Mutations in tetrapyrrole biosynthesis pathway uncouple nuclear WUSCHEL expression from de novo shoot development in Arabidopsis

Plant Cell, Tissue and Organ Culture (PCTOC) - The effect of several parameters on trans-resveratrol extracellular production in Vitis vinifera cv Monastrell suspension cultured cells elicited with...     

VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis.

Most plant cells are characterized by the presence of a large central vacuole that in differentiated cells accounts for more than 90% of the total volume. We have undertaken a genetic screen to look for mutants that are affected in the formation of vacuoles in plants. In this study, we report that inactivation of the Arabidopsis gene VACUOLELESS1 (VCL1) blocks vacuole formation and alters the pattern of cell division orientation and cell elongation in the embryo. Consistent with a role in vacuole biogenesis, we show that VCL1 encodes the Arabidopsis ortholog of yeast Vps16p. In contrast to yeast mutants that lack a vacuolar compartment but are viable and morphologically normal, loss of the plant vacuole leads to aberrant morphogenesis and embryonic lethality.     

'Generic' physical mechanisms of morphogenesis and pattern formation

The role of 'generic' physical mechanisms in morphogenesis and pattern formation of tissues is considered. Generic mechanisms are defined as those physical processes that are broadly applicable to living and non-living systems, such as adhesion, surface tension and gravitational effects, viscosity, phase separation, convection and reaction-diffusion coupling. They are contrasted with 'genetic' mechanisms, a term reserved for highly evolved, machine-like, biomolecular processes. Generic mechanisms acting upon living tissues are capable of giving rise to morphogenetic rearrangements of cytoplasmic, tissue and extracellular matrix components, sometimes leading to 'microfingers', and to chemical waves or stripes. We suggest that many morphogenetic and patterning effects are the inevitable outcome of recognized physical properties of tissues, and that generic physical mechanisms that act on these properties are complementary to, and interdependent with genetic mechanisms. We also suggest that major morphological reorganizations in phylogenetic lineages may arise by the action of generic physical mechanisms on developing embryos. Subsequent evolution of genetic mechanisms could stabilize and refine developmental outcomes originally guided by generic effects.     

Whorl morphogenesis in the dasycladalean algae: the pattern formation viewpoint

The dasycladalean algae produce diverse whorled structures, among which the best known are the vegetative and reproductive whorls of Acetabularia acetabulum. In this paper, we review the literature pertaining to the origin of these structures. The question is addressed in terms of the necessary pattern-forming events and the possible mechanisms involved, an outlook we call the pattern formation viewpoint. The pattern-forming events involved in the morphogenesis of the vegetative and reproductive whorls of Acetabularia have been used to define five and six morphogenetic stages, respectively. We discuss three published mechanisms which account, at least in part, for the pattern-forming events. The mechanisms are mechanical buckling of the cell wall, reaction-diffusion of morphogen molecules along the cell membrane, and mechanochemical interactions between Ca2+ ions and the cytoskeleton in the cytosol. The numerous differences between these mechanisms provide experimental grounds to test their validity. To date, the results of these experiments point towards reaction diffusion as the most likely patterning mechanism. Finally, we consider the evolutionary origin of the vegetative and reproductive whorls and provide mechanistic explanations for some of the major evolutionary advances.     

Mutations in the pilz group genes disrupt the microtubule cytoskeleton and uncouple cell cycle progression from cell division in Arabidopsis embryo and endosperm

Organised cell division and expansion play important roles in plant embryogenesis. To address their cellular basis, we have analysed Arabidopsis abnormal-embryo mutants which were isolated for their characteristic phenotype: mutant embryos are small, mushroom-shaped ("pilz") and consist of only one or few large cells each containing one or more variably enlarged nuclei and often cell wall stubs. These 23 mutants represent four genes, PFIFFERLING, HALLIMASCH, CHAMPIGNON, and PORCINO, which map to different chromosomes. All four genes have very similar mutant phenotypes although porcino embryos often consisted of only one large cell. The endosperm did not cellularise and contained a variably reduced number of highly enlarged nuclei. By contrast, genetic evidence suggests that these genes are not required for gametophyte development. Expression of cell cycle genes, Cdc2a, CyclinA2 and CyclinB1, and the cytokinesis-specific KNOLLE gene was not altered in mutant embryos. However, KNOLLE syntaxin accumulated in patches but no KNOLLE-positive structure resembling a forming cell plate occurred in mitotic cells. A general defect in microtubule assembly was observed in all mutants. Interphase cells lacked cortical microtubules, and spindles were absent from mitotic nuclei although in rare cases, short stubs of microtubules were attached to partially condensed chromosomes. Our results suggest that the cellular components affected by the pilz group mutations are necessary for continuous microtubule organisation, mitotic division and cytokinesis but do not mediate cell cycle progression.     

