Interactions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate.

Upon floral induction, the primary shoot meristem of an Arabidopsis plant begins to produce flower meristems rather than leaf primordia on its flanks. Assignment of floral fate to lateral meristems is primarily due to the cooperative activity of the flower meristem identity genes LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER. We present evidence here that AP1 expression in lateral meristems is activated by at least two independent pathways, one of which is regulated by LFY. In lfy mutants, the onset of AP1 expression is delayed, indicating that LFY is formally a positive regulator of AP1. We have found that AP1, in turn, can positively regulate LFY, because LFY is expressed prematurely in the converted floral meristems of plants constitutively expressing AP1. Shoot meristems maintain an identity distinct from that of flower meristems, in part through the action of genes such as TERMINAL FLOWER1 (TFL1), which bars AP1 and LFY expression from the influorescence shoot meristem. We show here that this negative regulation can be mutual because TFL1 expression is downregulated in plants constitutively expressing AP1. Therefore, the normally sharp phase transition between the production of leaves with associated shoots and formation of the flowers, which occurs upon floral induction, is promoted by positive feedback interactions between LFY and AP1, together with negative interactions of these two genes with TFL1.     

Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time

Citrus trees have a long juvenile phase that delays their reproductive development by between 6 and 20 years, depending on the species. With the aim of accelerating their flowering time, we transformed juvenile citrus seedlings to constitutively express the Arabidopsis LEAFY (LFY) or APETALA1 (AP1) genes, which promote flower initiation in Arabidopsis. Both types of transgenic citrus produced fertile flowers and fruits as early as the first year, notably through a mechanism involving an appreciable shortening of their juvenile phase. Furthermore, expression of AP1 was as efficient as LFY in the initiation of flowers, and did not produce any severe developmental abnormality. Both types of transgenic trees flowered in consecutive years, and their flowering response was under environmental control. In addition, zygotic and nucellar derived transgenic seedlings had a very short juvenile phase and flowered in their first spring, demonstrating the stability and inheritance of this trait. These results open new possibilities for domestication, genetic improvement, and experimental research in citrus and other woody species.     

Evolution of floral meristem identity genes. Analysis of Lolium temulentum genes related to APETALA1 and LEAFY of Arabidopsis

Flowering (inflorescence formation) of the grass Lolium temulentum is strictly regulated, occurring rapidly on exposure to a single long day (LD). During floral induction, L. temulentum differs significantly from dicot species such as Arabidopsis in the expression, at the shoot apex, of two APETALA1 (AP1)-like genes, LtMADS1 and LtMADS2, and of L. temulentum LEAFY (LtLFY). As shown by in situ hybridization, LtMADS1 and LtMADS2 are expressed in the vegetative shoot apical meristem, but expression increases strongly within 30 h of LD floral induction. Later in floral development, LtMADS1 and LtMADS2 are expressed within spikelet and floret meristems and in the glume and lemma primordia. It is interesting that LtLFY is detected quite late (about 12 d after LD induction) within the spikelet meristems, glumes, and lemma primordia. These patterns contrast with Arabidopsis, where LFY and AP1 are consecutively activated early during flower formation. LtMADS2, when expressed in transgenic Arabidopsis plants under the control of the AP1 promoter, could partially complement the organ number defect of the severe ap1-15 mutant allele, confirming a close relationship between LtMADS2 and AP1.     

The Rho1 GTPase acts together with a vacuolar glutathione S-conjugate transporter to protect yeast cells from oxidative stress

