Petal-specific subfunctionalization of an APETALA3 paralog in the Ranunculales and its implications for petal evolution

? The petals of the lower eudicot family Ranunculaceae are thought to have been derived many times independently from stamens. However, investigation of the genetic basis of their identity has suggested an alternative hypothesis: that they share a commonly inherited petal identity program. This theory is based on the fact that an ancient paralogous lineage of APETALA3 (AP3) in the Ranunculaceae appears to have a conserved, petal-specific expression pattern. ? Here, we have used a combination of approaches, including RNAi, comparative gene expression and molecular evolutionary studies, to understand the function of this petal-specific AP3 lineage. ? Functional analysis of the Aquilegia locus AqAP3-3 has demonstrated that the paralog is required for petal identity with little contribution to the identity of the other floral organs. Expanded expression studies and analyses of molecular evolutionary patterns provide further evidence that orthologs of AqAP3-3 are primarily expressed in petals and are under higher purifying selection across the family than the other AP3 paralogs. ? Taken together, these findings suggest that the AqAP3-3 lineage underwent progressive subfunctionalization within the order Ranunculales, ultimately yielding a specific role in petal identity that has probably been conserved, in stark contrast with the multiple independent origins predicted by botanical theories.     

The MIXTA-like Transcription factor MYB16 is a major regulator of cuticle formation in vegetative organs

Cuticle secreted on the surface of the epidermis of aerial organs protects plants from the external environment. We recently found that Arabidopsis MIXTA-like R2R3-MYB family members MYB16 and MYB106 regulate cuticle formation in reproductive organs and trichomes. However, the artificial miRNA (amiRNA)-mediated knockdown plants showed no clear phenotypic abnormality in vegetative tissues. In this study, we used RNA interference (RNAi) targeting MYB16 to produce plants with reduced expression of both MYB16 and MYB106. The rosette leaves of RNAi plants showed more severe permeable cuticle phenotypes than the myb106 mutants expressing the MYB16 amiRNA in the previous study. The RNAi plants also showed reduced expression of cuticle biosynthesis genes LACERATA and ECERIFERUM1. By contrast, expression of a gain-of-function MYB16 construct induced over-accumulation of waxy substances on leaves. These results suggest that MYB16 functions as a major regulator of cuticle formation in vegetative organs, in addition to its effect in reproductive organs and trichomes.     

Spindle Assembly Checkpoint Protein Dynamics Reveal Conserved and Unsuspected Roles in Plant Cell Division

Background

In eukaryotes, the spindle assembly checkpoint (SAC) ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the spindle.

Methodology/Principal Findings

We investigated the mechanism underlying this surveillance mechanism in plants, by characterising the orthogolous SAC proteins BUBR1, BUB3 and MAD2 from Arabidopsis. We showed that the cell cycle-regulated BUBR1, BUB3.1 and MAD2 proteins interacted physically with each other. Furthermore, BUBR1 and MAD2 interacted specifically at chromocenters. Following SAC activation by global defects in spindle assembly, these three interacting partners localised to unattached kinetochores. In addition, in cases of ‘wait anaphase’, plant SAC proteins were associated with both kinetochores and kinetochore microtubules. Unexpectedly, BUB3.1 was also found in the phragmoplast midline during the final step of cell division in plants.

Conclusions/Significance

We conclude that plant BUBR1, BUB3.1 and MAD2 proteins may have the SAC protein functions conserved from yeast to humans. The association of BUB3.1 with both unattached kinetochore and phragmoplast suggests that in plant, BUB3.1 may have other roles beyond the spindle assembly checkpoint itself. Finally, this study of the SAC dynamics pinpoints uncharacterised roles of this surveillance mechanism in plant cell division.     

