Altered tissue specificity of the revertant shrunken allele upon Dissociation (Ds) excision is associated with loss of expression and molecular rearrangement at the corresponding non-allelic isozyme locus in maize

Summary Transposable element Activator (Ac) induced wild-type stable revertants, derived from McClintock's Dissociation (Ds) insertion shrunken (sh) mutant sh-m5933, have been examined for sucrose synthases, SS1 and SS2, encoded by the revertant (Sh) locus and the non-allelic gene Sus (previously designated as Ss2), respectively. A structurally normal Sh locus has been previously described in these revertants. Immuno-blot (Western) and Southern hybridization analyses reported here identify one of the nine alleles, Sh-r5, as unique for several features. It showed altered tissue specificity, as the SS1 protein encoded by the Sh-r5 allele was readily detectable in the immature embryo which is otherwise characterized by the Sus expression only. The level of Sh-r5 expression at the protein and enzyme level was marked by endosperm specific SS1 abundance and a significant down-regulation in the embryo similar to the standard Sh and Sus loci in endosperm and embryo, respectively. We infer that tissue specific levels of gene expression among maize Ss genes is significantly determined by trans-regulatory factors present in these two tissues. The Sh-r5 strain also exhibited a complete loss of the Sus expression in all tissues tested in the plant. Lack of any detectable phenotypic abnormality in the Sh-r5 strain due to the loss of SS2 protein indicated that either the SS2 protein is nonessential or that the two SS isozymes are functionally compensatory. Genomic filter hybridizations with the Sus cDNA clone indicated that the Sus locus in the Sh-r5 strain was not deleted and was, in fact, unique among these revertants. Together, these data provide an unusual insight into the regulation and function of the two SS isozymes in the maize plant.     

Evidence for plasma membrane-associated forms of sucrose synthase in maize

Plasma membrane fractions were isolated from maize (Zea mays L.) endosperms and etiolated kernels to investigate the possible membrane location of the sucrose synthase (SS) protein. Endosperms from seedlings at both 12 and 21 days after pollination (DAP), representing early and mid-developmental stages, were used, in addition to etiolated leaf and elongation zones from seedlings. Plasma membrane fractions were isolated from this material using differential centrifugation and aqueous two-phase partitioning. The plasma membrane-enriched fraction obtained was then analyzed for the presence of sucrose synthase using protein blots and activity measurements. Both isozymes SS1 and SS2, encoded by the lociSh1 andSus1, respectively, were detected in the plasma membrane-enriched fraction using polyclonal and monoclonal antisera to SS1 and SS2 isozymes. In addition, measurements of sucrose synthase activity in plasma membrane fractions of endosperm revealed high levels of specific activity. The sucrose synthase enzyme is tightly associated with the membrane, as shown by Triton X-100 treatment of the plasma membrane-enriched fraction. It is noteworthy that the gene products of bothSh1 andSus1 were detectable as both soluble and plasma membrane-associated forms.     

The Maize Invertase-Deficient miniature-1 Seed Mutation Is Associated with Aberrant Pedicel and Endosperm Development

Genetic evidence is presented to show that the developmental stability of maternal cells in the pedicel at the base of maize seeds is determined by the genotype of the developing endosperm. An early degeneration and withdrawal of maternal cells from the endosperm of homozygous miniature (mn mn) seed mutants were arrested if mn plants were pollinated by the wild-type Mn pollen. Similarly, the stability of the wild-type, Mn mn, maternal cells was also dependent on whether or not these cells were associated with the normal (Mn) or the mutant (mn) endosperm on the same ear. Biochemical and cellular analyses indicated that developing mn kernels have extremely low (<0.5% of the wild type) to undetectable levels of both soluble and wall-bound invertase activities. Extracts from endosperm with a single copy of the Mn gene showed a significant increase in both forms of invertases, and we suggest it is the causal basis of the wild-type seed phenotype. Collectively, these data provide evidence that invertase-mediated maintenance of a physiological gradient of photosynthate between pedicel and endosperm constitutes the rate-limiting step in structural stability of maternal cells as well as normal development of endosperm and seed.     

Genetic control of sucrose synthetase in maize endosperm

Summary Sucrose synthetase activity in endosperm extracts of seven shrunken(sh) mutants of spontaneous origin and three similar mutants due to the association of the controlling element Ds with the Sh locus is examined. A residual level of 3 to 5% as compared to the normal (Sh) endosperm is seen in all the mutants. The residual activity is similar to that of the Sh locus encoded endosperm sucrose synthetase by several criteria including an identical size of polypeptides and a similarity in antigenic properties. These two enzymes are, however, distinguishable by a slight difference in electrophoretic mobility in native gels and a difference in the relative abundance of enzyme molecules. The latter property is a reflection of a marked difference seen in the developmental profile of enzyme activity in the two genotypes. The earlier hypothesis (Chourey and Nelson 1976) that these two sucrose synthetases are encoded by two separate genes is strengthened by: (a) the presence of the residual enzyme in a sh deletion mutant and (b) an electrophoretic demonstration of two proteins, corresponding to the major and minor sucrose synthetase proteins, in the wild type (Sh) genotype. The two sucrose synthetase genes seem to provide a model system in plants for studying the molecular basis of temporal specificity of genes.Cooperative Investigation, United States Department of Agriculture and Institute of Food and Agricultural Sciences, University of Florida, Florida Agricultural Experiment Station Journal Series No. 3288. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

