Increased Transcript Abundance Research Articles

Expression of three serine protease genes from the South East Asian malaria vector, Anopheles dirus, in relation to blood feeding and parasite infection

Three serine protease cDNA clones were isolated from Anopheles dirus, a major vector of malaria in Southeast Asia. Transcript abundance was examined following infection by Plasmodium falciparum by RT-PCR analysis. SerF3 exhibited increased transcript abundance in the whole body at 10 days post-infection with P. falciparum. All three genes are candidates for further investigation to determine their roles in mosquito immune responses.

Sodium butyrate stimulates monoclonal antibody over‐expression in CHO cells by improving gene accessibility

Sodium butyrate treatment can increase the specific productivity of recombinant proteins in mammalian cells; however, it dramatically decreases cell growth and frequently leads to apoptosis. We have studied the responses of several Chinese hamster ovary (CHO) cells lines with different specific productivities (qP) to sodium butyrate treatment. Cell clones with lower productivities exhibited greater enhancement from butyrate treatment than cells with higher productivities. As we observed previously in cell clone characterization (Jiang et al., 2006. Biotechnol Prog 22: 313-318), heavy chain (HC) mRNA levels correlate very well with specific productivity and are amplified by butyrate treatment, indicating that sodium butyrate regulates the HC transcription. Sodium butyrate is an inhibitor of histone deacetylation, and possibly, increases gene transcription by enhancing gene accessibility to transcription factors. In this study, we applied DNase I footprinting to probe the HC and LC gene accessibility. We determined that more HC and LC gene copies are accessible by DNase I in sodium butyrate-treated CHO cells than in untreated controls, demonstrating that sodium butyrate regulates gene transcription by improving gene accessibility. However, the increase in accessibility did not correlate with the increase in transcript abundance, suggesting that butyrate enhances transcription by other mechanisms as well.

Transcript profiling of a conifer pathosystem: response of Pinus sylvestris root tissues to pathogen (Heterobasidion annosum) invasion

The mechanisms underlying defence reactions to a pathogen attack, though well studied in crop plants, are poorly understood in conifers. To analyze changes in gene transcript abundance in Pinus sylvestris L. root tissues infected by Heterobasidion annosum (Fr.) Bref. s.l., a cDNA microarray containing 2109 ESTs from P. taeda L. was used. Mixed model statistical analysis identified 179 expressed sequence tags differentially expressed at 1, 5 or 15 days post inoculation. In general, the total number of genes differentially expressed during the infection increased over time. The most abundant group of genes up-regulated upon infection coded for enzymes involved in metabolism (phenylpropanoid pathway) and defence-related proteins with antimicrobial properties. A class III peroxidase responsible for lignin biosynthesis and cell wall thickening had increased transcript abundance at all measurement times. Real-time RT-PCR verified the microarray results with high reproducibility. The similarity of the expression profiling pattern observed in this pathosystem to those documented in crop pathology suggests that angiosperms and gymnosperms use similar genetic programs in responding to invasive growth by microbial pathogens.

Functional identification and differential expression of 1-deoxy-d-xylulose 5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies)

Conifers produce terpenoid-based oleoresins as constitutive and inducible defenses against herbivores and pathogens. Much information is available about the genes and enzymes of the late steps of oleoresin terpenoid biosynthesis in conifers, but almost nothing is known about the early steps which proceed via the methylerythritol phosphate (MEP) pathway. Here we report the cDNA cloning and functional identification of three Norway spruce (Picea abies) genes encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, and their differential expression in the stems of young saplings. Among them are representatives of both types of plant DXS genes. A single type I DXS gene is constitutively expressed in bark tissue and not affected by wounding or fungal application. In contrast, two distinct type II DXS genes, PaDXS2A and PaDXS2B, showed increased transcript abundance after these treatments as did two other genes of the MEP pathway tested, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxyl 3-methylbutenyl diphosphate reductase (HDR). We also measured gene expression in a Norway spruce cell suspension culture system that, like intact trees, accumulates monoterpenes after treatment with methyl jasmonate. These cell cultures were characterized by an up-regulation of monoterpene synthase gene transcripts and enzyme activity after elicitor treatment, as well as induced formation of octadecanoids, including jasmonic acid and 12-oxophytodienoic acid. Among the Type II DXS genes in cell cultures, PaDXS2A was induced by treatment with chitosan, methyl salicylate, and Ceratocystis polonica (a bark beetle-associated, blue-staining fungal pathogen of Norway spruce). However, PaDXS2B was induced by treatment with methyl jasmonate and chitosan, but was not affected by methyl salicylate or C. polonica. Our results suggest distinct functions of the three DXS genes in primary and defensive terpenoid metabolism in Norway spruce.

