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.     

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.     

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.     

Localization of Superoxide in the Root Apex of Arabidopsis

Reactive oxygen species (ROS) fulfil many functions in plants. They have a signaling role in several physiological mechanisms, but they are also directly involved as substrates in important reactions, especially in the apoplast. Two ROS, superoxide and hydrogen peroxide, were shown to exhibit a typical accumulation pattern in the Arabidopsis root apex. While hydrogen peroxide is mainly present in the cell wall of fully elongated cells in the region of root hair formation, superoxide accumulation roughly coincides with the transition zone, between the meristem and the fast elongating zone. Developing lateral roots also exhibit a strong superoxide labeling with the same localization.Key Words: superoxide, hydrogen peroxide, cell elongation, transition zone, nitroblue tetrazoliumIn a recent work,¹ we have shown that superoxide radical and hydrogen peroxide have different accumulation sites in Arabidopsis root tip. Hydrogen peroxide is mainly present in a region identified as “differentiation zone”, according to the nomenclature used by Scheres et al.² This localization fits well with the role that was assigned to this ROS in the formation of root hairs.³ This hypothesis was strengthened by the fact that umbelliferone, which promotes the in vitro and in vivo formation of hydrogen peroxide by peroxidases, induces the formation and the elongation of root hairs. In contrast, potassium iodide, a H₂O₂ scavenger, prevents the formation of root hairs, but does not completely abolished their initiation.As for superoxide radical, it accumulates mainly in apoplast of cells ranging from the proximal part of root meristem to the point where cells initiate their fast elongation. This localization is in agreement with a role of superoxide in the cell elongation process.¹ This conclusion can be refined, taking into account the work of Baluška and coll.^4,5 Using various functional and structural criteria, these authors identified four distinct zones in the root apex of Arabidopsis. They introduced an additional zone, between the meristem and the fast elongating cells, named “transition zone”. This region comprises cells which do not divide any more and are preparing their elongation. A reappraisal of the localization of superoxide accumulation in the light of this classification could suggest that this ROS is actually mainly associated with this transition zone, rather than with the beginning of the elongation zone. Figure 1 shows an Arabidopsis root stained for the presence of superoxide with nitroblue tetrazolium. It appears that the strong superoxide staining ranges from about 80 to 250 µm away from the root tip. The respective sizes of the various zones somewhat differ from the sizes reported (in ref. ⁵). It is difficult to precisely determine the border between the meristem and the transition zone, which should be around 120 µm. The fast elongation zone begins at about 240 µm. Fast elongating cells exhibit only a slight superoxide staining in their cell wall. Therefore, it appears that superoxide accumulates mainly in the wall of cells preparing their rapid elongation. It has been reported that cells in the transition zone undergo several modifications to prepare their growth. This includes reactions leading to cell wall loosening.^6,7 The presence of superoxide in the cell wall of those cells could participate in the onset of the loosening process, for example by interacting with peroxidases to produce hydroxyl radicals.⁸Open in a separate windowFigure 1Distribution of superoxide radical in the root of a 7-day old Arabidopsis seedling stained with nitroblue tetrazolium. Growth conditions and staining procedure were as described (in ref. ¹). The scale indicates µm, starting from the root cap junction. The picture was taken with a MZ 16 Leica stereomicroscope. Arrowheads point to root hairs in formation. Black arrow, basal limit of meristem. White arrow, onset of the fast elongation zone.When roots get older, the intensity of superoxide staining in the main root tip decreases, while the apex of the newly formed lateral roots exhibits a stronger reaction (Fig. 2). This could be related to the important growth potential of young lateral roots. The emerging root primordium is usually clearly positive (Fig. 2A) and in a fully formed lateral root, superoxide staining is concentrated in a zone between the meristem and elongated cells, most likely corresponding to the transition zone (Fig. 2B). In conclusion, superoxide radical seems to accumulate in the wall of cells preparing their elongation in the transition zone of Arabidopsis root apex.Open in a separate windowFigure 2Detection of superoxide radical by nitroblue tetrazolium in a lateral root primordium marked by an arrow (A) and in a developing lateral root (B). mr, main root. Scale bar: 100 µm.     

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.     

Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes

Apoptotic caspases, such as caspase-7, are stored as inactive protease zymogens, and when activated, lead to a fate-determining switch to induce cell death. We previously discovered small molecule thiol-containing inhibitors that when tethered revealed an allosteric site and trapped a conformation similar to the zymogen form of the enzyme. We noted three structural transitions that the compounds induced: (i) breaking of an interaction between Tyr-223 and Arg-187 in the allosteric site, which prevents proper ordering of the catalytic cysteine; (ii) pinning the L2′ loop over the allosteric site, which blocks critical interactions for proper ordering of the substrate-binding groove; and (iii) a hinge-like rotation at Gly-188 positioned after the catalytic Cys-186 and Arg-187. Here we report a systematic mutational analysis of these regions to dissect their functional importance to mediate the allosteric transition induced by these compounds. Mutating the hinge Gly-188 to the restrictive proline causes a massive ∼6000-fold reduction in catalytic efficiency. Mutations in the Arg-187–Tyr-223 couple have a far less dramatic effect (3–20-fold reductions). Interestingly, although the allosteric couple mutants still allow binding and allosteric inhibition, they partially relieve the mutual exclusivity of binding between inhibitors at the active and allosteric sites. These data highlight a small set of residues critical for mediating the transition from active to inactive zymogen-like states.Caspases are a family of dimeric cysteine proteases whose members control the ultimate steps for apoptosis (programmed cell death) or innate inflammation among others (for reviews, see Refs. 1 and 2). During apoptosis, the upstream initiator caspases (caspase-8 and -9) activate the downstream executioner caspases (caspase-3, -6, and-7) via zymogen maturation (3). The activated executioner caspases then cleave upwards of 500 key proteins (4–6) and DNA, leading to cell death. Due to their pivotal role in apoptosis, the caspases are involved both in embryonic development and in dysfunction in diseases including cancer and stroke (7). The 11 human caspases share a common active site cysteine-histidine dyad (8), and derive their name, cysteine aspartate proteases, from their exquisite specificity for cleaving substrate proteins after specific aspartate residues (9–13). Thus, it has been difficult to develop active site-directed inhibitors with significant specificity for one caspase over the others (14). Despite difficulties in obtaining specificity, there has been a long-standing correlation between efficacy of caspase inhibitors in vitro and their ability to inhibit caspases and apoptosis in vivo (for review, see Ref. 31). Thus, a clear understanding of in vitro inhibitor function is central to the ability control caspase function in vivo.Caspase-7 has been a paradigm for understanding the structure and dynamics of the executioner caspases (15–21). The substrate-binding site is composed of four loops; L2, L3, and L4 are contributed from one-half of the caspase dimer, and L2′ is contributed from the other half of the caspase dimer (Fig. 1). These loops appear highly dynamic as they are only observed in x-ray structures when bound to substrate or substrate analogs in the catalytically competent conformation (17–19, 22) (Fig. 1B).Open in a separate windowFIGURE 1.Allosteric site and dimeric structure in caspase-7. A, the surface of active site-bound caspase-7 shows a large open allosteric (yellow) site at the dimer interface. This cavity is distinct from the active sites, which are bound with the active site inhibitor DEVD (green sticks). B, large subunits of caspase-7 dimers (dark green and dark purple) contain the active site cysteine-histidine dyad. The small subunits (light green and light purple) contain the allosteric site cysteine 290. The conformation of the substrate-binding loops (L2, L2′, L3, and L4) in active caspase-7 (Protein Data Bank (PDB) number 1f1j) is depicted. The L2′ loop (spheres) from one-half of the dimer interacts with the L2 loop from the other half of the dimer. C, binding of allosteric inhibitors influences the conformation of the L2′ loop (spheres), which folds over the allosteric cavity (PDB number 1shj). Subunit rendering is as in panel A. Panels A, B, and C are in the same orientation.A potential alternative to active site inhibitors are allosteric inhibitors that have been seeded by the discovery of selective cysteine-tethered allosteric inhibitors for either apoptotic executioner caspase-3 or apoptotic executioner caspase-7 (23) as well as the inflammatory caspase-1 (24). These thiol-containing compounds bind to a putative allosteric site through disulfide bond formation with a thiol in the cavity at the dimer interface (Fig. 1A) (23, 24). X-ray structures of caspase-7 bound to allosteric inhibitors FICA³ and DICA (Fig. 2) show that these compounds trigger conformational rearrangements that stabilize the inactive zymogen-like conformation over the substrate-bound, active conformation. The ability of small molecules to hold mature caspase-7 in a conformation that mimics the naturally occurring, inactive zymogen state underscores the utility and biological relevance of the allosteric mechanism of inhibition. Several structural changes are evident between these allosterically inhibited and active states. (i) The allosteric inhibitors directly disrupt an interaction between Arg-187 (next to the catalytic Cys-186) and Tyr-223 that springs the Arg-187 into the active site (Fig. 3), (ii) this conformational change appears to be facilitated by a hinge-like movement about Gly-188, and (iii) the L2′ loop folds down to cover the allosteric inhibitor and assumes a zymogen-like conformation (Fig. 1C) (23).Open in a separate windowFIGURE 2.Structure of allosteric inhibitors DICA and FICA. DICA and FICA are hydrophobic small molecules that bind to an allosteric site at the dimer interface of caspase-7. Binding of DICA/FICA is mediated by a disulfide between the compound thiol and Cys-290 in caspase-7.Open in a separate windowFIGURE 3.Movement of L2′ blocking arm. The region of caspase-7 encompassing the allosteric couple Arg-187 and Tyr-223 is boxed. The inset shows the down orientation of Arg-187 and Tyr-223 in the active conformation with DEVD substrate mimic (orange spheres) in the active site. In the allosteric/zymogen conformation, Arg-187 and Tyr-223 are pushed up by DICA (blue spheres).Here, using mutational analysis and small molecule inhibitors, we assess the importance of these three structural units to modulate both the inhibition of the enzyme and the coupling between allosteric and active site labeling. Our data suggest that the hinge movement and pinning of the L2-L2′ are most critical for transitioning between the active and inactive forms of the enzyme.     

