Faculty Publications from the Center for Plant Science Innovation Copyright (c) 2013 University of Nebraska - Lincoln All rights reserved. http://digitalcommons.unl.edu/plantscifacpub Recent documents in Faculty Publications from the Center for Plant Science Innovation en-us Fri, 03 May 2013 01:42:22 PDT 3600 Radiative Neutron β-Decay in Effective Field Theory http://digitalcommons.unl.edu/plantscifacpub/81 http://digitalcommons.unl.edu/plantscifacpub/81 Wed, 01 May 2013 19:41:02 PDT In summary, we have computed the photon energy spectrum and photon polarization in neutron radiative β-decay in an effective field theory approach, utilizing HBCHPT and the SSE, including all terms in O(1/M). The leading contribution to the photon energy spectrum has been calculated previously [4]; we agree with the expression in Ref. [4] for Σspins |M|2, though we disagree with their numerical results for the photon energy spectrum. Moreover, we find that the O(1/M) terms are numerically quite small, generating contributions no larger than O(0.5 %), so that radiative neutron β-decay is quite insensitive to nucleon structure effects beyond those encoded in gV and gA . We have found that nucleon structure effects have a similarly negligible role in the determination of the photon polarization, so that a precise measurement of the photon polarization may well offer a crisp diagnostic of non-SM effects.

]]> Susan Gardner et al. <i>Pseudomonas syringae</i> Catalases Are Collectively Required for Plant Pathogenesis http://digitalcommons.unl.edu/plantscifacpub/79 http://digitalcommons.unl.edu/plantscifacpub/79 Fri, 21 Sep 2012 11:56:17 PDT The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 must detoxify plant-produced hydrogen peroxide (H2O2) in order to survive in its host plant. Candidate enzymes for this detoxification include the monofunctional catalases KatB and KatE and the bifunctional catalase-peroxidase KatG of DC3000. This study shows that KatG is the major housekeeping catalase of DC3000 and provides protection against menadione-generated endogenous H2O2. In contrast, KatB rapidly and substantially accumulates in response to exogenous H2O2. Furthermore, KatB and KatG have nonredundant roles in detoxifying exogenous H2O2 and are required for full virulence of DC3000 in Arabidopsis thaliana. Therefore, the nonredundant ability of KatB and KatG to detoxify plant-produced H2O2 is essential for the bacteria to survive in plants. Indeed, a DC3000 catalase triple mutant is severely compromised in its ability to grow in planta, and its growth can be partially rescued by the expression of katB, katE, or katG. Interestingly, our data demonstrate that although KatB and KatG are the major catalases involved in the virulence of DC3000, KatE can also provide some protection in planta. Thus, our results indicate that these catalases are virulence factors for DC3000 and are collectively required for pathogenesis.

]]> Ming Guo et al. Regulation of miRNA abundance by RNA binding protein TOUGH in <i>Arabidopsis</i> http://digitalcommons.unl.edu/plantscifacpub/78 http://digitalcommons.unl.edu/plantscifacpub/78 Thu, 02 Aug 2012 09:40:48 PDT MicroRNAs (miRNAs) are regulators of gene expression in plants and animals. The biogenesis of miRNAs is precisely controlled to secure normal development of organisms. Here we report that TOUGH (TGH) is a component of the DCL1–HYL1–SERRATE complex that processes primary transcripts of miRNAs [i.e., primary miRNAs (pri-miRNAs)] into miRNAs in Arabidopsis. Lack of TGH impairs multiple DCL activities in vitro and reduces the accumulation of miRNAs and siRNAs in vivo. TGH is an RNA-binding protein, binds pri-miRNAs and precursor miRNAs in vivo, and contributes to pri-miRNA–HYL1 interaction. These results indicate that TGH might regulate abundance of miRNAs through promoting DCL1 cleavage efficiency and/or recruitment of pri-miRNAs.

Includes Supporting Information.