A time-course comparative microarray analysis of an incompatible and compatible response by Glycine max (soybean) to Heterodera glycines (soybean cyst nematode) infection

The development of an infection in soybean [Glycine max L. cultivar (cv.) Peking] roots by incompatible (I) and compatible (C) populations of soybean cyst nematode (SCN) (Heterodera glycines) was assayed using an AffymetriX soybean GeneChip. This time-course microarray analysis, using 37,744 probe sets, measured transcript abundance during I and C. These analyses reveal that infection by individual I and C H. glycines populations influence the transcription of G. max genes differently. A substantial difference in gene expression is present between I and C at 12 h post infection. Thus, G. max can differentiate between I and C nematode populations even before they have begun to select their feeding sites. The microarray analysis identified genes induced earlier in infection during I than C. MA also identified amplitude differences in transcript abundance between I and C reactions. Some of the probe sets measuring increased transcript levels during I represented no apical meristem (NAM) and WRKY transcription factors as well as NBS-LRR kinases. Later during I, heat shock protein (HSPs) probe sets (i.e. HSP90, HSP70, ClpB/HSP101) measured increased transcript abundance. These results demonstrate that G. max roots respond very differently to the different H. glycines races even before their feeding site selection has occurred. The ability of G. max to engage an I reaction, thus, appears to be dependent on the ability of root cells to recognize the different races of H. glycines because these experiments were conducted in the identical G. max genetic background.

Sucrose prevents up-regulation of senescence-associated genes in carnation petals

cDNA microarrays were used to characterize senescence-associated gene expression in petals of cut carnation (Dianthus caryophyllus) flowers, sampled from anthesis to the first senescence symptoms. The population of PCR fragments spotted on these microarrays was enriched for flower-specific and senescence-specific genes, using subtractive hybridization. About 90% of the transcripts showed a large increase in quantity, approximately 25% transiently, and about 65% throughout the 7 d experiment. Treatment with silver thiosulphate (STS), which blocks the ethylene receptor and prevented the normal senescence symptoms, prevented the up-regulation of almost all of these genes. Sucrose treatment also considerably delayed visible senescence. Its effect on gene expression was very similar to that of STS, suggesting that soluble sugars act as a repressor of ethylene signal transduction. Two fragments that encoded a carnation EIN3-like (EIL) protein were isolated, some of which are key transcription factors that control ethylene response genes. One of these (Dc-EIL3) was up-regulated during senescence. Its up-regulation was delayed by STS and prevented by sucrose. Sucrose, therefore, seems to repress ethylene signalling, in part, by preventing up-regulation of Dc-EIL3. Some other transcription factors displayed an early increase in transcript abundance: a MYB-like DNA binding protein, a MYC protein, a MADS-box factor, and a zinc finger protein. Genes suggesting a role in senescence of hormones other than ethylene encoded an Aux/IAA protein, which regulate transcription of auxin-induced genes, and a cytokinin oxidase/dehydrogenase, which degrades cytokinin. Taken together, the results suggest a master switch during senescence, controlling the co-ordinated up-regulation of numerous ethylene response genes. Dc-EIL3 might be (part of) this master switch.