The H+-ATPase in the Plasma Membrane of Saccharomyces cerevisiae Is Activated during Growth Latency in Octanoic Acid-Supplemented Medium Accompanying the Decrease in Intracellular pH and Cell Viability

Saccharomyces cerevisiae plasma membrane H⁺-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pH_i). The cell population used as inoculum exhibited a significant heterogeneity of pH_i, and the fall of pH_i correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.The biological target sites of octanoic acid in Saccharomyces cerevisiae may be related to processes of transport across membranes, particularly the plasma membrane (21). Like other weak acids at low pH, octanoic acid, a highly toxic by-product of yeast alcoholic fermentation (23) and an antimicrobial food additive (6), leads to the reduction of cytosolic pH (pH_i) due to its dissociation in the approximately neutral cytoplasm following the entrance of the undissociated toxic form into the cell by passive diffusion (5, 20, 23). It is likely that this highly liposoluble weak acid significantly affects the spatial organization of the plasma membrane, affecting its function as a matrix for enzymes and as a selective barrier, thereby leading to the dissipation of the proton motive force across the plasma membrane and to intracellular acidification (16, 18). Significantly, the H⁺-ATPase in the plasma membrane in yeast, which creates the electrochemical proton gradient that drives the secondary transport of solutes and is implicated in the maintenance of pH_i around neutrality, has been pointed out as a critical component of yeast adaptation to weak acids (8, 19, 24). Indeed, yeast plasma membrane H⁺-ATPase is activated during exponential growth with octanoic acid (19, 24), and the duration of lag phase before yeast cells enter exponential growth in the presence of sorbic acid is significantly extended in a mutant with reduced levels of plasma membrane ATPase activity (8). The activation of the H⁺-ATPase in the plasma membrane in yeast cells exposed to other stresses that also lead to the dissipation of the H⁺ gradient and intracellular acidification (such as subcritical inhibitory concentrations of ethanol [12, 14, 15], supraoptimal temperatures below 40°C [25], presence of other organic acids at low pH [1, 5, 8], and deprivation of nitrogen source [2]) have also been observed. Several lines of evidence indicate that ATPase activation is due to posttranslational modifications of the PMA1 ATPase (2, 12, 24, 25). Considerable information has been obtained on the variation of plasma membrane ATPase activity during exponential growth and early stationary phase of yeast cells cultivated in media, at low pH, supplemented or not with octanoic acid (24). However, this is not the case during the period of latency preceding exponential growth at concentrations of octanoic acid close to the maximal concentration allowing growth. The main objective of the present work was to obtain information about the pattern of ATPase activity and the changes in pH_i and cell viability during the lag phase necessary for yeast adaptation to the physiological effects of octanoic acid before exponential growth.

