ABSTRACT
The main goal of this study was to develop a method for the extraction and indirect estimation of the quantity of calcium oxalate (CaOx) in the foliage of trees. Foliar tissue was collected from a single tree of each species (five conifers and five hardwoods) for comparison of extractions in different solvents using 10 replicates per species from the same pool of tissue. For each species, calcium (Ca) and oxalate were extracted sequentially in double deionized water and 2N acetic acid, and finally, five replicate samples were extracted in 5% (0.83N) perchloric acid (PCA) and the other five in 2N hydrochloric acid (HCl); three cycles of freezing and thawing were used for each solvent. Total ions were extracted by microwave digestion. Calcium was quantified with an inductively coupled plasma emission spectrophotometer method and oxalate was eluted and quantified using a high performance liquid chromatography method. This experiment was repeated again with two conifer and two hardwood species using four trees per species, and two analytical replicates for each tree. We report here that, regardless of age of individual trees within a species, time of collection or species type, the third extraction in PCA or HCl resulted in near equimolar quantities of Ca and oxalate (r(2) ≥ 0.99). This method provides an easy estimate of the quantity of CaOx crystals using a small sample of foliar tissue. An additional benefit of PCA is that it precipitates the nucleic acids and proteins, allowing the quantification of several free/soluble metabolites such as amino acids, polyamines, organic acids and inorganic elements all from a single sample extract.
Subject(s)
Botany/methods , Calcium Oxalate/analysis , Trees/chemistry , Calcium Oxalate/isolation & purification , Calcium Oxalate/metabolism , Plant Leaves/chemistry , Plant Leaves/metabolism , Trees/metabolismABSTRACT
The evolutionarily conserved PUF proteins stimulate CCR4 mRNA deadenylation through binding to 3' untranslated region sequences of specific mRNA. We have investigated the mechanisms by which PUF3 in Saccharomyces cerevisiae accelerates deadenylation of the COX17 mRNA. PUF3 was shown to affect PAN2 deadenylation of the COX17 mRNA independent of the presence of CCR4, suggesting that PUF3 acts through a general mechanism to affect deadenylation. Similarly, eIF4E, the cap-binding translation initiation factor known to control CCR4 deadenylation, was shown to affect PAN2 activity in vivo. PUF3 was found to be required for eIF4E effects on COX17 deadenylation. Both eIF4E and PUF3 effects on deadenylation were shown, in turn, to necessitate a functional poly(A)-binding protein (PAB1) in which removal of the RRM1 (RNA recognition motif 1) domain of PAB1 blocked both their effects on deadenylation. While removal of the proline-rich region (P domain) of PAB1 substantially reduces CCR4 deadenylation at non-PUF3-controlled mRNA and correspondingly blocked eIF4E effects on deadenylation, PUF3 essentially bypassed this P domain requirement. These results indicate that the PAB1-mRNP structure is critical for PUF3 action. We also found that multiple components of the CCR4-NOT deadenylase complex, but not PAN2, interacted with PUF3. PUF3 appears, therefore, both to act independently of CCR4 activity, possibly through effects on PAB1-mRNP structure, and to be capable of retaining the CCR4-NOT complex.
Subject(s)
Poly(A)-Binding Proteins/metabolism , RNA-Binding Proteins/metabolism , Ribonucleases/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Copper Transport Proteins , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Genes, Fungal , Kinetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Multiprotein Complexes , Poly(A)-Binding Proteins/chemistry , Poly(A)-Binding Proteins/genetics , Protein Structure, Tertiary , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribonucleases/chemistry , Ribonucleases/genetics , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
CCR4, a poly(A) deadenylase of the exonuclease III family, is a component of the multiprotein CCR4-NOT complex of Saccharomyces cerevisiae that is involved in mRNA degradation. CCR4, unlike all other exonuclease III family members, contains a leucine-rich repeat (LRR) motif through which it makes contact to CAF1 and other factors. The LRR residues important in contacting CAF1 were identified by constructing 29 CCR4 mutations encompassing a majority (47 of 81) of residues interstitial to the conserved structural residues. Two-hybrid and immunoprecipitation data revealed that physical contact between CAF1 and the LRR is blocked by mutation of just two alpha-helix/beta-helix strand loop residues linking the first and second repeats. In contrast, CAF16, a potential ligand of CCR4, was abrogated in its binding to the LRR by mutations in the N terminus of the second beta-strand. The LRR domain was also found to contact the deadenylase domain of CCR4, and deletion of the LRR region completely inhibited CCR4 enzymatic activity. Mutations throughout the beta-sheet surface of the LRR, including those that did not specifically interfere with contacts to CAF1 or CAF16, significantly reduced CCR4 deadenylase activity. These results indicate that the CCR4-LRR, in addition to binding to CAF1, plays an essential role in the CCR4 deadenylation of mRNA.
