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.     

Triple X Egyptian woman and a Down's syndrome offspring

The 47, XXX karyotype (triple X) has a frequency of 1 in 1000 female newborns. However, this karyotype is not usually suspected at birth or childhood. Female patients with a sex chromosome abnormality may be fertile. In patients with a 47, XXX cell line there appears to be an increased risk of a cytogenetically abnormal child but the extent of this risk cannot yet be determined; it is probably lower in the non-mosaic 47, XXX patient than the mosaic 46, XX/47, XXX one. We describe a new rare case of triple X woman and a Down''s syndrome offspring. The patient is 26 years of age. She is a housewife, her height is 160 cm and weight is 68 kg and her physical features and mentality are normal. She has had one pregnancy at the age of 25 years resulted in a girl with Down''s syndrome. The child had 47 chromosomes with trisomy 21 (47, XX, +21) Figure 1. The patient also has 47 chromosomes with a triple X karyotype (47, XX, +X) Figure 2. The patient''s husband (27 years old) is physically and mentally normal. He has 46 chromosomes with a normal XY karyotype (46, XY). There are neither Consanguinity between her parent''s nor she and her husband.Open in a separate windowFigure 1Karyotype 47, XX + 21 of the daughter of Triple X syndromeOpen in a separate windowFigure 2Karyptype 47, XX + X of the Down syndrome''s mother     

Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity.

The evolution of modern cells is arguably the most challenging and important problem the field of biology has ever faced …—Carl R. Woese(Woese 2002)

Current models may accept that all modern eukaryotic cells arose from a single common ancestor (the cenancestral eukaryote), the nature of which is—owing to the lack of direct living or fossil descendants—still highly under debate (de Duve 2007). The chimeric nature of eukaryotic genomes with eubacterial and archaebacterial shares led to a discussion about the origin of this first “proto-eukaryote.” Several models exist (see Fig. 1), which either place the evolution of the nucleus before or after the emergence of the mitochondrion (outlined in Koonin 2010; Martijn and Ettema 2013). According to the different postulated scenarios (summarized in Embley and Martin 2006), eukaryotes in the latter case might have evolved by endosymbiosis between a hydrogen-producing, oxygen-producing, or sulfur-dependent α-proteobacterium and an archaebacterial host (Fig. 1C). The resulting mitochondriate prokaryote would have evolved the nucleus subsequently. In other scenarios (Fig. 1B), the cenancestral eukaryote emerged by cellular fusion or endosymbiosis of a Gram-negative, maybe hydrogen-producing, eubacterium and a methanogenic archaebacterium or eocyte, leading to a primitive but nucleated amitochondrial (archezoan) cell (Embley and Martin 2006, and references therein). As a third alternative, Cavalier-Smith (2002) suggested a common eubacterial ancestor for eukaryotes and archaebacteria (the Neomuran hypothesis) (Fig. 1A).Open in a separate windowFigure 1.Evolution of the last common ancestor of all eukaryotic cells. A schematic depiction of the early eukaryogenesis. Because of the lack of living and fossil descendants, several opposing models are discussed (A–C). The anticipated order of events is shown as a flow chart. For details, see text. (Derived from Embley and Martin 2006; Koonin 2010.)     

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.     

Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT.Key words: Testicular neoplasm, Sertoli cell tumor, Sclerosing Sertoli cell tumorA 23-year-old man was referred to the Cleveland Clinic Department of Urology (Cleveland, OH) for an incidentally detected right testicular mass. The mass was identified during a work-up for transient left testicular discomfort. His only notable medical history was nephrolithiasis. There was no personal or family history of testicular cancer or cryptorchidism. On physical examination, he was a well-nourished, well-masculinized young man without gynecomastia. Testicular examination revealed normal volume and consistency bilaterally without other relevant findings. Testicular ultrasonography demonstrated an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle with peripheral flow on color Doppler (Figure 1).Open in a separate windowFigure 1Testicular ultrasound demonstrating an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle (blue arrows).The remainder of the ultrasound examination yielded normal results. Lactic dehydrogenase, B-human chorionic gonadotropin, and α-fetoprotein levels were all within the normal range. After a thorough review of the options, the patient was then taken to the operating room for inguinal exploration. Intraoperative ultrasound confirmed a superficial 8-mm hypoechoic testis lesion. A whiteyellow, well-demarcated nodule was widely excised and a frozen section was sent to pathology for examination. The frozen section examination revealed the lesion to be a neoplasm with differential diagnosis including sclerosing Sertoli cell tumor (SSCT), adenomatoid tumor, and a variant of Leydig cell tumor. Because the final diagnosis could not be determined from frozen section, the decision was made to perform a right radical orchiectomy. Pathologic examination revealed a grossly unifocal, well-circumscribed, white, firm mass of 0.8 cm. Microscopically the lesion was composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Although the lesion was somewhat well circumscribed, entrapped seminiferous tubules with Sertoli-only cells were present within the tumor (Figure 2). Tumor cells had pale or eosinophilic cytoplasm with small and dark nuclei with inconspicuous nucleoli. The tumor was confined to the testis and margins were negative. A diagnosis of SSCT was reached, supported by positive immunostain results for steroidogenic factor 1, focal inhibin, and calretinin expression, and negative stain results for cytokeratin AE1/AE3 and epithelial membrane antigen in the tumor (Figure 3). The postoperative course was unremarkable. Computed tomography scan of the abdomen and pelvis and chest radiograph were negative for metastatic disease.Open in a separate windowFigure 2Low-power examination revealing a well-circumscribed tumor composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Hematoxylin and eosin stain, original magnification ×40. (B) High-power examination. Note entrapped seminiferous tubules lacking spermatogenesis. Hematoxylin and eosin stain, original magnification ×100.Open in a separate windowFigure 3Nuclear expression of steroidogenic factor 1 in the tumor as well as benign Sertoli cells in entrapped seminiferous tubules (original magnification ×200). (B) Focal calretinin expression in the tumor (inhibin had a similar staining pattern; original magnification ×100).     

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.     

Mutational Biosynthesis of Novel Rapamycins by a Strain of Streptomyces hygroscopicus NRRL 5491 Disrupted in rapL,Encoding a Putative Lysine Cyclodeaminase

The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added l-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific l-lysine cyclodeaminase important for the production of the l-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.Rapamycin is a 31-member macrocyclic polyketide produced by Streptomyces hygroscopicus NRRL 5491 which, like the structurally related compounds FK506 and immunomycin (Fig. ​(Fig.1),1), has potent immunosuppressive properties (24). Such compounds are potentially valuable in the treatment of autoimmune diseases and in preventing the rejection of transplanted tissues (16). The biosynthesis of rapamycin requires a modular polyketide synthase, which uses a shikimate-derived starter unit (11, 20) and which carries out a total of fourteen successive cycles of polyketide chain elongation that resemble the steps in fatty acid biosynthesis (2, 27). l-Pipecolic acid is then incorporated (21) into the chain, followed by closure of the macrocyclic ring, and both these steps are believed to be catalyzed by a pipecolate-incorporating enzyme (PIE) (18), the product of the rapP gene (8, 15). Further site-specific oxidations and O-methylation steps (15) are then required to produce rapamycin. Open in a separate windowFIG. 1Structures of rapamycin, FK506, and immunomycin.The origin of the pipecolic acid inserted into rapamycin has been previously established (21) to be free l-pipecolic acid derived from l-lysine (although the possible role of d-lysine as a precursor must also be borne in mind) (9). Previous work with other systems has suggested several alternative pathways for pipecolate formation from lysine (22), but the results of the incorporation of labelled lysine into the pipecolate moiety of immunomycin (Fig. ​(Fig.1)1) clearly indicate loss of the α-nitrogen atom (3). More recently, the sequencing of the rap gene cluster revealed the presence of the rapL gene (Fig. ​(Fig.2),2), whose deduced gene product bears striking sequence similarity to two isoenzymes of ornithine deaminase from Agrobacterium tumefaciens (25, 26). Ornithine deaminase catalyzes the deaminative cyclization of ornithine to proline, and we have proposed (15) that the rapL gene product catalyzes the analogous conversion of l-lysine to l-pipecolate (Fig. ​(Fig.3).3). Open in a separate windowFIG. 2A portion of the rapamycin biosynthetic gene cluster which contains ancillary (non-polyketide synthase) genes (15, 27). PKS, polyketide synthase.Open in a separate windowFIG. 3(A) The conversion of l-ornithine to l-proline by ornithine cyclodeaminase (17). (B) Proposed conversion of l-lysine to l-pipecolic acid by the rapL gene product.Here, we report the use of ΦC31 phage-mediated gene replacement (10) to introduce a frameshift mutation into rapL and the ability of the mutant to synthesize rapamycins in the absence or presence of added pipecolate or pipecolate analogs.     