Duration of yeast growth latency in octanoic acid-supplemented media.

When cells of S. cerevisiae IGC3507III grown, at 30°C, in medium that had not been supplemented with octanoic acid were used to inoculate buffered YG media (30 g of glucose liter⁻¹, 6.7 g of Yeast Nitrogen Base [Difco] liter⁻¹) (pH 4.0) supplemented with increasing concentrations of this toxic acid up to around 0.35 mM total acid (19, 23), exponential growth was initiated without significant delay (Fig. ​(Fig.1a),1a), although a dose-dependent decrease in specific growth rate was observed (Fig. ​(Fig.1b).1b). However, for higher concentrations up to the maximal that allowed growth (0.42 mM), a lag phase was observed and its duration strongly increased with the severity of octanoic acid stress (Fig. ​(Fig.1a).1a). The duration of latency was drastically reduced when exponential cells used as inoculum were grown in medium with an identical concentration of octanoic acid (Fig. ​(Fig.1a),1a), but the specific growth rate was not modified (Fig. ​(Fig.1b).1b). At a concentration of total octanoic acid of 0.39 mM, a lag phase of around 55 h was necessary for yeast cells, which had been cultivated under nonstressing conditions, to adapt to the deleterious effects of octanoic acid and to initiate inhibited exponential growth (Fig. ​(Fig.2).2). Open in a separate windowFIG. 1Effect of the addition to the growth medium of increasing concentrations of octanoic acid on the duration of lag phase (a) and the specific growth rate of S. cerevisiae IGC 3507III (b) for exponentially growing cells (used as inoculum) cultivated at 30°C at pH 4.0 in the absence (○) or presence (•) of concentrations of toxic lipophilic acid identical to those present in the growth medium. Results are representative of the many growth experiments carried out.Open in a separate windowFIG. 2Specific activity of plasma membrane H⁺-ATPase (filled symbols) and growth curve (open symbols) of S. cerevisiae IGC 3507III during cultivation in the presence (a) or absence (b) of 0.39 mM total octanoic acid (at pH 4.0, 30°C). The data are averages with standard deviations for at least three enzyme assays using cells from at least two independent growth experiments. OD, optical density.

Activation of plasma membrane ATPase during octanoic acid-induced latency.

The specific activity of plasma membrane ATPase assayed in crude membrane suspensions prepared from nonadapted cells, as previously reported (19, 25), during cultivation in buffered medium (at pH 4.0) supplemented with 0.39 mM octanoic acid, increased during the 55 h of latency (Fig. ​(Fig.2a).2a). A peak of activity was reached during the early stages of exponential growth and values of ATPase activity were consistently higher (twofold) in cells grown under octanoic acid stress (Fig. ​(Fig.2),2), as described by Viegas et al. (24). Yeast cells must adapt to the physiological effects of octanoic acid during an extended lag period, the length of which depended on the severity of acid stress (Fig. ​(Fig.1a),1a), before eventually recovering and entering exponential growth; the activation of plasma membrane H⁺-ATPase observed during this period of latency reinforces the idea that this proton pump is an important component of this adaptative response (5, 8, 19, 24). In fact, the ability of yeast cells to grow in the presence of lipophilic acids at a low pH reflects their capacity to maintain control over their internal pH by excluding protons. This adaptative phenomenon, reported for the first time in the present work, complements the observation of Holyoak et al. (8) that a strain with reduced plasma membrane H⁺-ATPase activity displayed increased lag phase in the presence of the weak-acid preservative sorbic acid. Significantly, plasma membrane H⁺-ATPase activity was also pointed out to play a critical role in yeast tolerance of ethanol (15) or supraoptimal temperatures (13, 25). The mechanism underlying plasma membrane ATPase activation during octanoic acid-induced latency remains obscure at the present time, but it is likely that this is due to a posttranslational modification of ATPase, as proposed for ATPase activation during octanoic acid-stressed exponential growth (24). It is likely that during lag phase the amount of H⁺-ATPase in the plasma membrane slightly decreases, as found by Benito et al. (2) in yeast cells deprived of nitrogen source where ATPase activation also occurred (2), as the estimated half-life of the enzyme is about 11 h (2). ATPase activation during latency can hardly be attributed to the adaptative modification of the ATPase lipid environment in cells grown under lipophilic acid stress, as suggested by Alexandre et al. (1).

