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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Abstract-Increased phosphorylation of the cardiac ryanodine receptor (RyR)2 by protein kinase A (PKA) at the phosphoepitope encompassing Ser2808 has been advanced as a central mechanism in the pathogenesis of cardiac arrhythmias and heart failure. In this scheme, persistent activation of the sympathetic system during chronic stress leads to PKA "hyperphosphorylation" of RyR2-S2808, which increases Ca 2ϩ release by augmenting the sensitivity of the RyR2 channel to diastolic Ca 2ϩ . This gain-of-function is postulated to occur with the unique participation of RyR2-S2808, and other potential PKA phosphorylation sites have been discarded. Although it is clear that RyR2 is among the first proteins in the heart to be phosphorylated by ␤-adrenergic stimulation, the functional impact of phosphorylation in excitation-contraction coupling and cardiac performance remains unclear. We used gene targeting to produce a mouse model with complete ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Whole-heart and isolated cardiomyocyte experiments were performed to test the role of ␤-adrenergic stimulation and PKA phosphorylation of Ser2808 in heart failure progression and cellular Ca 2ϩ handling. We found that the RyR2-S2808A mutation does not alter the ␤-adrenergic response, leaves cellular function almost unchanged, and offers no significant protection in the maladaptive cardiac remodeling induced by chronic stress. Moreover, the RyR2-S2808A mutation appears to modify single-channel activity, although modestly and only at activating [Ca 2ϩ ]. Taken together, these results reveal some of the most important effects of PKA phosphorylation of RyR2 but do not support a major role for RyR2-S2808 phosphorylation in the pathogenesis of cardiac dysfunction and failure.

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.

Cotton, an important natural fiber, is a differentiated epidermal cell. The number of genes that are active in fiber cells is similar to those in leaf, ovule, or root tissues. Through differential screening of a fiber cDNA library, we isolated five cDNA clones that are preferentially expressed in fiber. One of the cDNA clones, pCKE6, corresponded to an abundant mRNA in fiber. Transcripts for E6 were detected throughout the development of the fiber. Immunoprecipitation of in vitro translation products and Western blot analysis of fiber proteins showed two polypeptides in the range of 30-32 kDa as the products of E6 mRNA. Sequence analysis and hybrid-selected RNA translation also suggest that E6 mRNAs encode two polypeptides. Concentrations of E6 mRNA and protein are highest during the late primary cell wall and early secondary cell wall synthesis stages. Sequence comparison of E6 with other known eukaryotic and prokaryotic genes reveals no significant homology (GenBank; December 1991). E6 or a homologous gene(s) is conserved in several members of Malvaceae as well as in one other fiber-producing plant, kapok, but is not found in several other plants examined or in Acetobacter xylinum. A genomic clone corresponding to pCKE6 was isolated, and the promoter element of the E6 gene was shown to direct the expression of a carrot extensin mRNA in a tissuespecific and developmentally regulated fashion in transgenic cotton plants.Cotton is the most important textile fiber crop; therefore, the development of cotton fiber and the biosynthesis of cellulose are significant areas of research. Cotton fiber is a differentiated single epidermal cell of the outer integument of the ovule. It has four distinct growth phases; initiation, elongation (primary cell wall synthesis), secondary cell wall synthesis, and maturation (for a review, see ref. 1). Initiation of fiber development appears to be triggered by hormones, auxins, and gibberellins (for a review, see ref.2). The primary cell wall is laid down during the elongation phase, lasting up to 25 days postanthesis (DPA). Synthesis of the secondary wall commences prior to the cessation of the elongation phase and continues to -40 DPA, forming a wall (5-10 ,um) ofalmost pure cellulose. Upon maturity, cotton fiber is -87% cellulose. Thus cell wall biosynthesis is a major synthetic activity in fiber cells. The cell wall components are synthesized and transported by a functionally integrated membrane system of endoplasmic reticulum, Golgi complex, and plasmalemma. Newly synthesized cell wall polypeptides are released into the endoplasmic reticulum lumen before transportation and incorporation into cell walls. Glycosylation of structural proteins, as well as polymerization of hemicellulose and pectin, takes place in the Golgi complex from which the products are released into plasmalemma through the fusion of vesicles (1, 3).Plants, as well as certain bacteria, synthesize cellulose through enzyme systems localized in plasma membranes that use UDP-glucose as a substrate (1). The en...