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.     

Negative effects of desiccation on the protein sorting and post-translational modification

Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress.¹ In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition.Key words: AP-1 protein, cell ultrastructure, desiccation, golgi bodies, physcomitrella, proteomeThe plant Golgi apparatus is composed of many small stacks of cisternae, sometimes known as dictyosomes. The Golgi is a complex polarized organelle consisting of both a cis and trans side, containing compartments with functionally different capacities for directing cellular components. The plant Golgi apparatus synthesis a wide range of cell wall polysaccharides and proteoglycans, and also carries out O-linked glycosylation and N-linked glycan processing.^2–5 Moreover, the Golgi is involved in returning escaped proteins back to the endoplasmic reticulum, sorting of proteins and polysaccharides to the cell wall or vacuoles, and in organizing the compartmentation of its own enzymes by retention or retrieval mechanisms.⁶ In conclusion, The Golgi apparatus is central to the growth and division of the plant cell through its roles in protein glycosylation, protein sorting and cell wall synthesis.The transit of proteins and lipids from the trans-Golgi network (TGN) and the plasma membrane to endosomes within eucaryotic cells occurs via the budding and fusion of clathrin-coated vesicles (CCVs).^7,8 At the TGN, this process is mediated by the heterotetrameric AP-1 adaptor complex, which consists of two large subunits, β and γ1; a medium subunit, µ1; and a small σ1 subunit. Recruitment of AP-1 to the TGN membrane is regulated by a small GTPase, ADP-ribosylation factor 1 (ARF1), which cycles between an inactive GDP-bound form in cytosol and an active GTP-bound form that associates with the membrane like other small GTPase.⁹ There is also evidence that phosphorylation/dephosphorylation events are involved in the regulation of the function of AP-1. Ghosh and Kornfeld demostrated that AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its β1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated β1 compared with phosphorylated µ1. Once on the membrane, the µ1 subunit undergoes phosphorylation, which results in a conformation change. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of µ1 (and µ2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.¹⁰Plants experience desiccation stress either as part of a developmental programme, such as during seed maturation, or because of reductions in air humid and water availability in the soil. Underlying the ability of bryophytes to withstand periods of desiccation are morphological and biochemical adaptations. Plants respond to stress as individual cells and synergistically as a whole organism. Scanning electron microscopy observation showed that the P. patens gametophore cells were shrunk upon the treatment of desiccation, and the shrinking started from the edge of the leaves (Fig. 1). We could clearly observe some dark granula in the untreated cells, but these granula disappeared post-desiccation treatment (Fig. 1). Transmission electron microscopy also revealed that the large stacks of Golgi bodies and numerous coated vesicles are typically visible in the hydrated cells (Fig. 2), but these are absent in the desiccative cells (data not shown). The plant Golgi apparatus plays an important role in protein glycosylation and sorting. Therefore, this event means that the protein sorting and the cargo transporting are disrupted by desiccation stress. During desiccation, the absentness of Golgi bodies reduce the leaf activities of cell, and this is expected to similar to plant dormancy which is a phenomenon in resurrection plants and some drought-tolerant plants. In addition, through two-dimensional gel electrophoresis (2-DE) and LC-MS/MS analysis, AP-1 large subunit was identified as downregulated protein during the course of dehydration (Fig. 3). AP-1 is ubiquitously expressed and participates in the budding of clathrin-coated vesicles from the trans-Golgi network (TGN) and endosomes. AP-1 also recognizes sorting motifs in cargo molecules. Our results suggested that desiccation led to a marked disrupt in protein posttranslational modification, protein sorting and cell walls synthesis.Open in a separate windowFigure 1Scanning Electron microscopy images of normal and dehydrated P. patens gametophores. (A) the fresh leaf; (B) enlargement of the rectangle area of (A); (C) dehydrated gametophores of P. patens. Bar = 5 µm.Open in a separate windowFigure 2Transmission electron microscopy images of cell in fresh game-tophores. The arrows indicate Golgi body, Bar = 2 µm.Open in a separate windowFigure 3Part protein profile of the control and desiccation plants. The arrows indicate the AP-1 large subunit.     

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.