Changes in yeast pH_i and viability during octanoic acid-stressed cultivation.

The change in pH_i during cultivation of nonadapted cells with 0.39 mM octanoic acid was monitored by using an adaptation of the fluorescence microscopic image processing technique developed by Imai and Ohno (9); 5- (and 6)-carboxyfluorescein (cF) was used as the internal pH-dependent fluoroprobe. Cells washed and resuspended in cold CF buffer (citrate-phosphate buffer [at pH 4.0] with 50 mM glycine [Sigma], 110 mM NaCl, 5 mM KCl, and 1 mM MgCl₂) to a cellular density of 2 × 10⁸ ml⁻¹ were loaded with cF by adding 20 μM of 5 (and 6)-carboxyfluorescein-diacetate (Sigma) and vortexing in two bursts of 1 min each, interspersed with 15 min on ice (9). After being washed twice with cold CF buffer, cF-loaded cells were immediately examined with a Zeiss Axioplan microscope equipped with adequate epifluorescence interference filters (Zeiss BP450-490 and Zeiss LP520) and connected to a video camera and to a computer with an image- analysis program (gel documentation system SW2000; UVP, San Gabriel, Calif.). Following a cell-by-cell analysis, the value of fluorescence intensity (fI) emitted by each cell, measured by direct densitometry, corresponded to the arithmetical mean value of fI measured in two or three different regions in the cytoplasm of the same cell, with the less fluorescent vacuole excluded. To estimate average pH_i, an in vivo calibration curve was prepared (Fig. ​(Fig.3)3) by using cell suspensions grown in the absence of toxics which were loaded with cF as described above and incubated, at 30°C, with 0.5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to dissipate the plasma membrane pH gradient (4), before adjustment of external pH (in the range 3.5 to 7.5) by the addition of HCl or NaOH at 2 M. Fluorescence images were fixed 15 s after the occurrence of the excitation radiation in order to minimize interferences due to leakage of cF as well as fluorescence quenching (3, 7). Cells were kept on ice throughout the procedure, and CF buffer lacked glucose; therefore, the active efflux of cF (3) was minimized as confirmed by measuring the fluorescence in the medium surrounding the cells, which was negligible. Under the experimental conditions used and for the purpose of the study, this technique proved to be highly useful and suitable despite the limitations that might be raised (3, 7). It allowed a clear-cut picture of the pH_i of individual cells, giving information about the distribution of pH_i values of a yeast population (Fig. ​(Fig.44 and ​and5a5a to c), instead of solely an estimation of the average value of the whole population, as is the case with techniques based on the distribution of radioactive propionic acid (20) or on the in vivo ³¹P nuclear magnetic resonance (5). Moreover, values calculated for the average pH_i of the whole yeast population during latency and exponential growth in medium with octanoic acid (Fig. ​(Fig.5d)5d) were close to, although slightly lower than, the values previously obtained based on the distribution of [¹⁴C]propionic acid (20, 22). Results revealed that the cell population used to inoculate octanoic acid-supplemented medium exhibited a significant heterogeneity (Fig. ​(Fig.4);4); around 31% showed a pH_i in the optimal range (above 6.5) (Fig. ​(Fig.4),4), with the average pH_i value of the whole population estimated to be approximately 6.0. This low pH_i value results from cell cultivation in a rich medium with high production of organic acids (11) (external pH, 3.6), followed by washing of the cells with YG medium buffered at pH 4.0 (17). The introduction of the inoculum in octanoic acid-supplemented medium led to the very rapid (5-min) increase in the percentage of the cell population with pH_i below 5.5, consistent with the rapid kinetics of cytosol acidification when yeast cells are exposed to weak acids (5). During extended incubation with octanoic acid and until the end of latency, the percentage of the population with a very low pH_i (below 5.5) continued to increase, reaching 80% of the cell population, while the percentage of cell population with a pH_i above 6.0 suffered a corresponding decrease (Fig. ​(Fig.5).5). During exponential growth, the opposite pH_i modification was observed, consistent with a recovery of pH_i to physiological levels (Fig. ​(Fig.5).5). The time-dependent pattern of internal acidification during lag phase correlated with plasma membrane ATPase activation (Fig. ​(Fig.2a2a and ​and5),5), suggesting that this activation was triggered by intracellular acidification, as proposed for acetic acid (5)- or nitrogen starvation (2)-induced activation. Immediately before yeast cells entered exponential growth, 80% of the initial viable population had lost viability, as assessed by the number of CFU (21) (Fig. ​(Fig.6),6), suggesting that octanoic acid-induced death during latency is related to internal acidification down to critical values (Fig. ​(Fig.55 and ​and6),6), in agreement with the relationship established by Imai and Ohno (10) between yeast viability and intracellular pH. Only about 20% of the initial population was able to start cell division in octanoic acid-supplemented medium, presumably those cells that in the inoculum exhibited pH_i values around neutrality (Fig. ​(Fig.55 and ​and6).6). These results suggest that despite plasma membrane H⁺- ATPase activation, this system of pH homeostasis may not be able to fully counteract the physiological effects of increasing octanoic acid concentrations and eventually fails at very severe acid stress. Open in a separate windowFIG. 3In vivo calibration curve, showing the pH dependence of the fI of cF-loaded-cells of S. cerevisiae IGC 3507III. Intracellular and extracellular pHs were equilibrated by incubation of cF-loaded cells, for 10 min at 30°C, with 0.5 mM CCCP. At each pH, values of fI correspond to the average fI of about 20 cells. The data are averages with standard deviations for three independent experiments.Open in a separate windowFIG. 4Distribution of cells with different pH_i values present in the inoculum of S. cerevisiae IGC 3507III prepared in growth medium without octanoic acid supplementation.Open in a separate windowFIG. 5Percentage of yeast cells with pH_i below 5.5 (a), between 5.5 and 6.0 (b), or above 6.0 (c); average pH_i of the whole cell population (▴) during S. cerevisiae IGC3507III cultivation in medium supplemented with 0.39 mM total octanoic acid (pH 4.0, 30°C); and the optical density (OD) of the culture at 600 nm (▪). The average pH_i values estimated for the whole cell population are the arithmetical mean values of the various average pH_i values calculated for individual cells. The percentage of cells present in the inoculum with pH_i values within the three ranges (○) and the average pH_i of the inoculum cell population (▵) are indicated.Open in a separate windowFIG. 6Concentration of viable cells (▴) and culture optical density (O.D.) at 600 nm (□) during lag and exponential phases of S. cerevisiae IGC 3507III growth in medium supplemented with 0.39 mM octanoic acid, at pH 4.0 and 30°C.