Maintenance of redox homeostasis is critical for the survival of all aerobic organisms. In the budding yeast Saccharomyces cerevisiae, as in other eukaryotes, reactive oxygen species (ROS) are generated during metabolism and upon exposure to environmental stresses. The abnormal production of ROS triggers defense mechanisms to avoid the deleterious consequence of ROS accumulation. Here, we show that the Rho1 GTPase is necessary to confer resistance to oxidants in budding yeast. Temperature-sensitive rho1 mutants (rho1(ts)) are hypersensitive to oxidants and exhibit high accumulation of ROS even at a semipermissive temperature. Rho1 associates with Ycf1, a vacuolar glutathione S-conjugate transporter, which is important for heavy metal detoxification in yeast. Rho1 and Ycf1 exhibit a two-hybrid interaction with each other and form a bimolecular fluorescent complex on the vacuolar membrane. A fluorescent-based complementation assay suggests that the GTP-bound Rho1 associates with Ycf1 and that their interaction is enhanced upon exposure to hydrogen peroxide. The rho1(ts) mutants also exhibit hypersensitivity to cadmium, while cells carrying a deletion of YCF1 or mutations in a component of the Pkc1-MAP kinase pathway exhibit little or minor sensitivity to oxidants. We thus propose that Rho1 protects yeast cells from oxidative stress by regulating multiple downstream targets including Ycf1. Since both Rho1 and Ycf1 belong to highly conserved families of proteins, similar mechanisms may exist in other eukaryotes.     

Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex

The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC.     

Soluble E-selectin acts in synergy with platelet-activating factor to activate neutrophil beta 2-integrins. Role of tyrosine kinases and Ca2+ mobilization

Selectins play a critical role in neutrophil recruitment to sites of inflammation, in tethering and rolling of neutrophils on vascular endothelium, as well as triggering beta(2)-integrin-mediated adhesion. We have previously demonstrated potential pro-inflammatory effects of soluble E-selectin upon neutrophil effector functions, using a soluble recombinant molecule (E-zz), which increased beta(2)-integrin-mediated adhesion, decreased beta(2)-integrin-dependent migration, and triggered reactive oxygen species generation and release. In this study, we have examined the intracellular signals following neutrophil activation by soluble E-selectin. We show that exposure of neutrophils to E-selectin and platelet-activating factor (PAF) in combination induced a synergistic effect upon beta(2)-integrin-mediated adhesion. Although soluble E-selectin did not induce Ca(2+) mobilization in neutrophils by itself, elevation of intracellular Ca(2+) was specifically prolonged in response to PAF but not leukotriene B(4) or N-formyl-Met-Leu-Phe. The prolonged Ca(2+) mobilization observed in the presence of E-selectin was dependent on Ca(2+) influx from intracellular stores rather than influx of extracellular Ca(2+) through SKF 96365-sensitive channels. The specific alteration of Ca(2+) mobilization reported here appears not to have a role in the synergistic effects of E-selectin and PAF upon neutrophil O(2) release but may be involved in augmentation of beta(2)-integrin-mediated adhesion.     

Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos

The maternal RNA-binding proteins Pumilio (Pum) and Nanos (Nos) act together to specify the abdomen in Drosophila embryos. Both proteins later accumulate in pole cells, the germline progenitors. Nos is required for pole cells to differentiate into functional germline. Here we show that Pum is also essential for germline development in embryos. First, a mutation in pum causes a defect in pole-cell migration into the gonads. Second, in such pole cells, the expression of a germline-specific marker (PZ198) is initiated prematurely. Finally, pum mutation causes premature mitosis in the migrating pole cells. We show that Pum inhibits pole-cell division by repressing translation of cyclin B messenger RNA. As these phenotypes are indistinguishable from those produced by nos mutation, we conclude that Pum acts together with Nos to regulate these germline-specific events.     

APETALA1 and SEPALLATA3 interact to promote flower development

In Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS-box genes share overlapping roles in promoting flower meristem identity. Later in flower development, the AP1 gene is required for normal development of sepals and petals. Studies of MADS-domain proteins in diverse species have shown that they often function as heterodimers or in larger ternary complexes, suggesting that additional proteins may interact with AP1 and CAL during flower development. To identify proteins that may interact with AP1 and CAL, we used the yeast two-hybrid assay. Among the five MADS-box genes identified in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study. Mutations in the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes that closely resemble intermediate alleles of AP1. Furthermore, the early flowering phenotype of plants constitutively expressing AP1 is significantly enhanced by constitutive SEP3 expression. Taken together, these studies suggest that SEP3 interacts with AP1 to promote normal flower development.