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Dark-induced leaf senescence is an extreme example of leaf senescence induced by light deprivation. Prolonged dark treatments of individual leaves result in chlorophyll degradation, macromolecule catabolism, and reduction of photosynthesis. In this work, we described an at-home Dark-induced Leaf Senescence laboratory exercise for a junior-level undergraduate Plant Physiology course. To perform the dark-induced senescence assay on attached leaves, students may cover individual leaves of an outdoor plant with aluminum foils and record the leaf morphology with controlled vocabularies for ~9 days. To perform senescence assays on detached leaves, the students may incubate detached leaves in various aqueous solutions (e.g., tap water, sucrose solution, alkali solution, and acid solution) either in the dark or under natural light, and then record the leaf morphology with controlled vocabularies for ~9 days. This laboratory exercise provides hands-on opportunities for students to understand the relationships among sunlight, chlorophyll, and photosynthesis, in the comfort of students' own homes. Specifically, it helps students to comprehend intrinsic and dark-induced leaf senescence mechanisms, the effects of sugars on leaf senescence, and the importance of optimal pH to plant health. This laboratory exercise can be adapted to support inquiry-based learning or be implemented in a middle or high school classroom. 

Abstract We investigated the flowering plant salicylic acid methyl transferase (SAMT) enzyme lineage to understand the evolution of substrate preference change. Previous studies indicated that a single amino acid replacement to the SAMT active site (H150M) was sufficient to change ancestral enzyme substrate preference from benzoic acid to the structurally similar substrate, salicylic acid (SA). Yet, subsequent studies have shown that the H150M function-changing replacement did not likely occur during the historical episode of enzymatic divergence studied. Therefore, we reinvestigated the origin of SA methylation preference here and additionally assessed the extent to which epistasis may act to limit mutational paths. We found that the SAMT lineage of enzymes acquired preference to methylate SA from an ancestor that preferred to methylate benzoic acid as previously reported. In contrast, we found that a different amino acid replacement, Y267Q, was sufficient to change substrate preference with others providing small positive-magnitude epistatic improvements. We show that the kinetic basis for the ancestral enzymatic change in substate preference by Y267Q appears to be due to both a reduced specificity constant, kcat/KM, for benzoic acid and an improvement in KM for SA. Therefore, this lineage of enzymes appears to have had multiple mutational paths available to achieve the same evolutionary divergence. While the reasons remain unclear for why one path was taken, and the other was not, the mutational distance between ancestral and descendant codons may be a factor.