Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) leaf

The aim of the present study was to isolate clones of genes which are likely to be involved in wax deposition on barley leaves. Of particular interest were those genes which encode proteins that take part in the synthesis and further modification of very long chain fatty acids (VLCFAs), the precursors of waxes. Previously, it had been shown that wax deposition commences within a spatially well-defined developmental zone along the growing barley leaf (Richardson et al. in Planta 222:472–483, 2005). In the present study, a barley microarray approach was used to screen for candidate contig-sequences (www.barleybase.org) that are expressed particularly in those leaf zones where wax deposition occurs and which are expressed specifically within the epidermis, the site of wax synthesis. Candidate contigs were used to screen an established in-house cDNA library of barley. Six full-length coding sequences clones were isolated. Based on sequence homologies, three clones were related to Arabidopsis CER6/CUT1, and these clones were termed HvCUT1;1, HvCUT1;2 and HvCUT1;3. A fourth clone, which was related to Arabidopsis Fiddlehead (FDH), was termed HvFDH1;1. These clones are likely to be involved in synthesis of VLCFAs. A fifth and sixth clone were related to Arabidopsis CER1, and were termed HvCER1;1 and HvCER1;2. These clones are likely to be involved in the decarbonylation pathway of VLCFAs. Semi-quantitative RT-PCR confirmed microarray expression data. In addition, expression analyses at 10-mm resolution along the blade suggest that HvCUT1;1 (and possibly HvCUT1;2) and HvCER1;1 are involved in commencement of wax deposition during barley leaf epidermal cell development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The timing of maize leaf senescence and characterisation of senescence-related cDNAs

Leaf senescence was characterised in two Zea mays lines, earlier senescence (ES) and later senescence (LS). Loss of chlorophyll was delayed in LS compared with ES, but the decline in photosynthesis occurred simultaneously in the two lines. Western analysis detected transition points during senescence of both lines when major quantitative and qualitative changes occurred in a number of leaf proteins. Differences in the pattern of translatable mRNAs were apparent earlier than alterations in pigment or protein levels. A cDNA library was constructed using mRNA from ES leaves early in senescence and differential screening was employed to isolate senescence-related clones. Two senescence-enhanced cDNAs showed sequence homology with cDNAs for seed proteins - a cysteine protease and a protein-processing enzyme. These findings suggest that there are similarities between gene expression during seed maturation, germination and leaf senescence. Other senescence-enhanced cDNAs were related to genes implicated in gluconeogenesis and chlorophyll breakdown.     

Leaf senescence in Brassica napus: expression of genes encoding pathogenesis-related proteins

Genes that are expressed during leaf senescence in Brassica napus were identified by the isolation of representative cDNA clones. DNA sequence and deduced protein sequence from two senescence-related cDNAs, LSC94 and LSC222, representing genes that are expressed early in leaf senescence before any yellowing of the leaves is visible, showed similarities to genes for pathogenesis-related (PR) proteins: a PR-1a-like protein and a class IV chitinase, respectively. The LSC94 and LSC222 genes showed differential regulation with respect to each other; an increase in expression was detected at different times during development of healthy leaves. Expression of both genes was induced by salicylic acid treatment. These findings suggest that some PR genes, as well as being induced by pathogen infection, may have alternative functions during plant development, for example in the process of leaf senescence.     

Cloning and characterization of tomato leaf senescence-related cDNAs

Senescence-related cDNA clones designated SENU1, 4, 5 (senescence up-regulated) and SEND32, 33, 34, 35 and 36 (senescence down-regulated) isolated from a tomato leaf cDNA library [9] were characterized. Southern analysis showed that SEND32 is encoded by a single-copy gene while SEND33, 34, 35, 36 and SENU1 and SENU5 are members of small gene families. DNA and protein database searches revealed that SEND32, SEND35, SENU1 and SENU5 are novel cDNAs of unknown function. SEND33 encodes ferredoxin, SEND34 encodes a photosystem II 10 kDa polypeptide and SEND36 encodes catalase. The SENU4 sequence is identical to the P6 tomato protein previously reported to be pathogenesis-related [46]. The mRNA levels of SENU1, 4 and 5 increased during leaf senescence and SENU1 and SENU5 were also expressed at high levels during leaf development and in other plant organs. The SENU4 mRNA was associated more specifically with leaf senescence, although low expression was also detected in green fruit. The mRNAs for all SEND clones decreased during tomato leaf development and senescence and all except SEND32 were expressed at low levels in other plant organs. The accumulation of mRNA homologous to SENU4 and the decrease in abundance of SEND32 provide good molecular markers for leaf senescence.     

Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy.

As part of the ongoing human and mouse genome projects, the aim of this study was to isolate novel, previously uncharacterized, genes from mouse testis. Two approaches were compared for their effectiveness in isolating novel genes: random, vs differential, complementary DNA (cDNA) cloning methods. In the differential approach, only the cDNA clones containing rare sequences (as determined by preliminary clone hybridization) are further analyzed; in the random approach, cDNA clones are isolated at random from the cDNA library. More than two hundred cDNA clones altogether were analyzed, using a PCR-mediated amplification and sequencing strategy. A comparison of these sequences to nucleic acid and protein sequence databases, revealed that 84% of the isolated rare cDNA clones represented new, previously uncharacterized mouse genes. In contrast, less than 63% of the cDNA clones isolated at random from cDNA libraries, contained novel genes. Thus, the probability of isolating new, previously uncharacterized, mammalian genes from cDNA libraries can be markedly improved by focusing efforts on clones containing rare sequences.     

