Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron

Actin-depolymerizing factor (ADF) is one of the actin cytoskeleton-modulating proteins. We have characterized the accumulation pattern of petunia ADF proteins. PhADF proteins are accumulated in every petunia organ and their accumulation is differentially regulated by developmental signals. Their cellular localization is vascular tissue-preferential in vegetative organs, whereas somewhat different in reproductive organs. In reproductive organs, PhADFs are present in outer integument, endocarp of ovary wall, transmitting tissue of style, and epidermis and endothecium of young anther. From a petunia genomic library, we have isolated a genomic clone encoding PhADF1. Comparison to complementary DNA sequence revealed that the coding region of PhADF1 gene consists of three exons and two introns. Analysis of chimeric gene expression using beta-glucuronidase as a reporter gene in transgenic Arabidopsis revealed that PhADF1 was strongly expressed in every vegetative tissue except petal. In addition, expression of the gene was highly enhanced by its first intron. These results suggest that PhADF1 gene of petunia is mainly expressed in vascular tissues and its expression is regulated by intron-mediated enhancement mechanism.     

Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor

A cDNA clone, LMP131A, which is preferentially expressed in mature anther was isolated from a lily cDNA library. Northern blot analysis and plaque hybridization expriments showed that the LMP131A mRNA is present at ca. 0.3% of the mRNA in mature pollen and is not detectable in carpel, petal, floral bud, leaf, or root. The clone contains an open reading frame of 139 amino acid residues which shows greater than 40% sequence identity in a 91 amino acid overlap to animal actin-depolymerizing factors (ADF), cofilin and destrin. The sequences at and near the actin-binding site are most conserved. Using the lily clone as a probe, a cDNA clone, BMP1, was isolated from a mature anther library of Brassica napus. The expression pattern of the BMP1 clone was the same as that of the lily clone. The Brassica anther-preferential clone contains an open reading frame which is 79% identical to the lily LMP131A protein. Southern blot analysis showed that there are one or a few copies of the putative ADF genes in B. napus and Arabidopsis thaliana.     

Distinct Genes Produce the Alcohol Dehydrogenases of Pollen and Maternal Tissues in Petunia hybrida

Analysis of cDNA clones derived from hypoxic root mRNA of Petunia hybrida has revealed the existence of a third active gene encoding alcohol dehydrogenase in petunia. A combination of RT-PCR and ADH activity gels provide evidence for the selective tissue-specific expression of these three genes in multiple floral organs and hypoxically stressed roots. Expression of adh 1 in the plant appears to be restricted to immature pollen grains; the other two genes are expressed differentially in maternal anther tissues, stigma, petals, and hypoxic root. This work underscores the utility of RT-PCR for distinguishing expression patterns of closely related genes, clarifies the expression patterns exhibited by members of this gene family, and suggests multiple functions for the adh genes of petunia.     

Organ boundary NAC‐domain transcription factors are implicated in the evolution of petal fusion

• Research rationale: Evolution of fused petals (sympetaly) is considered to be an important innovation that has repeatedly led to increased pollination efficiency, resulting in accelerated rates of plant diversification. Although little is known about the underlying regulation of sympetaly, genetic pathways ancestrally involved in organ boundary establishment (e.g. CUP SHAPED COTYLEDON [CUC] 1–3 genes) are strong candidates. In sympetalous petunia, mutations in the CUC1/2‐like orthologue NO APICAL MERISTEM (NAM) inhibit shoot apical meristem formation. Despite this, occasional ‘escape shoots’ develop flowers with extra petals and fused inter‐floral whorl organs.
• Central methods: To To determine if petunia CUC‐like genes regulate additional floral patterning, we used virus‐induced silencing (VIGS) following establishment of healthy shoot apices to re‐examine the role of NAM in petunia petal development, and uniquely characterise the CUC3 orthologue NH16.
• Key results: Confirming previous results, we found that reduced floral NAM/NH16 expression caused increased petal–stamen and stamen–carpel fusion, and often produced extra petals. However, further to previous results, all VIGS plants infected with NAM or NH16 constructs exhibited reduced fusion in the petal whorl compared to control plants.
• Main conclusions: Together with previous data, our results demonstrate conservation of petunia CUC‐like genes in establishing inter‐floral whorl organ boundaries, as well as functional evolution to affect the fusion of petunia petals.

