Donna S. Williams

Miniature1-Encoded Cell Wall Invertase Is Essential for Assembly and Function of Wall-in-Growth in the Maize Endosperm Transfer Cell

The miniature1 (mn1) seed phenotype in maize (Zea mays) is due to a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase (INCW2) that localizes exclusively to the basal endosperm transfer cells (BETCs) of developing seeds. A common feature of all transfer cells is the labyrinth-like wall-in-growth (WIG) that increases the plasma membrane area, thereby enhancing transport capacity in these cells. To better understand WIG formation and roles of INCW2 in the BETC development, we examined wild-type and mn1 mutant developing kernels by cryofixation and electron microscopy. In Mn1 seeds, WIGs developed uniformly in the BETC layer during 7 to 17 d after pollination, and the secretory/endocytic organelles proliferated in the BETCs. Mitochondria accumulated in the vicinity of WIGs, suggesting a functional link between them. In the mn1 BETCs, WIGs were stunted and their endoplasmic reticulum was swollen; Golgi density in the mutant BETCs was 51% of the Mn1 Golgi density. However, the polarized distribution of mitochondria was not affected. INCW2-specific immunogold particles were detected in WIGs, the endoplasmic reticulum, Golgi stacks, and the trans-Golgi network in the Mn1 BETCs, while immunogold particles were extremely rare in the mutant BETCs. Levels of WIG development in the empty pericarp4 mutant was heterogeneous among BETCs, and INCW2 immunogold particles were approximately four times more abundant in the larger WIGs than in the stunted WIGs. These results indicate that polarized secretion is activated during WIG formation and that INCW2 is required for normal development of WIGs to which INCW2 is localized.

Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with “Candidatus Liberibacter asiaticus”

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.

The correlation of the gene csoS2 of the carboxysome operon with two polypeptides of the carboxysome in Thiobacillus neapolitanus

Abstract The carboxysomal polypeptides of Thiobacillus neapolitanus with apparent molecular masses of 85 and 130 kDa were isolated and subjected to N-terminal sequencing. The first 17 amino acids of the two peptides were identical. The sequence perfectly matched the deduced amino acid sequence of an open reading frame in the carboxysome operon. The gene was subsequently named csoS2. Expression of the gene in Escherichia coli resulted in the production of two peptides with apparent molecular masses of 85 and 130 kDa. Immunospecific antibodies generated against the smaller peptide recognized both peptides; the peptides were named CsoS2A and CsoS2B, respectively. A digoxigenin-hydrazide glycosylation assay revealed that both CsoS2A and CsoS2B are post-translationally modified by glycosylation. CsoS2 was localized to the edges of purified carboxysomes by immunogold electron microscopy using the monospecific CsoS2A antibodies. The molecular mass of CsoS2A calculated from the nucleotide sequence was 92.3 kDa.

CHARACTERIZATION OF PARAMYXOVIRUSES ISOLATED FROM THREE SNAKES

Multiple epizootics of pneumonia in captive snakes have been attributed to viruses which have been tentatively placed in the family Paramyxoviridae. Viruses isolated from an ill Neotropical rattlesnake (Crotalus durissus terrificus), from an Aruba Island rattlesnake (Crotalus unicolor), and from a bush viper (Atheris sp.) were propagated in Vero cells and characterized. Viral particles produced in Vero cells were pleomorphic, enveloped, and contained helical nucleocapsids. The viruses were sensitive to ether and to acidic and basic pH. Moreover, they had neuraminidase activity and were able to agglutinate erythrocytes from chicken and a variety of species of mammals. Hemagglutination was inhibited with rabbit antiserum raised against each virus. The buoyant densities of the three isolates ranged from 1.13/cm3 to 1.18/cm3, values consistent with that for an enveloped virus. The nucleic acid in the virion was determined to be RNA by [3H]uridine incorporation. Viral proteins characteristic of paramyxoviruses were immunoprecipitated from cells infected with each of the three isolates using rabbit anti-Neotropical virus serum. The morphologic appearance, physico- and biochemical properties, and cytopathologic effects of these snake viruses were consistent with those of certain members of the family Paramyxoviridae.

