Proteomic analysis of tomato (Lycopersicon esculentum) pollen

In flowering plants, pollen grains are produced in the anther and released to the external environment with the primary function of delivering sperm cells to the female gametophyte. This study was conducted to identify proteins in tomato pollen and to analyse their roles in relation to pollen function. Tomato is an important crop which is grown worldwide and is an excellent experimental system. Proteins were extracted from pollen, separated by two-dimensional gel electrophoresis (2-DE), and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting. Of the 960 spots observed on Colloidal Coomassie Blue (CCB)-stained 2-DE gels, 190 were selected for analysis. Of these, 158 spots, representing 133 distinct proteins, were identified by searching the NCBInr and Expressed Sequence Tag databases. The identified proteins were classified based on designated functions and the majority included those involved in defence mechanisms, energy conversions, protein synthesis and processing, cytoskeleton formation, Ca(2+) signalling, and as allergens. A number of proteins in tomato pollen were similar to those reported in the pollen of other species; however, several additional proteins with roles in defence mechanisms, metabolic processes, and hormone signalling were identified. The potential roles of the identified proteins in the survival strategy of the small, independent, two-celled pollen grain of tomato, and subsequently in pollen germination and tube growth are discussed

Proteome profile and functional classification of proteins in Arabidopsis thaliana (Landsberg erecta) mature pollen

Proteome analysis of mature Arabidopsis thaliana (Landsberg erecta ecotype) pollen was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 960 spots were resolved on pH 4–7 IPG strips and 110 distinct proteins were identified from 150 spots analyzed. The identified proteins were categorized based on their functional role in the pollen, which included proteins involved in energy regulation, defense-related mechanisms, calcium-binding and signaling, cytoskeletal formation, pollen allergens, glycine-rich proteins (GRPs), and late embryogenesis abundant (LEA) proteins. These proteins potentially play important roles in pollen function at maturity and during subsequent germination and tube growth. Some of the proteins identified were related to known pollen-specific transcripts, while some were similar to proteins found in the seed. In this study, 66 new proteins were identified which were not reported in two other recent studies on Arabidopsis pollen, 17 proteins were common in all three studies, and 35 or 26 proteins reported here had an overlap with one or the other two studies. These differences may be attributed to the methods of protein extraction, spot selection for analysis, and the ecotype used. Together, the three studies provide a broad spectrum of the Arabidopsis pollen proteome

Photosynthetic Carbon Fixation Characteristics of Fruiting Structures of Brassica campestris L.

Activities of key enzymes of the Calvin cycle and C(4) metabolism, rates of CO(2) fixation, and the initial products of photosynthetic (14)CO(2) fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv `Toria.' Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5-phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C(4) metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwall and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of (14)CO(2) assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO(2) during light. However, respiratory losses were very high during the dark period

Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target DNA

We designed MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of transcription factors Max and Myc, which bind with high DNA sequence specificity and affinity to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. ME47, however, displays propensity for instability and misfolding. We therefore sought to improve ME47\u27s structural and functional features. We used phage-assisted continuous evolution (PACE) to uncover "nonrational" changes to complement our rational design. PACE mutated Arg12 that contacts the DNA phosphodiester backbone. We would not have rationally made such a change, but this mutation improved ME47\u27s stability with little change in DNA-binding function. We mutated Cys29 to Ser and Ala in ME47\u27s HLH to eliminate undesired disulfide formation; these mutations reduced E-box binding activity. To compensate, we fused the designed FosW leucine zipper to ME47 to increase the dimerization interface and improve protein stability and E-box targeting activity. This "franken-protein" MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper—plus mutations that arose during PACE and rational design—and is a tractable, reliable protein in vivo and in vitro. Compared with ME47, MEF gives three-fold stronger binding to Ebox with four-fold increased specificity for E-box over nonspecific DNA. Generation of MEF demonstrates that combining rational design and continuous evolution can be a powerful tool for designing proteins with robust structure and strong DNA-binding function. <br /

Cluster Bean—A Ureide- or Amide-Producing Legume?

Xylem sap of cluster bean (Cyamopsis tetragonoloba L. cv FS-277) and pigeonpea (Cajanus cajan cv UPAS-120) were analyzed for total nitrogen, amide nitrogen, and ureide nitrogen at flowering stage. Nitrogenase, uricase, and allantoinase were compared in nodules of cluster bean and pigeonpea. Xylem sap of cluster bean exhibited higher amounts of amides as compared to ureides, and the activities of uricase and allantoinase (ureide-producing enzymes) in nodules were also low, whereas the reverse was the case for pigeonpea. Based on these investigations, it has been concluded that cluster bean is an amide-producing legume rather than ureide-producing as had been reported earlier