]]> Guodong Ren et al. A subgroup of SGS3-like proteins act redundantly in RNA-directed DNA methylation http://digitalcommons.unl.edu/plantscifacpub/77 http://digitalcommons.unl.edu/plantscifacpub/77 Tue, 03 Jul 2012 06:29:10 PDT Plant specific SGS3-like proteins are composed of various combinations of an RNA-binding XS domain, a zinc-finger zf-XS domain, a coil–coil domain and a domain of unknown function called XH. In addition to being involved in de novo 2 (IDN2) and SGS3, the Arabidopsis genome encodes 12 uncharacterized SGS3-like proteins. Here, we show that a group of SGS3-like proteins act redundantly in RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. Transcriptome co-expression analyses reveal significantly correlated expression of two SGS3-like proteins, factor of DNA methylation 1 (FDM1) and FDM2 with known genes required for RdDM. The fdm1 and fdm2 double mutations but not the fdm1 or fdm2 single mutations significantly impair DNA methylation at RdDM loci, release transcriptional gene silencing and dramatically reduce the abundance of siRNAs originated from high copy number repeats or transposons. Like IDN2 and SGS3, FDM1 binds dsRNAs with 50 overhangs. Double mutant analyses also reveal that IDN2 and three uncharacterized SGS3-like proteins FDM3, FDM4 and FDM5 have overlapping function with FDM1 in RdDM. Five FDM proteins and IDN2 define a group of SGS3-like proteins that possess all four-signature motifs in Arabidopsis. Thus, our results demonstrate that this group of SGS3-like proteins is an important component of RdDM. This study further enhances our understanding of the SGS3 gene family and the RdDM pathway.

Includes supplemental figures.

]]> Meng Xie et al. Ectopic expression of Rubisco subunits in maize mesophyll cells does not overcome barriers to cell type-specific accumulation http://digitalcommons.unl.edu/plantscifacpub/76 http://digitalcommons.unl.edu/plantscifacpub/76 Tue, 03 Jul 2012 06:12:57 PDT In Zea mays, ribulose bisphosphate carboxylase (Rubisco) accumulates in bundle sheath but not mesophyll chloroplasts, but the mechanisms that underlie cell-type specific expression are poorly understood. To explore the coordinated expression of the chloroplast rbcL gene, which encodes the Rubisco large subunit (LS), and the two nuclear RBCS genes which encode the small subunit (SS), RNAi was used to reduce RBCS expression. This resulted in Rubisco deficiency, and was correlated with translational repression of rbcL. Thus, as in C3 plants, LS synthesis depends on the presence of its assembly partner SS. To test the hypothesis that the previously documented transcriptional repression of RBCS in mesophyll cells is responsible for repressing LS synthesis in mesophyll chloroplasts, a ubiquitin promoter-driven RBCS gene was expressed in both bundle sheath and mesophyll cells. This did not lead to Rubisco accumulation in the mesophyll, suggesting that LS synthesis is impeded even in the presence of ectopic SS expression. To attempt to bypass this putative mechanism, a ubiquitin promoter-driven nuclear version of the rbcL gene was created, encoding an epitope-tagged LS, which was expressed in the presence or absence of the Ubi-RBCS construct. Both transgenes were robustly expressed, and the tagged LS was readily incorporated into Rubisco complexes. However, neither immunolocalization nor biochemical approaches revealed significant accumulation of Rubisco in mesophyll cells, suggesting a continuing cell type-specific impairment of its assembly or stability. We conclude that additional cell type-specific factors limit Rubisco expression to bundle sheath chloroplasts.

Includes supplemental data (Table 1).

]]> Katia Wostrikoff et al. Evidence against equimolarity of large repeat arrangements and a predominant master circle structure of the mitochondrial genome from a monkeyflower (<i>Mimulus guttatus</i>) lineage with cryptic CMS http://digitalcommons.unl.edu/plantscifacpub/75 http://digitalcommons.unl.edu/plantscifacpub/75 Tue, 01 May 2012 14:41:29 PDT Despite intense investigation for over 25 years, the in vivo structure of plant mitochondrial genomes remains uncertain. Mapping studies and genome sequencing generally produce large circular chromosomes, whereas electrophoretic and microscopic studies typically reveal linear and multi-branched molecules. To more fully assess the structure of plant mitochondrial genomes, the complete sequence of the monkeyflower (Mimulus guttatus DC. line IM62) mtDNA was constructed from a large (35 kb) paired-end shotgun sequencing library to a high depth of coverage (~30x). The complete genome maps as a 525,671 bp circular molecule and exhibits a fairly conventional set of features including 62 genes (encoding 35 proteins, 24 tRNAs, 3 rRNAs), 22 introns, 3 large repeats (2.7, 9.6, 29 kb), and 96 small repeats (40–293 bp). Most paired-end reads (71%) mapped to the consensus sequence at the expected distance and orientation across the entire genome, validating the accuracy of assembly. Another 10% of reads provided clear evidence of alternative genomic conformations due to apparent rearrangements across large repeats. Quantitative assessment of these repeat-spanning read pairs revealed that all large repeat arrangements are present at appreciable frequencies in vivo, although not always in equimolar amounts. The observed stoichiometric differences for some arrangements are inconsistent with a predominant master circular structure for the mitochondrial genome of M. guttatus IM62. Finally, because IM62 contains a cryptic cytoplasmic male-sterility (CMS) system, an in silico search for potential CMS genes was undertaken. The three chimeric ORFs identified in this study, in addition to the previously identified ORFs upstream of the nad6 gene, are the most likely CMS candidate genes in this line.