Gonadotropin-dependent regulation of bovine pituitary adenylate cyclase-activating polypeptide in ovarian follicles prior to ovulation

To study the regulation of bovine pituitary adenylate cyclase-activating polypeptide (PACAP) in preovulatory follicles prior to ovulation, PACAP cDNA was isolated by RT-PCR. Its open reading frame (ORF) is composed of 531 bp, and encodes for a 176-amino acid protein that bears 76-90% identity with other PACAP homologs. Using bovine preovulatory follicles obtained between 0 and 24 h after human chorionic gonadotropin (hCG) and semiquantitative RT-PCR/Southern blot, we demonstrate that levels of PACAP mRNA were low at 0 h, markedly increased at 6 and 12 h (P<0.05), and declined 18 and 24 h after hCG. Levels of PACAP mRNA were high in the bovine pituitary, testis, intestine and uterus, but moderate to low in other tissues. Analyses performed on isolated preparations of granulosa and theca cells showed a significant increase of PACAP transcripts in both cell types after hCG, whereas primary granulosa cell cultures revealed high levels of PACAP as well as its receptors PAC-1 and VPAC-2 mRNA after forskolin treatment. Overexpression of the catalytic subunit of protein kinase A (PKA) in granulosa cells stimulated, but treatment with H89 or PKA inhibitor protein inhibited PACAP mRNA expression, whereas PACAP overexpression stimulated an increase in abundance of transcripts for PGHS-2, PGES, EP2 receptor, progesterone receptor, and ADAMTS-1, but not for P450-side chain cleavage and P450 aromatase. Thus, this study demonstrates the gonadotropin-dependent regulation of PACAP mRNA in bovine preovulatory follicles, the importance of PKA activation in the expression of PACAP in granulosa cells, and stimulating effect of PACAP on gene expression during the ovulatory process.

51 CHARACTERIZATION OF HISTONE H3 LYSINE 27 TRI-METHYLATION AND POLYCOMB GROUP GENE EXPRESSION IN BOVINE FERTILIZED, PARTHENOGENETIC, AND CLONED EMBRYOS

The low developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos has been attributed to aberrant or incomplete epigenetic reprogramming. Tri-methylation of Histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes (Ezh2, Eed, and Suz12) and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes during bovine preimplantation development and to compare the dynamics of H3K27me3 after IVF, parthenogenetic activation (PG), and SCNT. Embryos were produced using our standard protocols (Ross et al. 2006 Biotechniques 41, 741–750). MII oocytes and pronuclear-, 2-cell-, 4-cell-, 8-cell-, morula-, and blastocyst-stage embryos were collected for quantitative RT-PCR or immunofluorescence (IF). IF analysis of embryos was performed using primary antibodies against H3K27me3, Eed, Ezh2 (Abcam, Cambridge, MA, USA), and Suz12 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and imaged by confocal microscopy. The average background-corrected nuclear fluorescence intensity for each embryo was analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Ezh2, Eed, Suz12, and control transcript levels from oocytes and IVF-derived embryos were determined by absolute quantification as described by Bettegowda et al. (2006 Mol. Reprod. Dev. 73, 267–278). RNA levels were normalized to the external control and analyzed by ANOVA (mixed model). A steady decrease in total transcripts was observed from the MII to the 8-cell stage (P &lt; 0.05). During this period, the levels of polyadenylated Suz12 and Eed transcripts remained steady, while Ezh2 transcripts increased at the 2-cell stage (P &lt; 0.05) and then decreased from the 2- to 4-cell stage (P = 0.10). After embryonic genome activation, all three genes displayed an increase in transcript abundance by the blastocyst stage (P &lt; 0.05). Concurrent IF analysis revealed that Ezh2 localized to the nucleus throughout preimplantation development, Eed localized to the cytoplasm at the 2-, 4- and 8-cell stages (expression confirmed by westernblot) and to the nucleus at morula and blastocyst stages, and Suz12 was exclusively nuclear and detected only at morula and blastocyst stages. IF analysis of H3K27me3 revealed that, for all embryo types, nuclear fluorescent intensity was highest at the MII stage, steadily decreased to reach a nadir at the 8-cell stage (P &lt; 0.05), and then significantly increased at the blastocyst stage (P &lt; 0.05). H3K27me3 fluorescent intensity was significantly higher in SCNT blastocysts than in IVF and PG blastocysts (P &lt; 0.05). We conclude that H3K27me3 is efficiently removed from the embryonic and somatic cell chromatin, but is abnormally reestablished in SCNT embryos.

