SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1.     

The duplicated B-class MADS-box genes display dualistic characters in orchid floral organ identity and growth

Orchidaceae are an excellent model to examine perianth development because of their sophisticated floral architecture. In this study, we identified 24 APETALA3 (AP3)-like and 13 PISTILLA (PI)-like genes from 11 species of orchids and characterized them into four AP3- and two PI-duplicated homologs. The first duplication event in AP3 homologs occurring in the early evolutionary history of the Orchidaceae gave rise to AP3A and AP3B clades. Further duplication events resulted in four subclades, namely AP3A1, AP3A2, AP3B1 and AP3B2, during the evolution of Orchidaceae. The AP3 paralogous genes were expressed throughout inflorescence and floral bud development. From the in situ hybridization results, we noticed that the transition timings from ubiquitous to constrained expression in floral organs for both clades are different. The transition point of expression of the AP3A clade (clades 3 and 4) was at the late floral organ primordia stage. In contrast, that for the AP3B clade (clades 1 and 2) was not observed until the late inflorescence and floral bud stages. In addition, the AP3 orthologous genes revealed diverse expression patterns in various species of orchids, whereas the PI homologs were uniformly expressed in all floral whorls. AP3A2 orthologs play a noticeable role in lip formation because of their exclusive expression in the lip. Further evidence comes from the ectopic expression of AP3A2 detected in the lip-like petals extending from the lip in four sets of peloric mutants. Finally, a Homeotic Orchid Tepal (HOT) model is proposed, in which dualistic characters of duplicated B-class MADS-box genes are involved in orchid perianth development and growth.     

Genetic basis for innovations in floral organ identity

Of the many innovations associated with the radiation of the angiosperms, the evolution of a petal identity program is among the best understood from a genetic standpoint. Although the existing data do indicate that similar genetic mechanisms control petal development across diverse taxa, there is also considerable evidence for variability in petal identity programs, likely due to a number of factors. These points are illustrated through a review of our current knowledge on the subject, integrating phylogenetic, morphological, and genetic studies. Comparative studies of petal identity highlight the complex nature of homology in plants and stand as a cautionary tale for the interpretation of gene expression data.     

Candidate reference genes for gene expression studies in water lily

The selection of an appropriate reference gene(s) is a prerequisite for the proper interpretation of quantitative Real-Time polymerase chain reaction data. We report the evaluation of eight candidate reference genes across various tissues and treatments in the water lily by the two software packages geNorm and NormFinder. Across all samples, clathrin adaptor complexes medium subunit (AP47) and actin 11 (ACT11) emerged as the most suitable reference genes. Across different tissues, ACT11 and elongation factor 1-alpha (EF1α) exhibited a stable expression pattern. ACT11 and AP47 also stably expressed in roots subjected to various treatments, but in the leaves of the same plants the most stably expressed genes were ubiquitin-conjugating enzyme 16 (UBC16) and ACT11.     

The ABC model and the diversification of floral organ identity

Broad studies of the ABC program across angiosperms have found that interactions between gene duplication, biochemical evolution, shifts in gene expression and modification of existing identity programs have been critical to the evolution of floral morphology. Several themes can be recognized in this context. First, the original concept of “A” function applies only very narrowly to Arabidopsis and its close relatives. Second, while many types of petaloid organs are associated with the expression of AP3/PI homologs, there is growing evidence that there are other genetic mechanisms for producing petaloidy, especially in first whorl organs. Third, pre-existing organ identity programs can be modified to yield novel organ types, often in association with gene duplications. Lastly, there are many aspects of ABC gene function outside the major model systems that remain a mystery, perhaps none more so than the C-terminal amino acid motifs that distinguish specific ABC gene lineages.     

