David R. Quilici

Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development

BackgroundGrape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip®Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine.ResultsOver the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (≥2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as véraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from véraison to mature berries.ConclusionThese results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalog of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing.

Water deficit alters differentially metabolic pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay

BackgroundWater deficit has significant effects on grape berry composition resulting in improved wine quality by the enhancement of color, flavors, or aromas. While some pathways or enzymes affected by water deficit have been identified, little is known about the global effects of water deficit on grape berry metabolism.ResultsThe effects of long-term, seasonal water deficit on berries of Cabernet Sauvignon, a red-wine grape, and Chardonnay, a white-wine grape were analyzed by integrated transcript and metabolite profiling. Over the course of berry development, the steady-state transcript abundance of approximately 6,000 Unigenes differed significantly between the cultivars and the irrigation treatments. Water deficit most affected the phenylpropanoid, ABA, isoprenoid, carotenoid, amino acid and fatty acid metabolic pathways. Targeted metabolites were profiled to confirm putative changes in specific metabolic pathways. Water deficit activated the expression of numerous transcripts associated with glutamate and proline biosynthesis and some committed steps of the phenylpropanoid pathway that increased anthocyanin concentrations in Cabernet Sauvignon. In Chardonnay, water deficit activated parts of the phenylpropanoid, energy, carotenoid and isoprenoid metabolic pathways that contribute to increased concentrations of antheraxanthin, flavonols and aroma volatiles. Water deficit affected the ABA metabolic pathway in both cultivars. Berry ABA concentrations were highly correlated with 9-cis-epoxycarotenoid dioxygenase (NCED1) transcript abundance, whereas the mRNA expression of other NCED genes and ABA catabolic and glycosylation processes were largely unaffected. Water deficit nearly doubled ABA concentrations within berries of Cabernet Sauvignon, whereas it decreased ABA in Chardonnay at véraison and shortly thereafter.ConclusionThe metabolic responses of grapes to water deficit varied with the cultivar and fruit pigmentation. Chardonnay berries, which lack any significant anthocyanin content, exhibited increased photoprotection mechanisms under water deficit conditions. Water deficit increased ABA, proline, sugar and anthocyanin concentrations in Cabernet Sauvignon, but not Chardonnay berries, consistent with the hypothesis that ABA enhanced accumulation of these compounds. Water deficit increased the transcript abundance of lipoxygenase and hydroperoxide lyase in fatty metabolism, a pathway known to affect berry and wine aromas. These changes in metabolism have important impacts on berry flavor and quality characteristics. Several of these metabolites are known to contribute to increased human-health benefits.

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

In eukaryotes, 14‐3‐3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation‐dependent interactions. To uncover new clients, 14‐3‐3 omega (At1g78300) from Arabidopsis was engineered with a “tandem affinity purification” tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14‐3‐3 omega can dimerize with at least 10 of the 12 14‐3‐3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14‐3‐3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K+ channel (GORK), a Cl− channel (CLCg), Ca2+ channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS‐6, ‐7 and ‐8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (β and γ). More than 80% (101) of these putative clients represent previously unidentified 14‐3‐3 interactors. These results raise the number of putative 14‐3‐3 clients identified in plants to over 300.

A novel yeast two‐hybrid approach to identify CDPK substrates: Characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein1

Calcium‐dependent protein kinases (CDPKs) are sensor‐transducer proteins capable of decoding calcium signals in diverse phosphorylation‐dependent calcium signaling networks in plants and some protists. Using a novel yeast two‐hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11‐interacting protein. AtDi19 is a member of a small family of stress‐induced genes. The interaction was confirmed using pull‐down assays with in vitro translated AtCPK11 and GST–AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca2+‐dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase–substrate interactions without affecting specificity.

Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORFs with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite, Zootermopsis nevadensis

Abstract In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis . The in vivo incorporation of [1,4- 14 C]succinate and [2,3- 14 C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4- 13 C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3- 14 C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.

Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs

The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA1. Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress2,3 and metabolic disorders4–6. How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat-diet-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (m5C, m2G) in sperm 30–40 nt RNA fractions that are induced by a high-fat diet. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs and rRNA-derived small RNAs, which might be essential in composing a sperm RNA ‘coding signature’ that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated m5C contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.Zhang et al. report that tRNA methyltransferase Dnmt2 is required for sperm small-non-coding-RNA-mediated transmission of paternal metabolic disorders to the offspring.