Coordination of PsAS1 and PsASPG expression controls timing of re-allocated N utilization in hypocotyls of pine seedlings

During pine seed germination, a large amount of N mobilized from the storage proteins is re-allocated in the hypocotyl as free asparagine, as a result of the high levels of asparagine synthetase (AS) encoded by the PsAS1 gene. To determine the role of this re-allocated N reserve, a full-length cDNA encoding L: -asparaginase (ASPG) has been cloned from Scots pine (Pinus sylvestris L.) seedlings and characterized. Like other N-terminal nucleophile hydrolases, pine ASPG requires a post-translational processing to exhibit enzymatic activity. However, in contrast to previous reports on other plant ASPGs, purified recombinant pine ASPG does not undergo autoproteolytic cleavage in vitro. Our results suggest that the processing requires accessory proteins to assist in the proteolysis or in the proper folding before autocleavage in a divalent cation-dependent manner. Sequence comparison analysis revealed that the pine protein is included in the K+-dependent subfamily of plant ASPGs. The expression of the ASPG-encoding gene (PsASPG) was higher in organs with extensive secondary development of the vascular system. The increase in transcript abundance observed at advanced stages of hypocotyl development was concomitant with a decrease of PsAS1 transcript abundance and a remarkable increase in the number of xylem elements and highly lignified cell walls. These results, together with the precise localization of PsASPG transcripts in cells of the cambial region, suggest that the expression of PsAS1 and PsASPG is temporally coordinated, to control the re-allocation of N from seed storage proteins toward the hypocotyl to be later used during early development of secondary vascular system.

Phototropin involvement in the expression of genes encoding chlorophyll and carotenoid biosynthesis enzymes and LHC apoproteins in Chlamydomonas reinhardtii

Phototropin (PHOT) is a photoreceptor involved in a variety of blue-light-elicited physiological processes including phototropism, chloroplast movement and stomatal opening in plants. The work presented here tests whether PHOT is involved in expression of light-regulated genes in Chlamydomonas reinhardtii. When C. reinhardtii was transferred from the dark to very low-fluence rate white light, there was a substantial increase in the level of transcripts encoding glutamate-1-semialdehyde aminotransferase (GSAT), phytoene desaturase (PDS) and light-harvesting polypeptides (e.g. LHCBM6). Increased levels of these transcripts were also elicited by low-intensity blue light, and this blue-light stimulation was suppressed in three different RNAi strains that synthesize low levels of PHOT. The levels of GSAT and LHCBM6 transcripts also increased following exposure of algal cells to low-intensity red light (RL). The red-light-dependent increase in transcript abundance was not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, implying that the influence of RL on transcript accumulation was not controlled by cytoplasmic redox conditions, and that a red-light photoreceptor(s) may be involved in regulating the levels of transcripts from specific photosynthesis-related genes in C. reinhardtii. Interestingly, elevated GSAT and LHCBM6 transcript levels in RL were significantly reduced in the PHOT RNAi strains, which raises the possibility of co-action between blue and RL signaling pathways. Microarray experiments indicated that the levels of several transcripts for photosystem (PS) I and II polypeptides were also modulated by PHOT. These data suggest that, in C. reinhardtii, (i) PHOT is involved in blue-light-mediated changes in transcript accumulation, (ii) synchronization of the synthesis of chlorophylls (Chl), carotenoids, Chl-binding proteins and other components of the photosynthetic apparatus is achieved, at least in part, through PHOT-mediated signaling, and (iii) a red-light photoreceptor can also influence levels of certain transcripts associated with photosynthetic function, although its action requires normal levels of PHOT.

