Elena R. Alvarez-Buylla

A Gene Regulatory Network Model for Cell-Fate Determination during Arabidopsis thaliana Flower Development That Is Robust and Recovers Experimental Gene Expression Profiles

Flowers are icons in developmental studies of complex structures. The vast majority of 250,000 angiosperm plant species have flowers with a conserved organ plan bearing sepals, petals, stamens, and carpels in the center. The combinatorial model for the activity of the so-called ABC homeotic floral genes has guided extensive experimental studies in Arabidopsis thaliana and many other plant species. However, a mechanistic and dynamical explanation for the ABC model and prevalence among flowering plants is lacking. Here, we put forward a simple discrete model that postulates logical rules that formally summarize published ABC and non-ABC gene interaction data for Arabidopsis floral organ cell fate determination and integrates this data into a dynamic network model. This model shows that all possible initial conditions converge to few steady gene activity states that match gene expression profiles observed experimentally in primordial floral organ cells of wild-type and mutant plants. Therefore, the network proposed here provides a dynamical explanation for the ABC model and shows that precise signaling pathways are not required to restrain cell types to those found in Arabidopsis, but these are rather determined by the overall gene network dynamics. Furthermore, we performed robustness analyses that clearly show that the cell types recovered depend on the network architecture rather than on specific values of the models gene interaction parameters. These results support the hypothesis that such a network constitutes a developmental module, and hence provide a possible explanation for the overall conservation of the ABC model and overall floral plan among angiosperms. In addition, we have been able to predict the effects of differences in network architecture between Arabidopsis and Petunia hybrida.

Conversion of leaves into petals in Arabidopsis.

More than 200 years ago, Goethe proposed that each of the distinct flower organs represents a modified leaf [1]. Support for this hypothesis has come from genetic studies, which have identified genes required for flower organ identity. These genes have been incorporated into the widely accepted ABC model of flower organ identity, a model that appears generally applicable to distantly related eudicots as well as monocot plants. Strikingly, triple mutants lacking the ABC activities produce leaves in place of flower organs, and this finding demonstrates that these genes are required for floral organ identity [2]. However, the ABC genes are not sufficient for floral organ identity since ectopic expression of these genes failed to convert vegetative leaves into flower organs. This finding suggests that one or more additional factors are required [3, 4]. We have recently shown that SEPALLATA (SEP) represents a new class of floral organ identity genes since the loss of SEP activity results in all flower organs developing as sepals [5]. Here we show that the combined action of the SEP genes, together with the A and B genes, is sufficient to convert leaves into petals.

Demography and allometry of Cecropia obtusifolia, a neotropical pioneer tree - an evaluation of the climax-pioneer paradigm for tropical rain forests

Summary 1. Demography and allometry of the dioecious tree Cecropia obtusifolia were studied in a 5-ha permanent plot at Los Tuxtlas rain forest, Mexico. Treefall gaps were aged and a significant positive correlation was found between gap age and tree size. Active regeneration of the species was suggested by the high proportion (>35%) of young individuals. The population was highly clumped among young juvenile trees and became less aggregated as density within-gaps decreased among older trees. New and successful recruitment was restricted to recently formed ( 100-M2) gaps. Oldest individuals (35 m high) were growing in 37-year-old gaps. 2. Tree height and stem diameter increased allometrically with a slope 0-3 m tall. The margin between the actual tree diameter (d) and the theoretical minimum buckling diameter (dmin.) declined exponentially from c. 20 in juvenile trees to almost 1 in the oldest trees (>30 years old). Whilst the number of leaves remains fairly constant before branching, it increases linearly as the number of branches increase. Changes in area per leaf during the life cycle yield an overall allometric pattern between tree height and total leaf area. 3. Seed and seedling mortality was >99%. Reduced light, falling debris, movement of rotting logs and soil dryness were important mortality factors in pre-reproductive stages. Oldest adults died standing, apparently due to senescence. Maximum life expectancy is reached when trees attain a non-suppressed canopy position (c. 20 m high). Individuals showed high growth rates (a stem-diameter increment of up to 2 cm year-1). Growth-rate variation in juvenile stages seem to be related to high environmental heterogeneity. In adult stages the number of conspecifics growing in the same gap seems to affect diameter growth rate negatively. First reproduction occured at about 3 years of age (when 10 cm in diameter) and increased with age. Annual fecundity rates of individuals ranged from 1-4 x 104 to 1-4 x 107 seeds and increased allometrically with stem diameter and number of branches. 4. The most important environmental filter in the life-cycle of C. obtusifolia operates from the seedling to the juvenile stage. This finding does not support the suggestion that pioneer limitation to gap regeneration is determined during germination. The allometric and demographic traits of C. obtusifolia correspond to those expected for a shade-intolerant species that colonizes ephemeral sites, and they contrast with those of purported shade-tolerant tree species reported in the literature. However, rather than finding two clearly distinguishable types of lifehistories, a continuum of demographic life-cycles was found. Therefore, the dichotomous pioneer-climax framework should be viewed as a means of identifying the extremes of a continuum of tree life histories.

