Transforming petals into sepaloid organs in<Emphasis Type="Italic"> Arabidopsis</Emphasis> and oilseed rape: implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner

Oilseed rape (Brassica napus L.) genotypes with no or small petals are thought to have advantages in photosynthetic activity. The flowers of field-grown oilseed rape form a bright-yellow canopy that reflects and absorbs nearly 60% of the photosynthetically active radiation (PAR), causing a severe yield penalty. Reducing the size of the petals and/or removing the reflecting colour will improve the transmission of PAR to the leaves and is expected to increase the crop productivity. In this study the hairpin RNA-mediated (hpRNA) gene silencing technology was implemented in Arabidopsis thaliana (L.) Heynh. and B. napus to silence B-type MADS-box floral organ identity genes in a second-whorl-specific manner. In Arabidopsis, silencing of B-type MADS-box genes was obtained by expressing B. napus APETALA3 (BAP3) or PISTILLATA (BPI) homologous self-complementary hpRNA constructs under control of the Arabidopsis A-type MADS-box gene APETALA1 (AP1) promoter. In B. napus, silencing of the BPI gene family was achieved by expressing a similar hpRNA construct as used in Arabidopsis under the control of a chimeric promoter consisting of a modified petal-specific Arabidopsis AP3 promoter fragment fused to the AP1 promoter. In this way, transgenic plants were generated producing male fertile flowers in which the petals were converted into sepals (Arabidopsis) or into sepaloid petals (B. napus). These novel flower phenotypes were stable and heritable in both species.Abbreviations PAR photosynthetically active radiation - ST-LS1 potato light-inducible tissue-specific ST-LS1 gene - GUS -glucuronidase     

Floral MADS-box Genes in Trioecious Papaya: Characterization of AG and AP1 Subfamily Genes Revealed a Sex-type-specific Gene

The trioecious papaya is a unique system to study the roles of flower organ identity genes of the ABC model in a multi-sex-type plant species. We have cloned two Agamous ( AG) subfamily genes, CpPLE and CpSTK, and one AP1 subfamily gene, CpFUL—a FRUITFUL homolog. CpPLE, CpSTK, and CpFUL are grouped into the PLE, D, and euFUL sublineages, respectively. Both CpPLE and CpSTK were expressed only in flowers, not in roots and leaves based on Northern and RT-PCR analyses. Specifically, CpPLE was detected only in the stamens and carpels of flowers of all three sex types, from a very early stage of flower development through full maturity. CpSTK expression was detected in female and hermaphrodite flowers, but completely absent in male flowers. This is the only gene found so far that shows sex-type-specific expression in papaya but this is likely to be an indirect effect of sex determination rather than a causative agent. CpFUL was expressed in leaves and all parts of the flowers except stamens. The genomic structures and expression patterns of CpPLE and CpSTK are consistent with their potential functions as C and D class genes, respectively. CpPLE belongs to the PLE lineage and is therefore an ortholog of SHP1/2 rather than AG. However, CpPLE is likely to perform ancestral functions in carpel and stamen identity, whereas SHP1/2 are involved in fruit development. These findings demonstrate that the evolution of gene function within the AG and PLE lineages has been quite dynamic, even over relatively short phylogenetic distances.     

Nucleotide sequence of a flower-specific MADS box cDNA clone from orchid

An orchid (Aranda deborah) mature flower cDNA library was screened with an agamous cDNA probe from Arabidopsis. One positive clone for agamous gene was isolated, cloned and sequenced. This cDNA clone (om1) has a full length open reading frame of 750 bp corresponding to 250 amino acid residues. Comparison of om1 MADS box with that of its counterparts in tomato and Arabidopisis reveals significantly high homology (>95%). Northern analysis indicated this gene is expressed in mature flowers and not in young developing inflorescences or young floral buds. In the mature flowers, it is only expressed in petals and weakly in sepals but not in the column (gynostemium).     

A study of integrative transformation in Schizosaccharomyces pombe

Summary Organ-specific and constitutive expression of the Arabidosis HSP18.2 gene under normal growth conditions (22° C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for -glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.     