Increased CUG Triplet Repeat-binding Protein-1 Predisposes to Impaired Adipogenesis with Aging

Preadipocyte differentiation capacity declines between middle and old age. Expression of the adipogenic transcription factors, CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor gamma (PPARgamma), is lower in differentiating preadipocytes from old than young animals, although no age-related changes occur in C/EBPbeta mRNA, which is upstream of C/EBPalpha and PPARgamma. C/EBPbeta-liver-enriched inhibitory protein (C/EBPbeta-LIP), a truncated C/EBPbeta isoform that is a dominant inhibitor of differentiation, increases with aging in rat fat tissue and preadipocytes. CUG triplet repeat-binding protein-1 (CUGBP1) binds to C/EBPbeta mRNA, increasing C/EBPbeta-LIP translation. Abundance and nucleotide binding activity of CUGBP1 increased with aging in preadipocytes. CUGBP1 overexpression in preadipocytes from young animals increased C/EBPbeta-LIP and impaired adipogenesis. Decreasing CUGBP1 in preadipocytes from old rats by RNA interference reduced C/EBPbeta-LIP abundance and promoted adipogenesis. Tumor necrosis factor-alpha, levels of which are elevated in fat tissue with aging, increased CUGBP1 protein, CUGBP1 binding activity, and C/EBPbeta-LIP in preadipocytes from young rats. Thus, CUGBP1 contributes to regulation of adipogenesis in primary preadipocytes and is responsive to tumor necrosis factor-alpha. With aging, preadipocyte CUGBP1 abundance and activity increases, resulting in enhanced translation of the C/EBPbeta-LIP isoform, thereby blocking effects of adipogenic transcription factors, predisposing preadipocytes from old animals to resist adipogenesis. Altered translational processing, possibly related to changes in cytokine milieu and activation of stress responses, may contribute to changes in progenitor differentiation and tissue function with aging.

Nitrogen Fixation by White Lupin under Phosphorus Deficiency

White lupin is highly adapted to growth in a low-P environment. The objective of the present study was to evaluate whether white lupin grown under P-stress has adaptations in nodulation and N2 fixation that facilitate continued functioning. Nodulated plants were grown in silica sand supplied with N-free nutrient solution containing 0 to 0.5 mm P. At 21 and 37 d after inoculation (DAI) growth, nodulation, P and N concentration, N2 fixation (15N2 uptake and H2 evolution), root/nodule net CO2 evolution and CO2 fixation (14CO2 uptake) were measured. Furthermore, at 21 DAI in-vitro activities and transcript abundance of key enzymes of the C and N metabolism in nodules were determined. Moreover, nodulation in cluster root zones was evaluated. Treatment without P led to a lower P concentration in shoots, roots, and nodules. In both treatments, with or without P, the P concentration in nodules was greater than that in the other organs. At 21 DAI nitrogen fixation rates did not differ between treatments and the plants displayed no symptoms of P or N deficiency on their shoots. Although nodule number at 21 DAI increased in response to P-deficiency, total nodule mass remained constant. Increased nodule number in P-deficient plants was associated with cluster root formation. A higher root/nodule CO2 fixation in the treatment without P led to a lower net CO2 release per unit fixed N, although the total CO2 released per unit fixed N was higher in the treatment without P. The higher CO2 fixation was correlated with increased transcript abundance and enzyme activities of phosphoenolpyruvate carboxylase and malate dehydrogenase in nodules. Between 21 and 37 DAI, shoots of plants grown without P developed symptoms of N- and P-deficiency. By 37 DAI the P concentration had decreased in all organs of the plants treated with no P. At 37 DAI, nitrogen fixation in the treatment without P had almost ceased. Enhanced nodulation in cluster root zones and increased potential for organic acid production in root nodules appear to contribute to white lupin's resilience to P-deficiency.

Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering

Taxus plant suspension cell cultures provide a sustainable source of paclitaxel (Taxol ®) for the treatment of many cancers. To develop an optimal bioprocess for paclitaxel supply, taxane biosynthetic pathway regulation must be better understood. Here we examine the expression profile of paclitaxel biosynthetic pathway genes by RNA gel blot analysis and RT-PCR in the Taxus cuspidata cell line P991 and compare with taxane metabolite levels. Upon methyl jasmonate (MJ) elicitation (100 μM), paclitaxel accumulates to 3.3 mg/L and cephalomannine to 2.2 mg/L 7 days after elicitation but neither are observed before this time. 10-deacetylbaccatin III accumulates to 3.3 mg/L and baccatin III to 1.2 mg/L by day 7 after elicitation. The early pathway enzyme genes GGPPS, TASY, and T5αH are up-regulated by MJ elicitation within 6 h and continue through 24 h before their abundances decrease. This study reveals the preference for one side of the biosynthetic pathway branch in early taxane synthesis, where transcripts coding for T αH are abundant after elicitation with MJ but transcripts encoding the two enzymes for the alternative branch ( TDAT and T10βH) are not highly expressed following elicitation. Transcripts encoding the enzymes DBBT and DBAT are up-regulated upon MJ elicitation. Their products, 10-deacetylbaccatin III and baccatin III, respectively, accumulate within 6 h of the initial increase in transcript abundance. Importantly, the steady-state levels of the two terminal enzyme transcripts ( BAPT and DBTNBT) are much lower than transcripts of early pathway steps. These are potential steps in the pathway for targeted metabolic engineering to increase accumulation of paclitaxel in suspension cell culture.

Correction: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

A correction to Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress by AD Aragon, GA Quiñones, EV Thomas, S Roy and M Werner-Washburne. Genome Biology 2006, 7:R9

Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

A rapid transcript increase due to the release of extraction-resistant mRNAs from yeast cells in response to stress is described.

Ethylene-sensitivity regulates proteolytic activity and cysteine protease gene expression in petunia corollas*

To investigate ethylene's role in petal senescence, a comparative analysis of age-related changes in total protein, protease activity, and the expression of nine cysteine protease genes in the corollas of ethylene-sensitive Petuniaxhybrida cv. Mitchell Diploid (MD) and ethylene-insensitive (35S:etr1-1; line 44568) transgenic petunias was conducted. The later stages of corolla senescence in MD flowers were associated with decreased fresh weight, decreased total protein, and increased proteolytic activity. Corolla senescence was delayed by approximately 8 d in etr-44568 transgenic petunias, and decreases in corolla fresh weight, protein content, and maximum proteolytic activity were similarly delayed. Protease inhibitor studies indicated that the majority of the protease activity in senescing petals was due to cysteine proteases. Nine cysteine proteases expressed in petals were subsequently identified. Northern blot analysis indicated that six of the nine cysteine proteases showed increased transcript abundance during petal senescence. One of these cysteine proteases, PhCP10, was detected only in senescing tissues. Expression of four of the senescence-associated cysteine proteases was delayed, but not prevented in etr-44568 flowers. The other two senescence associated cysteine proteases had high levels of transcript accumulation in etr-44568 corollas at 8 d after flower opening, when MD flowers were senescing. These patterns suggest that age-related factors, other than ethylene, were regulating the up-regulation of these genes during flower ageing. The delay in visible symptoms and biochemical and molecular indicators of senescence in ethylene-insensitive flowers is consistent with the concept that ethylene modulates the timing of senescence pathways in petals.

The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promotes high-level expression by increasing transcript abundance and translation efficiency

The involvement of regions located upstream of the translation start site in the expression of two Arabidopsis thaliana nuclear COX5c genes encoding subunit 5c of mitochondrial cytochrome c oxidase has been analysed. It was observed that these regions, which include a leader intron, direct the tissue-specific expression of the gus reporter gene, mainly in root and shoot meristems, actively growing tissues and vascular strands. Expression was also observed in flowers, specifically localized in anthers, stigma, and the receptacle, and in developing seeds. GUS activity measurements in protein extracts from transformed plants indicated that expression levels are higher than those observed with the constitutive CaMV 35S promoter. Removal of the leader intron produced a significant decrease in expression to values only slightly higher than those observed with a promoterless gus gene. Histochemical staining of plants transformed with the intronless construct revealed expression only in pollen, suggesting that regulatory elements capable of directing pollen-specific expression are present upstream of the intron. The COX5c-2 intron also increased GUS expression levels when fused in the correct orientation with the promoter of the unrelated COX5b-1 gene. Comparison of GUS activity values with the transcript levels suggests that the intron also increases translation efficiency of the corresponding mRNA. The results obtained point to an essential role of the intron present in the 5'-non-coding region of all known COX5c genes in directing the expression of these genes in plants.