The expression level of anthocyanidin synthase determines the anthocyanin content of crabapple (<Emphasis Type="Italic">Malus</Emphasis> sp.) petals

In addition to contributing to the coloration of plant organs and their defense against herbivores, the consumption of anthocyanins in the human diet has a number of health benefits. Crabapple (Malus sp.) represents a valuable experimental model system to research the mechanisms and regulation of anthocyanin accumulation, in part due to the often vivid and varied petal and leaf coloration that is exhibited by various cultivars. The enzyme anthocyanidin synthase (ANS) plays a pivotal role in anthocyanin biosynthesis; however, the relationship between ANS expression and petal pigmentation has yet to be established in crabapple. To illuminate the mechanism of anthocyanin accumulation in crabapple petals, we evaluated the expression of two crabapple ANS allelic genes (McANS-1 and McANS-2) and the levels of anthocyanins in petals from cultivars with dark red (‘Royalty’) and white (‘Flame’) petals, as well as another (‘Radiant’) whose petals have an intermediate pink color. We determined that the expression of McANS in the three cultivars correlated with the variation of anthocyanin accumulation during different petal developmental stages. Furthermore, transgenic tobacco plants constitutively overexpressing one of the two McANS genes, McANS-1, had showed elevated anthocyanin accumulation and a deeper red coloration in their petals than those from untransformed control lines. In conclusion, we propose that McANS are responsible for anthocyanin accumulation during petal coloration in different crabapple cultivars.     

A petal‐specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal‐specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal‐specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal‐specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β‐glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal‐specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA‐like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal‐specific promoter in molecular breeding of floricultural crops.     

Arabidopsis genes AS1, AS2, and JAG negatively regulate boundary-specifying genes to promote sepal and petal development

Boundary formation is crucial for organ development in multicellular eukaryotes. In higher plants, boundaries that separate the organ primordia from their surroundings have relatively low rates of cell proliferation. This cellular feature is regulated by the actions of certain boundary-specifying genes, whose ectopic expression in organs can cause inhibition of organ growth. Here, we show that the Arabidopsis thaliana ASYMMETRIC LEAVES1 and 2 (AS1 and AS2) and JAGGED (JAG) genes function in the sepal and petal primordia to repress boundary-specifying genes for normal development of the organs. Loss-of-function as1 jag and as2 jag double mutants produced extremely tiny sepals and petals. Analysis of a cell-cycle marker HISTONE4 revealed that cell division in sepal primordia of the double mutant was inhibited. Moreover, these abnormal sepals and petals exhibited ectopic overexpression of the boundary-specifying genes PETAL LOSS (PTL) and CUP-SHAPED COTYLEDON1 [corrected] and 2 (CUC1 and CUC2). Loss of PTL or CUC1 and CUC2 functions in the as1 jag background could partially rescue the tiny sepal and petal phenotypes, supporting the model that the tiny sepal/petal phenotypes are caused, at least in part, by ectopic expression of boundary-specifying genes. Together, our data reveal a previously unrecognized fundamental regulation by which AS1, AS2, and JAG act to define sepal and petal from their boundaries.     

Spatial distribution of the RABBIT EARS protein and effects of its ectopic expression in Arabidopsis thaliana flowers

In many flowering plants, flowers consist of two peripheral organs, sepals and petals, occurring in outer two whorls, and two inner reproductive organs, stamens and carpels. These organs are arranged in a concentric pattern in a floral meristem, and the organ identity is established by the combined action of floral homeotic genes expressed along the whorls. Floral organ primordia arise at fixed positions in the floral meristem within each whorl. The RABBIT EARS (RBE) gene is transcribed in the petal precursor cells and primordia, and regulates petal initiation and early growth in Arabidopsis thaliana. We investigated the spatial and temporal expression pattern of a RBE protein fused to the green fluorescent protein (GFP). Expression of the GFP:RBE fusion gene under the RBE cis-regulatory genomic fragment rescues the rbe petal defects, indicating that the fusion protein is functional. The GFP signal is located to the cells where RBE is transcribed, suggesting that RBE function is cell-autonomous. Ectopic expression of GFP:RBE under the APETALA1 promoter causes the homeotic conversion of floral organs, resulting in sterile flowers. In these plants, the class B homeotic genes APETALA3 and PISTILLATA are down-regulated, suggesting that the restriction of the RBE expression to the petal precursor cells is crucial for flower development.