 (Lan-0; with functional ER) to low light was indistinguishable from the response of Landsberg erecta (Ler) (Fig. 1A). However, complemented Ler (ER-Ler) showed an enhanced response compared to Ler (Fig. 1B). The response of mutant er105 was slightly attenuated compared to the wild type Columbia-0 (Fig. 1C). Mutant er104, however, showed an indistinguishable hyponastic growth phenotype to low light compared to the wild type Wassilewskija-2 (Ws-2) (Fig. 1D). Complementation of the natural occurring erecta mutant accession Vancouver-0 (Van-0) resulted in an enhanced hyponastic growth response to low light (Fig. 1E), whereas this was not the case for Hiroshima-1 (Hir-1) (Fig. 1F). Together, these data suggest that ER acts as positive regulator of low light-induced hyponastic growth and therefore may be part of the shared signaling cascade towards differential petiole growth. Yet, the effect is strongly dependent on the genetic background since the effects were not observed in every accession tested.Open in a separate windowFigure 1ERECTA involvement in low light-induced hyponasty. Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth in Arabidopsis thaliana. (A) mutant (circles) Ler and wild type (dashed line) Lan-0, (B) Ler and Ler complemented (ER-; squares) with the Col-0 ERECTA allele (ER-Ler), (C) er105 and Col-0 wild type, (D) er104 and Ws-2 wild type, (E) natural mutant Van-0 and Van-0 complemented with the Col-0 ER allele (ER-Van-0), (F) natural mutant Hir-1 and Hir-1 complemented with the Col-0 ER allele (ER-Hir-1). Petiole angles were measured using time-lapse photography and subsequent image analysis. Data is pairwise subtracted, which corrects for diurnal petiole movement in control conditions. For details on this procedure, growth conditions and materials, transformation protocol, treatments, data acquirement and all analyses see.^1,8 Error bars represent standard errors; n ≥ 12.Phytochrome B (PhyB) and Cryptochrome 2 (Cry2) photoreceptor proteins are required for a full induction of low light-induced hyponastic growth.² We transformed the phyb5 cry2 mutant9 (Ler genetic background) with Col-0 derived ER. This complementation did not restore the ability of phyb5 cry2 to induce hyponastic growth to neither ethylene (data not shown) nor low light conditions (Fig. 2A). Mutant phyb5 cry2 plants have a typical constitutive shade avoidance phenotype, reflected by severely elongated organs. This includes enhanced inflorescence and silique length and thin inflorescences (Fig. 2B-D). Complementation with ER resulted in a significant additional effect on these parameters (Fig. 2B-D). Together, this suggests that ER is not an integral part of PhyB nor Cry2 signaling with respect to (hyponastic) growth. Moreover, PhyB and Cry2 control of plant architecture does not require ER action. Rather, ER seems to mediate growth via genetic interaction with light-reliant growth mechanisms, instead of being downstream of photoreceptor action. Studies on the effects of ER on shade avoidance responses and various hormone responses, including cytokinin and auxin, led to the similar conclusion, suggesting a possible role for ER as a molecular hub coordinating light- and hormone-mediated plant growth.^10,11 One could speculate that ER fine-tunes other (than light) environmental clues with light signaling components. A comparable conclusion was drawn previously for gibberellin (GA) reliant growth mechanisms, as er enhanced the negative effect on plant size of the short internode (shi) mutation12 and er represses the positive effect of the spindly mutation in a GA independent manner.¹³Open in a separate windowFigure 2Effects of ERECTA on light signaling. (A) Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth of Ler (dashed lines), the photoreceptor double mutant phyb5 cry2 (circles) and this mutant complemented with the Col-0 ERECTA (ER-phyb cry2; squares). For details see legend Figure 1. (B) Plant height, (C) silique length and (D) inflorescence stem thickness of the above mentioned lines. These parameters were measured when the last flower on the plant developed a silique. Plant height was measured from root/shoot junction to inflorescence top. Stem thickness was measured ∼1 cm above the root/shoot junction with a caliper and silique lengths were measured from representative pedicels in the top ∼10 cm of the main inflorescence stem. Error bars represent standard errors; n ≥ 12. Significance levels; *p < 0.05; **p < 0.01; ***p < 0.001; ns = non significant, by Students t-test.     