蝴蝶兰花发育的分子生物学研究进展

蝴蝶兰花非常独特且高度进化,如萼片瓣化、瓣片特化为唇瓣、雌雄蕊合生成合蕊柱及子房发育须由授粉启动等,是单子叶植物花发育研究的理想材料。近年来蝴蝶兰花发育分子生物学取得了重要进展。该文就近年来国内外有关蝴蝶兰开花转换及花器官发育相关基因研究以及B类基因与兰花花被的进化发育关系方面的研究进展进行综述。研究表明:MADS基因在蝴蝶兰开花转换及花器官发育过程中起重要作用,推测其中的DEF(DE-FICIENS)-like基因早期经过2轮复制,形成了4类不同的DEF-like基因,进而决定兰花花被属性。蝴蝶兰花发育分子生物学的深入研究,将极大地利于通过基因工程手段提高蝴蝶兰花品质如花色改良及花期调控等,推动分子育种进程。     

Expression of AODEF,a B-functional MADS-box gene,in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A. officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.     

Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana)

In higher eudicotyledonous angiosperms the floral organs are typically arranged in four different whorls, containing sepals, petals, stamens and carpels. According to the ABC model, the identity of these organs is specified by floral homeotic genes of class A, A+B, B+C and C, respectively. In contrast to the sepal and petal whorls of eudicots, the perianths of many plants from the Liliaceae family have two outer whorls of almost identical petaloid organs, called tepals. To explain the Liliaceae flower morphology, van Tunen et al. (1993) proposed a modified ABC model, exemplified with tulip. According to this model, class B genes are not only expressed in whorls 2 and 3, but also in whorl 1. Thus the organs of both whorls 1 and 2 express class A plus class B genes and, therefore, get the same petaloid identity. To test this modified ABC model we have cloned and characterized putative class B genes from tulip. Two DEF- and one GLO-like gene were identified, named TGDEFA, TGDEFB and TGGLO. Northern hybridization analysis showed that all of these genes are expressed in whorls 1, 2 and 3 (outer and inner tepals and stamens), thus corroborating the modified ABC model. In addition, these experiments demonstrated that TGGLO is also weakly expressed in carpels, leaves, stems and bracts. Gel retardation assays revealed that TGGLO alone binds to DNA as a homodimer. In contrast, TGDEFA and TGDEFB cannot homodimerize, but make heterodimers with PI. Homodimerization of GLO-like protein has also been reported for lily, suggesting that this phenomenon is conserved within Liliaceae plants or even monocot species.these authors contributed equally to this work     

Analysis of B-Class Genes NAP3L3 and NAP3L4 in Narcissus tazetta var. chinensis

Very few flower organ identity genes have been characterized in Chinese narcissus (Narcissus tazetta var. chinensis), which has petaloid sepals. Here, we report the cloning of two full-length B-class genes, namely NAP3L3 and NAP3L4, that are orthologs of the DEFICIENS lineage. Both genes are highly expressed in the second whorl of the perianth and in the stamens. NAP3L4 is also expressed strongly in the ovule. The functions of these two genes were further analyzed using transgenic plants. Ectopic expression of either gene in Arabidopsis gave no obvious floral organ transformation phenotypes. In yeast two-hybrid assays, NAP3L3 and NAP3L4 failed to homodimerize and interacted weakly with each other. The data suggest that these two genes might not be involved in the formation of petaloid sepals. Isolation and functional analysis of other B-class paralogs should be conducted to fully understand petaloid tepal development in Chinese narcissus.     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.     

Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana

An Arabidopsis thaliana gene (UBC6) encoding a homologue to ubiquitin-conjugating enzymes has been isolated which is capable of encoding a protein of 183 amino acids of ca. 21 kDa. Northern analysis indicates that the gene is expressed in flowers, seeds and, to a somewhat lesser extent, in 10-day seedlings but not in mature leaves, callus and pre-flowering plants. This pattern of expression is confirmed using transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion construct. These plants displey GUS activity in mature anthers prior to dehiscence, in developing embryos, sepals and the style after pollination.     

Sex specific expression and distribution of small RNAs in papaya

Background

Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored.

Results

We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot.

Conclusion

By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Y^h chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-20) contains supplementary material, which is available to authorized users.     

