Edward Himelblau

Molecular aspects of leaf senescence

Senescence is the last stage of leaf development and one type of programmed cell death that occurs in plants. The relationships among senescence programs that are induced by a variety of factors have been addressed at a molecular level in recent studies. Furthermore, an overlap between the pathogen-response and senescence programs is beginning to be characterized. The complexity of the senescence program is also evident in studies of senescence-specific gene regulation and the role of photosynthesis and plant hormones in senescence regulation. New molecular-genetic approaches are expected to be useful in unraveling the molecular mechanisms of the leaf senescence program.

Nutrients mobilized from leaves of Arabidopsis thaliana during leaf senescence

Summary During leaf senescence nutrients are mobilized to seeds, storage organs or new vegetative growth. Here we determine the changes in the levels of various nutrients during leaf senescence in Arabidopsis thaliana . Levels of C, Cr, Cu, Fe, K, Mo, N, P, S and Zn drop by greater than 40 percnt; during senescence indicating that these nutrients are mobilized from senescing leaves. Thus, for some elements, Arabidopsis is an effective model for the study of mobilization during senescence. Mutations in two senescence-induced genes involved in copper transport, COPPER CHAPERONE and RESPONSIVE-TO-ANTAGONIST1 were identified. Nutrient mobilization is not affected in the cch-1 mutant indicating that the CCH gene product does not play a role in this process or that a gene with redundant function exists. It was not possible to analyze nutrient mobilization in the ran1-4 mutant due to the numerous pleiotropic effects of this mutation. Nevertheless, these experiments provide an approach for future studies of the molecular genetics of nutrient mobilization.

Integration of Flowering Signals in Winter-Annual Arabidopsis

Photoperiod is the primary environmental factor affecting flowering time in rapid-cycling accessions of Arabidopsis (Arabidopsis thaliana). Winter-annual Arabidopsis, in contrast, have both a photoperiod and a vernalization requirement for rapid flowering. In winter annuals, high levels of the floral inhibitor FLC (FLOWERING LOCUS C) suppress flowering prior to vernalization. FLC acts to delay flowering, in part, by suppressing expression of the floral promoter SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1). Vernalization leads to a permanent epigenetic suppression of FLC. To investigate how winter-annual accessions integrate signals from the photoperiod and vernalization pathways, we have examined activation-tagged alleles of FT and the FT homolog, TSF (TWIN SISTER OF FT), in a winter-annual background. Activation of FT or TSF strongly suppresses the FLC-mediated late-flowering phenotype of winter annuals; however, FT and TSF overexpression does not affect FLC mRNA levels. Rather, FT and TSF bypass the block to flowering created by FLC by activating SOC1 expression. We have also found that FLC acts as a dosage-dependent inhibitor of FT expression. Thus, the integration of flowering signals from the photoperiod and vernalization pathways occurs, at least in part, through the regulation of FT, TSF, and SOC1.

Delivering copper within plant cells.

Two genes recently identified in Arabidopsis thaliana may be involved in sequestering free copper ions in the cytoplasm and delivering copper to post-Golgi vesicles. The genes COPPER CHAPERONE and RESPONSIVE TO ANTAGONIST1 are homologous to copper-trafficking genes from yeast and humans. This plant copper-delivery pathway is required to create functional ethylene receptors. The pathway may also facilitate the transport of copper from senescing leaf tissue. In addition, several other genes have been identified recently that may have a role in copper salvage during senescence.

Forward and Reverse Genetics of Rapid-Cycling Brassica oleracea

Seeds of rapid-cycling Brassica oleracea were mutagenized with the chemical mutagen, ethylmethane sulfonate. The reverse genetics technique, TILLING, was used on a sample population of 1,000 plants, to determine the mutation profile. The spectrum and frequency of mutations induced by ethylmethane sulfonate was similar to that seen in other diploid species such as Arabidopsis thaliana. These data indicate that the mutagenesis was effective and demonstrate that TILLING represents an efficient reverse genetic technique in B. oleracea that will become more valuable as increasing genomic sequence data become available for this species. The extensive duplication in the B. oleracea genome is believed to result in the genetic redundancy that has been important for the evolution of morphological diversity seen in today’s B. oleracea crops (broccoli, Brussels sprouts, cauliflower, cabbage, kale and kohlrabi). However, our forward genetic screens identified 120 mutants in which some aspect of development was affected. Some of these lines have been characterized genetically and in the majority of these, the mutant trait segregates as a recessive allele affecting a single locus. One dominant mutation (curly leaves) and one semi-dominant mutation (dwarf-like) were also identified. Allelism tests of two groups of mutants (glossy and dwarf) revealed that for some loci, multiple independent alleles have been identified. These data indicate that, despite genetic redundancy, mutation of many individual loci in B. oleracea results in distinct phenotypes.