Seed bank versus seed rain in the regeneration of a tropical pioneer tree

SummaryWe used the tropical pioneer tree, Cecropia obtusifolia to evaluate the relative importance of different sources of seeds in the regeneration of species that depend on ephemeral sites. We studied seed production in a population established in a 5 ha plot, and dispersal, dormancy and seed predation in two recent treefall gaps (<1 year-old), two building or successional forest patches (10–15 since disturbed), and two mature forest patches (>35 years since disturbed) for a one year period at Los Tuxtlas (Mexico). Flowers and fruits were counted at monthly intervals. Annual fecundity per tree ranged from 1.4×104 to 1.4×107 seeds. Seeds were continuously available on the trees and on the ground. Average annual seed rain per m2 (as measured by 0.5×0.5 m seed traps) varied from 184 to 1925 among the six sites. Distance to nearest seed source and patch type explained more than 60% of the seed rain variation among sites. Soil seed density, estimated by counting seeds from ten samples (78.5 cm2×10 cm deep) collected from each site in October and January, ranged among the six sites from 269 to 4485 seeds per m2 in January and from 204 to 5073 in October. Soil seed viabilities were much lower (17.1% in October and 5.1% in January) than those of rain seeds (48.26%). Annual survivorships of 2.2% were estimated for seeds artificially sown on the soil surface of a gap and a mature patch, and 3.75% in a building patch. In two other experiments seed removal rates ranged from 27% to 98% in 4 days. Removal rates were significantly higher in gap and mature patches than in building patches. Ants (Paratrechina vividula) and grasshopper nymphs (Hygronemobius. sp.) were the main predators. We draw three main conclusions from our data: (1) Pathogens and predators determine low survivorship of C. obtusifolias seeds in the soil and a rapid turnover rate (1.07 to 1.02 years) of its seed bank; (2) a continuous and copious seed production and an abundant and extensive seed rain replenish the soil seed pool in patches with different disturbance ages at least up to 86 m from nearest source; (3) more than 90% of the seeds contributing to C. obtusifolia seedling recruitment in gaps are less than one year-old. We discuss our results in the context of previous similar studies for tropical forests.

Critical Dynamics in Genetic Regulatory Networks: Examples from Four Kingdoms

The coordinated expression of the different genes in an organism is essential to sustain functionality under the random external perturbations to which the organism might be subjected. To cope with such external variability, the global dynamics of the genetic network must possess two central properties. (a) It must be robust enough as to guarantee stability under a broad range of external conditions, and (b) it must be flexible enough to recognize and integrate specific external signals that may help the organism to change and adapt to different environments. This compromise between robustness and adaptability has been observed in dynamical systems operating at the brink of a phase transition between order and chaos. Such systems are termed critical. Thus, criticality, a precise, measurable, and well characterized property of dynamical systems, makes it possible for robustness and adaptability to coexist in living organisms. In this work we investigate the dynamical properties of the gene transcription networks reported for S. cerevisiae, E. coli, and B. subtilis, as well as the network of segment polarity genes of D. melanogaster, and the network of flower development of A. thaliana. We use hundreds of microarray experiments to infer the nature of the regulatory interactions among genes, and implement these data into the Boolean models of the genetic networks. Our results show that, to the best of the current experimental data available, the five networks under study indeed operate close to criticality. The generality of this result suggests that criticality at the genetic level might constitute a fundamental evolutionary mechanism that generates the great diversity of dynamically robust living forms that we observe around us.