DFL, a FLORICAULA/LEAFY homologue gene from Dendranthema lavandulifolium is expressed both in the vegetative and reproductive tissues

FLO/LFY homologue genes were initially characterized as floral meristem identity genes and play a key role in flower development among diverse species. The inflorescence organization of chrysanthemum differs from typical dicotyledons such as Arabidopsis and Antirrhinum as clear sepals are absent, and instead, a pappus, a rudimentary sepal, is formed. To understand the mechanism of reproduction of chrysanthemum at the molecular level, DFL, a FLORICAULA/LEAFY homologous gene, was cloned from Dendranthema lavandulifolium, which is one of the original species of chrysanthemum. The DFL gene consists of a 1,236-bp open reading frame and encodes a putative protein of 412 amino acids, which is 63% identical to LFY and 70% to FLO. The expression patterns of DFL during the flower development were analyzed, and RT-PCR results showed that DFL was strongly expressed in the flower bud. In situ hybridization experiments showed that it is strongly expressed in the inflorescence bract, petal and stamen primordial tissues throughout the inflorescence development. Its expression signals were also detected in stems, leaf primordial tissues and developing inflorescence bracts.     

Characterization,expression and phylogenetic study of R2R3-MYB genes in orchid

cDNA fragments representing 21 R2R3-MYB genes were isolated by RT-PCR from the Dendrobiumorchid hybrid Woo Leng. Six full-length cDNA clones were obtained from a flower cDNA library, four of which, DwMYB1, DwMYB2, DwMYB8 and DwMYB10, represent typical plant R2R3-MYB genes. The conceptual DwMYB4 protein is truncated at the C-terminal region and contains the R2 repeat and the N-terminal half of the R3 repeat (R2R3). DwMYB4 expression is restricted to flowers. DwMYB9 contains an 8 amino acid N-terminal deletion in the R2 repeat (R2R3) and is expressed at high levels in mature flower and inflorescence, but at very low levels in young flower buds. DwMYB8 and DwMYB10 show similar expression patterns and share very high sequence similarity in the N-terminal part of the MYB domain. Analysis of amino acid substitution indicated that the pattern and type of substitution between Arabidopsis and maize are quite different. Maize may have more conserved substitution in the MYB^BRH domain than Arabidopsis.     

Isolation,expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana

We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu). This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes. However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu. Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves. Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus. As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.     

The study of the E-class SEPALLATA3-like MADS-box genes in wild-type and mutant flowers of cultivated saffron crocus (Crocus sativus L.) and its putative progenitors

To further understand flowering and flower organ formation in the monocot crop saffron crocus (Crocus sativus L.), we cloned four MIKC^c type II MADS-box cDNA sequences of the E-class SEPALLATA3 (SEP3) subfamily designated CsatSEP3a/b/c/c_as as well as the three respective genomic sequences. Sequence analysis showed that cDNA sequences of CsatSEP3 c and c_as are the products of alternative splicing of the CsatSEP3c gene. Bioinformatics analysis with putative orthologous sequences from various plant species suggested that all four cDNA sequences encode for SEP3-like proteins with characteristic motifs and amino acids, and highlighted intriguing sequence features. Phylogenetically, the isolated sequences were closest to the SEP3-like genes from monocots such as Asparagus virgatus, Oryza sativa, Zea mays, and the dicot Arabidopsis SEP3 gene. All four isolated C. sativus sequences were strongly expressed in flowers and in all flower organs: whorl1 tepals, whorl2 tepals, stamens and carpels, but not in leaves. Expression of CsatSEP3a/b/c/c_as cDNAs was compared in wild-type and mutant flowers. Expression of the isolatedCsatSEP3-like genes in whorl1 tepals together with E-class CsatAP1/FUL subfamily and B-class CsatAP3 and CsatPI subfamilies of genes, fits the ABCE “quartet model,” an extended form of the original ABC model proposed to explain the homeotic transformation of whorl1 sepals into whorl1 tepals in Liliales and Asparagales plants such as C. sativus. This conclusion was also supported by the interaction of the CsatSEP3b protein with CsatAP1/FUL and CsatAP3 proteins. In contrast, expression of both B-class CsatAP3 and CsatPI genes and the C-class CsatAGAMOUS genes together with E-class CsatSEP3-like genes in carpels, without any phenotypic effects on carpels, raises questions about the role of these gene classes in carpel formation in this non-grass monocot and requires further experimentation. Finally, taking advantage of the size and sequence differences in amplified genomic sequences of the triploid C. sativus and comparing them with the respective sequences from C. tomasii, C. hadriaticus and C. cartwrightianus, three putative wild-type diploid progenitor species, we examined the origin of CsatSEP3a sequence.