Antagonism by L-glutamine of toxicity and growth inhibition caused by other amino acids in suspension cultures of Nicotiana silvestris

Abstract The toxicity of l -amino acids toward exponentially dividing cells of Nicotiana silvestris in suspension culture was monitored by following growth rates throughout a span of 8 days. Except for l -glutamine, all 19 protein amino acids inhibited cell growth. Inhibition progressed with an initial stage of slowed exponential growth. Cells in this stage were receptive to complete recovery under some conditions. Otherwise an irreversible stage of total growth inhibition and progressive cell deterioration followed. Electron microscopy showed that amino acids triggered a state of cell shrinkage which eventually degenerated to total cellular disorganization. An apparent exocytotic deposition of condensed and blackened cellular debris occurred between the cell wall and plasmalemma. l -Glutamine was not only an effective agent for prevention of amino acid toxicity, but enhanced the final growth yield. l -Glutamine also was able to completely reverse inhibitor effects in cells which had been in the slowed exponential phase for up to 3 days. We propose that any amino acid inhibition which can be completely antagonized by l -glutamine be called ‘general amino acid inhibition’. ‘Specific amino acid inhibition’, resulting from particular pathway imbalances caused by certain exogenous amino acids, can be recognized and studied in the presence of l -glutamine which abolishes the complicating effects of general amino acid inhibition. It is hypothesized that the l -glutamine: amino-acid ratio established in vivo might influence susceptibility to apoptosis, a eukaryotic process of programmed cell death which has been characterized most extensively in animals.

Defective chloroplast development inhibits maintenance of normal levels of abscisic acid in a mutant of the Arabidopsis RH3 DEAD‐box protein during early post‐germination growth

The plastid has its own translation system, and its ribosomes are assembled through a complex process in which rRNA precursors are processed and ribosomal proteins are inserted into the rRNA backbone. DEAD-box proteins have been shown to play roles in multiple steps in ribosome biogenesis. To investigate the cellular and physiological roles of an Arabidopsis DEAD-box protein, RH3, we examined its expression and localization and the phenotypes of rh3-4, a T-DNA insertion mutant allele of RH3. The promoter activity of RH3 is strongest in the greening tissues of 3-day and 1-week-old seedlings but reduced afterwards. Cotyledons were pale and seedling growth was retarded in the mutant. The most obvious abnormality in the mutant chloroplasts was their lack of normal ribosomes. Electron tomography analysis indicated that ribosome density in the 3-day-old mutant chloroplasts is only 20% that of wild-type chloroplasts, and the ribosomes in the mutant are smaller. These chloroplast defects in rh3-4 were alleviated in 2-week-old cotyledons and true leaves. Interestingly, rh3-4 seedlings have lower amounts of abscisic acid prior to recovery of their chloroplasts, and were more sensitive to abiotic stresses. Transcriptomic analysis indicated that nuclear genes for chloroplast proteins are down-regulated, and proteins mediating chloroplast-localized steps of abscisic acid biosynthesis are expressed to a lower extent in 1-week-old rh3-4 seedlings. Taken together, these results suggest that conversion of eoplasts into chloroplasts in young seedlings is critical for the seedlings to start carbon fixation as well as for maintenance of abscisic acid levels for responding to environmental challenges.

Characterization of Arabidopsis 6-Phosphogluconolactonase T-DNA Insertion Mutants Reveals an Essential Role for the Oxidative Section of the Plastidic Pentose Phosphate Pathway in Plant Growth and Development

Arabidopsis PGL1, PGL2, PGL4 and PGL5 are predicted to encode cytosolic isoforms of 6-phosphogluconolactonase (6PGL), whereas PGL3 is predicted to encode a 6PGL that has been shown to localize in both plastids and peroxisomes. Therefore, 6PGL may exist in the cytosol, plastids and peroxisomes. However, the function of 6PGL in these three subcellular locations has not been well defined. Here we show that PGL3 is essential, whereas PGL1, PGL2 and PGL5 are individually dispensable for plant growth and development. Knockdown of PGL3 in the pgl3 mutant leads to a dramatic decrease in plant size, a significant increase in total glucose-6-phosphate dehydrogenase activity and a marked decrease in cellular redox potential. Interestingly, the pgl3 plants exhibit constitutive pathogenesis-related gene expression and enhanced resistance to Pseudomonas syringae pv. maculicola ES4326 and Hyaloperonospora arabidopsidis Noco2. We found that although pgl3 does not spontaneously accumulate elevated levels of free salicylic acid (SA), the constitutive defense responses in pgl3 plants are almost completely suppressed by the npr1 and sid2/eds16/ics1 mutations, suggesting that the pgl3 mutation activates NPR1- and SID2/EDS16/ICS1-dependent defense responses. We demonstrate that plastidic (not peroxisomal) localization and 6PGL activity of the PGL3 protein are essential for complementing all pgl3 phenotypes, indicating that the oxidative section of the plastidic pentose phosphate pathway (PPP) is required for plant normal growth and development. Thus, pgl3 provides a useful tool not only for defining the role of the PPP in different subcellular compartments, but also for dissecting the SA/NPR1-mediated signaling pathway.