Includes Supplementary Information.

]]> Jeffrey P. Mower et al. Folate synthesis in plants: The <i>p</i>-aminobenzoate branch is initiated by a bifunctional PabA–PabB protein that is targeted to plastids http://digitalcommons.unl.edu/plantscifacpub/74 http://digitalcommons.unl.edu/plantscifacpub/74 Fri, 14 Oct 2011 09:39:39 PDT It is not known how plants synthesize the p-aminobenzoate (PABA) moiety of folates. In Escherichia coli, PABA is made from chorismate in two steps. First, the PabA and PabB proteins interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC). The PabC protein then mediates elimination of pyruvate and aromatization to give PABA. Fungi, actinomycetes, and Plasmodium spp. also synthesize PABA but have proteins comprising fused domains homologous to PabA and PabB. These bipartite proteins are commonly called ‘‘PABA synthases,’’ although it is unclear whether they produce PABA or ADC. Genomic approaches identified Arabidopsis and tomato cDNAs encoding bipartite proteins containing fused PabA and PabB domains, plus a putative chloroplast targeting peptide. These cDNAs encode functional enzymes, as demonstrated by complementation of an E. coli pabA pabB double mutant and a yeast PABA-synthase deletant. The partially purified recombinant Arabidopsis protein did not produce PABA unless the E. coli PabC enzyme was added, indicating that it forms ADC, not PABA. The enzyme behaved as a monomer in size-exclusion chromatography and was not inhibited by physiological concentrations of PABA, its glucose ester, or folates. When the putative targeting peptide was fused to GFP and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that PABA synthesis is plastidial. In the pericarp of tomato fruit, the PabA–PabB mRNA level fell drastically as ripening advanced, but there was no fall in total PABA content, which stayed between 0.7 and 2.3 nmol·g-1 fresh weight.

]]> Gilles J. C. Basset et al. Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I http://digitalcommons.unl.edu/plantscifacpub/73 http://digitalcommons.unl.edu/plantscifacpub/73 Fri, 14 Oct 2011 09:30:05 PDT GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of ≈26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying ≈50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover.

]]> Gilles J. C. Basset et al. Folate Synthesis and Metabolism in Plants and Prospects For Biofortification http://digitalcommons.unl.edu/plantscifacpub/72 http://digitalcommons.unl.edu/plantscifacpub/72 Fri, 14 Oct 2011 09:21:41 PDT Folates are essential cofactors for one-carbon transfer reactions in most living organisms and are required for the biosynthesis of nucleic acids, amino acids, and pantothenate. Unlike plants and microorganisms, humans cannot synthesize folates de novo and must acquire them from the diet, primarily from plant foods. However, lack of folates is the most common vitamin deficiency in the world and has serious health consequences, including increased risk of neural tube defects in infants, cancers, and vascular diseases. Consequently, there is much interest in engineering plants with enhanced folate content (biofortification). In this review, we outline progress in defining the plant folate synthesis pathway and its unique progress in defining the plant folate synthesis pathway and its unique compartmentation and point out sectors of folate metabolism that have yet to be elucidated, including transport and catabolism. We also consider possible strategies to enhance plant folate synthesis and accumulation by metabolic engineering.

