Brassicaceae INDEHISCENT genes specify valve margin cell fate and repress replum formation

Members of the Brassicaceae family, including Arabidopsis thaliana and oilseed rape (Brassica napus), produce dry fruits that open upon maturity along a specialised tissue called the valve margin. Proper development of the valve margin in Arabidopsis is dependent on the INDEHISCENT (IND) gene, the role of which in genetic and hormonal regulation has been thoroughly characterised. Here we perform phylogenetic comparison of IND genes in Arabidopsis and Brassica to identify conserved regulatory sequences that are responsible for specific expression at the valve margin. In addition we have taken a comparative development approach to demonstrate that the BraA.IND.a and BolC.IND.a genes from B. rapa and B. oleracea share identical function with Arabidopsis IND since ethyl methanesulphonate (EMS) mutant alleles and silenced transgenic lines have valve margin defects. Furthermore we show that the degree of these defects can be fine‐tuned for crop improvement. Wild‐type Arabidopsis produces an outer replum composed of about six cell files at the medial region of the fruits, whereas Brassica fruits lack this tissue. A strong loss‐of‐function braA.ind.a mutant gained outer replum tissue in addition to its defect in valve margin development. An enlargement of replum size was also observed in the Arabidopsis ind mutant suggesting a general role of Brassicaceae IND genes in preventing valve margin cells from adopting replum identity.     

CRABS CLAW and SPATULA, two Arabidopsis genes that control carpel development in parallel with AGAMOUS

To help understand the process of carpel morphogenesis, the roles of three carpel development genes have been partitioned genetically. Mutants of CRABS CLAW cause the gynoecium to develop into a wider but shorter structure, and the two carpels are unfused at the apex. Mutants of a second gene, SPATULA, show reduced growth of the style, stigma, and septum, and the transmitting tract is absent. Double mutants of crabs claw and spatula with homeotic mutants that develop ectopic carpels demonstrate that CRABS CLAW and SPATULA are necessary for, and inseparable from, carpel development, and that their action is negatively regulated by A and B organ identity genes. The third carpel gene studied, AGAMOUS, encodes C function that has been proposed to fully specify carpel identity. When AGAMOUS function is removed together with the A class gene APETALA2, however, the organs retain many carpelloid properties, suggesting that other genes are also involved. We show here that further mutant disruption of both CRABS CLAW and SPATULA function removes remaining carpelloid properties, revealing that the three genes together are necessary to generate the mature gynoecium. In particular, AGAMOUS is required to specify the identity of the carpel wall and to promote the stylar outgrowth at the apex, CRABS CLAW suppresses radial growth of the developing gynoecium but promotes its longitudinal growth, and SPATULA supports development of the carpel margins and tissues derived from them. The three genes mostly act independently, although there is genetic evidence that CRABS CLAW enhances AGAMOUS and SPATULA function.     

Brassinosteroids interact with auxin to promote lateral root development in Arabidopsis

Plant hormone brassinosteroids (BRs) and auxin exert some similar physiological effects likely through their functional interaction, but the mechanism for this interaction is unknown. In this study, we show that BRs are required for lateral root development in Arabidopsis and that BRs act synergistically with auxin to promte lateral root formation. BR perception is required for the transgenic expression of the beta-glucuronidase gene fused to a synthetic auxin-inducible promoter (DR5::GUS) in root tips, while exogenous BR promotes DR5::GUS expression in the root tips and the stele region proximal to the root tip. BR induction of both lateral root formation and DR5::GUS expression is suppressed by the auxin transport inhibitor N-(1-naphthyl) phthalamic acid. Importantly, BRs promote acropetal auxin transport (from the base to the tip) in the root. Our observations indicate that BRs regulate auxin transport, providing a novel mechanism for hormonal interactions in plants and supporting the hypothesis that BRs promote lateral root development by increasing acropetal auxin transport.     