Isolation of high quality RNA and DNA from plants especially cotton (Gossypium hirsutum; family Malvaceae) is notoriously difficult. This problem has been attributed to high content of phenolic terpenoids and tannins present in cotton cells (Katterman, and Shattuck, 1983). These compounds bind to RNA and DNA upon cell lysis and cannot be removed by conventional extraction procedures. Such RNA is not amenable to in vitro translation or cDNA cloning. We have encountered the same problem in many plants from family Malvaceae as well as from related family Bombacaceae. The methodology described here is a modified protocol of Chirgwin, et al., (1979) to include higher buffering capacity, alkaline pH, and most importantly polyvinyl pyrrolidone, which through hydrogen bonding, complexes with polyphenolics, effectively removing them from the homogenate. The homogenate is then subjected to ultracentrifugation to isolate RNA or DNA. The isolated RNA is suitable for cDNA cloning, in vitro translation or polymerase chain reaction. Plant tissues (leaf, fiber, root, stem, ovules or flowers) were quick frozen in liquid nitrogen. The frozen tissue was powdered in a mortar in liquid nitrogen and then homogenized in a buffer for 1.5 minutes using a polytron at full speed. The homogenization buffer was added at a ratio of 1:2 of tissue (weight) to buffer (volume). In some instances it was found that increasing the ratio to 1:4 enhances the yield. Homogenization buffer was: 5 M guanidine isothiocyanate, 0.2 M Tris-acetate

We describe a family of stress-induced, developmentally regulated soybean genes for which cDNAs have been obtained from two different cultivars (Glycine max cv. Mandarin and Glycine max cv. Williams). The mRNAs corresponding to these cDNAs, called SAM22 and H4, respectively, accumulate predominantly in the roots of soybean seedlings but are present at high levels in the roots and leaves of mature plants. SAM22 accumulation is especially dramatic in senescent leaves. In addition, SAM22 accumulation can be induced on young leaves by wounding or by transpiration-mediated uptake of salicylic acid, methyl viologen, fungal elicitor, hydrogen peroxide or sodium phosphate (pH 6.9). Taken together, these data indicate that the genes corresponding to SAM22 and H4 are induced by various stresses and developmental cues. Southern blot analysis indicates that multiple copies of sequences related to SAM22 exist in the soybean genome. We also show that the nucleotide sequences of the cDNAs corresponding to SAM22 and H4 are 86% identical at the nucleotide level to each other and 70% identical at the amino acid level to the 'disease resistance response proteins' of Pisum sativum.

To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for remote memory consolidation, we generated conditional knockout mice in which the L-VGCC isoform Ca V 1.2 was postnatally deleted in the hippocampus and cortex. In the Morris water maze, both Ca V 1.2 conditional knockout mice (Ca V 1.2 cKO ) and control littermates displayed a marked decrease in escape latencies and performed equally well on probe trials administered during training. In distinct contrast to their performance during training, Ca V 1.2 cKO mice exhibited significant impairments in spatial memory when examined 30 d after training, suggesting that Ca V 1.2 plays a critical role in consolidation of remote spatial memories.In recent years, a wealth of experimental evidence has emerged suggesting that activity-dependent changes in neuronal gene expression are regulated in large part by calcium influx via L-VGCCs. For example, calcium influx through L-VGCCs activates calcium-dependent calmodulin kinase IV and Ras/mitogenactivated kinase, which, in turn, phosphorylate the transcription factor CREB at serine 133 (Wu et al. 2001). Once phosphorylated, CREB becomes part of an active transcriptional complex that binds to cAMP-response element DNA sequences to regulate transcription of a number of gene products. Importantly, calciummediated activation of CREB has been implicated as a key subcellular signaling cascade in a wide range of behavioral processes including long-term memory consolidation (West et al. 2002;Deisseroth et al. 2003). Given the prominent role that L-VGCCs play in the activation of these signaling cascades, we have begun to examine the impact of altering L-VGCC function on memory consolidation. Consistent with the idea that L-VGCCs are a key component of this process, we have recently demonstrated that deletion of the L-VGCC isoform Ca V 1.3 produces a deficit in consolidation of contextual fear conditioning (McKinney and Murphy 2006). Here we report here that forebrain deletion of the alternate L-VGCC isoform Ca V 1.2 specifically disrupts remote spatial memories while sparing recently acquired spatial memories.To obtain regionally restricted deletion of Ca V 1.2, mice were generated using a BAC clone containing Cacna1c (the mouse Ca V 1.2 gene) obtained by screening a murine strain SV/129J BAC library (Genome Systems Inc.) with a probe representing exon 2 of Cacna1c. DNA fragments containing portions of Cacna1c were subcloned and mapped. Three adjacent XbaI fragments (4, 0.6, and 1.9 kb) were subcloned into a modified pflox vector (Chui et al. 1997) in which the thymidine kinase (TK) cassette had been removed. The MC1-TK cassette (Thomas and Capecchi 1987) was introduced at the junction of the 3Ј homology unit and the plasmid vector. The final targeting vector (Fig. 1A) had 4 kb of 5Ј homology, a loxP site introduced into intron 1, a 0.6-kb region containing exon 2, a loxP-Neo-loxP cassette introduced into intron 2, 1.9 kb of 3Ј homology, and the MC1-TK cassette.The targeting vector was linearized and e...