An at-home Plant Physiology laboratory applied to dark-induced leaf senescence by college students and science teachers

18 Dark-induced leaf senescence is an extreme example of leaf senescence induced by light deprivation. 19 Prolonged dark treatments of individual leaves result in chlorophyll degradation , macromolecule 20 catabolism, and reduction of photosynthesis. In this work, we described an at-home “Dark-induced Leaf 21 Senescence” laboratory exercise for a junior-level undergraduate Plant Physiology course. To perform the 22 dark-induced senescence assay on attached leaves, students may cover individual leaves of an outdoor plant 23 with aluminum foils and record the leaf morphology with controlled vocabularies for ~9 days. To perform 24 senescence assays on detached leaves , the students may incubate detached leaves in various aqueous 25 solutions (e.g., tap water, sucrose solution, alkali solution, and acid solution) either in the dark or under 26 natural light, and then record the leaf morphology with controlled vocabularies for ~9 days. This laboratory 27 exercise provides hands -on opportunities for students to understand the relationships among sunlight, 28 chlorophyll, and photosynthesis, in the comfort of students’ own homes . Specifically, it helps students to 29 comprehend intrinsic and dark -induced leaf senescence mechanisms , the effects of sugars on l eaf 30 senescence, and the importance of optimal pH to plant health . This laboratory exercise can be adapted to 31 support inquiry-based learning or be implemented in a middle or high school classroom. 32 33

34 Plant physiology, dark-induced leaf senescence , at-home laboratory, college students, middle and high 35 school science teachers 36 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 3

37 Senescence is an energy -dependent, self -digesting process controlled by the interactions between 38 environmental cues and developmental programs (Taiz et al., 2023 ). It is a universal c haracteristic in 39 biological systems. According to the level of the senescing unit, plant senescence could be classified into: 40 programmed cell death, organ senescence, and whole plant senescence (Taiz et al., 2023 ). All leaves, 41 including those of evergreens (e.g., blue spruce), undergo senescence, in response to developmental factors 42 (e.g., flowering and seeding), environmental factors (e.g., seasonal daylength and temperature changes), 43 biotic stresses (e.g., pathogen attacks), or abiotic stresses (e.g., shading and wounding) (Taiz et al., 2023). 44 Intrinsic leaf senescence is a specialized form of programmed cell death, which permits remobilization of 45 nutrients from source leaves to vegetative or reproductive sinks (Keskitalo et al., 2005 ). The earlies t 46 structural change during intrinsic leaf senescence is chloroplast breakdown (Taiz et al., 2023 ). Carbon 47 fixation is thus replaced by the degradation and conversion of chlorophyll, proteins, and other 48 macromolecules to exportable nutrients. Intrinsic leaf senescence is a normal developmental process 49 (Kanojia et al., 2020). 50 Dark-induced leaf senescence is an extreme example of leaf senescence induced by shading (Sobieszczuk-51 Nowicka et al., 2018). Similar to intrinsic leaf senescence, dark-induced leaf senescence results in increased 52 degradation of chlorophyll, disassembly of cellular elements (e.g., nucleic acids and proteins), and a loss 53 of photosynthetic activity (Paluch-Lubawa et al., 2021 ). Dark-induced leaf senescence assays could be 54 performed on whole plants, attached leaves, or detached leaves (Weaver and Amasino, 2001). This could 55 be achieved by covering whole plants or individual leaves or by placing whole plants or detached leaves in 56 the dark. Unlike whole plants or attac hed leaves, detached leaves are subjected to mechanical wounding 57 and water-soaking (Iakimova and Woltering, 2018), as they need to be excised from th e plant and kept in 58 an aqueous solution. Mechanical wounds may act as additional entry points to detached leaves for 59 substances present in the aqueous solution (Savatin et al., 2014). 60 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 4 During the social distancing imposed by COVID19 in Fall 2020 - Spring 2021, we developed an at-home 61 laboratory topic – Dark-induced Leaf Senescence, for a junior-level undergraduate Plant Physiology course 62 at Western Michigan University (WMU). 