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PubMed2004

Nodulin 22 from Phaseolus vulgaris protects Escherichia coli cells from oxidative stress.

Plant genes that are induced during the formation and function of a root nodule are called nodulin genes. Cloning and functional analysis of nodule-specific gene products are of valuable help in establishing the role and requirements of the host plant for the specificity and effectiveness of the symbiosis. A cDNA clone (nod22) was isolated from Phaseolus vulgaris L. (common bean) cDNA library derived from Rhizobium-infected roots. Nodulin 22 (Nod22) transcripts are accumulated from early to late stages in root nodule development. RT-PCR in situ studies indicated that Nod22 transcripts are highly accumulated in cortical, vascular bundle and infected cells. The deduced Nod22 protein contains a highly hydrophobic N-terminus, with signal peptide characteristics, and a C-terminal extension with high identity to the alpha-crystallin domains found in alpha-crystallin lens chaperone, and other small heat-shock proteins. These domains have not been previously described in other known nodulins, but have been observed in small heat-shock proteins found in plant tissues exposed to elevated temperature and oxidative stress. Nod22, when it is over-expressed in Escherichia coli, cells confers protection against oxidative stress suggesting its possible role in plant host protection from oxidative toxicity during the Rhizobium-legume symbiosis.

Amino Acid Sequence↗
PubMed1999

NADH-glutamate synthase in alfalfa root nodules. Genetic regulation and cellular expression.

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric beta-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

Amino Acid Oxidoreductases↗
PubMed1998

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus.

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of approximately 1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.

Crosses, Genetic↗
PubMed2005

The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses.

Plants sense phosphate (Pi) deficiency and initiate signaling that controls adaptive responses necessary for Pi acquisition. Herein, evidence establishes that AtSIZ1 is a plant small ubiquitin-like modifier (SUMO) E3 ligase and is a focal controller of Pi starvation-dependent responses. T-DNA insertional mutated alleles of AtSIZ1 (At5g60410) cause Arabidopsis to exhibit exaggerated prototypical Pi starvation responses, including cessation of primary root growth, extensive lateral root and root hair development, increase in root/shoot mass ratio, and greater anthocyanin accumulation, even though intracellular Pi levels in siz1 plants were similar to wild type. AtSIZ1 has SUMO E3 ligase activity in vitro, and immunoblot analysis revealed that the protein sumoylation profile is impaired in siz1 plants. AtSIZ1-GFP was localized to nuclear foci. Steadystate transcript abundances of Pi starvation-responsive genes AtPT2, AtPS2, and AtPS3 were moderate but clearly greater in siz1 seedlings than in wild type, where Pi is sufficient. Pi starvation induced the expression of these genes to the same extent in siz1 and wild-type seedlings. However, two other Pi starvation-responsive genes, AtIPS1 and AtRNS1, are induced more slowly in siz1 seedlings by Pi limitation. PHR1, a MYB transcriptional activator of AtIPS1 and AtRNS1, is an AtSIZ1 sumoylation target. These results indicate that AtSIZ1 is a SUMO E3 ligase and that sumoylation is a control mechanism that acts both negatively and positively on different Pi deficiency responses.

Anthocyanins↗
PubMed2004

Development of extraradical mycelium of Scutellospora reticulata under root-organ culture: spore production and function of auxiliary cells.

The development of the extraradical mycelium and auxiliary cells and spore production of Scutellospora reticulata in association with Ri T-DNA transformed carrot roots was followed under root-organ culture conditions. Extraradical mycelium development followed classical lag-exponential-plateau phases, with an additional late decline phase in number of auxiliary cells. Spore production started in parallel with a critical extraradical mycelium biomass produced, continued long after root growth ceased and during the late decline in auxiliary cells number. Isolated auxiliary cells were shown to exhibit hyphal re-growth, but not root colonization, either in situ or in vitro. These results showed that root and extraradical mycelium development were intimately associated in a sequence where both grew together during active root growth, followed during root aging by a period in which only the fungus developed. Spore production appeared dependent on a critical extraradical mycelium biomass and on the re-allocation of resources from both the intraradical mycelium and the auxiliary cells via the hyphal network.

