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.     

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.     

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.     

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).     

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.     

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.     

CLE polypeptide signaling gene expression in Arabidopsis embryos

The CLAVATA3 (CLV3)/ESR-related (CLE) family of small polypeptides mediate intercellular signaling events in plants. The biological roles of several CLE family members have been characterized, but the function of the majority still remains elusive. We recently performed a systematic expression analysis of 23 Arabidopsis CLE genes to gain insight into the developmental processes they may potentially regulate during vegetative and reproductive growth. Our study revealed that each Arabidopsis tissue expresses one or more CLE genes, suggesting that they might play roles in many developmental and/or physiological processes. Here we determined the expression patterns of nine Arabidopsis CLE gene promoters in mature embryos and compared them to the known expression patterns in seedlings. We found that more than half of these CLE genes have similar expression profiles at the embryo and seedling stages, whereas the rest differ dramatically. The implications of these findings in understanding the biological processes controlled by these CLE genes are discussed.Key words: arabidopsis, CLE, embryo, polypeptide, signalingThe CLE genes encode small, secreted polypeptides characterized by a highly conserved 14 amino-acid region at their carboxyl termini called the CLE domain.¹ To date 32 family members have been identified in Arabidopsis, yet only three have been assigned functions: CLV3, CLE40 and CLE41 have been implicated in stem cell homeostasis in shoot, root and vascular meristems, respectively.^2–5 Overexpression studies indicated that CLE genes may regulate additional biological processes as diverse as root and shoot growth, phyllotaxis, apical dominance and leaf shape and size control.^6,7 This hypothesis is consistent with our recent expression analysis of Arabidopsis A-type CLE genes,⁸ in which we found that all examined tissues expressed one or more CLE genes, in overlapping patterns. Each CLE promoter exhibited a highly distinct and specific activity profile, and many showed complex expression dynamics during vegetative and reproductive growth.Consistent with their roles in meristem maintenance, CLV3 and CLE40 are expressed early in embryogenesis when meristem initiation and organization take place.^3,5 Yet there are no other reports of CLE gene expression in Arabidopsis embryos, and therefore it is not known to what extent this family of small peptides regulates intercellular signaling events during embryogenesis. We addressed this question by analyzing the expression patterns of selected CLE promoters in mature embryos and compared them with those in 11-day-old seedlings. We chose nine CLE genes whose promoters are active in different tissues of the seedling.⁸ Transgenic dried seeds carrying a single CLE promoter sequence driving the expression of the uidA reporter gene were imbibed in water for four days, the embryos dissected out of their seed coats, and beta-glucuronidase (GUS) reporter assays performed.⁹ Stained embryos were cleared with chloral hydrate¹⁰ and visualized using a Zeiss Axiophot microscope.Five of the CLE genes analyzed showed similar promoter expression patterns in mature embryos and in seedlings. In embryos, the CLE11, 13, 16 and 17 promoters drove GUS activity in specific patterns in the root. CLE11 and CLE13 promoter activity was detected in the root cap and root apical meristem (Fig. 1A and B), CLE16 promoter activity was observed in the root cap and above the root apical meristem (Fig. 1C), and CLE17 promoter activity was seen weakly in the root apical meristem (Fig. 1D). Each of these CLE genes exhibited a similar expression pattern in seedling roots.⁸ CLE17 was additionally expressed in the embryo shoot apex and at the cotyledon margins (Fig. 1D). Similarly, in seedlings CLE17 was expressed in the vegetative shoot apex, and at the margins of the cotyledons and fully expanded leaves.⁸ In embryos, CLE27 promoter activity was strong in the hypocotyl, as well as in the medial region of the cotyledons along the main vein (Fig. 1E). In seedlings, CLE27 was strongly expressed in the hypocotyl and exhibited patchy expression in both cotyledons and leaves.⁸ Our analysis reveals that the expression of these CLE genes is established early during development and remains constant at later stages, suggesting that they may perform the same function throughout the Arabidopsis life cycle.Open in a separate windowFigure 1GUS reporter activity driven by the promoters of (A) CLE11, (B) CLE13, (C) CLE16, (D) CLE17, (E) CLE27, (F) CLE1, (G) CLE12, (H) CLE18 and (I) CLE25 in mature Arabidopsis embryos. Arrowhead indicates GUS activity in the root cap and the arrow indicates GUS activity in the root apical meristem. Scale bar, 100 µm.Remarkably, the other four CLE promoters drove embryo expression patterns that were strongly divergent from what was observed in seedlings. We found that the CLE1 promoter was active in the embryo throughout the hypocotyl and in the central region of the cotyledons (Fig. 1F), but was observed in seedlings solely in the vasculature of fully differentiated roots and at the root tips.⁸ CLE12 promoter activity in embryos was observed throughout the hypocotyl and the cotyledons (Fig. 1G), whereas in seedlings it was detected weakly in the leaf vasculature and more strongly in the root vasculature.⁸ In contrast, the CLE18 and CLE25 promoters did not drive reporter activity in mature embryos (Fig. 1H and I), despite being broadly and strongly expressed in seedlings.⁸These four CLE gene promoters show dynamic shifts in their activity between different developmental stages. From our data we infer that CLE1 activity in hypocotyls and cotyledons is required solely during embryogenesis, and that the gene then acquires a distinct function in post-embryonic root development. Similarly CLE12 appears to acquire a post-embryonic function in the root vasculature, and its broad activity in the embryonic leaves becomes restricted to the leaf vasculature following germination. Finally, the absence of CLE18 and CLE25 promoter activity in mature embryos suggests that they may be dispensable for embryo formation, and might either specifically regulate post-embryonic signaling events in certain tissues or be involved in mediating responses to environmental stimuli to which embryos are not subjected. Alternatively, they may be expressed earlier during embryogenesis and become repressed during seed dormancy.Our spatio-temporal expression analysis of a small group of CLE genes in mature embryos and seedlings indicates that the majority of these signaling molecules exert their roles beginning early in development, potentially contributing to tissue patterning and organization. Yet whereas some appear to contribute to the same biological processes throughout the plant life cycle, others seem to function in different tissues at different developmental stages. In addition, each CLE promoter studied here is active in vegetative and/or reproductive tissues that are not present in embryos, such as trichomes (CLE16 and CLE17) and style (CLE1).⁸ This observation suggests that CLE genes are widely recruited to new tissue-specific signaling functions during the course of plant development.     