Treefall age determination and gap dynamics in a tropical forest

(1) Most individuals of Astrocaryum mexicanum, a monopodial neotropical understorey palm, endure treefalls that form gaps in the forest, by bending under falling trees and limbs. After one year, a bent palm recovers vertical growth at its terminal meristem and forms a permanent kink in its stem. Previous detailed demographic studies have allowed us to determine with accuracy the passage of time, based on the age-constant rate of stem elongation of the palm. (2) Using this morphological feature of the palm, together with its high density (3001230 mature individuals ha-1) in a 5-ha tract of rainforest at Los Tuxtlas, Veracruz, Mx more than 50% of the quadrats suffered disturbance in the last thirty years and 28% suffered more than one disturbance in the last seventy years. These results provide evidence that canopy disturbances capable of promoting the release of suppressed seedlings and saplings of forest trees may occur at the small scale of some tens of square metres. (4) The long-term treefall patterns analysed (up to seventy years) show that canopy disturbances are a permanent ecological factor in the rainforest environment. Annual rainfall explains more than 50% of the annual variation of the proportion of forest opened to gaps per year. (5) The yearly disturbance does not indicate the gap availability for species regeneration: for example, a year of high disturbance (e.g. 6 1 % of the forest opened to gaps) may have a similar number of sizeable gaps suitable for pioneer regeneration as a year of low disturbance (e.g. 1 5% of the forest opened to gaps). (6) The results obtained in this study show that the gap-formation process operating at Los Tuxtlas forest promotes a strong temporally and spatially random variation in the physical environment of plants. This heterogeneity may be one of the factors involved in determining the high biological diversity found in most tropical rainforests.

Adaptive evolution in the Arabidopsis MADS-box gene family inferred from its complete resolved phylogeny

Gene duplication is a substrate of evolution. However, the relative importance of positive selection versus relaxation of constraints in the functional divergence of gene copies is still under debate. Plant MADS-box genes encode transcriptional regulators key in various aspects of development and have undergone extensive duplications to form a large family. We recovered 104 MADS sequences from the Arabidopsis genome. Bayesian phylogenetic trees recover type II lineage as a monophyletic group and resolve a branching sequence of monophyletic groups within this lineage. The type I lineage is comprised of several divergent groups. However, contrasting gene structure and patterns of chromosomal distribution between type I and II sequences suggest that they had different evolutionary histories and support the placement of the root of the gene family between these two groups. Site-specific and site-branch analyses of positive Darwinian selection (PDS) suggest that different selection regimes could have affected the evolution of these lineages. We found evidence for PDS along the branch leading to flowering time genes that have a direct impact on plant fitness. Sites with high probabilities of having been under PDS were found in the MADS and K domains, suggesting that these played important roles in the acquisition of novel functions during MADS-box diversification. Detected sites are targets for further experimental analyses. We argue that adaptive changes in MADS-domain protein sequences have been important for their functional divergence, suggesting that changes within coding regions of transcriptional regulators have influenced phenotypic evolution of plants.

Length Variation in the Nuclear Ribosomal DNA Internal Transcribed Spacer Region of Non-flowering Seed Plants