Mycelium↗
PubMed2001

Spatial and temporal distribution of sucrose synthase in the radish hypocotyl in relation to thickening growth.

Sucrose synthase (SuSy) is a key enzyme in the development of storage root of radish. Clarification of its spatial and temporal expression during the thickening growth of radish hypocotyl, which later develops into storage root, was carried out immunologically using light microscopy. Sequential harvests at 3, 7, 11 and 13 d after sowing (DAS) were performed on two radish cultivars having different sink capacity. A very low level of SuSy was observed 3 DAS for both cultivars. White Cherrish (WC; strong storage root) showed the maximum level of SuSy between 7 and 11 DAS with increased cell development (thickening), while in Kosena (K; low storage root) the level remained high after 13 d of growth. A high level of SuSy was found in companion cells, which was consistent with previous observations, but SuSy was also found in the xylem parenchyma and in some cortical cells. The level of SuSy differed according to the localization and depended highly on cell development. Both cell division and cell enlargement were stimulated in WC compared with K. The role of SuSy during thickening growth of radish hypocotyl is discussed in terms of utilizing photosynthates.

Brassicaceae↗
PubMed2002

Accuracy of reporting endocervical component adequacy--a continuous quality improvement project.

Inaccurate reporting of the absence of an endocervical (EC) component on Pap smears often results in slide rescreens, amended reports, clinician dissatisfaction, and sometimes unnecessary repeat smears. Therefore, the accuracy of reporting EC component adequacy was selected as a quality indicator for the laboratory continuous quality improvement program (CQI). The process consisted of problem identification, analysis of the situation, collection of data, implementation of solutions, and evaluation of results. The objective of the study was to determine if the accuracy of reporting EC component adequacy on Pap smears improved after application of such a program. During the first phase, 150 Pap smears originally reported with the absence of an adequate EC component and 150 smears reported with the presence of an adequate EC component were rescreened to measure the baseline accuracy of EC component adequacy reporting. The improvement process was then implemented. A cause-and-effect diagram was developed and root cause was determined. A presentation was then made to the cytology staff. Criteria for EC component adequacy were reviewed, examples were shown, and standardized marking of EC component was implemented. Following improvement actions, a second audit of 150 Pap smears reported with the absence of an adequate EC component as well as 150 smears reported with the presence of an adequate EC component was undertaken to measure change in performance in assessing EC component adequacy. For the baseline rescreening, before initiation of the CQI program, 98% accuracy was achieved with smears that were reported as adequate for EC component present. However, the accuracy with smears reported as absence of an adequate EC component was only 71%, i.e., an adequate EC component was identified in almost 1/3 of these cases on rescreen. After the implementation of improvement actions, the accuracy with smears reported with the presence of EC component remained high (98%) and the accuracy of reporting the absence of EC component was 90%. The difference of the latter before and after the implementation was statistically significant (P = 0.015, z-test). The accuracy of reporting EC component adequacy increased following the CQI process. Using reporting EC component adequacy as an example, we demonstrate that by treating clinical problems as quality control issues and applying basic quality improvement tools, a positive outcome can be effected.

Cell Biology↗
PubMed2006

Evolution and development of Hertwig's epithelial root sheath.

Periodontal regeneration and tissue engineering has re-awakened interest in the role of Hertwig's Epithelial Root Sheath (HERS), an epithelial tissue layer first discovered in amphibians more than a century ago. Using developmental, evolutionary, and cell biological approaches, we have, therefore, performed a careful analysis of the role of HERS in root formation and compared our data with clinical findings. Our developmental studies revealed HERS as a transient structure assembled in the early period of root formation and elongation and, subsequently, fenestrated and reduced to epithelial rests of Malassez (ERM). Our comparative evolutionary studies indicated that HERS fenestration was closely associated with the presence of a periodontal ligament and a gomphosis-type attachment apparatus in crocodilians and mammals. Based on these studies, we are proposing that HERS plays an important role in the regulation and maintenance of periodontal ligament space and function. Additional support for this hypothesis was rendered by our meta-analysis of recent clinical reports related to HERS function.

Animals↗
PubMed2003

Relative effects of the insecticide thiamethoxam on the predator Podisus nigrispinus and the tobacco whitefly Bemisia tabaci in nectaried and nectariless cotton.