Identification of class B and class C floral organ identity genes from rice plants

The functions of two rice MADS-box genes were studied by the loss-of-function approach. The first gene, OsMADS4, shows a significant homology to members in the PISTILLATA (PI) family, which is required to specify petal and stamen identity. The second gene, OsMADS3, is highly homologous to the members in the AGAMOUS (AG) family that is essential for the normal development of the internal two whorls, the stamen and carpel, of the flower. These two rice MADS box cDNA clones were connected to the maize ubiquitin promoter in an antisense orientation and the fusion molecules were introduced to rice plants by the Agrobacterium-mediated transformation method. Transgenic plants expressing antisense OsMADS4 displayed alterations of the second and third whorls. The second-whorl lodicules, which are equivalent to the petals of dicot plants in grasses, were altered into palea/lemma-like organs, and the third whorl stamens were changed to carpel-like organs. Loss-of-function analysis of OsMADS3 showed alterations in the third and fourth whorls. In the third whorl, the filaments of the transgenic plants were changed into thick and fleshy bodies, similar to lodicules. Rather than making a carpel, the fourth whorl produced several abnormal flowers. These phenotypes are similar to those of the agamous and plena mutants in Arabidopsis and Antirrhinum, respectively. These results suggest that OsMADS4 belongs to the class B gene family and OsMADS3 belongs to the class C gene family of floral organ identity determination.     

New members of the floral organ identity AGAMOUS pathway

The Arabidopsis floral organ identity gene AGAMOUS (AG) specifies stamen and carpel development as well as floral determinacy. Recent reports suggest that the HUA1, HUA2, HEN1 and HEN2 genes function redundantly as components of the AG pathway. The HUA1, HUA2, HEN1 and HEN2 genes encode nuclear proteins that perhaps play a role in RNA metabolism. The HUA and HEN genes function not only on the AG pathway, but also in vegetative development.     

Two lily SEPALLATA-like genes cause different effects on floral formation and floral transition in Arabidopsis

Two AGL2-like MADS-box genes, Lily MADS Box Gene (LMADS) 3 and LMADS4, with extensive homology of LMADS3 to the Arabidopsis SEPALLATA3 were characterized from the lily (Lilium longiflorum). Both LMADS3 and LMADS4 mRNA were detected in the inflorescence meristem, in floral buds of different developmental stages, and in all four whorls of the flower organ. LMADS4 mRNA is also expressed in vegetative leaf and in the inflorescence stem where LMADS3 expression is absent. Transgenic Arabidopsis, which ectopically expresses LMADS3, showed novel phenotypes by significantly reducing plant size, flowering extremely early, and loss of floral determinacy. By contrast, 35S::LMADS4 transgenic plants were morphologically indistinguishable from wild-type plants. The early-flowering phenotype in 35S::LMADS3 transgenic Arabidopsis plants was correlated with the up-regulation of flowering time genes FT, SUPPRESSOR OF OVEREXPRESSION OF CO 1, LUMINIDEPENDENS, and flower meristem identity genes LEAFY and APETALA1. This result was further supported by the ability of 35S::LMADS3 to rescue the late-flowering phenotype in gigantea-1 (gi-1), constans-3 (co-3), and luminidependens-1 but not for ft-1 or fwa-1 mutants. The activation of these flowering time genes is, however, indirect because their expression was unaffected in plants transformed with LMADS3 fused with rat glucocorticoid receptor in the presence of both dexamethasone and cycloheximide.     

Differential expression of carotenoid-related genes determines diversified carotenoid coloration in floral tissues of Oncidium cultivars

Three cultivars of Oncidium orchid with varied coloration, such as Oncidium Gower Ramsey (yellow), Sunkist (orange), and White Jade (white), were analyzed for carotenoid metabolites and gene expression of carotenoid-biosynthetic genes. The HPLC analysis revealed that yellow Gower Ramsey accumulates violaxanthin, 9-cis-violaxanthin and neoxanthin, orange Sunkist accumulates an additional β-carotene, and White Jade is devoid of carotenoid compounds. Molecular characterization indicated that the three Oncidium cultivars exhibited varied expression pattern and level in carotenoid-biosynthetic pathway. Among them, high expression level of β-hydroxylase (OgHYB) and zeaxanthin epoxidase (OgZEP) was displayed in yellow Gower Ramsey, relative to the down-regulation of OgHYB and OgZEP exhibited in orange Sunkist, which results in the accumulation of β-carotene and orange coloration in floral tissues. However, White Jade is caused by the up-regulation of OgCCD1 (Carotenoid Cleavage Dioxygenase 1), which catabolizes carotenoid metabolites. Methylation assay of OgCCD1 promoter in White Jade and Gower Ramsey revealed that a high level of DNA methylation was present in OgCCD1 promoter region of Gower Ramsey. Transient expression of OgCCD1 in yellow lip tissues of Gower Ramsey by bombardment confirmed its function of disintegrating carotenoid compounds. Our results suggest an evolutionary significance that genetic variation of carotenoid-related genes in Oncidium generates the complexity of floral pigmentation and consequently provides the profound varieties in Oncidium population.     