Analytical treatment of pattern formation in the Gierer-Meinhardt model of morphogenesis

Summary We first perform a linear stability analysis of the Gierer-Meinhardt model to determine the critical parameters where the homogeneous distribution of activator and inhibitor concentrations becomes unstable. There are two kinds of instabilities, namely, one leading to spatial patterns and another one leading to temporal oscillations. Focussing our attention on spatial pattern formation we solve the corresponding nonlinear equations by means of our previously introduced method of generalized Ginzburg-Landau equations. We explicitly consider the two-dimensional case and find both rolls and hexagon-like structures. The impact of different boundary conditions on the resulting patterns is also discussed. The occurrence of the new patterns has all the features of nonequilibrium phase transitions.     

The epidermis-specific extracellular BODYGUARD controls cuticle development and morphogenesis in Arabidopsis

The outermost epidermal cell wall is specialized to withstand pathogens and natural stresses, and lipid-based cuticular polymers are the major barrier against incursions. The Arabidopsis thaliana mutant bodyguard (bdg), which exhibits defects characteristic of the loss of cuticle structure not attributable to a lack of typical cutin monomers, unexpectedly accumulates significantly more cell wall-bound lipids and epicuticular waxes than wild-type plants. Pleiotropic effects of the bdg mutation on growth, viability, and cell differentiation are also observed. BDG encodes a member of the alpha/beta-hydrolase fold protein superfamily and is expressed exclusively in epidermal cells. Using Strep-tag epitope-tagged BDG for mutant complementation and immunolocalization, we show that BDG is a polarly localized protein that accumulates in the outermost cell wall in the epidermis. With regard to the appearance and structure of the cuticle, the phenotype conferred by bdg is reminiscent of that of transgenic Arabidopsis plants that express an extracellular fungal cutinase, suggesting that bdg may be incapable of completing the polymerization of carboxylic esters in the cuticular layer of the cell wall or the cuticle proper. We propose that BDG codes for an extracellular synthase responsible for the formation of cuticle. The alternative hypothesis proposes that BDG controls the proliferation/differentiation status of the epidermis via an unknown mechanism.     

The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis

The plant leaf provides an ideal system to study the mechanisms of organ formation and morphogenesis. The key factors that control leaf morphogenesis include the timing, location and extent of meristematic activity during cell division and differentiation. We identified an Arabidopsis mutant in which the regulation of meristematic activities in leaves was aberrant. The recessive mutant allele blade-on-petiole1-1 (bop1-1) produced ectopic, lobed blades along the adaxial side of petioles of the cotyledon and rosette leaves. The ectopic organ, which has some of the characteristics of rosette leaf blades with formation of trichomes in a dorsoventrally dependent manner, was generated by prolonged and clustered cell division in the mutant petioles. Ectopic, lobed blades were also formed on the proximal part of cauline leaves that lacked a petiole. Thus, BOP1 regulates the meristematic activity of leaf cells in a proximodistally dependent manner. Manifestation of the phenotypes in the mutant leaves was dependent on the leaf position. Thus, BOP1 controls leaf morphogenesis through control of the ectopic meristematic activity but within the context of the leaf proximodistality, dorsoventrality and heteroblasty. BOP1 appears to regulate meristematic activity in organs other than leaves, since the mutation also causes some ectopic outgrowths on stem surfaces and at the base of floral organs. Three class I knox genes, i.e., KNAT1, KNAT2 and KNAT6, were expressed aberrantly in the leaves of the bop1-1 mutant. Furthermore, the bop1-1 mutation showed some synergistic effect in double mutants with as1-1 or as2-2 mutation that is known to be defective in the regulation of meristematic activity and class I knox gene expression in leaves. The bop1-1 mutation also showed a synergistic effect with the stm-1 mutation, a strong mutant allele of a class I knox gene, STM. We, thus, suggest that BOP1 promotes or maintains a developmentally determinate state in leaf cells through the regulation of class I knox genes.     

