Genetic analysis of floral symmetry in Van Gogh's sunflowers reveals independent recruitment of CYCLOIDEA genes in the Asteraceae

The genetic basis of floral symmetry is a topic of great interest because of its effect on pollinator behavior and, consequently, plant diversification. The Asteraceae, which is the largest family of flowering plants, is an ideal system in which to study this trait, as many species within the family exhibit a compound inflorescence containing both bilaterally symmetric (i.e., zygomorphic) and radially symmetric (i.e., actinomorphic) florets. In sunflower and related species, the inflorescence is composed of a single whorl of ray florets surrounding multiple whorls of disc florets. We show that in double-flowered (dbl) sunflower mutants (in which disc florets develop bilateral symmetry), such as those captured by Vincent van Gogh in his famous nineteenth-century sunflower paintings, an insertion into the promoter region of a CYCLOIDEA (CYC)-like gene (HaCYC2c) that is normally expressed specifically in WT rays is instead expressed throughout the inflorescence, presumably resulting in the observed loss of actinomorphy. This same gene is mutated in two independent tubular-rayed (tub) mutants, though these mutations involve apparently recent transposon insertions, resulting in little or no expression and radialization of the normally zygomorphic ray florets. Interestingly, a phylogenetic analysis of CYC-like genes from across the family suggests that different paralogs of this fascinating gene family have been independently recruited to specify zygomorphy in different species within the Asteraceae.     

Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and L-allo-Thr cleavage

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyses the conversion of L-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially L-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via Calpha proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5'-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5'-phosphate. However, there was an alteration in the lambda max value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with Calpha and Cbeta of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the Calpha proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via Calpha proton abstraction by SHMT is proposed.     

X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry

X-ray crystallography, although a powerful technique for determining the three-dimensional structure of proteins, poses inherent problems in assigning the primary structure in residues Asp/Asn and Glu/Gln since these cannot be distinguished decisively in the electron density maps. In our recently published X-ray crystal structure of the Sclerotium rolfsii lectin (SRL) at 1.1 A resolution, amino acid sequence was initially deduced from the electron density map and residues Asp/Asn and Glu/Gln were assigned by considering their hydrogen bonding potential within their structural neighborhood. Attempts to verify the sequence by Edman sequencing were not successful as the N terminus of the protein was blocked. Mass spectrometry was applied to verify and resolve the ambiguities in the SRL X-ray crystal structure deduced sequence. From the Matrix assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI TOF-MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis of tryptic and chymotryptic peptides of SRL, we could confirm and correct the sequence at five locations with respect to Asp/Asn and Glu/Gln. Analysis data also confirmed the positions of Leu/Ile, Gln/Lys residues and the sequence covering 118 of the total 141 residues accounting to 83.68% of the earlier deduced sequence of SRL.     

Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR

Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of gene expression of E. coli gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of E. coli, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. RT-PCR experiment was done to investigate the down regulation of gyrase A gene. RT-PCR results demonstrated a significant decrease of gyrase A gene after 18 h of induction of the transformants. These experiments showed that the down regulation of the gene was seen after 18 h of induction than earlier hours of induction with IPTG suggesting inhibition of gyrase A gene with profound effect on cell viability. These results demonstrate the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins.     

Crystal structure of binary and ternary complexes of serine hydroxymethyltransferase from Bacillus stearothermophilus: insights into the catalytic mechanism

Serine hydroxymethyltransferase (SHMT), a member of the alpha-class of pyridoxal phosphate-dependent enzymes, catalyzes the reversible conversion of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate. We present here the crystal structures of the native enzyme and its complexes with serine, glycine, glycine, and 5-formyl tetrahydrofolate (FTHF) from Bacillus stearothermophilus. The first structure of the serine-bound form of SHMT allows identification of residues involved in serine binding and catalysis. The SHMT-serine complex does not show any significant conformational change compared with the native enzyme, contrary to that expected for a conversion from an "open" to "closed" form of the enzyme. However, the ternary complex with FTHF and glycine shows the reported conformational changes. In contrast to the Escherichia coli enzyme, this complex shows asymmetric binding of the FTHF to the two monomers within the dimer in a way similar to the murine SHMT. Comparison of the ternary complex with the native enzyme reveals the structural basis for the conformational change and asymmetric binding of FTHF. The four structures presented here correspond to the various reaction intermediates of the catalytic pathway and provide evidence for a direct displacement mechanism for the hydroxymethyl transfer rather than a retroaldol cleavage.     

Photophysics of nanographenes: from polycyclic aromatic hydrocarbons to graphene nanoribbons

Photosynthesis Research - Graphene quantum dots (GQDs) and nanoribbons (GNRs) are classes of nanographene molecules that exhibit highly tunable photophysical properties. There have been great...     