The predaceous stinkbug Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) feeds on plants as well as on arthropod prey. The question arises whether feeding on plants might expose the predator to systemic insecticide via ingestion of the active ingredient or its metabolites through plant sap of treated plants. This interaction was investigated with nectaried and nectariless cotton plants cropped in pots and treated with the systemic insecticide thiamethoxam at 0.5, 1.0 and 2.0 mg per plant as a root drench. Development of P. nigrispinus fed on prey and on treated nectaried and nectariless cotton plants and confined at 15, 30 and 45 days after insecticide application, and adult reproduction from nymphs caged 30 days after treatment were determined. Podisus nigrispinus life history traits were unaffected by the type of cotton plant, nectaried or nectariless, but were significantly affected by insecticide dose and time after application. Developmental time was extended and fresh adult body weight was reduced by feeding on prey and treated plants. Nymphs caged on treated plants with the highest thiamethoxam concentration at 15 days after application produced only 13.2% of adults. Females emerged from nymphs caged on both plants and at all thiamethoxam concentrations at day 30 after application presented similar reproductive characteristics, except for age of first oviposition, which was delayed on plants treated with the highest thiamethoxam concentration. Thiamethoxam at 0.5 mg per plant restrained tobacco whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) colonization only during the first 15 days after application to either cotton plant, and similar immature densities were sampled at day 35 after application on treated and untreated plants. However, plants treated with 1.0 and 2.0 mg per plant as a drench and cropped in pots were protected from tobacco whitefly for up to 45 days after exposure to a whitefly colony.

Animals↗
PubMed2000

The Ets domain transcription factor Erm distinguishes rat satellite glia from Schwann cells and is regulated in satellite cells by neuregulin signaling.

Distinct glial cell types of the vertebrate peripheral nervous system (PNS) are derived from the neural crest. Here we show that the expression of the Ets domain transcription factor Erm distinguishes satellite glia from Schwann cells beginning early in rat PNS development. In developing dorsal root ganglia (DRG), Erm is present both in presumptive satellite glia and in neurons. In contrast, Erm is not detectable at any developmental stage in Schwann cells in peripheral nerves. In addition, Erm is downregulated in DRG-derived glia adopting Schwann cell traits in culture. Thus, Erm is the first described transcription factor expressed in satellite glia but not in Schwann cells. In culture, the Neuregulin1 (NRG1) isoform GGF2 maintains Erm expression in presumptive satellite cells and reinduces Erm expression in DRG-derived glia but not in Schwann cells from sciatic nerve. These data demonstrate that there are intrinsic differences between these glial subtypes in their response to NRG1 signaling. In neural crest cultures, Erm-positive progenitor cells give rise to two distinct glial subtypes: Erm-positive, Oct-6-negative satellite glia in response to GGF2, and Erm-negative, Oct-6-positive Schwann cells in the presence of serum and the adenylate cyclase activator forskolin. Thus, Erm-positive neural crest-derived progenitor cells and presumptive satellite glia are able to acquire Schwann cell features. Given the in vivo expression of Erm in peripheral ganglia, we suggest that ganglionic Erm-positive cells may be precursors of Schwann cells.

Animals↗
PubMed2001

Platelet-derived growth factor receptor-alpha in ventricular zone cells and in developing neurons.

Cells in the early neuroepithelium differentiate and give rise to all cells in the central nervous system (CNS). The ways from a multipotent CNS stem cell to specialized neurons and glia are not fully understood. Using immunohistochemistry we found that neuroepithelial cells express the platelet-derived growth factor receptor-alpha (PDGFR-alpha) in the neural plate at embryonic day 8.5 and onwards in the neural tube. The protein was polarized to ventricular endfeet. Furthermore, PDGFR-alpha expression was localized to cells undergoing early neuronal development. We also found PDGFR-alpha expression in developing granule cells in the postnatal cerebellum, in Purkinje cells in the adult cerebellum and on processes of developing dorsal root ganglion cells. Previous reports mainly describe PDGFR-alpha expression in oligodendrocyte precursors and glial cells. We believe, in line with a few previous reports, that the PDGFR-alpha in addition marks a pool of undifferentiated cells, which are able to differentiate into neurons.

