Michael L. Christianson

A Dwarf Mutant of Arabidopsis Generated by T-DNA Insertion Mutagenesis.

Most plant genes that control complex traits of tissues, organs, and whole plants are uncharacterized. Plant height, structure of reproductive organs, seed development and germination, for example, are traits of great agronomic importance. However, in the absence of knowledge of the gene products, current molecular approaches to isolate these important genes are limited. Infection of germinatng seeds of Arabidopsis thaliana with Agrobacterium results in transformed lines in which the integrated T-DNA from Agrobacterium and its associated kanamycin-resistance trait cosegregate with stable, phenotypic alterations. A survey of 136 transformed lines produced plants segregating in a manner consistent with Mendelian predictions for phenotypes altered in height, flower structure, trichomes, gametogenesis, embryogenesis, and seedling development. This report is the characterization of a dwarf mutant in which the phenotype is inherited as a single recessive nuclear mutation that cosegregates with both the kanamycin-resistance trait and the T-DNA insert.

ABA prevents the second cytokinin-mediated event during the induction of shoot buds in the moss Funaria hygrometrica.

The induction of shoot buds in the moss Funaria hygrometrica is a classic and quantitative bioassay for cytokinin. This cytokinin-stimulated response can be inhibited by the plant hormone abscisic acid, ABA; the inhibition is concentration dependent and was proposed for use as a bioassay for ABA. This paper characterizes the ABA inhibition of the cytokinin-stimulated formation of shoot buds. Experiments transferring protonema between cytokinin and cytokinin plus ABA show that ABA does not interfere with the initial perception of cytokinin. Other experiments compare the results of transferring protonema from cytokinin to cytokinin-free medium or to medium with cytokinin plus ABA and reveal that ABA acts by blocking the cytokinin-mediated stable commitment of nascent buds. Extension of the technique of double-reciprocal plots to this whole-organism bioassay finds that ABA is not a competitive inhibitor of cytokinin. Analysis of the ABA inhibition of bud formation identifies a new regulatory step in the developmental process of bud formation in mosses.

Phenylurea cytokinins assayed for induction of shoot buds in the moss Funaria hygrometrica

The induction of shoot buds from the filamentous protonema of moss is a classic bioassay for cytokinin. While a large literature documents this response in many species of moss and for a wide range of natural and synthetic cytokinins, to date only substituted adenine cytokinins have been examined in detail. This paper shows that at least some of the novel phenylurea cytokinins will induce bud formation in mosses. Funaria responds to thidiazuron much as it responds to benzyladenine. Exposure to either substance results in log-linear dose-dependent increases in bud number that reach similar maximal numbers of buds at the optimal concentration of compound. The related compound chloro-pyridyl-phenylurea (CPPU) is slightly less active, but induces buds over a wider range of concentration. Carbanilide (diphenylurea or DPU), an active cytokinin in other systems, induces very few buds in Funaria, but does so over a wide range of concentration. Bioassay of mixtures of benzyladenine and DPU finds no evidence of competition for cytokinin receptors. That result could support suggestions that the phenylurea cytokinins act indirectly, by altering endogenous cytokinin metabolism, but we favor another interpretation. Unlike other cytokinin-responsive systems, the induction of buds from moss protonema involves two cytokinin-mediated events. The number of buds is controlled by the second cytokinin-mediated event. If DPU has little or no affinity for the receptor triggering this second event, DPU treatments will produce few to no buds, and kinetic analysis using bud number would find no evidence for competition with benzyladenine. Our results would support the hypothesis that bud induction in Funaria involves two chemically distinct cytokinin receptors.

A SIMPLE PROTOCOL FOR CRYOPRESERVATION OF MOSSES

Maintaining numbers of aseptic cultures of bryophytes by serial transfer not only consumes supplies and labor but can result in loss of vigor or the appearance of other abnormalities in long-term cultures. Cryopreservation of cultures solves the problems associated with long-term culture, and is of particular importance for cultures or mutant cell lines incapable of making spores or where spores are short-lived. Dramatic simplification of a published protocol for moss cryopreservation is accomplished by preconditioning cultures for 3-4 days in medium supplemented with 10-5 M ABA and 100 mM proline. This proline/ABA protocol can be successfully used with Ceratodon purpureus, Funaria hygrometrica, Physcomitrella patens, and two species of Sphagnum; cryopreserved cultures remain viable for a minimum of one year at -800C. Experiments demonstrate a differential response by cell types to cryopreservation, as well as differences between species or for particular selected cell lines within a species. Other experiments suggest that successful cryopreservation is not a simple consequence of brood cells in ABA-treated

Codon usage patterns distort phylogenies from or of DNA sequences

Papers reporting phylogenetic reconstructions often include discussion of the nature of third position substitutions and have often treated third position data differently from other data. This paper extends such considerations. Plant biotechnologists interested in high levels of expression of foreign proteins have accumulated information on preferences for otherwise synonymous codons. This paper presents a simple analysis for codon bias. Not only is bias frequent, but bias also varies between cohorts of proteins, both by amino acid and by taxon. Analysis of codon usage in the parallel divergence of phytochromes in three model plants finds identical bias for all family members within each taxon and increasingly divergent patterns of bias between increasingly divergent taxa. The molecular constraint of taxon-specific pools of tRNA molecules means individual triplets in a coding sequence are often not independent; algorithms designed to analyze independent characters are inappropriate for such data. Although a misestimate of the number of differences between taxa and groups of taxa can still generate an accurate description of the nesting of clades, other phylogenetic parameters will be strongly affected. Importantly, since codon bias produces smaller-than-expected within-taxon variance (common use of favored triplets) and larger-than-expected between-taxa variance (different favorites in different taxa), statistical support for nodes is certain to be wrong. The translational control of gene expression mediated by codon bias has implications for modern molecular systematics.