63 In this exercise, the students were asked to perform dark-induced leaf senescence assays on attached and 64 detached leaves. For attached -leaf assays, the students may cover both sides of a few leaves (e.g., four) 65 from a plant of their choice with aluminum foils. For detached leaf -assays, the students may excise some 66 morphological and developmental similar leaves from a plant, keep them in aqueous solution, and plac e 67 half of the leaves in the dark and the other half under natural light (e.g., by a window). The students were 68 also asked to supplement the aqueous solution with sucrose, alkali (e.g., sodium bicarbonate/baking soda), 69 or acid (e.g., acetic acid in vinegar and citric acid in lemon juice). Exogenous sugar treatments have been 70 found to delay dark-induced leaf senescence in detached leaves (Wingler and Roitsch, 2008; Schippers et 71 al., 2015; Li et al., 2020 ) and accelerate the senescence of detached leaves under light (Khudairi, 1970; 72 Wingler et al., 2004; Wingler et al., 2006). A 6% sucrose solution was reported to be suitable for detached 73 leaves or leaf segments (Li et al., 2020). Therefore, the students were asked to test whether supplying 6% 74 sucrose to detached leaves delays or accelerate leaf senescence. Most plants thrive in the pH 6.0 -7.0 75 (slightly acidic to neutral) range (Osman, 2018). The tap w ater in the Kalamazoo area has a pH of 7.0. 76 Hence, the students were also asked to investigate the effect of pH on detached leaves by supplementing 77 the aqueous solution with baking soda, which is sodium bicarbonate, or vinegar/lemon juice, which contains 78 acetic acid or citric acid, respectively. A 6% baking soda solution has a pH of 8.0. A 6% vinegar solution 79 has a pH of ~3.2. A 6% lemon juice solution has a pH of ~4.0. Before and during the treatments, the students 80 were required to use controlled vocabulary to describe leaf morphology. 81 In Summer 2022, we modified this exercise slightly and showed it to 9 middle and high school science 82 teachers from Southwest Michigan. They were participants of the Summer 2022 BIORETS (Research 83 Experiences for Teachers Sites in Biological Sciences) program at WMU. 84 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 5 LEARNING OBJECTIVES 85 The activities in this exercise should allow students to: 86 1. Understand leaf senescence mechanisms (e.g., intrinsic vs dark-induced leaf senescence) and the effects 87 of sugars on leaf senescence. 88 2. Understand the importance of optimal pH to plant health. 89 3. Learn the basic techniques of dark-induced leaf senescence assays. 90 4. Use controlled vocabularies to record leaf morphology. 91 92

93

94 In the lab manual (Supplemental Material 1), the students were provided with a list of m aterials used in 95 this at-home laboratory exercise: outdoor plants with green leaves; aluminum foil; tap water; a measuring 96 glass; eight glass/plastic jars/containers (e.g., Mason jars, jam jars, yeast jars, baby food jars, water glasses, 97 small food storage containers made of clear plastics); a set of measuring spoons (e.g., one tablespoon); table 98 sugar (i.e., sucrose); baking soda (i.e., sodium bicarbonate); vinegar, which contains acetic acid, or lemon 99 juice, which contains citric acid; and a pair of scissors. 100 Dark-Induced Leaf Senescence Assay with Attached Leaves 101 In the lab manual ( Supplemental Material 1 ), the students were also provided with step -by-step 102 instructions on how to perform dark-induced leaf senescence assays with attached and detached leaves. For 103 the assay with attached leaves, the students c hose four non-senescing green leaves from a plant and took 104 pictures of each dark -treatment leaf, with at least one control leaf in the same picture. The eight leaves 105 should be developmentally and morphologically similar. The students needed to use controlled vocabularies 106 (Table 1) to record the initial leaf morphology (leaf color, presence or absence of necrotic spots or lesions) 107 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 6 of the eight leaves (Table 2). The students then covered both sides of dark-treatment leaves with aluminum 108 foils, secured the foils on the lea ves by folding the foil near the ti p and the base of the leaf inward, and 109 labeled the leaves by tying a string on the petiole. If the students were concerned that the aluminum foil 110 blocks the air and water vapor movements, they may replace the aluminum foil with black -colored fabric 111 and secure the fabric with safety pins. The students also needed to l abel the four control leaves (e.g., by 112 tying a string on each petiole). One day later, the students removed the aluminum foils and took pictures of 113 each uncovered dark-treatment leaf, with at le ast one control leaf in the same picture. The students then 114 recorded the leaf morphology of the eight leaves, re-covered the same four leaves with aluminum foils, and 115 secured the foils. This process (morphology recording and imaging) may be repeated for 9 days for the 116 dark-treated leaves to develop visible symptoms. 