We have analyzed macromolecular changes that are associated with natural leaf senescence in Arabidopsis thaliana. The loss of chlorophyll that is characteristic of leaf senescence is accompanied by a specific pattern of decline of total RNA and protein levels. We have constructed two cDNA libraries representing mRNAs from Arabidopsis leaves at different senescence stages. Six cDNA clones corresponding to mRNAs that substantially increase in abundance during senescence were isolated. The levels of these mRNAs remain elevated into the late stages of senescence when most of the chlorophyll and protein of the leaf has been degraded. Sis cDNA clones that correspond to mRNAs that exhibit contrasting behavior were also identified: the levels of these mRNAs decrease to undetectable levels during senescence. The changes in the levels of these specific mRNAs during the course of senescence are presented. The results indicate that major changes in gene expression occur in Arabidopsis leaves during the process of senescence.

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Molecular analysis of natural leaf senescence in Arabidopsis ihaliana. -Physiol. Plant. 92: .^22-328,We have analyzed macromoleeuiar changes that are associated with natural leaf senescence in Arabidopsis thaliana. The loss of chlorophyll that is characteristic of leaf senescence is accompanied by a specific pattem of decline of total RN.A and protein ievels. We have construcled two cDNA libraries representing mR.NAs from .\rabidopsis leaves at different senescence stages. Six cDNA clones corresponding to mRNAs that substantially increase in abundance during senescence were isolated. The levels of these niRiN As remain e!e\ ated into the tate stages of senescence when most of the chlorophyll and protein of the leaf has been degraded. Sis cDNA clones that correspond to mRNAs that e,xhibit contrasting behavior were also identified: the levels of these mRNAs decrease to undetectable levels during senescence. The changes in the levels of these specific mRNAs during the course of senescence are presented. The results indicate that major ehanges in gene expression occur in ,Arahidiipsis leaves during the process of senescence.

SummaryPhotoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr form initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal

Five active site residues, Thr11, Glu12, Lys15, Lys37, and Ser41, implicated by the protein crystal structure studies of Escherichia coli DTBS, were mutated to determine their function in catalysis and substrate binding. Nine mutant enzymes, T11V, E12A, E12D, K15Q, K37L, K37Q, K37R, S41A, and S41C, were overproduced in an E. coli strain lacking a functional endogenous DTBS gene and purified to homogeneity. Replacement of Thr11 with valine resulted in a 24,000-fold increase in the Km(ATP) with little or no change in the Kd(ATP), KM(DAPA) and DTBS k(cat), suggesting an essential role for this residue in the steady-state affinity for ATP. The two Glu12 mutants showed essentially wild-type DTBS activity (slightly elevated k(cat)'s). Unlike wild-type DTBS, E12A had the same apparent KM(DAPA) at subsaturating and saturating ATP concentrations, indicating a possible role for Glu12 in the binding synergy between DAPA and ATP. The mutations in Lys15 and Lys37 resulted in loss of catalytic activity (0.01% and <0.9% of wild-type DTBS k(cat) for K15Q and the Lys37 mutant enzymes, respectively) and higher KM's for both DAPA (40-fold and >100-fold higher than wild-type for the K15Q and Lys37 mutant enzymes, respectively) and ATP (1800-fold and >10-fold higher than wild-type for K15Q and the K37 mutant enzymes, respectively). These results strongly suggest that Lys15 and Lys37 are crucial to both catalysis and substrate binding. S41A and S41C had essentially the same k(cat) as wild-type and had moderate increases in the DAPA and ATP KM and Kd (ATP) values. Replacement of Ser41 with cysteine resulted in larger effects than replacement with alanine. These data suggest that the H-bond between N7 of DAPA and the Ser41 side chain is not very important for catalysis. The catalytic behavior of these mutant enzymes was also studied by pulse-chase experiments which produced results consistent with the steady-state kinetic analyses. X-ray crystallographic studies of four mutant enzymes, S41A, S41C, K37Q, and K37L, showed that the crystals were essentially isomorphous to that of the wild-type DTBS. The models of these mutant enzymes were well refined (1.9 -2.6 A) and showed good similarity to the wild-type enzyme (rmsd of C alpha atoms: 0.16-0.24 A). The crystal structure of S41C complexed with DAPA, Mn2+/Mg2+, and AMPPCP revealed a localized conformational change (rotations of side chains of Cys41 and Thr11) which can account for the changes in the kinetic parameters observed for S41C. The crystal structures of the Lys37 mutant enzymes showed that the positive charge of the side chain of Lys37 is indispensable. Mutations of Lys37 to either glutamine or leucine resulted in a shift of the metal ion (up to 0.5 A) together with side chains of other active site residues which could disrupt the subtle balance between the positive and negative charges in the active site. The conformational change of the phosphate binding loop (Gly8-X-X-X-X-X-Gly14-Lys15-Thr16) upon nucleotide binding observed previously [Huang, W., Jia, J., Gibson, K. J., Taylo...