Variation in freezing tolerance,water content and carbohydrate metabolism of floral buds during deacclimation of contrasting blackcurrant cultivars

As a result of climate change, temperature patterns are expected to become increasingly irregular with longer and more frequent episodes of unseasonable warm spells during the winter season. Warm spells may promote premature loss of freezing tolerance and bud burst in woody perennials, thereby increasing the risk of tissue damage by subsequent frosts. This study investigated the variation in kinetics of deacclimation and bud break and associated changes in carbohydrate metabolism and water status in floral buds of six blackcurrant (Ribes nigrum) cultivars in response to a simulated warm spell (16/11 °C day/night). In three of the cultivars, the rate of deacclimation showed an almost logarithmic course, whereas the other three cultivars exhibited greater deacclimation resistance and a sigmoid deacclimation pattern. The timing and rate of bud development, and their relationship with deacclimation varied greatly amongst cultivars, indicating genotypic variation in time-dependent responses of freezing tolerance and bud break to warm temperatures. In all six cultivars, deacclimation and growth resumption were strongly associated with rehydration. In contrast, changes in carbohydrate metabolism were mostly associated with deacclimation. Evaluation of phenological responses of the same cultivars under field conditions showed that cultivars which were fast flushing in response to an experimental warm spell also exhibited early bud break under natural conditions, indicating that cultivar differences in phenological responses are consistent under different temperature conditions.     

The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity

The ABC model of flower organ identity is widely recognized as providing a framework for understanding the specification of flower organs in diverse plant species. Recent studies in Arabidopsis thaliana have shown that three closely related MADS-box genes, SEPALLATA1 (SEP1), SEP2 and SEP3, are required to specify petals, stamens, and carpels because these organs are converted into sepals in sep1 sep2 sep3 triple mutants. Additional studies indicate that the SEP proteins form multimeric complexes with the products of the B and C organ identity genes. Here, we characterize the SEP4 gene, which shares extensive sequence similarity to and an overlapping expression pattern with the other SEP genes. Although sep4 single mutants display a phenotype similar to that of wild-type plants, we find that floral organs are converted into leaf-like organs in sep1 sep2 sep3 sep4 quadruple mutants, indicating the involvement of all four SEP genes in the development of sepals. We also find that SEP4 contributes to the development of petals, stamens, and carpels in addition to sepals and that it plays an important role in meristem identity. These and other data demonstrate that the SEP genes play central roles in flower meristem identity and organ identity.     

Benzylaminopurine induces phenocopies of floral meristem and organ identity mutants in wild-typeArabidopsis plants

Arabidopsis thaliana (L.) Heynh. has been used as a model system to investigate the regulatory genes that control and coordinate the determination, differentiation and morphogenesis of the floral meristem and floral organs. We show here that benzylaminopurine (BAP), a cytokinin, influences flower development inArabidopsis and induces partial phenocopies of known floral homeotic mutants. Application of BAP to wild-type inflorescences at three developmental stages results in: (i) increase in floral organ number; (ii) formation of abnormal floral organs and (iii) induction of secondary floral buds in the axils of sepals. These abnormalities resemble the phenotypes of mutants,clv1 (increase in organ number),ap1,ap2,ap3 (abnormal floral organs) andap1 (secondary floral buds in the axils of first-whorl organs). In addition, BAP induces secondary floral buds in the axils of perianth members ofapt2-6, ap3-1 andag mutants, and accentuates the phenotype of theapt2-1 mutant to resemble theapt2-6 mutant. These observations suggest that exogenous BAP suppresses the normal functioning of the genes for floral meristem identity and thereby affects flower development and the later stages of floral organ differentiation.Abbreviations BAP N6-benzylaminopurine - CK cytokinin