REVEILLE8 and PSEUDO-REPONSE REGULATOR5 Form a Negative Feedback Loop within the Arabidopsis Circadian Clock

Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE–binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE–containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms

Characterization of the promoter of SmCP, the gene encoding Solanum melongena cysteine proteinase

published_or_final_versionabstractBotanyDoctoralDoctor of Philosoph

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Not AvailableThermophilic Aspergillus terreus RWY produced cellulases and xylanases in optimal concentrations at 45 °C in solid state fermentation process, though enzyme production was also observed at 50 and 55 °C. Filter paper cellulase (FP), endoglucanase (EG), b-glucosidase (BGL), cellobiohydrolase (CBH), xylanase, b-xylosidase, a-L-arabinofuranosidase and xylan esterase activities for A. terreus RWY at 45 °C in 72 h were 11.3 0.65, 103 6.4, 122.5 8.7, 10.3 0.66, 872 22.5, 22.1 0.75, 126.4 8.4 and 907 15.5U (g-ds) 1, respectively. Enzyme was optimally active at temperatures and pH ranging between 50–60 °C and 4.0–6.0, respectively. The half life (T1/2) of 270 and 240 min at 70 and 75 °C, respectively for the enzyme indicates its stability at higher temperatures. The addition of MnCl2, CoCl2, and FeCl3 significantly enhanced cellulase activity. Enzyme demonstrated multiplicity by having seven, one and three isoform(s) for EG, CBH and BGL, respectively. Significant production of functionally active consortium of cellulolytic and xylanolytic enzymes from A. terreus RWY makes it a potential candidate in bioprocessing applications.Not Availabl

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Not AvailableSolid state fermentation with pea pod waste and Aspergillus niger HN-1 resulted in filter paper cellu-lase (FP) and -glucosidase (BGL) activity of 30 FPU/gds and 270 U/gds, respectively. Validation withthe response surface optimized parameters (moisture content: 65%, pH 6.0, temperature: 33◦C, time:84 h) in a solid-state tray fermentation enhanced FP and BGL activity by about 40 and 28%, respectively.Multi-component enzyme from A. niger HN-1 produced FP, BGL, endoglucanase (EG), cellobiohy-drolase (CBHI), xylanase, -l-arabinofuranosidase, -xylosidase and xylan esterase with activities of41.07 ± 2.11 FPU/gds, 345.69 ± 17.1, 480.3 ± 21.5, 52.1 ± 1.5, 2800.5 ± 88.4, 88.1 ± 9.3, 280.8 ± 11.4 and3321.7 ± 14.8 U/gds, respectively. Enzyme was optimally active at temperature and pH of 55◦C and 5.0,respectively and demonstrated thermostability by retaining >95% activity for 6 h at 55◦C. SDS-PAGEshowed the presence of 11 protein bands with molecular mass ranging between 20 and 200 kDa, whilezymogram revealed the presence of multiple forms of EG, CBH and BGL with varying molecular mass.Hydrolysis of sweet sorghum bagasse at relatively high substrate loading (15%, w/v) with crude enzymeat 20 FPU/gds in thermostatically controlled glass reactor led to conversion of 82–91% of holocellulose tofermentable sugars in just 24 h as evident from HPLC analysis, showing promise for the reported enzymein bioprocessing applications.AMAAS sub-project (NBAIM/AMAAS/2008-09/AMBPH-05/HSO/BG/3/5982) from the Indian Council of Agricul-tural Research (ICAR), Government of India

The first crystal structures of a family 19 class IV chitinase : the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible.  Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity