Genetic mapping of a major codominant QTL associated with β-carotene accumulation in watermelon

The common flesh color of commercially grown watermelon is red due to the accumulation of lycopene. However, natural variation in carotenoid composition that exists among heirloom and exotic accessions results in a wide spectrum of flesh colors. We previously identified a unique orange flesh watermelon accession (NY0016) that accumulates mainly β-carotene and no lycopene. We hypothesized this unique accession could serve as a viable source for increasing provitamin A content in watermelon. Here we characterize the mode of inheritance and genetic architecture of this trait. Analysis of testcrosses of NY0016 with yellow and red fruited lines indicated a codominant mode of action as F₁ fruits exhibited a combination of carotenoid profiles from both parents. We combined visual color phenotyping with genotyping-by-sequencing of an F_2:3 population from a cross of NY0016 by a yellow fruited line, to map a major locus on chromosome 1, associated with β-carotene accumulation in watermelon fruit. The QTL interval is approximately 20 cM on the genetic map and 2.4 Mb on the watermelon genome. Trait-linked marker was developed and used for validation of the QTL effect in segregating populations across different genetic backgrounds. This study is a step toward identification of a major gene involved in carotenoid biosynthesis and accumulation in watermelon. The codominant inheritance of β-carotene provides opportunities to develop, through marker-assisted breeding, β-carotene-enriched red watermelon hybrids.     

Genetic code symmetry and efficient design of GC-constrained coding sequences

MOTIVATION: Cloning of long DNA sequences (40-60 bases) into phage display libraries using polymerase chain reaction (PCR) is a low efficiency process, in which PCR is used to incorporate a DNA insert, coding for a certain peptide, into the amplified sequence. The PCR efficiency in this process is strongly affected by the distribution of G-C bases in the amplified sequence. As any DNA insert coding for the target peptide may be attempted, there is a flexibility in choosing part of the amplified sequence. Since the number of inserts coding for the same peptide is exponential in the peptide length, a computational problem naturally arises--that of efficiently finding an insert, whose parameters are optimal for PCR cloning. RESULTS: The GC distribution requirements are formulated as a search problem. We developed an efficient, linear time 'one pass' algorithm for this problem. Interestingly, our algorithm strongly relies on an interesting symmetry, which we observed in the standard genetic code. Most non-standard genetic codes examined possess this symmetry as well, yet some do not. We generalize the search problem and consider the case of a non-standard, or arbitrary, genetic code where this symmetry does not necessary hold. We solve the generalized problem in polynomial, but nonlinear, time. AVAILABILITY: An implementation of the proposed algorithm is available upon request from the authors.     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Evolutionary Aspects of Enzyme Dynamics

The role of evolutionary pressure on the chemical step catalyzed by enzymes is somewhat enigmatic, in part because chemistry is not rate-limiting for many optimized systems. Herein, we present studies that examine various aspects of the evolutionary relationship between protein dynamics and the chemical step in two paradigmatic enzyme families, dihydrofolate reductases and alcohol dehydrogenases. Molecular details of both convergent and divergent evolution are beginning to emerge. The findings suggest that protein dynamics across an entire enzyme can play a role in adaptation to differing physiological conditions. The growing tool kit of kinetics, kinetic isotope effects, molecular biology, biophysics, and bioinformatics provides means to link evolutionary changes in structure-dynamics function to the vibrational and conformational states of each protein.     

Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

The hydride transfer reaction catalyzed by dihydrofolate reductase (DHFR) is a model for examining how protein dynamics contribute to enzymatic function. The relationship between functional motions and enzyme evolution has attracted significant attention. Recent studies on N23PP Escherichia coli DHFR (ecDHFR) mutant, designed to resemble parts of the human enzyme, indicated a reduced single turnover rate. NMR relaxation dispersion experiments with that enzyme showed rigidification of millisecond Met-20 loop motions (Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J., Benkovic, S. J., and Wright, P. E. (2011) Science 332, 234–238). A more recent study of this mutant, however, indicated that fast motions along the reaction coordinate are actually more dispersed than for wild-type ecDHFR (WT). Furthermore, a double mutant (N23PP/G51PEKN) that better mimics the human enzyme seems to restore both the single turnover rates and narrow distribution of fast dynamics (Liu, C. T., Hanoian, P., French, T. H., Hammes-Schiffer, S., and Benkovic, S. J. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 10159–11064). Here, we measured intrinsic kinetic isotope effects for both N23PP and N23PP/G51PEKN double mutant DHFRs over a temperature range. The findings indicate that although the C-H→C transfer and dynamics along the reaction coordinate are impaired in the altered N23PP mutant, both seem to be restored in the N23PP/G51PEKN double mutant. This indicates that the evolution of G51PEKN, although remote from the Met-20 loop, alleviated the loop rigidification that would have been caused by N23PP, enabling WT-like H-tunneling. The correlation between the calculated dynamics, the nature of C-H→C transfer, and a phylogenetic analysis of DHFR sequences are consistent with evolutionary preservation of the protein dynamics to enable H-tunneling from well reorganized active sites.     