The human uterine smooth muscle S-nitrosoproteome fingerprint in pregnancy, labor, and preterm labor

Molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor at term are incompletely known. More than 10% of babies born worldwide are premature and 1,000,000 die annually. Preterm labor results in preterm delivery in 50% of cases in the United States explaining 75% of fetal morbidity and mortality. There is no Food and Drug Administration-approved treatment to prevent preterm delivery. Nitric oxide-mediated relaxation of human uterine smooth muscle is independent of global elevation of cGMP following activation of soluble guanylyl cyclase. S-nitrosation is a likely mechanism to explain cGMP-independent relaxation to nitric oxide and may reveal S-nitrosated proteins as new therapeutic targets for the treatment of preterm labor. Employing S-nitrosoglutathione as an nitric oxide donor, we identified 110 proteins that are S-nitrosated in 1 or more states of human pregnancy. Using area under the curve of extracted ion chromatograms as well as normalized spectral counts to quantify relative expression levels for 62 of these proteins, we show that 26 proteins demonstrate statistically significant S-nitrosation differences in myometrium from spontaneously laboring preterm patients compared with nonlaboring patients. We identified proteins that were up-S-nitrosated as well as proteins that were down-S-nitrosated in preterm laboring tissues. Identification and relative quantification of the S-nitrosoproteome provide a fingerprint of proteins that can form the basis of hypothesis-directed efforts to understand the regulation of uterine contraction-relaxation and the development of new treatment for preterm labor.

Altered Protein Composition and Gene Expression in Strabismic Human Extraocular Muscles and Tendons

Purpose To determine whether structural protein composition and expression of key regulatory genes are altered in strabismic human extraocular muscles. Methods Samples from strabismic horizontal extraocular muscles were obtained during strabismus surgery and compared with normal muscles from organ donors. We used proteomics, standard and customized PCR arrays, and microarrays to identify changes in major structural proteins and changes in gene expression. We focused on muscle and connective tissue and its control by enzymes, growth factors, and cytokines. Results Strabismic muscles showed downregulation of myosins, tropomyosins, troponins, and titin. Expression of collagens and regulators of collagen synthesis and degradation, the collagenase matrix metalloproteinase (MMP)2 and its inhibitors, tissue inhibitor of metalloproteinase (TIMP)1 and TIMP2, was upregulated, along with tumor necrosis factor (TNF), TNF receptors, and connective tissue growth factor (CTGF), as well as proteoglycans. Growth factors controlling extracellular matrix (ECM) were also upregulated. Among 410 signaling genes examined by PCR arrays, molecules with downregulation in the strabismic phenotype included GDNF, NRG1, and PAX7; CTGF, CXCR4, NPY1R, TNF, NTRK1, and NTRK2 were upregulated. Signaling molecules known to control extraocular muscle plasticity were predominantly expressed in the tendon rather than the muscle component. The two horizontal muscles, medial and lateral rectus, displayed similar changes in protein and gene expression, and no obvious effect of age. Conclusions Quantification of proteins and gene expression showed significant differences in the composition of extraocular muscles of strabismic patients with respect to important motor proteins, elements of the ECM, and connective tissue. Therefore, our study supports the emerging view that the molecular composition of strabismic muscles is substantially altered.

De novo molecular modeling and biophysical characterization of Manduca sexta eclosion hormone.

Eclosion hormone (EH) is an integral component in the cascade regulating the behaviors culminating in emergence of an insect from its old exoskeleton. Little is known regarding the EH solution structure; consequently, we utilized a computational approach to generate a hypothetical structure for Manduca sexta EH. The de novo algorithm exploited the restricted conformational space of disulfide bonds (Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49) and predicted secondary structure elements to generate a thermodynamically stable structure characterized by 55% helical content, an unstructured N-terminus, a helical C-terminus, and a solvent-exposed loop containing Trp28 and Phe29. Both the strain and pseudo energies of the predicted peptide compare favorably with those of known structures. The 62-amino acid peptide was synthesized, folded, assayed for activity, and structurally characterized to confirm the validity of the model. The helical content is supported by circular dichroism and hydrogen-deuterium exchange mass spectrometry. Fluorescence emission spectra and acrylamide quenching are consistent with the solvent exposure predicted for Trp28, which is shielded by Phe29. Furthermore, thermodynamically stable conformations that deviated only slightly from the predicted Manduca EH structure were generated in silico for the Bombyx mori and Drosophila melanogaster EHs, indicating that the conformation is not species-dependent. In addition, the biological activities of known mutants and deletion peptides were rationalized with the predicted Manduca EH structure, and we found that, on the basis of sequence conservation, functionally important residues map to two conserved hydrophobic clusters incorporating the C-terminus and the first loop.