Adaptative response to octanoic acid.

The adaptation of yeast cells to octanoic acid at a low pH appears to depend on their H⁺-exporting ability, but this requires not only a highly active H⁺-ATPase in the plasma membrane but the provision of sufficient ATP to drive this energy-demanding process as indicated by the results of Holyoak et al. (8). It is likely that increased ATPase activity under octanoic acid stress may reduce cellular ATP levels and that ATP depletion contributes to the failure of the maintenance of pH_i homeostasis, particularly among the subpopulation that in the inoculum exhibited the lowest pH_i values. The loss of viability might occur for those cells where pH_i decreased down to nonphysiological values. The eventual recovery of growth therefore depends on the remaining viable population, in agreement with the well-known critical role played by the physiology and number of viable cells in the inoculum in the duration of latency under acid stress. The observation that octanoic acid-adapted cells reinoculated into the same fresh medium can resume growth after a much shorter latency (Fig. ​(Fig.1a)1a) is a good example of the importance of the physiology of the inoculum cells. Besides the increased plasma membrane H⁺-ATPase activity of octanoic acid-adapted cells, other mechanisms may underlie the adaptation to acid stress, such as the increased cellular buffering capacity of octanoic acid-grown cells due to their lower intracellular volume (20), the more favorable plasma membrane lipid composition (1), and the possible induction of the active efflux of the anion (26).     

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.