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     

Auxin and Monocot Development

Monocots are known to respond differently to auxinic herbicides; hence, certain herbicides kill broadleaf (i.e., dicot) weeds while leaving lawns (i.e., monocot grasses) intact. In addition, the characters that distinguish monocots from dicots involve structures whose development is controlled by auxin. However, the molecular mechanisms controlling auxin biosynthesis, homeostasis, transport, and signal transduction appear, so far, to be conserved between monocots and dicots, although there are differences in gene copy number and expression leading to diversification in function. This article provides an update on the conservation and diversification of the roles of genes controlling auxin biosynthesis, transport, and signal transduction in root, shoot, and reproductive development in rice and maize.Auxinic herbicides have been used for decades to control dicot weeds in domestic lawns (Fig. 1A), commercial golf courses, and acres of corn, wheat, and barley, yet it is not understand how auxinic herbicides selectively kill dicots and spare monocots (Grossmann 2000; Kelley and Reichers 2007). Monocots, in particular grasses, must perceive or respond differently to exogenous synthetic auxin than dicots. It has been proposed that this selectivity is because of either limited translocation or rapid degradation of exogenous auxin (Gauvrit and Gaillardon 1991; Monaco et al. 2002), altered vascular anatomy (Monaco et al. 2002), or altered perception of auxin in monocots (Kelley and Reichers 2007). To explain these differences, there is a need to further understand the molecular basis of auxin metabolism, transport, and signaling in monocots.Open in a separate windowFigure 1.Differences between monocots and dicots. (A) A dicot weed in a lawn of grasses. Note the difference in morphology of the leaves. (B) Germinating dicot (bean) seedling. Dicots have two cotyledons (cot). Reticulate venation is apparent in the leaves. The stem below the cotyledons is called the hypocotyl (hyp). (C) Germinating monocot (maize) seedling. Monocots have a single cotyledon called the coleoptile (col) in grasses. Parallel venation is apparent in the leaves. The stem below the coleoptile is called the mesocotyl (mes).Auxin, as we have seen in previous articles, plays a major role in vegetative, reproductive, and root development in the model dicot, Arabidopsis. However, monocots have a very different anatomy from dicots (Raven et al. 2005). Many of the characters that distinguish monocots and dicots involve structures whose development is controlled by auxin: (1) As the name implies, monocots have single cotyledons, whereas dicots have two cotyledons (Fig. 1B,C). Auxin transport during embryogenesis may play a role in this difference as cotyledon number defects are often seen in auxin transport mutants (reviewed in Chandler 2008). (2) The vasculature in leaves of dicots is reticulate, whereas the vasculature in monocots is parallel (Fig. 1). Auxin functions in vascular development because many mutants defective in auxin transport, biosynthesis, or signaling have vasculature defects (Scarpella and Meijer 2004). (3) Dicots often produce a primary tap root that produces lateral roots, whereas, in monocots, especially grasses, shoot-borne adventitious roots are the most prominent component of the root system leading to the characteristic fibrous root system (Fig. 2). Auxin induces lateral-root formation in dicots and adventitious root formation in grasses (Hochholdinger and Zimmermann 2008).Open in a separate windowFigure 2.The root system in monocots. (A) Maize seedling showing the primary root (1yR), which has many lateral roots (LR). The seminal roots (SR) are a type of adventitious root produced during embryonic development. Crown roots (CR) are produced from stem tissue. (B) The base of a maize plant showing prop roots (PR), which are adventitious roots produced from basal nodes of the stem later in development.It is not yet clear if auxin controls the differences in morphology seen in dicots versus monocots. However, both conservation and diversification of mechanisms of auxin biosynthesis, homeostasis, transport, and signal transduction have been discovered so far. This article highlights the similarities and the differences in the role of auxin in monocots compared with dicots. First, the genes in each of the pathways are introduced (Part I, Table I) and then the function of these genes in development is discussed with examples from the monocot grasses, maize, and rice (Part II).