Colonial development in Pandorina morum. II. Colony morphogenesis and formation of the extracellular matrix

The colonial green alga, Pandorina morum, resembles its unicellular relative Chlamydomonas in both intracellular architecture and the composition of the extracellular matrix. Despite these similarities, cell division in Pandorina leads to the formation of a colony instead of the 8 or 16 single cells produced by cell division in Chlamydomonas. To study colony formation, partially synchronized cultures of P. morum were sampled periodically and stained with ruthenium red for electron microscopy. The cells of the colony were found to be held together during development by medial and basal connections between cells; the basal connections include strands of chloroplast. Studies of cells removed from the parental matrix before division confirmed that the cytoplasmic connections are strong enough to maintain the colonial configuration. After the medial connections break, the cells of the plate of the developing colony swing outward and attain the nearly spherical colonial configuration; the basal connections are still present. After this inversion, the formation of the extracellular matrix begins, with the colonial boundary appearing first. Capsule and sheath then form on the outer and inner faces of the colonial boundary until the extracellular matrix is complete. The process is compared to previous observations of Volvox, and possible evolutionary implications are discussed.     

Trichome morphogenesis in Arabidopsis

Trichomes (plant hairs) in Arabidopsis thaliana are large non-secreting epidermal cells with a characteristic three-dimensional architecture. Because trichomes are easily accessible to a combination of genetic, cell biological and molecular methods they have become an ideal model system to study various aspects of plant cell morphogenesis. In this review we will summarize recent progress in the understanding of trichome morphogenesis.     

Cell morphogenesis in Arabidopsis

Cell morphogenesis encompasses all processes required to establish a three-dimensional cell shape. Cells acquire the architecture specific to their developmental context by using the spatial information provided by internal or external cues. As a response to these signals, cells become reorganized and establish functionally distinct subcellular domains that ultimately lead to morphological changes. In its simplest form, cell morphogenesis results in the establishment of asymmetry along one axis, a cell polarity. Although cell polarity has been studied intensively in budding yeast and epithelial cells, little is known about more complex modes of cell morphogenesis involving multiple axes. In this review we compare the regulation of cell morphogenesis of different genetically well-characterized cell types in Arabidopsis thaliana. BioEssays 20:20–29, 1998. © 1998 John Wiley & Sons, Inc.     

Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development.

Phytochromes are a family of plant photoreceptors that mediate physiological and developmental responses to changes in red and far-red light conditions. In Arabidopsis, there are genes for at least five phytochrome proteins. These photoreceptors control such responses as germination, stem elongation, flowering, gene expression, and chloroplast and leaf development. However, it is not known which red light responses are controlled by which phytochrome species, or whether the different phytochromes have overlapping functions. We report here that previously described hy3 mutants have mutations in the gene coding for phytochrome B (PhyB). These are the first mutations shown to lie in a plant photoreceptor gene. A number of tissues are abnormally elongated in the hy3(phyB) mutants, including hypocotyls, stems, petioles, and root hairs. In addition, the mutants flower earlier than the wild type, and they accumulate less chlorophyll. PhyB thus controls Arabidopsis development at numerous stages and in multiple tissues.     

The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development.

The vascular tissues of flowering plants form networks of interconnected cells throughout the plant body. The molecular mechanisms directing the routes of vascular strands and ensuring tissue continuity within the vascular system are not known, but are likely to depend on general cues directing plant cell orientation along the apical-basal axis. Mutations in the Arabidopsis gene MONOPTEROS (MP) interfere with the formation of vascular strands at all stages and also with the initiation of the body axis in the early embryo. Here we report the isolation of the MP gene by positional cloning. The predicted protein product contains functional nuclear localization sequences and a DNA binding domain highly similar to a domain shown to bind to control elements of auxin inducible promoters. During embryogenesis, as well as organ development, MP is initially expressed in broad domains that become gradually confined towards the vascular tissues. These observations suggest that the MP gene has an early function in the establishment of vascular and body patterns in embryonic and post-embryonic development.     

Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity

The deletion of residues 239-272 from the hamster beta-adrenergic receptor resulted in a loss of the ability of the receptor, expressed in mouse L cells, to stimulate adenylate cyclase (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). This mutant receptor (D(239-272)beta AR) bound the agonist isoproterenol with a single class of binding sites, in contrast to the wild-type beta-adrenergic receptor, which exhibited two classes of agonist affinity sites. We now report that the affinity of D(239-272)beta AR for isoproterenol is relatively insensitive to detergent solubilization or to treatment with either GTP or NaF, indicating the absence of a receptor-Gs interaction. Whereas deletions within the region of amino acids 229-258 did not reduce the ability of the receptor to couple to Gs or to stimulate adenylate cyclase, the deletion of either of the regions 222-229 or 258-270 resulted in receptors which were unable to couple to Gs. The affinities of D(222-229)beta AR, D(239-272)beta AR, and D(258-270)beta AR toward isoproterenol were greater than that observed for the low affinity, uncoupled form of the wild-type receptor. These results suggest a role for the regions of the beta-adrenergic receptor encompassing amino acids 222-229 and 258-270, which are predicted to form amphiphilic helices, in the agonist-promoted activation of Gs.     

Mutations in the F-box gene SNEEZY result in decreased Arabidopsis GA signaling

We previously reported that the SLEEPY1 (SLY1) homolog, F-box gene SNEEZY/SLEEPY2 (SNE/SLY2), can partly replace SLY1 in gibberellin (GA) hormone signaling through interaction with DELLAs RGA and GAI. To determine whether SNE normally functions in GA signaling, we characterized the phenotypes of two T-DNA alleles, sne-t2 and sne-t3. These mutations result in no apparent vegetative phenotypes, but do result in increased ABA sensitivity in seed germination. Double mutants sly1-t2 sne-t2 and sly1-t2 sne-t3 result in a significant decrease in plant fertility and final plant height compared to sly1-t2. The fact that sne mutations have an additive effect with sly1 suggests that SNE normally functions as a redundant positive regulator of GA signaling.Key words: gibberellin signaling, GA, SLEEPY1, SNEEZY, DELLA, F-box proteinThis paper describes genetic evidence that the SLEEPY1 (SLY1) homolog SNEEZY/SLEEPY2 (SNE/SLY2) functions redundantly with SLY1 to stimulate gibberellin signaling. GA responses such as seed germination, stem elongation and fertility are promoted by proteolysis of DELLA proteins, negative regulators of the GA signaling.¹ In the classic GA signaling model, GA binding to the GA receptor GID1 increases GID1 affinity for DELLA protein. This GID1-GA binding to DELLA causes SLY1, the F-box subunit of an SCF E3 ubiquitin ligase complex, to recognize, bind and ubiquitinate DELLA proteins thereby targeting them for destruction by the 26S proteasome. Thus, loss of SLY1 function results in decreased GA responses, causing dwarfism, delayed flowering, infertility and seed dormancy. The sly1 mutants over-accumulate DELLA proteins due to failure to destroy them through the ubiquitin-proteasome pathway.Overexpression of the SLY1 homolog, SNE, partially rescues the germination, dwarfism and infertility of the sly1-10 mutant.^2–4 SNE overexpression in the sly1-10 background is associated with reduced accumulation of DELLA proteins RGA and GAI, but not of DELLA RGL2. Co-immunoprecipitation assays demonstrated that SNE directly binds RGA protein as well as the cullin subunit of the SCF E3 complex. These recently published data suggest that SNE forms a functional SCF E3 ubiquitin ligase complex that negatively regulates a subset of the DELLA proteins regulated by SLY1.⁴The finding that SNE overexpression rescues sly1-10 phenotypes through down-regulation of DELLA RGA and GAI suggests that SNE is normally a positive regulator of GA signaling. If this is true, then we expect sne mutations to cause phenotypes resulting from reduced GA response including reduced germination, stature and fertility. To examine this hypothesis, three sne T-DNA mutants were identified: sne-t1, sne-t2 and sne-t3. The sne-t1 allele is a SALK line containing a T-DNA insertion 183-bp upstream of the coding region.⁵ This line showed no apparent phenotype and was not further characterized. The sne-t2 allele is a Sussman T-DNA line^4,6 containing a T-DNA insertion immediately before the ATG that is the SNE translational start codon (Fig. 1A). While this insertion does not disrupt the coding region, it likely disrupts SNE protein translation as the T-DNA contains multiple stop codons. The sne-t3 allele contains a T-DNA insertion within the SNE ORF before amino acid 146 of the 157 amino acid predicted protein (FLAG_461E03).