Leaf Senescence: Gene Expression and Regulation

Each autumn leaf senescence leaves its mark on the planet in the form of dramatic changes in color that can be seen from space. Annually, leaf senescence mediates the breakdown of 300 million tons of chlorophyll while changing green forests and fields to yellow and orange (1). The drama of these color changes is matched by the dramatic nature of the cellular events that underlie them. In 1961, Leopold (2) described leaf senescence as “distinctive changes in morphology, pigmentation, and internal nutrition of the plant” with “positive ecological or physiological value.” The benefit to the plant is the reclamation of nitrogen and other nutrients from leaves that have reached the end of their photosynthetic productivity. This reclamation entails the breakdown of the chloroplasts, the conversion of breakdown products into translocatable molecules and the movement of those molecules from the senescing leaf to growing regions of the plant, including the younger leaves and developing fruits. Together, these events are known as the “senescence syndrome.” Currently, numerous laboratories are expanding the definition of the senescence syndrome to encompass the molecular and genetic components of this important process. Leaf senescence is now understood to be a nuclear-regulated, developmental pathway facilitating the recovery of nutrients from spent leaves.

A Study Assessing the Potential of Negative Effects in Interdisciplinary Math–Biology Instruction

There is increasing enthusiasm for teaching approaches that combine mathematics and biology. The call for integrating more quantitative work in biology education has led to new teaching tools that improve quantitative skills. Little is known, however, about whether increasing interdisciplinary work can lead to adverse effects, such as the development of broader but shallower skills or the possibility that math anxiety causes some students to disengage in the classroom, or, paradoxically, to focus so much on the mathematics that they lose sight of its application for the biological concepts in the center of the unit at hand. We have developed and assessed an integrative learning module and found disciplinary learning gains to be equally strong in first-year students who actively engaged in embedded quantitative calculations as in those students who were merely presented with quantitative data in the context of interpreting biological and biostatistical results. When presented to advanced biology students, our quantitative learning tool increased test performance significantly. We conclude from our study that the addition of mathematical calculations to the first year and advanced biology curricula did not hinder overall student learning, and may increase disciplinary learning and data interpretation skills in advanced students.

FLOWERING LOCUS C influences the timing of shoot maturation in Arabidopsis thaliana.

Many plant species undergo changes in leaf morphology as the vegetative shoot meristem matures (Poethig, 2003; Telfer et al., 1997). In Arabidopsis, juvenile leaves are produced before shoot maturation and are relatively round and lack trichomes on the abaxial side (underside) of the leaves. Adult leaves formed after the shoot meristem matures are more elliptical and have abaxial trichomes. An important difference between the juvenilevegetative and adult-vegetative phases is that only during the adult-vegetative phase can the meristem become competent to respond to environmental and endogenous signals that promote flowering (Telfer et al., 1997). Genetic backgrounds and environmental conditions that delay flowering also delay shoot maturation (Telfer et al., 1997). For example, both flowering and shoot maturation occur later in the Columbia accession (Col) than in the Landsberg errecta accession (Ler) (Lee et al., 1993; Telfer et al., 1997). An important difference between these two accessions occurs at the FLC locus. FLC levels are slightly elevated in Col compared to Ler, which has a weak allele of FLC (Michaels et al., 2003). FLC encodes a MADS-domain transcription factor that acts as a repressor of flowering (Michaels and Amasino, 1999a; Sheldon et al., 1999). Flowering is delayed under conditions or in genetic backgrounds in which FLC expression is elevated (Michaels and Amasino, 1999a). The observation that shoot maturation is delayed in Col relative to Ler suggests that FLC influences the rate of shoot maturation. However, there are many genetic differences between Col and Ler making it difficult to determine whether FLC expression is solely responsible for the delay in shoot maturation observed in Col. To further explore the potential role of FLC in the regulation of shoot maturation, we performed experiments in the Col background using mutations that eliminate FLC function, as well as mutations in known regulators of FLC. This allowed us to create a series of lines containing various levels of FLC expression that could be used to determine if there is a correlation between FLC expression and the timing of shoot maturation. The appearance of abaxial trichomes was used to determine when the transition from juvenile to adult leaves occurred (Telfer et al., 1997). The first leaf with four or more abaxial trichomes was scored as the first adult-vegetative leaf. Leaves were photographed at the time of trichome counting, and those images confirm that the appearance of abaxial trichomes corresponds to leaf shape changes associated with shoot maturation in Arabidopsis (Fig. 1) (Telfer et al., 1997). The number of leaves produced before flowering was also recorded for each genotype and treatment (Table 1). In the Col background, shoot maturation occurred earlier in an flc null mutant (flc-3) than in wild type (ttest, P < 0.0001) (Table 1, Fig. 2a). Thus, FLC acts to delay shoot maturation, and a reduction in FLC expression is sufficient to promote shoot maturation. To test whether increased FLC expression would further delay shoot maturation, we examined the timing of shoot maturation in late-flowering autonomous pathway mutants that have elevated levels of FLC expression (Michaels et al., 2003). Previous work in the Ler background has shown that shoot maturation is slightly delayed by autonomous pathway mutants (Telfer et al., 1997). In the Ler background, lacking a strong allele of FLC, autonomous-pathway mutants are known to have little effect on flowering time (Michaels et al., 2003). Therefore, we examined the effect of autonomous-pathway mutants in Col. FCA is part of the autonomous pathway, and loss of function of FCA delays flowering (Sheldon et al., 2000). Shoot maturation was delayed in the presence of an fca null allele (fca-9) relative to wild type (t-test, P < 0.0001) (Table 1, Fig. 2a). In the flc-3 background, fca-9 did not influence the timing of shoot maturation (flc-3, FCA was indistinguishable from flc-3, fca-9) (t-test, P 5 0.373) indicating that the delay induced by fca was FLC-dependent. Loss-of-function of a different