Co-localization of mitochondria with chloroplasts is a light-dependent reversible response

Co-localization of mitochondria with chloroplasts in plant cells has long been noticed as beneficial interactions of the organelles to active photosynthesis. Recently, we have found that mitochondria in mesophyll cells of Arabidopsis thaliana expressing mitochondrion-targeted green fluorescent protein (GFP) change their distribution in a light-dependent manner. Mitochondria occupy the periclinal and anticlinal regions of palisade cells under weak and strong blue light, respectively. Redistributed mitochondria seem to be rendered static through co-localization with chloroplasts. Here we further demonstrated that distribution patterns of mitochondria, together with chloroplasts, returned back to those of dark-adapted state during dark incubation after blue-light illumination. Reversible association of the two organelles may underlie flexible adaptation of plants to environmental fluctuations.Key words: Arabidopsis thaliana, blue light, chloroplast, green fluorescent protein, mesophyll cell, mitochondrion, organelle positioningHighly dynamic cell organelles, mitochondria, are responsible not only for energy production, but also for cellular metabolism, cell growth and survival as well as gene regulations.^1,2 Appropriate intracellular positioning and distribution of mitochondria contribute to proper organelle functions and are essential for cell signaling.^3,4 In plant cells operating photosynthesis, the co-localization of mitochondria with chloroplasts has been a well known phenomenon for a long period of time.^5,6,7 Physical contact of mitochondria with chloroplasts may provide a means to transfer genetic information from the organelle genome,⁸ as well as to exchange metabolite components; a process required for the maintenance of efficient photosynthesis.^9,10,11Using Arabidopsis thaliana stably expressing mitochondrion-targeted GFP,¹² we have recently examined a different aspect of mitochondria positioning. Although mitochondria in leaf mesophyll cells are highly motile under dark condition, mitochondria change their intracellular positions in response to light illumination.¹³ The pattern of light-dependent positioning of mitochondria seems to be essentially identical to that of chloroplasts.¹⁴ Mitochondria occupy the periclinal regions under weak blue light (wBL; 470 nm, 4 µmol m⁻²s⁻¹) and the anticlinal regions under strong blue light (sBL; 100 µmol m⁻²s⁻¹), respectively. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts in the periclinal regions with time period of wBL illumination clearly demonstrates that the co-localization of these two organelles is a light-induced phenomenon.¹³In the present study, to ask whether the light-dependent positioning of mitochondria is reversible or not, a time course of mitochondria redistribution was examined transferring the sample leaves from light to dark conditions. The representative results (Fig. 1) clearly show that mitochondria re-changed their positions within several hours of dark treatment. Immediately after dark adaptation, mitochondria in the palisade mesophyll cells were distributed randomly throughout the cytoplasm (Fig. 1A and ^{ref. 13}). Chloroplasts were distributed along the inner periclinal walls and the lower half of the anticlinal walls. On the contrary, mitochondria accumulated along the outer (Fig. 1B) and inner periclinal walls when illuminated with wBL. Chloroplast position was also along the outer and inner periclinal walls. Many of the mitochondria located near the chloroplasts lost their motility. When wBL-illuminated leaves were transferred back to dark condition, the numbers of mitochondria and chloroplasts present on the periclinal regions began to decrease within several hours (Fig. 1C). After 10 h dark treatment, distribution patterns of mitochondria as well as chloroplasts almost recovered to those of dark-adapted cells (Fig. 1D).Open in a separate windowFigure 1Distribution of mitochondria and chloroplasts on the outer periclinal regions of palisade mesophyll cells of A. thaliana under different light conditions. Mitochondria (green; GFP) and chloroplasts (red; chlorophyll autofluorescence) were visualized with confocal microscopy after dark adaptation (A), immediately after wBL (470 nm, 4 µmol m⁻²s⁻¹) illumination for 4 h (B), after dark treatment for 6 h (C) and 10 h (D) following the 4-h wBL illumination, respectively. Bar = 50 µm.To our knowledge, this may be the first report that directly demonstrates that wBL regulates mitochondria and chloroplast positioning in a reversible manner, though the nuclei in A. thaliana leaf cells were also found to reverse their positions when transferred from sBL to dark conditions.¹⁵ Reversible regulation of organelle positioning in leaf cells should play critical roles in adaptation of plants to highly fluctuating light conditions in the nature. Since distribution patterns of mitochondria under wBL and sBL are identical to those of chloroplasts, we can assume that phototropins, the BL receptors for chloroplast photo-relocation movement,¹⁶ may have some role in the redistribution of mitochondria. On the other hand, we also found that red light exhibited a significant effect on mitochondria positioning (Islam et al. 2009), suggesting an involvement of photosynthesis. These possibilities are now under investigation.