Dose-Dependent Effect of Salicylates in a Moss, Funaria hygrometrica

AbstractPlant growth regulators now include more than the classic examples auxin, cytokinin, ethylene, and gibberellin, but little is known about the activity of these additional classes of molecules in nonvascular plants. The formation of buds by protonema of the moss Funaria hygrometrica is perhaps the best known and most fully characterized developmental system in the nonvascular plants. Examination of the effects of exogenously supplied salicylic acid and acetylsalicylate on this bioassay system shows that salicylates can regulate growth and development in mosses, producing a dose-dependent inhibition of bud formation. Other experiments show that this action is distinct from any direct effect on the well-known cytokinin stimulation of bud formation, occurs late in the process of bud formation, occurs prior to the stable commitment of nascent buds, and is not an effect on the outgrowth of young shoots. Because mosses are the sister clade to the vascular plants, these results suggest that the ability to perceive and transduce salicylate signals is an ancient feature of land plant physiology.

Carbon isotope discrimination correlates with a range of ratios of phosphoenolpyruvate to total carboxylase activities found in two C3species

Summary The most widely used model that relates leaf carbon isotopic ratios to {ie489-1} ratios in C 3 plants suggests that PEPcase contributes approximately 6% of the carbon pool in these species. Variability in this contribution during leaf development is not only possible, but has been shown in studies where PEP-case activity is highest during the heterotrophic (carbon gained from import) stages of leaf development and declines as the leaf attains greater amounts of autotrophy (carbon gained from photosynthesis). We tested the hypothesis that variation in PEPcase: total carboxylase activities associated with carbon sources for leaf growth could affect carbon isotopic discrimination values on mixotrophic (carbon gained from import and photosynthesis) and autotrophic Nicotiana tabacum and Salix interior plants. A range of mean ratios of PEPcase: total carboxylase activities was achieved in these two C 3 species that correlated negatively with mean leaf carbon isotopic discrimination. These results indicate that the proportion of carbon fixed by PEPcase to total carboxylation could vary between different leaf developmental stages within and among different species and that this contribution can exceed 6%.

The quantitative response to cytokinin in the moss Funaria hygrometrica does not reflect differential sensitivity of initial target cells.

Exposure to sufficient cytokinin induces the formation of buds from responsive cells in the protonema of Funaria hygrometrica. Initial perception of the phytohormone results in a Ca+2 cascade within minutes. A second cytokinin-mediated event occurs some days later, and converts incipient buds into stably committed buds. The concentration of exogenous cytokinin also regulates the total number of buds produced from a protonemal colony. This concentration-dependent production of buds has been thought to reflect differential sensitivity of target cells. Under that hypothesis, the regulation of bud number occurs during initial perception of hormone. This paper presents direct experimental evidence to the contrary and supports the alternate hypothesis that bud formation involves the gating of large numbers of responding cells by later events. Experiments transferring protonema between media with different levels of cytokinin show that the cytokinin concentration during the initial perception of cytokinin is unimportant in controlling bud number. Instead, bud number is found to be regulated by the concentration of exogenous cytokinin as incipient buds or bud initials become stably committed buds.

Reproductive short-shoots of Ginkgo biloba: A quantitative analysis of the disposition of axillary structures

Ginkgo biloba, the only living representative in an otherwise extinct clade, is of pivotal importance to understanding seed plant phylogeny. Although G. biloba and its fossil relatives have been studied for over two centuries, there are both gaps and contradictions in the information available. We present data documenting the distributions of strobili and consider what an understanding of the disposition of strobili along short-shoots in Ginkgo adds to knowledge of the evolution of reproductive structures in seed plants in general. The megasporangiate strobili are found at and around the boundary between bracts and foliage leaves, while the expanse of microsporangiate strobili centers on the fifth bract back from that boundary. Quantitative analysis of the locations of the strobili along the short-shoot finds that increases in numbers of strobili are the result of recruitment of adjacent axils into morphogenetic activity. Gaps in the series of strobili are exceedingly rare. Further, while increased numbers of megasporangiate strobili arise from the symmetrical addition of axils into the fertile zone, increased numbers of microsporangiate strobili arise from a distinctly asymmetrical, basipetally biased, addition of axillary positions. This accurate morphological framework should orient molecular genetic studies that probe gymnosperm development itself or that consider gymnosperms as the proximate sources of gene expression redeployed in the origin of the angiosperm flower.

Differences in the scaling of area and mass of Ginkgo biloba (Ginkgoaceae) leaves and their relevance to the study of specific leaf area.

PREMISE OF THE STUDY The manner in which the area of the leaf lamina (A) scales with respect to the dry mass of the lamina (M) is an important functional trait that is correlated with whole-plant growth rates and habitat preferences across diverse species. However, the extent to which the scaling between these two variables differs among leaves collected from different types of shoots within the canopy of a tree is poorly understood. Should they exist, significant differences in the A vs. M scaling relationship within canopies would raise a number of important questions, in particular what constitutes an adequate sampling procedure to determine the whole-canopy A vs. M relationship. METHODS To address this issue, we used a large data set representing 13 biologically distinct categories of leaves sampled from mega- and microsporangiate trees of the dioecious gymnosperm Ginkgo biloba. KEY RESULTS Analyses of the data for these 13 categories of leaves identify seven statistically significantly different modes of A vs. M scaling that result in significant differences in how specific leaf area (SLA) changes as M varies within the canopies of Ginkgo. CONCLUSIONS These results indicate that the protocols used to sample leaves for the analysis of foliar functional traits such as specific leaf area need to acknowledge and cope with the effects of leaf and shoot polymorphisms on the quantification of functional traits (and on the construction and testing of hypotheses about these traits).