117 Table 1. A list of controlled vocabularies to be used when recording leaf morphology 118 Category Controlled vocabulary Leaf color green, blue green, yellow green, yellow, brown, etc. Leaf anatomy leaf blade, petiole, leaf margin, leaf tip, leaf base Color of wounding sites Brown, not brown Brown necrotic spots Presence, absence Water-soaked spots presence or absence; translucent or not translucent Size of wounding sites, necrotic spots, or water-soaked spots small, medium, large, larger, even larger Percent leaf area (estimation) 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 0% Leaf location in solution floating, sunken Fungal infection Moldy, not moldy Turbidity of solution Clear, cloudy Color of solution No color, light yellow, yellow 119 Table 2. Daily morphology of dark-treated attached leaves. 120 Treatment Leaf # Category Day 1 (exemplary) Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Light 1 A. Leaf colors and percent leaf areas 100% green; 0% yellow B. Number of brown necrotic spots and their percent leaf area 0; 0% 2 A. Leaf colors and percent leaf areas 95% green; 5% yellow B. Number of brown necrotic spots and their percent leaf area 0; 0% 3 A. Leaf colors and percent leaf areas 95% green; 0% yellow B. Number of brown necrotic spots and their percent leaf area 1; 5% .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 7 4 A. Leaf colors and percent leaf areas 100% green; 0% yellow B. Number of brown necrotic spots and their percent leaf area 0; 0% Dark 1 A. Leaf colors and percent leaf areas B. Number of brown necrotic spots and their percent leaf area 2 A. Leaf colors and percent leaf areas B. Number of brown necrotic spots and their percent leaf area 3 A. Leaf colors and percent leaf areas B. Number of brown necrotic spots and their percent leaf area 4 A. Leaf colors and percent leaf areas B. Number of brown necrotic spots and their percent leaf area 121 Dark-Induced Leaf Senescence Assay with Detached Leaves 122 For the assay with detached leaves, the students l abeled 8 clear glass/plastic jars/glasses/containers with 123 “H2O Light”, “H2O Dark”, “Sucrose Light”, “Sucrose Dark”, “Alkali Light”, “Alkali Dark”, “Acid Light”, 124 and “Acid Dark”. For tap-water treatments, the students poured 1/2 cup (118 mL) of tap water into the jars 125 labeled “H2O Light” and “H2O Dark”. For sucrose treatments, t he students added 1 cup (237 mL) of tap 126 water and 1 tablespoon (15 g) of table sugar (sucrose) into the jar labeled “Sucrose Light”, stir red with a 127 stirring spoon to dissolve sucrose completely, and then transferred 1/2 cup of the resulting 6% sucrose 128 solution into the jar labeled “Sucrose Dark”. After this, the students needed to w ash the tablespoon, the 129 stirring spoon, and the measuring glass with tap water and blot dry them with paper towels. For alkali 130 treatments, the students added 1 cup (237 mL) of tap water and 1 tablespoon (15 g) of baking soda (sodium 131 bicarbonate) into the jar labeled “Alkali Light” , stirred to dissolve the baking soda completely, and then 132 transferred 1/2 cup of the resulting 6% baking soda solution into the jar labeled “Alkali Dark”. Again, the 133 students needed to wash the tablespoon, the stirring spoon, and the measuring glass with tap water, and blot 134 dry them with paper towels, after this step. For acid treatments, the students added 1 cup (237 mL) of tap 135 water and 1 tablespoon (15 mL) of vinegar or lemon juice into the jar labeled “Acid Light” , stir to mix 136 completely, and then transferred 1/2 cup of the resulting 6% acid solution into the jar labeled “Acid Dark”. 137 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 8 After washing the tablespoon, the stirring spoon, and the measuring glass with tap water , the students set 138 the eight jars aside (Figure 1A). 