The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis

Partitioning of the cytoplasm during cytokinesis or cellularisation requires syntaxin-mediated membrane fusion [1-3]. Whereas in animals, membrane fusion promotes ingression of a cleavage furrow from the plasma membrane [4,5], somatic cells of higher plants form de novo a transient membrane compartment, the cell plate, which is initiated in the centre of the division plane and matures into a new cell wall and its flanking plasma membranes [6,7]. Cell plate formation results from the fusion of Golgi-derived vesicles delivered by a dynamic cytoskeletal array, the phragmoplast. Mutations in two Arabidopsis genes, KNOLLE (KN) and KEULE (KEU), cause abnormal seedlings with multinucleate cells and incomplete cell walls [1,8]. The KN gene encodes a cytokinesis-specific syntaxin which localises to the cell plate [9]. Here, we show that KN protein localisation is unaffected in keu mutant cells, which, like kn, display phragmoplast microtubules and accumulate ADL1 protein in the plane of cell division but vesicles fail to fuse with one another. Genetic interactions between KN and KEU were analysed in double mutant embryos. Whereas the haploid gametophytes gave rise to functional gametes, the embryos behaved like single cells displaying multiple, synchronously cycling nuclei, cell cycle-dependent microtubule arrays and ADL1 accumulation between pairs of daughter nuclei. This complete inhibition of cytokinesis from fertilisation indicates that KN and KEU, have partially redundant functions and interact specifically in vesicle fusion during cytokinesis of somatic cells.     

MADS-box protein complexes control carpel and ovule development in Arabidopsis

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.     

Cytoplasmic and cortical determinants interact to specify ectoderm and mesoderm in the leech embryo.

In leech embryos, segmental ectoderm and mesoderm are produced by a pair of sister cells located near the animal and vegetal poles, respectively. We have investigated the mechanism that localizes ectodermal and mesodermal fates along the animal-vegetal axis. The results of cleavage arrest and cell ablation experiments suggest that the full range of normal cell interactions are not required for this process. However, when the animal and vegetal hemispheres are separated by re-orientation of the first cleavage plane from meridional to equatorial, the ectodermal fate co-segregates with the animal hemisphere and the mesodermal fate with the vegetal hemisphere. Two pools of yolk-deficient cytoplasm, called teloplasm, are located at the animal and vegetal poles of the zygote, but separation of the animal and vegetal teloplasms is not sufficient for the segregation of ectodermal and mesodermal fates. Rather, complete segregation of fates requires an equatorial cleavage orientation that separates not only the two teloplasms, but also the animal and vegetal cortical regions. This, in conjunction with previous findings, indicates that ectodermal determinants are localized to the cell cortex in the animal hemisphere of the zygote. We propose that these determinants segregate to the ectodermal precursor and interact with factors in teloplasm to transform the fate of this cell from a mesodermal ground state to ectoderm.     

Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis

Angiosperm seeds integrate various environmental signals, such as water availability and light conditions, to make a proper decision to germinate. Once the optimal conditions are sensed, gibberellin (GA) is synthesized, triggering germination. Among environmental signals, light conditions are perceived by phytochromes. However, it is not well understood how phytochromes regulate GA biosynthesis. Here we investigated whether phytochromes regulate GA biosynthesis through PIL5, a phytochrome-interacting bHLH protein, in Arabidopsis. We found that pil5 seed germination was inhibited by paclobutrazol, the ga1 mutation was epistatic to the pil5 mutation, and the inhibitory effect of PIL5 overexpression on seed germination could be rescued by exogenous GA, collectively indicating that PIL5 regulates seed germination negatively through GA. Expression analysis revealed that PIL5 repressed the expression of GA biosynthetic genes (GA3ox1 and GA3ox2), and activated the expression of a GA catabolic gene (GA2ox) in both PHYA- and PHYB-dependent germination assays. Consistent with these gene-expression patterns, the amount of bioactive GA was higher in the pil5 mutant and lower in the PIL5 overexpression line. Lastly, we showed that red and far-red light signals trigger PIL5 protein degradation through the 26S proteasome, thus releasing the inhibition of bioactive GA biosynthesis by PIL5. Taken together, our data indicate that phytochromes promote seed germination by degrading PIL5, which leads to increased GA biosynthesis and decreased GA degradation.     