LED-Fluorescence Microscopy for Diagnosis of Pulmonary Tuberculosis under Programmatic Conditions in India

Background

Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.

Methods

This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.

Results

The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012 (P value <0.01) in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged (13.0%versus 12.6%;P value0.31).

Conclusion

Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond.     

Insights into the Structure and Metabolic Function of Microbes That Shape Pelagic Iron-Rich Aggregates (“Iron Snow”)

Microbial ferrous iron [Fe(II)] oxidation leads to the formation of iron-rich macroscopic aggregates (“iron snow”) at the redoxcline in a stratified lignite mine lake in east-central Germany. We aimed to identify the abundant Fe-oxidizing and Fe-reducing microorganisms likely to be involved in the formation and transformation of iron snow present in the redoxcline in two basins of the lake that differ in their pH values. Nucleic acid- and lipid-stained microbial cells of various morphologies detected by confocal laser scanning microscopy were homogeneously distributed in all iron snow samples. The dominant iron mineral appeared to be schwertmannite, with shorter needles in the northern than in the central basin samples. Total bacterial 16S rRNA gene copies ranged from 5.0 × 10⁸ copies g (dry weight)⁻¹ in the acidic central lake basin (pH 3.3) to 4.0 × 10¹⁰ copies g (dry weight)⁻¹ in the less acidic (pH 5.9) northern basin. Total RNA-based quantitative PCR assigned up to 61% of metabolically active microbial communities to Fe-oxidizing- and Fe-reducing-related bacteria, indicating that iron metabolism was an important metabolic strategy. Molecular identification of abundant groups suggested that iron snow surfaces were formed by chemoautotrophic iron oxidizers, such as Acidimicrobium, Ferrovum, Acidithiobacillus, Thiobacillus, and Chlorobium, in the redoxcline and were rapidly colonized by heterotrophic iron reducers, such as Acidiphilium, Albidiferax-like, and Geobacter-like groups. Metaproteomics yielded 283 different proteins from northern basin iron snow samples, and protein identification provided a glimpse into some of their in situ metabolic processes, such as primary production (CO₂ fixation), respiration, motility, and survival strategies.     

Biostimulation induces syntrophic interactions that impact C,S and N cycling in a sediment microbial community

Stimulation of subsurface microorganisms to induce reductive immobilization of metals is a promising approach for bioremediation, yet the overall microbial community response is typically poorly understood. Here we used proteogenomics to test the hypothesis that excess input of acetate activates complex community functioning and syntrophic interactions among autotrophs and heterotrophs. A flow-through sediment column was incubated in a groundwater well of an acetate-amended aquifer and recovered during microbial sulfate reduction. De novo reconstruction of community sequences yielded near-complete genomes of Desulfobacter (Deltaproteobacteria), Sulfurovum- and Sulfurimonas-like Epsilonproteobacteria and Bacteroidetes. Partial genomes were obtained for Clostridiales (Firmicutes) and Desulfuromonadales-like Deltaproteobacteria. The majority of proteins identified by mass spectrometry corresponded to Desulfobacter-like species, and demonstrate the role of this organism in sulfate reduction (Dsr and APS), nitrogen fixation and acetate oxidation to CO₂ during amendment. Results indicate less abundant Desulfuromonadales, and possibly Bacteroidetes, also actively contributed to CO₂ production via the tricarboxylic acid (TCA) cycle. Proteomic data indicate that sulfide was partially re-oxidized by Epsilonproteobacteria through nitrate-dependent sulfide oxidation (using Nap, Nir, Nos, SQR and Sox), with CO₂ fixed using the reverse TCA cycle. We infer that high acetate concentrations, aimed at stimulating anaerobic heterotrophy, led to the co-enrichment of, and carbon fixation in Epsilonproteobacteria. Results give an insight into ecosystem behavior following addition of simple organic carbon to the subsurface, and demonstrate a range of biological processes and community interactions were stimulated.     

Endophytes of opium poppy differentially modulate host plant productivity and genes for the biosynthetic pathway of benzylisoquinoline alkaloids

Planta - Endophytes reside in different parts of the poppy plant and perform the tissue-specific functions. Most leaf endophytes modulate photosynthetic efficiency, plant growth, and productivity...