139 140 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 9 Figure 1. Dark-induced leaf senescence assay with detached lea ves. (A) Eight jars with solutions. (B) 141 Exemplary outdoor plants in winter 2020. (C) Arrange the leaves (e.g., Chrysanthemum leaves) according 142 their size on a table. (D) Eight capless jars with detached leaves. 143 The students harvested ~12 green leaves from the plant of their choice (exemplary plants in winter are 144 shown in Figure 1B). These leaves should be non -senescing and developmentally and morphologically 145 similar to each other. The students were also encouraged to include leaves from another plant in the assay, 146 if they are interested. Multiple leaves could be incubated in each jar. The students then arranged the leaves 147 according their size on a table , select 8 leaves that are non -senescing and most similar to each other 148 developmentally and morphologically (Figure 1C), place one leaf per jar, and make sure all leaves face up. 149 The students needed to use controlled vocabularies to record the morphology (leaf color, percentage of the 150 leaf in that color, color of wounding sites, presence of brown necroti c spots and/or water -soaked spots, 151 floating or sunken, etc.) of each leaf that goes into each jar, and the turbidity and color of each solution, in 152 a table (see Table 3). After recording the morphology, the students took a group picture of the eight capless 153 jars with leaves (Figure 1D) and p laced the four jars labeled with “Light” under natural light (e.g., by a 154 window) and the four jars labeled with “Dark” in the dark (e.g., in a drawer, cabinet, or closet). Capping 155 the jars was optional during incubation. The students may repeat morphology recording and imaging every 156 day for 9 days for the detached leaves to develop visible symptoms. 157 Table 3. Daily morphology of dark-treated detached leaves. 158 Treatment Category Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 e.g., H2O Light 1. Leaf color and percentage 100% Green 2. Wounding site color Green 3. Number of brown necrotic or water - soaked spots and their percent leaf area 0; 0% 4. Translucent or not Not 5. Floating or sunken Floating 6. Moldy or not Not 7. Solution turbidity Clear 8. Solution color No color 159

160 Individually shaded, attached leaves displayed yellowing and senescence 161 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 10 We found that the attached leaves of various outdoor plants displayed yellowing and senescence after 7 162 days of shading with aluminum foils (Figure 2). Examples of such outplants include: common dandelions, 163 day lilies , f alse bindweeds , h ostas, p rairie milkweeds , and yews. Performing the dar k-induced leaf 164 senescence assay may help students visually understand the mechanisms of intrinsic and dark-induced leaf 165 senescence. 166 167 Figure 2. Examples of attached leaves or leaf sections of outdoor plants after 7 days of dark treatment in 168 summer 2022. (A -B) Common dandelions. (C -D) Day lilies. (E) False bindweeds. (F) Hostas. (G) Prairie 169 milkweeds. (H) Yews. Red rectangles indicate leaves or le af sections covered with aluminum foils for 7 170 days. 171 Detached leaves treated with “H 2O + Dark” showed signs of senescence earlier than those treated 172 with “H2O + Light” 173 We found that detached leaves placed in the dark in tap water showed signs of senescence earlier than those 174 placed under nature light in the same tap water (Figure 3). After being incubated in tap water under nature 175 light for 9 days (Figure 3A), the two lawn grass leaf sections and the poppy leaf section were still green. 176 Although the Chrysanthemum leaf had three black necrotic spots, it still floated on top of water. The tap 177 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 11 water was still clear. The leaves placed in the dark in the same tap water (Figure 3B) appeared less healthy. 178 One of two lawn grass leaf sections turned yellow completely. In addition, the Chrysanthemum leaf and the 179 poppy leaf section both sank to the bottom of the container, which is an extreme example of water-soaking. 180 Furthermore, the tap water turned yellow, a sign of chloroplast destruction and chlorophyll leakage. These 181 observations are consistent with the hypothesis that dark treatments result in leaf senescence. 182 183 Figure 3. Detached leaves after 9 days of light or dark treatment in tap water or 6% sucrose. 