]]> Gilles J. C. Basset et al. Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis http://digitalcommons.unl.edu/plantscifacpub/53 http://digitalcommons.unl.edu/plantscifacpub/53 Fri, 07 Oct 2011 11:28:10 PDT Plants are the main source of folate in human diets, but many fruits, tubers, and seeds are poor in this vitamin, and folate deficiency is a worldwide problem. Plants synthesize folate from pteridine, p-aminobenzoate (PABA), and glutamate moieties. Pteridine synthesis capacity is known to drop in ripening tomato fruit; therefore, we countered this decline by fruit-specific overexpression of GTP cyclohydrolase I, the first enzyme of pteridine synthesis. We used a synthetic gene based on mammalian GTP cyclohydrolase I, because this enzyme is predicted to escape feedback control in planta. This engineering maneuver raised fruit pteridine content by 3- to 140-fold and fruit folate content by an average of 2-fold among 12 independent transformants, relative to vector-alone controls. Most of the folate increase was contributed by 5-methyltetrahydrofolate polyglutamates and 5,10-methenyltetrahydrofolate polyglutamates, which were also major forms of folate in control fruit. The accumulated pteridines included neopterin, monapterin, and hydroxymethylpterin; their reduced forms, which are folate biosynthesis intermediates; and pteridine glycosides not previously found in plants. Engineered fruit with intermediate levels of pteridine overproduction attained the highest folate levels. PABA pools were severely depleted in engineered fruit that were high in folate, and supplying such fruit with PABA by means of the fruit stalk increased their folate content by up to 10-fold. These results demonstrate that engineering a moderate increase in pteridine production can significantly enhance the folate content in food plants and that boosting the PABA supply can produce further gains.

]]> Rocio Diaz de la Garza et al. Horizontal acquisition of multiple mitochondrial genes from a parasitic plant followed by gene conversion with host mitochondrial genes http://digitalcommons.unl.edu/plantscifacpub/52 http://digitalcommons.unl.edu/plantscifacpub/52 Wed, 14 Sep 2011 13:46:38 PDT Background: Horizontal gene transfer (HGT) is relatively common in plant mitochondrial genomes but the mechanisms, extent and consequences of transfer remain largely unknown. Previous results indicate that parasitic plants are often involved as either transfer donors or recipients, suggesting that direct contact between parasite and host facilitates genetic transfer among plants.

Results: In order to uncover the mechanistic details of plant-to-plant HGT, the extent and evolutionary fate of transfer was investigated between two groups: the parasitic genus Cuscuta and a small clade of Plantago species. A broad polymerase chain reaction (PCR) survey of mitochondrial genes revealed that at least three genes (atp1, atp6 and matR) were recently transferred from Cuscuta to Plantago. Quantitative PCR assays show that these three genes have a mitochondrial location in the one species line of Plantago examined. Patterns of sequence evolution suggest that these foreign genes degraded into pseudogenes shortly after transfer and reverse transcription (RT)- PCR analyses demonstrate that none are detectably transcribed. Three cases of gene conversion were detected between native and foreign copies of the atp1 gene. The identical phylogenetic distribution of the three foreign genes within Plantago and the retention of cytidines at ancestral positions of RNA editing indicate that these genes were probably acquired via a single, DNA-mediated transfer event. However, samplings of multiple individuals from two of the three species in the recipient Plantago clade revealed complex and perplexing phylogenetic discrepancies and patterns of sequence divergence for all three of the foreign genes.

Conclusions: This study reports the best evidence to date that multiple mitochondrial genes can be transferred via a single HGT event and that transfer occurred via a strictly DNA-level intermediate. The discovery of gene conversion between co-resident foreign and native mitochondrial copies suggests that transferred genes may be evolutionarily important in generating mitochondrial genetic diversity. Finally, the complex relationships within each lineage of transferred genes imply a surprisingly complicated history of these genes in Plantago subsequent to their acquisition via HGT and this history probably involves some combination of additional transfers (including intracellular transfer), gene duplication, differential loss and mutation-rate variation. Unravelling this history will probably require sequencing multiple mitochondrial and nuclear genomes from Plantago.

]]> Jeffrey P. Mower et al. Mining the bitter melon (<i>momordica charantia</i> l.) seed transcriptome by 454 analysis of nonnormalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes http://digitalcommons.unl.edu/plantscifacpub/51 http://digitalcommons.unl.edu/plantscifacpub/51 Wed, 14 Sep 2011 13:34:49 PDT Background: Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids.

Results: Deep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis.

Conclusions: 454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in developing bitter melon seeds. The transcriptomic data presented provide a resource for the study of novel fatty acid metabolism and for the biotechnological production of conjugated fatty acids and possibly other novel fatty acids in established oilseed crops.

]]> Peizhen Yang et al. Recombination between defective tombusvirus RNAs generates functional hybrid genomes http://digitalcommons.unl.edu/plantscifacpub/50 http://digitalcommons.unl.edu/plantscifacpub/50 Wed, 30 Mar 2011 13:39:19 PDT The tombusviruses represent a group of small Icosahedral plant viruses that contain monopartite positive-sense RNA genome. Tombusviruses are able to generate small replicating deletion mutants of their genomes (i.e., defective interfering RNAs) during infections via RNA recombination and/or rearrangment. To further study the process of RNA recombination and to determine whether tombusviruses were capable of tran-recombination, protoplasts were coinoculated with in vitro-generated transcripts of a nonreplicating 3'- truncated genomic RNA of cucumber necrosis tombusvirus and either replicative or replication-defective DI RNAs of tomato bushy stunt tombusvirus. After a 48-hr incubation, two dominant replicative chimeric recombinant viral RNA populations were detected that contained various large contiguous 5' segments of the cucumber necrosis tombusvirus genomic RNA fused to 3'-terminal regions of the tomato bushy stunt tombusvirus defective interfering RNA. Some of the larger chimeric recombinants formed in protoplasts were able to systemically infect plants and induce wild-type symptoms. In addition, a functional chimeric genome was generated in planta after direct coinoculation of whole plants with the defective RNA components. These results indicate that (i) RNA recombination can occur relatively efficiently in single-cell infections, (ii) trans-recombination can occur with nonreplicating viral RNA components, and (iii) functional chimeric genomes can be generated via recombination. Possible mechaisms for the formation of the recombinants are proposed, and evolutionary implications are disussed.

]]> K. Andrew White et al. Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors http://digitalcommons.unl.edu/plantscifacpub/49 http://digitalcommons.unl.edu/plantscifacpub/49 Thu, 28 Oct 2010 08:55:01 PDT In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large (n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genomewide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.

]]> Andrew K. Benson et al. γ-Zeins are essential for endosperm modification in quality protein maize http://digitalcommons.unl.edu/plantscifacpub/48 http://digitalcommons.unl.edu/plantscifacpub/48 Thu, 28 Oct 2010 08:39:49 PDT Essential amino acids like lysine and tryptophan are deficient in corn meal because of the abundance of zein storage proteins that lack these amino acids. A naturalmutant, opaque 2 (o2) causes reduction of zeins,anincreaseofnonzeinproteins,andas a consequence, adoubling of lysine levels.However, o2’s soft inferior kernels precluded its commercial use. Breeders subsequently overcame kernel softness, selectingseveral quantitative loci (QTLs), called o2modifiers,without losing the high-lysine trait. These maize lines are known as “quality protein maize” (QPM). One of the QTLs is linked to the 27-kDa γ-zein locus on chromosome 7S. Moreover, QPM lines have 2- to 3-fold higher levels of the 27-kDa γ-zein, but the physiological significance of this increase is not known. Because the 27- and 16-kDa γ-zein genes are highly conserved in DNA sequence, we introduced a dominant RNAi transgene into a QPM line (CM105Mo2) to eliminate expression of them both. Elimination of γ-zeins disrupts endosperm modification by o2 modifiers, indicating their hypostatic action to γ-zeins. Abnormalities in protein body structure and their interaction with starch granules in the F1 with Mo2/+; o2/o2; γRNAi/+ genotype suggests that γ-zeins are essential for restoring protein body density and starch grain interaction in QPM. To eliminate pleiotropic effects caused by o2, the 22-kDa α-zein, γ-zein, and β-zein RNAis were stacked, resulting in protein bodies forming as honeycomb-like structures. We are unique in presenting clear demonstration that γ-zeins play a mechanistic role in QPM, providing a previously unexplored rationale for molecular breeding.

]]> Yongrui Wu et al. Ribosomal protein L10 is encoded in the mitochondrial genome of many land plants and green algae http://digitalcommons.unl.edu/plantscifacpub/47 http://digitalcommons.unl.edu/plantscifacpub/47 Wed, 29 Sep 2010 06:51:31 PDT Background: The mitochondrial genomes of plants generally encode 30-40 identified protein-coding genes and a large number of lineage-specific ORFs. The lack of wide conservation for most ORFs suggests they are unlikely to be functional. However, an ORF, termed orf-bryo1, was recently found to be conserved among bryophytes suggesting that it might indeed encode a functional mitochondrial protein.

Results: From a broad survey of land plants, we have found that the orf-bryo1 gene is also conserved in the mitochondria of vascular plants and charophycean green algae. This gene is actively transcribed and RNA edited in many flowering plants. Comparative sequence analysis and distribution of editing suggests that it encodes ribosomal protein L10 of the large subunit of the ribosome. In several lineages, such as crucifers and grasses, where the rpl10 gene has been lost from the mitochondrion, we suggest that a copy of the nucleus-encoded chloroplast-derived rpl10 gene may serve as a functional replacement.

