Lab 05: Leaf development in Pisum sativum (pea) Pea leaf mutants demonstrate the plasticity of leaf development. A change in one gene product results in the conversion of compound leaves to simple leaves (unifoliata-uni), the conversion of leaflets to tendrils (afila-af), tendrils to leaflets (tendril-less-tl), partial loss of stipules (reduced stipules-st), change in leaflet and stipule shape (sinuate leaf-sil), and crinkling of leaves due to loss of adaxial identity (crispa-cri). More detailed descriptions and pictures of each mutant can be found on the last page of this lab. To prepare for this lab, you need to read sections 4.16-4.18 in your book (Cronk, 2010). In this lab you will: -describe wild-type pea leaves -identify which genes are defective in a series of mutant plants -make predictions of the phenotypes of additional mutant combinations -make predictions of the effects of mutations in other plants -contrast pea mutants with other plants with compound leaves Characterizing mutants Obtain a pot with a wild-type pea plant to share with your table. In the space below, sketch one leaf, including its attachment to the stem. Include the petiole, rachis, stipules, the correct number of leaflets and tendrils. Label each part of the leaf. 54 We will typically have five single mutants, but this may vary year-to-year. Describe and illustrate the plant’s appearance (leaf phenotype) and attempt to determine which of the genes described in the introduction is mutated (genotype) in each case. When you are done, you may check your answers with your TA. 1. Phenotype: Genotype: 2. Phenotype: Genotype: 3. Phenotype: Genotype: 4. Phenotype: Genotype: 5. Phenotype: Genotype: 55 The pots labeled with letters are mutant combinations. These may be a bit trickier. Refer to the descriptions in the introduction and the mutants you just observed. Attempt to identify at least 3 of the mutant combinations. A. Phenotype: Genotype: B. Phenotype: Genotype: C. Phenotype: Genotype: 56 As you examine the pea plants, answer the following questions: Do mutations that affect the compound leaf also affect the stipules? If so, which ones? ___________________________________________________ Do mutations that affect the stipules also affect the compound leaf? If so, which ones? ___________________________________________________ Do any mutations have similar effects in the leaflets and stipules? If so, which ones? ____________________________________________________ Do regions of stipules and the compound leaf share a developmental program? Compound leaves in other lineages Compound leaves are characteristic of the legume family (Fabaceae). Bean plants (Phaseolus vulgaris) have compound trifoliate leaves, but do not have stipules or tendrils. Given what you know about each of the pea leaf mutants observed, predict the effect of similar mutations on bean leaves. afila tendril-less unifoliata crispa 57 Simple leaves are most certainly ancestral in Angiosperms, and compound leaves have evolved many times. Compound leaf development has been well studied in the tomato family (Solanaceae). Observe available tobacco (Nicotiana tabacum) and two species of Solanum, one with simple leaves (Solanum cheesmanii) and compound-leaf tomato plants (S. lycopersicon). Use your lecture notes and readings to help answer the following questions: How is the development of compound leaves different in pea and tomato plants at a morphological level? How is the development of compound leaves different in pea and tomato plants at a molecular level? What are the underlying molecular mechanisms for compound leaf evolution? Is there evidence that small changes in gene expression can lead to large changes in leaf morphology? 58 afila (af) “The leaves on afila plants (af/af) look quite unlike those on plants without this mutation. Rather than having pinnately compound leaflets, afila leaves have only tendrils - and lots of them. The primary photosynthetic surfaces on an afila plant are the stipules, which are the "leafy" looking structures on the image below. While the photosynthetic efficiency of an afila plant is somewhat compromised, this mutation has been used to a limited extent in cultivar development, because afila plants have the virtue of "standing" well. When densely planted, the tendrils of neighboring plants vine together extensively, and the planting tends to be self-supporting in a tall canopy. This holds the pods well above the ground and out of reach of rodents and fungal pathogens.”1 Figure 1: afila mutant1 crispa(cri) “The crispa mutation is one of the most dramatic there is, on the overall appearance of the plant. Leaves, flowers and pods of plants expressing this gene are crinkled and folded. Peduncles and petioles are shortened, and leaflets are quite narrow and lanceolate, with their sides more or less parallel.”1 Cri is only available as a heterozygote and may not be available in lab. Figure 2: crispa mutant1 59 sinuate leaves (sil) sinuate= wavy leaf margins “In the af/af background, sil/sil plants are characterized by the presence of ectopic tendrils arising from a cleft in the distal one-fourth or one-third of the stipule. These morphological features are generally confined to the stipules located approximately between nodes 7 and 15; below or above this the stipules may be contorted or sinuate, but they are not incised, nor do they have adventitious tendrils. In Af/- background, sil/sil causes sinuate leaves and stipules.”2 st (reduced stipule) “The recessive allele of this gene conditions stipules that have an approximately 80% reduction in surface area. These smaller stipules are also notable in that they are more linear in shape, and are often somewhat bent, as seen in the image below. In concert with the af gene, which converts leaflets to tendrils, a pea plant with the st gene is called 'fully leafless'. Such plants have extremely reduced photosynthetic surface area, but are especially good at 'standing' in a field without the need of a trellis for support.”1 Figure 3: reduced stipule mutant (st)1 tendril-less (tl) “The 'tl' mutation is commonly called the 'acacia' mutant, because the leaf shape of this mutant resembles leaves of the genus Acacia. Tendrils of plants with this mutation are converted into leaflets, so that true tendrils are absent on such plants. Therefore, plants with the acacia phenotype do not "vine" the way regular pea plants do, and they therefore have problems "standing" in a field. This gene has been the subject of several studies of gene interaction, when studied with the afila (af) mutation. Afila conditions a seemingly opposite phenotype, when it converts leaflets to tendrils, which results in leaves composed entirely of tendrils. ‘Acacia’ leaves are composed entirely of leaflets.”1 Figure 4: tendril-less mutant1 60 unifoliata (uni) “The unifoliata (uni) mutants of Pisum sativum affect both leaf and flower development. The uni mutant converts compound leaf blades into a simple leaf or tri-lobed leaf with stipules (above). The adult leaves also lack a shoot-like rachis. UNI was shown to be the pea homologue of Floricaula from Antirrhinum and LEAFY (LFY) of Arabidopsis thaliana.”3,4 Unfortunately, because uni also affects flower development, this mutation is only available as a heterozygote and may not be available in lab. Figure 5: unifoliata mutant leaf3 1 USDA-ARS Pisum Genetic Stock Collection- mutant description (http://www.ars.usda.gov/Main/docs.htm?docid=14140) 2Marx, G.A. 1977. A Genetic Syndrome Affecting Leaf Development in Pisum. American Journal of Botany. 64: 273-277. 2Marx, G. 1987. A suite of mutants that modify pattern formation in pea leaves. Plant Molecular Biology Reporter. 5: 311-335 3Hofer, J., L. Turner, r. Hellnes, M. Ambrose, P. Matthews, A. Michael and N. Ellis 1997 UNIFOLIATA regulates leaf and flower morphogenesis in pea. Current Biology 7: 581- 587 4Springer, Susan. Carleton College Plant Development Course Materials, personal communication. 61 .