184 Detached leaves treated with “6% Sucrose + Light” showed sig ns of senescence earlier than those 185 treated with “H2O + Light” 186 We found that detached leaves incubated in a 6% sucrose solution under nature light showed signs of 187 senescence earlier than those incubated in tap water under the same nature light ( Figure 3). After being 188 incubated in 6% sucrose under natural light for 9 days (Figure 3C), the two lawn grass leaf sections, the 189 Chrysanthemum leaf, and the poppy leaf section all turned yellow green and had many brown -to-black 190 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 12 necrotic spots. These observations were initially surprising to the students because sucrose is a final product 191 of photosynthesis and it can enhance plant growth. Interestingly, sugars also act as signaling molecules and 192 regulate plant metabolism, development, and even senescence (Wingler et al., 2006). Sugar accumulations 193 have been found to induce leaf senescence (Khudairi, 1970; Wingler et al., 2004). 194 Detached leaves treated with “6% Sucrose + Dark” showed signs of senescence later than those 195 treated with “H2O + Dark” 196 We found that detached leaves incubated in a 6% sucrose solution in the dark showed signs of senescence 197 later than those incubated in tap water in the dark (Figure 3). After 9 days of dark treatment in 6% sucrose 198 (Figure 3D), the Chrysanthemum leaf and the poppy leaf section were mostly green. As mentioned above, 199 the Chrysanthemum leaf and the poppy leaf section subjected to 9 days of dark treatment in tap water sank 200 to the bottom of the container and the tap water turned yel low (a sign of chloroplast destruction and 201 chlorophyll leakage) (Figure 3B). These observations are consistent with the hypothesis that e xogenous 202 sugar may delay dark-induced leaf senescence in detached leaves (Wingler and Roitsch, 2008; Schippers 203 et al., 2015; Li et al., 2020). 204 Supplementing water with 6% baking soda caused damage to the detached leaves 205 We found that supplementing water with 6% baking soda caused damage to the detached leaves ( Figure 206 4). After 3 days of incubation in a 6% baking soda solution under natural light, a large brown -to-black 207 necrotic spot formed near the petiole of the Chrysanthemum leaf and the excision area of the poppy leaf 208 section (Figure 4C). These two necrotic spots covered about 25% of the leaf area. After 5 days of incubation 209 in a 6% baking soda solution under natural light, the necrotic spot covered about 50% of the poppy leaf 210 section (Figure 4D). The two lawn grass leaf sections also de veloped necrotic spots near the excisions. 211 Furthermore, the baking soda solution also became yellow, which is a sign of chloroplast destruction and 212 chlorophyll leakage into the solution. 213 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 13 214 Figure 4. Detached leaves after being incubated in tap water (A-B), 6% baking soda (C-D), or 6% lime juice 215 (E-F) under nature light for 3 (A, C, E) or 5 (B, D, F) days. 216 Supplementing water with 6% vinegar or lemon juice caused damage to the detached leaves 217 We also found that supplementing water with 6% lemon juice caused damage to the detached leaves (Figure 218 4). After 3 days of incubation in a 6% lemon juice solution under natural light, the Chrysanthemum leaf 219 and the poppy leaf section became translucent and sank to the bottom of the container ( Figure 4E). After 220 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 14 5 days of incubation in a 6% lemon juice solution under natural light, the Chrysanthemum leaf and the 221 poppy leaf section became dark brown (Figure 4F). 222 223

224 Effects of light on the senescence of attached and detached leaves 225 For many plant species, severe shading of leaves, especially when only applied to a part of the plant, results 226 in rapid senescence (Liebsch and Keech, 2016). In this at-home laboratory exercise, after an individual leaf 227 of an outdoor plant was covered with aluminum foils for 7 days, the leaf often turned yellow or even 228 senesced (Figure 2). On the contrary, the control leaves not subjected to the dark treatment stayed green 229 (Figure 2). Performing this at-home laboratory exercise allowed the students to visually understand that 230 light deprivation is essential to the success of dark-induced senescence assays of both attached and detached 231 leaves. 