Immunogold localization of coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase in Methanobacterium formicicum

The ultrastructural locations of the coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F420-dependent formate dehydrogenase activity or F420-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.

Deficiency in a cytosolic ribose-5-phosphate isomerase causes chloroplast dysfunction, late flowering and premature cell death in Arabidopsis.

The oxidative pentose phosphate pathway (oxPPP) is part of central metabolism, consisting of two distinct phases: the oxidative phase and the non-oxidative phase. The non-oxidative phase of the oxPPP generates carbon skeletons for the synthesis of nucleotides, aromatic amino acids, phenylpropanoids and their derivatives, which are essential for plant growth and development. However, it is not well understood how the non-oxidative phase of the oxPPP contributes to plant growth and development. Here, we report the characterization of Arabidopsis T-DNA knockout mutants of the RPI2 gene (At2g01290), which encodes a cytosolic ribose-5-phosphate isomerase (RPI) that catalyzes the reversible interconversion of ribulose-5-phosphate and ribose-5-phosphate in the non-oxidative phase of the oxPPP. Although recombinant Arabidopsis RPI2 protein exhibits marked RPI enzymatic activity, knockout of the RPI2 gene does not significantly change the total RPI activity in the mutant plants. Interestingly, knockout of RPI2 interferes with chloroplast structure and decreases chloroplast photosynthetic capacity. The rpi2 mutants accumulate less starch in the leaves and flower significantly later than wild-type when grown under short-day conditions. Furthermore, the rpi2 mutants display premature cell death in the leaves when grown at an above-normal temperature (26 degrees C). These results demonstrate that a deficiency in the non-oxidative phase of the cytosolic oxPPP has pleiotropic effects on plant growth and development and causes premature cell death.

Ultrastructure and life history of Myolaimus byersi n. sp. (Myolaimina: Myolaimidae), a phoretic associate of the crane fly, Limonia schwarzi (Alexander) (Limoniidae), in Florida

Myolaimus byersi n. sp., a phoretic associate of the crane fly, Limonia (Rhipidia) schwarzi (Diptera: Limoniidae), was recovered from moist and decaying tissue from the crown shaft of a living spindle palm, Hyophorbe verschaffeltii, in southern Florida and is described herein. Dauers were carried in the abdominal folds of male and female L. schwarzi. Examination of the highly mobile crane fly larvae and pupae confirmed that the dauers were externally associated with the cuticle. Dauers from crane flies were culturable to adults on 1/20 strength TSB agar. The association appears to be relatively host specific. SEM studies, early embryonic development, dauers, molecular data and TEM ultrastructural comparisons of the stoma, sensory structures and sperm are used to discuss the relative placement of Myolaimus within the Nematoda. The stoma resembles diplogastrids in being strongly anisomorphic with an enlarged dorsal sector of the stegostom, yet also resembles rhabditids in having three triangular flaps in the metastegostom and matches cephalobs and panagrolaims in having a pharyngeal collar with two sets of three interradial muscles followed by two sets of six adradial muscles. The ultrastructure of the cheilostom epidermis shows a high degree of conservation with several Rhabditida. The sperm of M. byersi n. sp. is nearly identical to that of Caenorhabditis elegans. In early cell division, M. byersi n. sp. is closest to Parascaris equorum followed by C. elegans. Myolaimus apparently represents a divergent lineage that has followed a non-coalescing trajectory for a long time, allowing it to retain some highly conserved characters while also developing some surprisingly unique features, such as a baggy cuticle and males that lack a gubernaculum or spicules.