(455) Differential Patterns of Expression and Regulation of Two Dehydrin Genes from Peach (Prunus persica) Bark Tissues

We have characterized the seasonal accumulation of transcripts and proteins in peach (Prunuspersica), particularly a 60 kDa dehydrin (PCA60; PpDhn1). Recently, we have isolated another dehydrin gene (PpDhn2). The present report compares the structural organization of the two dehydrin genes, their promoters, and the response of the genes to temperature, photoperiod, and water deficit. Trees were exposed for 3 or 5 weeks to either short day (SD) or long day (LD) photoperiods at either 25 or 5 °C. Additional experiments exposed trees to a period of water deficit followed by recovery. Transcript abundance of both genes, as assessed by RT-PCR, was determined, in response to the different photoperiods and temperatures as well as a prolonged SD/5 °C regime, from monthly-collected field samples, and trees subjected to water deficit. Results indicated that water deficit increased transcript abundance of both genes, but their abundance differed dramatically in response to low temperature and seasonal cues. Surprisingly, neither gene exhibited a significant elevation in transcript abundance in response to SD conditions. The lack of response of PpDhn1to SD is problematical given the observation that transcript levels in field-collected samples begin to increase substantially in September, prior to the onset of cold temperatures. Analysis of the promoter regions and cis-acting elements suggest that ABA may play an important role in seasonal expression, interacting with photoperiod in field conditions. Two CRT/DRE elements are present in the promoter region of PpDhn1, but absent in the promoter of PpDhn2.

Changes in Proteolytic Activity and Cysteine Proteinase Gene Expression during the Senescence of etr1-1 Transgenic Petunias

Corolla senescence in petunias was accompanied by a decrease in total proteins and a corresponding increase in proteolytic activity. Transgenic petunias that contain the mutated ethylene receptor (35S:etr1-1) have reduced sensitivity to ethylene and delayed flower senescence. Declines in total protein levels and increases in proteolytic activity were also delayed in etr1-1 flowers and corresponded with corolla wilting. Experiments using class-specific proteinase inhibitors indicated that proteolytic activity in petunia corollas was largely due to cysteine proteinases. Total nitrogen levels within the corollas of both wild type and etr1-1 flowers also decreased during senescence. Nine cDNAs encoding putative cysteine proteinases (CPs) were identified from a petunia EST database developed at the University of Florida. Six of these cysteine proteinases showed increased transcript abundance during corolla senescence (senescence-associated CPs) while three decreased in abundance. Of the six senescence-associated cysteine proteinases, only five showed delayed up regulation in etr1-1 flowers that corresponded with corolla wilting. The role of ethylene in the regulation of protein degradation during flower senescence will be discussed.

Variation in transcript abundance among the four members of the Arabidopsis thaliana RIBOSOMAL PROTEIN S15a gene family

As a ribonucleoprotein complex, the plant ribosome consists of four ribosomal RNAs (rRNAs) and 75–92 ribosomal proteins (r-proteins), depending on the species. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members that, at the amino acid level, share 87–100% identity. The 5′ regulatory regions of the four genes contain many of the same putative regulatory elements but share only 46–49% nucleotide sequence identity. Semi-quantitative RT-PCR (sqRT-PCR) was used to determine RPS15a transcript abundance patterns and identify possible regulatory differences among the four genes. RPS15aC expression was not detected in any tissue under any treatment while transcript abundance for RPS15aA, - D and - F was highest in mitotically active tissues e.g. bud and flower. Seedlings showed increased transcript abundance following treatment with the cytokinin 6-benzylaminopurine (BAP) while the auxin indole acetic acid (IAA) induced an increase in RPS15aF abundance only. Abscisic acid (ABA) treatment resulted in decreased transcript abundance while gibberellic acid (GA 3) had little effect on all four genes. Similar trends were established for RPS15aA, -D and - F transcript abundance as a result of temperature mechanical stress. This study reports the differences in transcript abundance of the four cytosolic RPS15a genes in Arabidopsis and we suggest that RPS15a genes should no longer be considered ‘housekeeping’ genes.