^7,8 This allele should result in loss of the last 11 SNE amino acids. We know that loss of the last 8 SLY1 amino acids in sly1-10 results in dwarfism, suggesting that loss of the last 11 SNE amino acids may also cause some loss of function in the small F-box protein. When the homozygous sne-t2 and sne-t3 lines were compared to wild-type Ws, no change was observed either in final plant height or fertility measured in seeds/silique (Fig. 1B). An ABA dose-response curve in seed germination detected a small but reproducible increase in ABA sensitivity during seed germination of sne-t2 and sne-t3 (Fig. 1C). The fact that the sne-t2 and sne-t3 mutants, like sly1-2 and sly1-10, show increased ABA sensitivity suggests that SNE and SLY1 may have similar functions in GA signaling during seed germination.²Open in a separate windowFigure 1The phenotypes of sne-t2 and sne-t3 T-DNA mutants. (A) Schematic diagram of the sne-t2 T-DNA insertion at position −1 bp and of sne-t3 at position +435 bp with respect to the translation start site. (B) Final plant height (upper) and fertility (lower) of indicated genotypes. Letters indicate statistically different classes as determine by t-test. Bars represent standard error. (C) sne mutants show increase in ABA sensitivity. Seeds of wild-type Ws, sne-t2 and sne-t3 were after-ripened for 2 weeks then sown on MS-agar containing indicated concentrations of ABA as described by Steber et al.¹¹ Germination was scored based on radical emergence after incubating 3 days at 4°C followed by 14 days at 22°C. (D) Mutations in SNE cause no significant effect on DELLA RGA, GAI and RGL2 protein accumulation. Total protein was extracted from leaves of 12-d-old seedlings (Top) or flower buds (FB, bottom) and detected as described in Ariizumi et al.⁴One possible explanation for the lack of apparent GA-insensitive phenotypes in sne T-DNA insertion lines, is that SNE function is redundant with SLY1 in GA signaling.⁹ If so, we would expect sly1 sne double mutants to show stronger GA-insensitive phenotypes than the sly1 single mutation. Double mutants were constructed containing either the sne-t2 or sne-t3 mutation in the sly-t2 null background. The sly1-t2 allele was chosen because sly1-t2, sne-t2 and sne-t3 are all in the Ws ecotype. The sly1-t2 allele contains a T-DNA insertion within the F-box domain resulting in severe GA-insensitive phenotypes including failure to germinate, reduced stature and infertility.¹⁰ The sly1-t2 sne-t2 and sly1-t2 sne-t3 double mutants showed a small but significant decrease in final plant height and fertility (seeds/silique) compared to sly1-t2 (Fig. 1B). This increase in phenotype severity was not associated with an apparent increase in DELLA RGA, GAI or RGL2 protein accumulation (Fig. 1D). It could be that DELLA protein levels in sly1-t2 are so high that any slight increase due to sne mutations is undetectable. Our previous study of SNE overexpression lines showed that SNE has the ability to downregulate RGA and GAI protein accumulation. Figure 1 shows that the chromosomal SNE gene contributes to GA signaling presumably through ubiquitination of DELLA protein.Taken together, the fact that sne mutants show only mild GA-insensitive phenotypes and that the natural SNE expression cannot compensate for lack of SLY1, indicate that SLY1 is the main E3 ubiquitin ligase stimulating GA signaling (this study, reviewed in ref. ⁴). We cannot rule out the possibility that stronger SNE alleles would show either stronger GA response phenotypes or phenotypes that are unrelated to GA signaling. Indeed, there is evidence to suggest that SNE may have unique functions. The sne-t3 (sne-1) allele results in a shortened root phenotype.⁸ That SNE is expressed in the endodermis and quiescent center of the root whereas SLY1 is expressed in the stele, suggests that SNE may function independently in the root.⁸ Moreover, SNE overexpression, but not SLY1 overexpression, results in decreased apical dominance and a prone growth habit suggesting that SNE may play a unique role in development.^2,4 Our model is that in addition to regulating DELLA proteins RGA and GAI, SNE may also regulate a yet unidentified target involved in apical dominance (Fig. 2). Future research will need to elucidate the role of SNE in Arabidopsis growth and development.Open in a separate windowFigure 2Model for SNE function in Arabidopsis. Both SLY1 and SNE act as positive regulators of GA responses via DELLA protein destruction. SNE may negatively regulate an unknown protein that maintains apical dominance.