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was PCR- amplified in 32 genera of non-flowering seed plants. Length of the ITS region was determined by restriction site mapping of PCR products and nucleotide sequences were obtained from the ITS-2 and 5.8S rDNA of selected genera. In contrast to the relatively narrow range of ITS region lengths reported from angiosperms (565-700 base pairs (bp)), substantial length variation (975-3125 bp) is observed in the ITS region of Coniferales, Cycadales, Ginkgoales, and Gnetales. Restriction site analyses indicate that the 5.8S rDNA + ITS-2 ranges from 375-450 bp, while the ITS-1 is responsible for most of the length variation found in gymnosperm ITS regions. The representatives of Pinaceae exhibit the greatest variation in ITS region length (1550-3125 bp), while those of sampled members of Cupressaceae, Taxodiaceae, Cephalotaxaceae, and Taxaceae are relatively stable (975-1125 bp). The observed ITS region lengths in Sciadopityaceae (1250 bp) and Araucariaceae (1325-1350 bp) are somewhat larger than those of Cupressaceae, Taxodiaceae, Cephalotaxa- ceae, and Taxaceae, while those of sampled Podocarpaceae (2000-2100 bp) fall into the range observed in the Pinaceae. Outside of the Coniferales, ITS region length is similar among Cycadales (1150- 1450 bp), Ginkgoales (1200 bp), and two of the three members of Gnetales, Ephedra (1500 bp) and Gnetum (1200 bp). In contrast, the ITS region of Welwitschia is only 750 bp long, ca. 50 bp longer than the longest known angiosperm ITS region. Levels of nucleotide sequence variation as estimated by restriction site mapping suggest that phylogenetic analysis of the ITS region will be informative at the intrageneric level in most non-flowering seed plants. In Cupressaceae, intergeneric comparisons may also be feasible. Because the ITS region is relatively long, it may also permit population-level phylogenetic analysis in many non-flowering seed plants.

Population genetic structure of Cecropia obtusifolia, a tropical pioneer tree species

Theoretical analyses of the genetic organization of pioneer species have postulated two very different scenarios. Some models have predicted that such species would show strong population substructuring, whereas other models have suggested that extinction and recolonization can augment gene flow and reduce interpopulation differentiation. We tested these alternative scenarios by analyzing the genetic structure of eight loci from populations of the pioneer dioecious tree, Cecropia obtusifolia, in the tropical rain forest region of Los Tuxtlas, México. The populations studied exhibit low overall FST values, no clear pattern of isolation by distance, and high estimates of gene flow. These results suggest either that the species is not at a genetic equilibrium under present levels of gene flow with populations derived from each other in the recent past, or that pollen and seed dispersal in this species occur over long distances (up to more than 100 km). Mating among relatives appears higher than expected by chance based on significantly positive fixation indices (F) and FIS values at some loci. However, no direct evidence for biparental inbreeding was found. The multilocus and single‐locus outcrossing rates for C. obtusifolia were estimated at tm = 0.974 (SE = 0.024) and ts = 0.980 (SE = 0.035), respectively. These are not significantly different from 1, and the difference, tm — ts = — 0.006 (SE = 0.018), is not significantly different from 0. These estimates, however, could be biased because in all enzymes, except PGM‐1, we found statistically significant departures from the mixed‐mating model used to estimate them. Two rare alleles were found only in seeds collected from the soil, and the greatest number of different alleles were found also in soil seeds. It is hypothesized that the seed bank may play an important role in the genetic buffering of C. obtusifolia. Significantly positive or negative fixation indices in adults at some loci and significantly different heterozygosities among different life stages (from seeds to adults) suggest the action of selection at some loci.

An AGAMOUS -Related MADS-Box Gene, XAL1 ( AGL12 ), Regulates Root Meristem Cell Proliferation and Flowering Transition in Arabidopsis

MADS-box genes are key components of the networks that control the transition to flowering and flower development, but their role in vegetative development is poorly understood. This article shows that the sister gene of the AGAMOUS (AG) clade, AGL12, has an important role in root development as well as in flowering transition. We isolated three mutant alleles for AGL12, which is renamed here as XAANTAL1 (XAL1): Two alleles, xal1-1 and xal1-2, are in Columbia ecotype and xal1-3 is in Landsberg erecta ecotype. All alleles have a short-root phenotype with a smaller meristem, lower rate of cell production, and abnormal root apical meristem organization. Interestingly, we also encountered a significantly longer cell cycle in the strongest xal1 alleles with respect to wild-type plants. Expression analyses confirmed the presence of XAL1 transcripts in roots, particularly in the phloem. Moreover, XAL1∷β-glucuronidase expression was specifically up-regulated by auxins in this tissue. In addition, mRNA in situ hybridization showed that XAL1 transcripts were also found in leaves and floral meristems of wild-type plants. This expression correlates with the late-flowering phenotypes of the xal1 mutants grown under long days. Transcript expression analysis suggests that XAL1 is an upstream regulator of SOC, FLOWERING LOCUS T, and LFY. We propose that XAL1 may have similar roles in both root and aerial meristems that could explain the xal1 late-flowering phenotype.