232 Effects of sugars on the senescence of detached leaves 233 Exogenous sugar treatments have been found to accelerate the senescence of detached leaves under light 234 but delay the senescence of detached leaves in the dark (Khudairi, 1970; Wingler et al., 2004; Wingler et 235 al., 2006; Wingler and Roitsch, 2008 ; Schippers et al., 2015 ; Li et al., 2020 ). In this at-home laboratory 236 exercise, detached leaves treated with 6% sucrose senesced earlier than those treated with tap water under 237 nature light and senesced later than those treated with tap water in the dark ( Figure 3 ). Therefore, 238 performing this at-home laboratory exercise provided a hands-on opportunity for the students to understand 239 the differential effects of sugars on the senescence of detached leaves under light or in the dark. 240 Effects of pH on detached leaves 241 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 15 Most plants thrive in the pH 6.0 -7.0 range (Osman, 2018). Treating plants with alkali or acidic solutions 242

in cell membrane leakage and water -soaking (Grant, 2024; Portland-Parks-and-Recreation, 2024). 243 In this at-home laboratory exercise, detached leaves treated with 6% baking soda or 6% lemon juice showed 244 signs of leaf damage (e.g., brown-to-black necrotic spots, translucent leaf coloration, sinking to the bottom) 245 after 3 days of treatments and the symptoms worsened after 5 days of treatments ( Figure 4). Therefore, 246 carrying out this at-home laboratory exercise helped the students understand the importance of optimal pH 247 to plant health. 248 Connection between this at-home laboratory exercise and the corresponding lecture 249 Intrinsic leaf senescence is covered in one of the last four chapters of the “ Plant Physiology and 250 Development” textbook (Taiz et al., 2023) for the BIOS 3190 Plant Physiology course. During the online 251 teaching of this chapter – “Plant Senescence and Developmental Cell Death”, students learned a number of 252 related topics, such as the leaf senescence syndrome, the regulatory network of leaf senescence, and whole 253 plant senescence. Therefore, having the students perform this at-home laboratory exercise near the end of 254 the spring semester is complementary to and in sync with what the students have learned from the lectures. 255 During the development stage of this laboratory module, we thought that performing dark -induced leaf 256 senescence assays may help students understand the mechanisms of intrinsic and dark -induced leaf 257 senescence. Indeed, one student stated in the final course evaluation that “ I really enjoy doing the last lab 258 at home as it was hands, which helped me learn and understand”. 259 Completion rate of this at-home laboratory exercise 260 In Spring 2021, there were 11 undergraduate students enrolled in this junior-level BIOS 3190 Plant 261 Physiology course. As a writing-intensive course, 32% of the overall grade came from lab reports and a 262 total of n ine lab reports were assigned. The first 8 lab topics were virtual and worth 16 points each 263 (Supplemental Material 2 ). Dark-induced leaf senescence is the only at -home laboratory exercise and 264 worth 40 points (Supplemental Material 2 ). Among the 11 students, 8 chose to complete this at -home 265 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 16 exercise and submit a lab report about this laboratory topic. Therefore, the completion rate of this at-home 266 exercise was 73%, similar to the average lab report completion rate of the 8 virtual topics (75%). This 267 suggested that the extra work associated with the at-home laboratory exercise didn’t discourage the students 268 from completing the lab and then submitting the lab report. 269 270 POTENTIAL MODIFICATIONS 271 This laboratory exercise was developed during the COVID19 pandemic for undergraduate students to 272 perform at home or in a classroom. If the students cannot find eight containers at home, they may drop the 273 alkali or acid treatment. If the students have other class duties on certain days , they may opt out leaf 274 morphology observation and photographing on these days. The students may also compare the images and 275 morphology of detached leaves incubated in aqueous solutions with those attached to the plant to investigate 276 the differences and similarities between detached and attached leaf senescence. 277 This at-home laboratory exercise can be easily adapted to a n in-person classroom setting. For example, 278 during the Summer 2022 BIORETS program, we had 9 middle and high school science teachers from 279 Southwest Michigan performed this exercise in a classroom and it went very well. The images of attached 280 leaf senescence assay shown in Figure 2 were actually taken in Summer 2022. 