Cell-wall damage activates DOF transcription factors to promote wound healing and tissue regeneration in Arabidopsis thaliana

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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.     

Bovine theca and granulosa cells interact to promote androgen production

Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Density-dependent processes influencing the evolutionary dynamics of dispersal: a functional analysis of seed dispersal in Arabidopsis thaliana (Brassicaceae)

We conducted a functional analysis of seed dispersal and its plasticity in response to density in Arabidopsis thaliana by growing morphologically diverse ecotypes under high and low density and measuring seed dispersion patterns under controlled conditions. Maternal plant architectural traits such as height and branching, and fruit traits such as dehiscence and silique length influenced various measures of seed dispersion patterns, including the average dispersal distance, kurtosis of the seed dispersion pattern, and post-dispersal seed density. The density at which plants grew determined which traits influenced dispersal. A change in density would therefore change which maternal characters would be subjected to natural selection through selection on dispersal. Density-mediated maternal effects on dispersal contributed to a negative correlation between parents and offspring for sibling density after dispersal, which could impede the response to selection on post-dispersal sibling density. Plant traits that influenced dispersal also influenced maternal fitness- sometimes opposing selection on dispersal and sometimes augmenting it-and the direction of the relationship sometimes depended on density. These density-dependent relationships between plant traits, dispersal, and maternal fitness can increase or reduce evolutionary constraints on dispersal, depending on the trait and depending on post-dispersal density itself.     

Fire and summer temperatures interact to shape seed dormancy thresholds

Background and AimsIn Mediterranean ecosystems, the heat shock of wildfire disrupts physical seed dormancy in many plant species. This triggers germination in the post-fire environment where seedling establishment is optimal due to decreased competition and increased resource availability. However, to maintain the soil seed bank until a fire occurs, the minimum heat capable of breaking seed dormancy (i.e. the lower heat threshold) must be above the maximum temperatures typically observed in the soil during the summer. We therefore hypothesized that summer temperatures have shaped heat requirements for physical dormancy release. Specifically, we predicted that seeds from populations growing under warmer summers will have higher values of the lower heat threshold.MethodsTo evaluate this prediction, we collected seeds from two Cistus species in 31 populations (20 Cistus albidus and 11 Cistus salviifolius) along a climate gradient of summer temperatures on the eastern coast of Spain. For each population, seeds were treated to 10 min heat shocks, from 30 to 120 °C in 5 °C increments (19 treatments), to simulate increasing heat doses from summer to fire-related temperatures. Seeds were then germinated in the lab.Key ResultsFor all populations, maximum germination was observed when applying temperatures associated with fire. Lower heat thresholds varied among populations, with a positive relationship between summer temperatures at seed population origin and the heat dose required to break dormancy.ConclusionsOur results suggest that fire drives maximum dormancy release for successful post-fire germination, while summer temperatures determine lower heat thresholds for ensuring inter-fire seed bank persistence. Significant among-population variation of thresholds also suggests that post-fire seeder species have some potential to modify their dormancy release requirements in response to changing climate.     

Arabidopsis relatives of the human lysine-specific Demethylase1 repress the expression of FWA and FLOWERING LOCUS C and thus promote the floral transition

The timing of the developmental transition to flowering is critical to reproductive success in plants. Here, we show that Arabidopsis thaliana homologs of human Lysine-Specific Demethylase1 (LSD1; a histone H3-Lys 4 demethylase) reduce the levels of histone H3-Lys 4 methylation in chromatin of the floral repressor FLOWERING LOCUS C (FLC) and the sporophytically silenced floral repressor FWA. Two of the homologs, LSD1-LIKE1 (LDL1) and LSD1-LIKE2 (LDL2), act in partial redundancy with FLOWERING LOCUS D (FLD; an additional homolog of LSD1) to repress FLC expression. However, LDL1 and LDL2 appear to act independently of FLD in the silencing of FWA, indicating that there is target gene specialization within this histone demethylase family. Loss of function of LDL1 and LDL2 affects DNA methylation on FWA, whereas FLC repression does not appear to involve DNA methylation; thus, members of the LDL family can participate in a range of silencing mechanisms.