Fine needle aspiration cytology of juvenile hyaline fibromatosis: a case report

BACKGROUND: Juvenile hyaline fibromatosis (YHF) is a rare inherited disorder characterized by tumorous growth of hyalinized fibrous tissue. No report on cytomorphology of this condition is available in English on MEDLINE. CASE REPORT: A 6-year-old girl had multiple nontender nodules on both ear lobes, nose and scalp. Fine needle aspiration of the nodule on the left ear revealed benign, spindle-shaped cells with an eosinophilic ground substance in the background. The diagnosis of JHF was made following cytologic and histopathologic studies. CONCLUSION: Fine needle aspiration cytology is reliable for the diagnosis of JHF.     

A change in reaction specificity of sheep liver serine hydroxymethyltransferase. Induction of NADH oxidation upon mutation of His230 to Tyr.

Both serine hydroxymethyltransferase and aspartate aminotransferase belong to the alpha-class of pyridoxal-5'-phosphate (pyridoxalP)-dependent enzymes but exhibit different reaction and substrate specificities. A comparison of the X-ray structure of these two enzymes reveals that their active sites are nearly superimposable. In an attempt to change the reaction specificity of serine hydroxymethyltransferase to a transaminase, His 230 was mutated to Tyr which is the equivalent residue in aspartate aminotransferase. Surprisingly, the H230Y mutant was found to catalyze oxidation of NADH in an enzyme concentration dependent manner instead of utilizing L-aspartate as a substrate. The NADH oxidation could be linked to oxygen consumption or reduction of nitrobluetetrazolium. The reaction was inhibited by radical scavengers like superoxide dismutase and D-mannitol. The Km and kcat values for the reaction of the enzyme with NADH were 74 microM and 5. 2 x 10-3 s-1, respectively. This oxidation was not observed with either the wild type serine hydroxymethyltransferase or H230A, H230F or H230N mutants. Thus, mutation of H230 of sheep liver serine hydroxymethyltransferase to Tyr leads to induction of an NADH oxidation activity implying that tyrosyl radicals may be mediating the reaction.     

His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis.

The three-dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3' of pyridoxal-5'-phosphate and other residues at the active site such as S203, K257, H357 and R402 (numbering as per the human enzyme). This and the conserved nature of H231 in all serine hydroxymethyltransferases highlights its importance in catalysis and/or maintenance of oligomeric structure of the enzyme. In an attempt to decipher the role of H230 (H231 of the human enzyme) in the catalytic mechanism and/or maintenance of oligomeric structure of sheep liver serine hydroxymethyltransferase, the residue was mutated to arginine, phenylalanine, alanine, asparagine or tyrosine. Our results suggest that the nature of the amino acid substitution has a marked effect on the catalytic activity of the enzyme. H230R and H230F mutant proteins were completely inactive, dimeric and did not bind pyridoxal-5'-phosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxal-5'-phosphate. These mutants had only 2-3% catalytic activity. The side reactions like transamination and 5,6,7, 8-tetrahydrofolate independent aldol cleavage were much more severely affected. They were able to form the external aldimine with glycine and serine but the quinonoid intermediate was not observed upon the addition of 5,6,7,8-tetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxal-5'-phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcat / Km values for L-serine and Lallothreonine were 10-fold and 174-fold less for this mutant enzyme compared to the wild-type protein. These results suggest that H230 is involved in the step prior to the formation of the quinonoid intermediate, possibly in orienting the pyridine ring of the cofactor, in order to facilitate effective proton abstraction.     

Overexpression of yeast spermidine synthase impacts ripening,senescence and decay symptoms in tomato

Polyamines (PAs) are ubiquitous, polycationic biogenic amines that are implicated in many biological processes, including plant growth and development, but their precise roles remain to be determined. Most of the previous studies have involved three biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), and their derivatives. We have expressed a yeast spermidine synthase (ySpdSyn) gene under constitutive (CaMV35S) and fruit‐ripening specific (E8) promoters in Solanum lycopersicum (tomato), and determined alterations in tomato vegetative and fruit physiology in transformed lines compared with the control. Constitutive expression of ySpdSyn enhanced intracellular levels of Spd in the leaf, and transiently during fruit development, whereas E8‐ySpdSyn expression led to Spd accumulation early and transiently during fruit ripening. The ySpdSyn transgenic fruits had a longer shelf life, reduced shriveling and delayed decay symptom development in comparison with the wild‐type (WT) fruits. An increase in shelf life of ySpdSyn transgenic fruits was not facilitated by changes in the rate of water loss or ethylene evolution. Additionally, the expression of several cell wall and membrane degradation‐related genes in ySpdSyn transgenic fruits was not correlated with an extension of shelf life, indicating that the Spd‐mediated increase in fruit shelf life is independent of the above factors. Crop maturity, indicated by the percentage of ripening fruits on the vine, was delayed in a CaMV35S‐ySpdSyn genotype, with fruits accumulating higher levels of the antioxidant lycopene. Notably, whole‐plant senescence in the transgenic plants was also delayed compared with WT plants. Together, these results provide evidence for a role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing post‐harvest senescence and decay.