281 This laboratory exercise, or part of this exercise, can also be simplified and implemented in a middle or 282 high school science classroom as a hands -on activity for teaching photosynthesis. Chlorophyll is an 283 essential component in photosynthesis. The simple and hands-on laboratory exercise described in this work 284 may help students to visually understand the relationship among sunlight, chlorophyll, and photosynthesis. 285 After performing this laboratory exercise during the Summer 2022 BIORETS program, some teacher 286 participants remarked that they “could see how to implement it in their own classrooms”. 287 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 17 This laboratory exercise can also be adapted to support other pedagogical approaches, such as inquiry-based 288 learning. For example, students may subject detached leaves to different concentrations of sucrose, baking 289 soda, and vinegar/lemon juice and investigate whether different concentrations of sugars, alkali, or acids 290 have differential effects on leaf health and senescence. Student may also place a set of detached leaves in 291 the refrigerator and compare them with those incubated at room temperature. For an inquiry -based 292 laboratory exercise, the students will be given a list of relevant references and will be asked to write a mini 293 research proposal that contains an introduction and an experimental design section. In the introduction, the 294 students are required to provide background information about their laboratory topic. In the experimental 295 design section, the students are required to state their hypothesis, propose appropriate experiments, describe 296 how to perform the experiments, list what equipment and materials they will need, define appropriate 297 controls, explain what data they plan to collect, and clarify how they plan to analyze the data. After the 298 students have finished the experiments and data collection, they will be asked to submit a lab report on this 299 inquiry-based laboratory exercise, according to the grading criteri a shown in Supplemental Material 2. 300 Such inquiry-based laboratory exercises are expected to improve students’ motivation, critical thinking 301 skills, and analysis skills (Buck et al., 2008; Díaz-Vázquez et al., 2012; Stefanou et al., 2013; Ambruso and 302 Riley, 2022). 303 304

305 In this work, we described an at-home laboratory exercise that was successfully implemented in a n 306 undergraduate Plant Physiology course. Materials needed for this exercise, such as aluminum foil , a 307 measuring glass , glass/plastic containers , and a tablespoon, are readily available at students’ home . 308 Therefore, performing this at-home laboratory exercise does not require shipping laboratory kits to students’ 309 home. The activities in volved this exercise should help students to: (1) u nderstand leaf senescence 310 mechanisms and the effects of sugars on leaf senescence ; (2) understand the importance of optimal pH to 311 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 18 plant health ; (3) l earn the basic techniques of dark -induced leaf senescence assays ; (4) u se controlled 312 vocabularies to record leaf morphology. Activities included in this laboratory exercise are very flexible; 313 students are encouraged to modify their experiments according to what they have at home. This laboratory 314 exercise can also be adapted to support inquiry-based learning or be implemented as a hands-on activity for 315 teaching photosynthesis in a middle or high school classroom. 316 317 SUPPLEMENTAL MATERIALS 318 Supplemental Material 1. BIOS 3190 Plant Physiology Lab Manual on Dark-Induced Leaf Senescence. 319 Supplemental Material 2. BIOS 3190 Plant Physiology Point Distribution and Grading Criteria for Lab 320 Reports. 321 ACKNOWLEDGMENTS 322 The authors thank all the Spring 2021 BIOS 3190 Plant Physiology students and all the Summer 2022 323 BIORETS (Research Experiences for Teachers Sites in Biological Sciences) teacher participants at Western 324 Michigan University (WMU). The authors also thank Mr. Christopher D. Jackson (WMU) for growth 325 chamber management. 326 DECLARATION OF INTEREST STATEMENT 327 The authors report there are no competing interests to declare. 328 ORCID 329 Yan Lu: 0000-0002-3374-7376 330 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2025. ; https://doi.org/10.1101/2025.01.07.630824doi: bioRxiv preprint 19

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