Pleiotropic effects of tobacco-mosaic-virus movement protein on carbon metabolism in transgenic tobacco plants

Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing wild-type or mutant forms of the 30-kDa movement protein of tobacco mosaic virus (TMV-MP) were employed to study the effects of the TMV-MP on carbon metabolism in source leaves. Fully expanded source leaves of transgenic plants expressing the TMV-MP were found to retain more newly fixed ¹⁴C compared with control plants. Analysis of ¹⁴C-export from young leaves of TMV-MP plants, where the MP is yet to influence plasmodesmal size exclusion limit, indicated a similar pattern, in that daytime ¹⁴C export was slower in TMV-MP plants as compared to equivalent-aged leaves on control plants. Pulse-chase experiments were used to monitor radioactivity present in the different carbohydrate fractions, at specified intervals following ¹⁴CO₂ labeling. These studies established that the-TMV-MP can cause a significant adjustment in short-term ¹⁴-C-photosynthate storage and export. That these effects of the TMV-MP on carbon metabolism and phloem function were not attributable to the effect of this protein on plasmodesmal size exclusion limits, per se, was established using transgenic tobacco plants expressing temperature-sensitive and C-terminal deletion mutant forms of the TMV-MP. Collectively, these studies establish the pleiotropic nature of the TMV-MP in transgenic tobacco, and the results are discussed in terms of potential sites of interaction between the TMV-MP and endogenous processes involved in regulating carbon metabolism and export.Abbreviations MP movement protein - SEL size exclusion limit - TMV tobacco mosaic virus - ts temperature sensitive This work was supported by United State-Israel Binational Agricultural Research Development Fund grant No. 90-00070 (S.W. and W.J.L). We thank Roger N. Beachy for generously providing some of the transgenic plant lines employed in this study. This paper is a contribution from the Uri Kinamon Laboratory. A.A.O. was supported by a scholarship from the Kinamon Foundation.     

Exposure to Hyperbaric Oxygen Intensified Vancomycin-Induced Nephrotoxicity in Rats

It has been suggested that oxidative stress is a potential mechanism for vancomycin-induced nephrotoxicity and hyperbaric oxygen therapy (HBO) has been shown to be effective in treating renal toxicity that has been pharmacologically induced in animal models. The aim of this study was to investigate the effect of HBO therapy on vancomycin-induced nephrotoxicity in rats. The study group comprised 36 Sprague Dawley male rats. We treated 30 with 500 mg/kg of intraperitoneal vancomycin once a day for 7 days. Half of these rats received a daily 1-hour treatment with HBO at 2 Atmospheres (ATM) on the same 7 days and formed the HBO+ group. The other 15 subjects received no HBO treatment (HBO- group). The remaining six rats served as the control group, three received HBO treatments alone and no treatment was administered to the other three rats. Laboratory results were obtained on day 8 and the intervention and control groups were compared. Rats in the HBO+ group gained less weight than the HBO- group (11.6 grams vs 22.6 grams; P = 0,008) and had significantly higher serum blood urea nitrogen (99.6 vs 52.6 mg/dL; P<0.001), serum creatinine (0.42 vs 0.16 mg/dL; P = 0.001) and magnesium (3.6 vs 3.1mg/dL; P = 0.014). The vancomycin blood levels were also higher in the HBO+ group (27.8 vs 6.7 μg/mL; P = 0.078). There were no pathological kidney changes in the control group. All the kidneys from the treated groups (vancomycin +HBO and vancomycin HBO-) showed moderate to severe histopathological changes with no statistical significance between them. This study demonstrated that exposure to hyperbaric oxygen intensified vancomycin-induced nephrotoxicity in rats.     

Guard cells of Commelina communis L. do not respond metabolically to osmotic stress in isolated epidermis: Implications for stomatal responses to drought and humidity

We investigated the hypothesis that stomatal aperture is regulated by epidermal water status. Detached epidermal peels of Commelina communis L. or leaf disks with epidermis attached were incubated in graded solutions of mannitol (0–1.2 M) containing KCl. In isolated epidermis, guard-cell solute content of open stomata did not decrease in response to desiccation. Guard cells of closed stomata accumulated solutes to the same extent in all levels of mannitol tested. There was no evidence of stress-induced hydroactive closure nor of inhibition of hydroactive opening, even when guard cells of closed stomata were initially plasmolyzed. Hydropassive, osmometer-like, changes in stomatal aperture in the isolated epidermis were induced by addition or removal of mannitol, but these did not involve changes in guard-cell solute content. In leaf disks, stomata exhibited clear hydroactive stomatal responses. Steady-state guard-cell solute content of initially open and initially closed stomata decreased substantially with increasing mannitol. Stomata were completely closed above approx. 0.4 M mannitol, near the turgor-loss point for the bulk leaf tissue. Stomata of Commelina did not exhibit direct hydroactive responses to environmental or epidermal water status. Stomatal responses to water deficit and low humidity may be indirect, mediated by abscisic acid or other signal metabolite(s) from the mesophyll.Abbreviations ABA abscisic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulfonic acid