Conclusion: Despite the fact that there are now over 20 mitochondrial genome sequences for land plants and green algae, this gene has remained unidentified and largely undetected until now because of the unlikely coincidence that most of the earlier sequences were from the few lineages that lack the intact gene. These results illustrate the power of comparative sequencing to identify novel genomic features.

]]> Jeffrey P. Mower et al. Prediction of antigenic epitopes on protein surfaces by consensus scoring http://digitalcommons.unl.edu/plantscifacpub/46 http://digitalcommons.unl.edu/plantscifacpub/46 Wed, 29 Sep 2010 06:46:13 PDT Background: Prediction of antigenic epitopes on protein surfaces is important for vaccine design. Most existing epitope prediction methods focus on protein sequences to predict continuous epitopes linear in sequence. Only a few structure-based epitope prediction algorithms are available and they have not yet shown satisfying performance.
Results: We present a new antigen Epitope Prediction method, which uses ConsEnsus Scoring (EPCES) from six different scoring functions - residue epitope propensity, conservation score, sidechain energy score, contact number, surface planarity score, and secondary structure composition. Applied to unbounded antigen structures from an independent test set, EPCES was able to predict antigenic eptitopes with 47.8% sensitivity, 69.5% specificity and an AUC value of 0.632. The performance of the method is statistically similar to other published methods. The AUC value of EPCES is slightly higher compared to the best results of existing algorithms by about 0.034.
Conclusion: Our work shows consensus scoring of multiple features has a better performance than any single term. The successful prediction is also due to the new score of residue epitope propensity based on atomic solvent accessibility.

]]> Shide Liang et al. Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS http://digitalcommons.unl.edu/plantscifacpub/45 http://digitalcommons.unl.edu/plantscifacpub/45 Wed, 29 Sep 2010 06:41:24 PDT The plant mitochondrial genome is retained in a multipartite structure that arises by a process of repeat-mediated homologous recombination. Low-frequency ectopic recombination also occurs, often producing sequence chimeras, aberrant ORFs, and novel subgenomic DNA molecules. This genomic plasticity may distinguish the plant mitochondrion from mammalian and fungal types. In plants, relative copy number of recombination-derived subgenomic DNA molecules within mitochondria is controlled by nuclear genes, and a genomic shifting process can result in their differential copy number suppression to nearly undetectable levels. We have cloned a nuclear gene that regulates mitochondrial substoichiometric shifting in Arabidopsis. The CHM gene was shown to encode a protein related to the MutS protein of Escherichia coli that is involved in mismatch repair and DNA recombination. We postulate that the process of substoichiometric shifting in plants may be a consequence of ectopic recombination suppression or replication stalling at ectopic recombination sites to effect molecule-specific copy number modulation.

]]> Ricardo V. Abdelnoor et al. A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco http://digitalcommons.unl.edu/plantscifacpub/44 http://digitalcommons.unl.edu/plantscifacpub/44 Wed, 29 Sep 2010 06:34:53 PDT In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239M gene constructs, with or without a mitochondrial targeting sequence, exhibited a semisterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the 0RF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that 0RF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.

]]> Shichuan He et al. Mitochondrial DNA rearrangement associated with fertility restoration and cytoplasmic reversion to fertility in cytoplasmic male sterile <i>Phaseolus vulgaris</i> L. http://digitalcommons.unl.edu/plantscifacpub/43 http://digitalcommons.unl.edu/plantscifacpub/43 Wed, 29 Sep 2010 06:30:31 PDT Restoration of pollen fertility to cytoplasmic male sterile (CMS) Phaseolus vulgaris by a nuclear restorer gene provides a system for studying nuclear-cytoplasmic interactions. Introduction of a nuclear restorer gene to this CMS line of P. vulgaris (CMS-Sprite) results in a mitochondrial genome rearrangement similar to that observed upon spontaneous cytoplasmic reversion to fertility. Three spontaneous heritable cytoplasmic revertants were derived from CMS-Sprite. Five fully fertile restored lines were also produced by using restorer line R-351 (BC3F3 populations). Comparison of the mitochondrial DNA restriction patterns of CMS-Sprite, the three fertile revertants, and the five restored lines revealed loss of a 6.0-kilobase (kb) Pst I fragment in all restored and revertant lines. Southern hybridizations with a 1.3-kb BamHI clone, internal to the 6.0-kb Pst I fragment, as a probe revealed two configurations of 6.0-kb homologous sequences in the sterile cytoplasm; one of the configurations was lost upon reversion or restoration. Mitochondrial DNA rearrangement has thus been observed upon restoration by a nuclear restorer gene in this CMS system.

]]> Sally MacKenzie et al.