     

Cloning and expression of flavonol synthase from Petunia hybrida

Flavonols are important co-pigments in flower colour and are also essential for pollen tube growth. In petunia, flavonol synthesis is controlled by the Fl locus. Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase family. Dioxygenase gene fragments were amplified by PCR on cDNA made from FlFl and flfl flowers using degenerate primers designed from conserved dioxygenase sequences. A petunia petal cDNA library was screened for clones that hybridized more strongly to the Fl PCR products than the fl PCR products. A full-length cDNA clone identified by this screening exhibited FLS activity when expressed in yeast. FLS gene expression is developmentally regulated during flower development. Antisense expression of an FLS cDNA clone in petunia markedly reduced flavonol synthesis in petals. RFLP mapping showed that the FLS gene is linked to Fl , suggesting that Fl is the structural gene for FLS.     

4个棉花ADF基因的分子鉴定及其差异表达

肌动蛋白解聚合因子(actin-depolymerizing factor, ADF)是一种在真核生物中广泛存在的低分子量的肌动蛋白结合蛋白,它在调控细胞内肌动蛋白纤丝的解聚合和再聚合中起着关键作用。我们在棉纤维cDNA文库中分离克隆了4个ADF基因(cDNAs),分别命名为GhADF2,GhADF3,GhADF4,GhADF5。GhADF2 cDNA 长度为705 bp,编码139个氨基酸;GhADF3 cDNA长度为819 bp,编码139个氨基酸;GhADF4 cDNA长度为804 bp,编码143个氨基酸;GhADF5 cDNA长度为644 bp,编码141个氨基酸。分析表明,GhADF2与GhADF3的氨基酸序列同源性为99%。而且,GhADF2/3与矮牵牛PeADF2之间的氨基酸序列同源性也高达89%。GhADF4与拟南芥AtADF6的亲缘关系较近,二者的氨基酸序列同源性为78%。GhADF5与拟南芥AtADF5的亲缘关系较近,氨基酸序列的同源性为83%。上述结果表明植物ADF基因在进化中具有高度保守性。RT-PCR分析表明,GhADF2在纤维中优势表达,而GhADF5基因则在子叶中表达量最高。另一方面,GhADF3和GhADF4似乎不具有组织特异性或偏爱性表达。同一组织中不同GhADF基因表达量有较大的差异,表明它们可能涉及棉花不同组织生长发育过程的调节。而且,在进化过程中,各ADF同分异构体之间可能发展形成某种功能上的差异性。     

A complex gene superfamily encodes actin in petunia.

We have shown by several independent criteria that actin is encoded by a very large and complex superfamily of genes in Petunia. Several cDNA and genomic probes encoding actins from diverse organisms (Dictyostelium, Drosophila, chicken and soybean) hybridize to hundreds of restriction fragments in the petunia genome. Actin-hybridizing sequences were isolated from a petunia genomic library at a rate of at least 200 per genome equivalent. Twenty randomly selected actin-hybridizing clones were characterized in more detail. DNA sequence data from four representative and highly divergent clones, PAc2, PAc3, PAc4 and PAc7, demonstrate that these actin-like sequences are related to functional actin genes. Intron positions typical of other known plant actin genes are conserved in these clones. Four of six clones analyzed (PAc1, PAc2, PAc3, PAc4) hybridize to leaf mRNA of the same size (1.7 kb) as that reported for other plant actin mRNAs and to a slightly smaller mRNA species (1.5 kb). Five distinct subfamilies of actin-related genes were characterized which varied in size from a few members to several dozen members. It is clear from our data that other actin gene subfamilies must also exist within the genome. Possible mechanisms of actin gene amplification and genome turnover are discussed.     

Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.     

Cloning and Characterization of One Member of the Chalcone Synthase Gene Family from Solanum tuberosum L.

We have isolated a chalcone synthase gene 2 (ST-CHS2) from potato by rapid amplification of cDNA ends by PCR. CHS2 cDNA had high homology to tomato LET-CHS2 (98%), petunia PHCHSJ (94%), potato ST-CHS1B (92%), petunia PHCHSA (92%), and LET-CHS1 (90%) at the overall 389-amino acid level. Genomic hybridization analysis indicated that CHS genes of potato comprise a family of at least six individual members.     

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.