Heavy metal stress and leaf senescence induce the barley gene HvC2d1 encoding a calcium-dependent novel C2 domain-like protein

By comparing cDNA populations derived from chromium-stressed primary leaves of barley (Hordeum vulgare L.) with controls, differentially expressed cDNA fragments could be identified. The deduced amino acid sequence of one of these cDNAs [named 'C2 domain 1' (HvC2d1)] exhibits a motif that is similar to the known C2 domain and a nuclear localization signal (NLS). Expression of this member of a novel class of plant C2 domain-like proteins was studied using real-time PCR, and subcellular localization was investigated using green fluorescent protein (GFP) fusion constructs. Calcium binding was analysed using a (45)Ca(2+) overlay assay. HvC2d1 was transiently induced after exposure to different heavy metals and its mRNA accumulated during the phase of leaf senescence. HvC2d1 expression responded to changes in calcium levels caused by the calcium ionophore A23187 and to treatment with methylviologen resulting in the production of reactive oxygen species (ROS). Using overexpressed and purified HvC2d1, the binding of calcium could be confirmed. Chimeric HvC2d1-GFP protein was localized in onion epidermal cells at the plasma membrane, cytoplasm and the nucleus. After addition of calcium ionophore A23187 green fluorescence was only visible in the nucleus. The data suggest a calcium-dependent translocation of HvC2d1 to the nucleus. A possible role of HvC2d1 in stress- and development-dependent signalling in the nucleus is discussed.     

Subtractive screening of genes involved in cellular senescence

We attempted to identify the genes involved in cellularsenescence, telomere maintenance and telomerase regulationthrough subtractive screening of cDNA libraries prepared froma human lung adenocarcinoma cell line A549 and its sublinesnamed A5DC7, CK and AST-9. Cell phenotypes of A5DC7, CK andAST-9 are normal cell-like, cancer cell-like and intermediate,respectively. These cell lines have different phenotypes interms of telomerase activity and telomere maintenance, andthus are thought to be useful for identifying the genesinvolved in cellular senescence and telomerase regulation. In this study, we identified 86 independent cDNA clones bysubtractive screening. Among these cDNA clones, subtractingA5DC7 cDNAs from A549 cDNAs and CK cDNAs gave 7 and 3 cDNAclones which highly and specifically expressed in tester celllines. Genes corresponding to these 10 cDNA clones mightparticipate in maintaining cancer-cell phenotypes. As aresult of database searching, each four of A549 specific cDNAclones are found to correspond to known cDNAs. Each two ofA549 specific and two of CK specific cDNA clones have highhomology to independent ESTs. Sequences having homology toeach one of A549 specific and one of CK specific cDNA cloneshave not been deposited in the Genbank database, indicatingthat these two cDNA clones are part of novel genes. Weanticipate that their involvement in telomerase regulationand/or senescence program can be clarified by functionalanalysis using each full-length cDNA.     

Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Identification of a gene encoding a senescence-specific metallothionein-like protein.

cDNA clones representing genes that are expressed during leaf senescence in Brassica napus were identified by differential screening of a cDNA library made from RNA isolated from leaves at different stages of senescence. The expression of these genes at different stages of leaf development was examined by northern blot analysis, and several different patterns of expression were observed. One of the clones, LSC54, represented a gene that is expressed at high levels during leaf senescence. Analysis of this gene indicated strong expression in flowers as well as in senescing leaves. DNA sequence analysis of the LSC54 cDNA indicated a similarity between the deduced amino acid sequence and several metallothionein-like proteins previously identified in plants.     

A barley gene (rsh1) encoding a ribonuclease S-like homologue specifically expressed in young light-grown leaves

 A group of frequent cDNA clones from a young-leaf cDNA library was found to code for a homologue of S-ribonucleases (S-RNases) involved in gametophytic incompatibility and the so-called S-like RNases active in flowers and in vegetative tissues. The derived amino acid sequence starts with a signal peptide and has a 27-amino-acid C-terminal extension of unknown function. The barley (Hordeum vulgare L.) gene, rsh1 (for RNase S-like homologue) corresponding to the cDNA clones was isolated. The gene has three introns and the position of one intron corresponds to the site of the single, small intron in the S-RNase genes. The deduced amino acid sequence of mature RSH1 shares 35% identical and 58% similar amino acid residues with an S-like RNase from tomato, RNase LE. However, two active-site histidine residues, conserved between all S and S-like RNases are replaced by serine residues in RSH1. The new barley RNase S-like homologue is clearly related to the family of active RNases but is probably not active as an RNase. Sequences from the same class of presumably inactive RNases have been recorded in maize, rice and sorghum. The barley gene is exclusively expressed in young leaf tissue and is substantially induced by light. Received: 26 July 1999 / Accepted: 26 October 1999     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Identification of early senescence-associated genes in rice flag leaves

Leaf senescence is one of the key stages of plant leaf development. It is a highly complex but ordered process involving expression of large scale senescence associated genes, and its molecular mechanisms still remain unclear. By using suppression subtractive hybridization, 815 ESTs that are up-regulated at the onset of rice flag leaf senescence have been isolated. A total of 533 unigenes have been confirmed by macroarray detection and sequencing. 183 of these unigenes have GO annotations, involved in macromolecule metabolism, protein biosynthesis regulation, energy metabolism, gene expression regulations, detoxification, pathogenicity and stress, cytoskeleton organization and flower development. Another 121 unigenes co-localized with previously reported known stay-green QTLS. RT-PCR analysis on the other novel genes indicated that they can be up-regulated in natural early senescence and induced by hormone. Our results indicate that senescence is closely related to various metabolic pathways, thus providing new insight into the onset of leaf senescence mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.