Age Factors↗
PubMed1993

Isolation of an Arabidopsis cDNA sequence encoding a 22 kDa calcium-binding protein (CaBP-22) related to calmodulin.

Complementary DNA sequences were isolated from a library of cloned Arabidopsis leaf mRNA sequences in lambda gt10 that encoded a 21.7 kDa polypeptide (CaBP-22), which shared 66% amino acid sequence identity with Arabidopsis calmodulin. The putative Ca(2+)-binding domains of CaBP-22 and calmodulin, however, were more conserved and shared 79% sequence identity. Ca2+ binding by CaBP-22, which was inferred from its amino acid sequence similarity with calmodulin, was demonstrated indirectly by Ca(2+)-induced mobility shifting of in vitro translated CaBP-22 during SDS-polyacrylamide gel electrophoresis. CaBP-22 is encoded by a ca. 0.9 kb mRNA that was detected by northern blotting of leaf poly(A)+ RNA; this mRNA was slightly larger than the 809 bp CaBP-22 cDNA insert, indicating that the deduced amino acid sequence of CaBP-22 is near full-length. CaBP-22 mRNA was detected in RNA fractions isolated from leaves of both soil-grown and hydroponically grown Arabidopsis, but below the limits of detection in RNA isolated from roots, and developing siliques. Thus, CaBP-22 represents a new member of the EF-hand family of Ca(2+)-binding proteins with no known animal homologue and may participate in transducing Ca2+ signals to a specific subset of response elements.

Amino Acid Sequence↗
PubMed1994

Signal molecules involved in plant embryogenesis.

In plant embryogenesis, inductive interactions mediated by diffusable signal molecules are most likely of great importance. Evidence has been presented that at late globular stages in plant embryogenesis, perturbation of the polar auxin transport results in abberrant embryo morphology. Rhizobium lipooligosaccharides or Nod factors are a newly discovered class of bacterial molecules that are able to trigger initial steps in root nodule development in legumes. Part of the activity of Nod factors may be directed towards alteration of endogenous plant growth regulator balance. The same bacterial Nod factors promoted the formation of globular embryos in the carrot cell line ts11. Whether there exist plant analogues of the Nod factors and whether these molecules are active as a more universal control system perhaps designed to initiate and or mediate gradients in auxin and cytokinin remains to be determined.

Cell Communication↗
PubMed1996

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings.

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development.

We took advantage of a subtractive hybridization procedure to isolate a set of cDNA clones of nodule-specific genes (nodulin genes) from developing soybean root nodules. Single-stranded 32P-labelled cDNA synthesized from nodule poly(A)+ RNA was hybridized with a large excess of uninfected root poly(A)+ RNA. Unhybridized cDNA was selected and used to screen nodule cDNA libraries. By this procedure we isolated several novel nodulin cDNA clones together with most of the nodulin cDNAs previously described. Four novel nodulin genes, which were expressed long before the onset of nitrogen fixation, were further characterized. GmN#36 and GmN#93 transcripts appeared in the roots less than 3 days after sowing and inoculation with Bradyrhizobium, but GmN#36 transcripts were also detected at very low levels in the stems of uninfected plants. Transcripts of GmN#315 and GmN#70 first appeared at 6-7 days, just before nodule emergence. Amino acid sequences of the predicted products of GmN#36, GmN#93 and GmN#70 exhibited no significant homology to proteins identified so far. The GmN#315 encoded protein has a limited but significant homology to some plant cyanins, suggesting that it is a metal-binding glycoprotein. In situ hybridization studies revealed that GmN#36 transcripts first appeared in the pericycle cells of the root stele near the infected site. During nodule emergence they were found in a few cell layers surrounding the vascular strands connecting the nodule meristem with the root stele, and in mature nodules they were present specifically in the pericycle cells in vascular bundles. These observations led us to hypothesize that GmN#36 gene products play a role in the transport and/or degradation of photosynthate. On the other hand, GmN#93 transcripts first appeared in the primary nodule meristem just below the root epidermis. In mature nodules they were only present in the infected cells.

Amino Acid Sequence↗