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A leaf drying assay to measure cuticular wax permeability in wild type and glossy mutant rapid cycling Brassica rapa

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Drought is a major limiting factor in crop yields, and as global warming becomes increasingly important, finding crop varieties which resist drought more effectively and understanding the genetics behind drought resistance is essential. One feature
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  A leaf drying assay to measure cuticular wax permeability in wild typeand  glossy mutant rapid cycling  Brassica rapa Heather Wick, Tufts University, Medford, MA 02155 ABSTRACT Drought is a major limiting factor in crop yields, and as global warming becomesincreasingly important, finding crop varieties which resist drought more effectively andunderstanding the genetics behind drought resistance is essential. One feature plants haveto resist drought is a cuticular wax coating which covers and water-proofs the aerialsurfaces of the plant. Fast-cycling  Brassica rapa was used in this experiment as arepresentative of important crop species in the  Brassica genus in order to study howcuticular wax mutants withstand water loss compared to wild type. Batches of wild typeand  glossy    B. rapa were reared in the laboratory. A leaf drying bioassay was conductedon cotyledons, first leaves, and second leaves of both strains in the summer and in the fallto compare the rates of cuticular water loss of both strains. The results were notconclusive, and further literary research suggests that confounding factors, particularlystomatal water loss, may have affected the experiment. Further exploration of stomataland cuticular water regulation in wild type and  glossy    B. rapa needs to be undertaken inorder to determine the role of cuticular wax mutations in  B. rapa . INTRODUCTION Global climate change is one of the most ecologically prevalent issues of thecentury, and increased water stress, or drought, is just one of the many expected results of this global climate change (Hurrell 2008). Because drought is already considered to bethe leading limiter of crop yield (Tang et al. 2005), determining how crop plants respondto drought and cultivating more drought-tolerant crop varieties is vital (Tang et al. 2005).All plants are covered in a waxy cuticular coating which helps protect againstmany different environmental stresses, including ultraviolet radiation, infection from bacteria and fungi, and predation (Post-Beittenmiller 1996). Cuticular waxes also regulatenon-stomatal water loss and are essential in protecting plants against drought (Post-Beittenmiller 1996). Mutations in the genes that regulate cuticular wax production canalter the permeability of the cuticular wax coating to water, usually adversely affectingthe plant’s ability to regulate water loss. The glossy  B. rapa strain is a putative singlegene recessive mutant with shiny leaves, stems, and seedpods, most likely due to analtered amount or composition of cuticular wax (Post-Beittenmiller 1996). Its appearancemakes it easily discernable from the glaucous, or powdery-looking, wild type.In this experiment we wanted to investigate whether   glossy fast cycling  B. rapa has increased or decreased cuticular wax permeability compared to the wild type. Rapidcycling  Brassica rapa , a mustard, is an ideal study model for this experiment because it isa representative member of the  Brassica genus which includes many globally importantcrop plants such as broccoli, Chinese cabbage, and turnips. It grows to maturity in a1  matter of weeks and has short generational cycles, and it is easy to care for in a laboratorysetting, requiring little more than water and light. METHODSPlanting and rearing  B. rapa seeds Six batches of   B. rapa were planted in total; 3 in the summer: 6/23, 6/30, and 7/7;and 3 in the fall: 9/22, 10/8, and 10/17. For each batch of plants, 2 plastic trays 57.8cm l x29.8cm w were lined with paper towels; one tray for wild type and one tray for glossy  B.rapa . Six pots 15.9cm l x 12.1cm w x 5.5cm d with drainage holes in the bottom were placed in each tray, lined with paper towels and filled 1/3 deep with moist potting soil(Peter’s brand, (Scotts Company, 14111 Scottslawn rd, Marysville, OH 43041) whichcontains composted bark, Canadian sphagnum peat moss, and vermiculite). Eight time-release fertilizer pellets 13:13:13 (N:P:K) were placed evenly on the surface of the soil.Pots were then filled 2/3 deep, 8 more pellets were added, and then pots were filledcompletely. Approximately 20-22  B. rapa seeds were planted 3-5mm deep, evenlyspaced, per pot. Wild type and glossy trays were placed on separate growth carts onopposite sides of the room to prevent cross-pollination (summer), or on the same growthcart with aluminum foil barriers to prevent cross-contamination (fall), under fluorescentfull spectrum growth lights for 24 hours a day. Each pot was watered from the top with20 ml tap water for 3 days (until seeds germinated and cotyledons unfurled), and wateredfrom the bottom each weekday by filling the tray several mm deep with tap water (depthdepended on dryness of soil). Leaf-drying bioassay of   B. rapa After 1 st and 2 nd leaves unfurled, but before flower buds opened (approximately 2weeks after planting), the leaf-drying bioassay was conducted. Three cotyledons of approximately the same size, 3 1 st leaves of approximately the same size, and 3 2 nd leavesof approximately the same size were selected from both wild type and glossy trays for the bioassay. Leaves were severed at the base of the petiole with a scalpel and massed at 0min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 210 min, 240 min, and300 min after severance. The bioassay was conducted once per batch: 3 times in thesummer and 3 times in the fall. Percent average srcinal mass was calculated and graphedwith standard error for each leaf type for the summer batches and the fall batches. Obtaining temperature and humidity Temperature and humidity were estimated for the summer bioassays by using aRadioshack brand temperature and humidity gauge on 8/11, 8/12, and 8/13. The gaugewas placed in the same room as the plants and checked twice a day during the time of daythat the experiment had taken place. Average temperature and humidity for all summer assays were then calculated. The same device was used to estimate temperature andhumidity during two of the fall bioassays, but readings were taken throughout the actualexperiments. Average temperature and humidity were calculated for all fall assays.2  RESULTSSummer data Overall, all leaves tended to lose water at a rapid but decreasing rate for the firstinitial 45-60 min before settling into a gentler, steady rate of water loss for the remainder of the assay.On average,  glossy cotyledons lost water at a faster rate than standard cotyledonsfor the first 45 min, and then both cotyledon types lost water at approximately equal ratesfor the remainder of the assay (Figure 1). However between the last two data points the  glossy cotyledon water rate loss decreased, so both cotyledons types had similar average percent initial weights by the end of the assay (standard: 52.78%,  glossy : 52.02%).On average, wild type and  glossy 1 st leaves lost water at nearly the same ratethroughout the assay (Figure 2). Both types ended with very similar average initial percent weight (standard: 52.78%,  glossy : 52.86%)Second leaves of both types lost water at nearly the same rate for approximately90 minutes, at which point both types were approximately 54% of their initial weight(Figure 3). After this point,  glossy 2 nd leaves lost water at a slightly faster rate on averagethan wild type. At the end of the assay,  glossy leaves had lost more water than the wildtype on average (Percent initial weights: wild type: 46.93%,  glossy : 42.07%) 020406080100050100150200250300350 Time (min.)    A  v  e  r  a  g  e   %   i  n   i   t   i  a   l   W  e   i  g   h   t Wild type cotyledonsGlossy cotyledons Figure 1. The average percent initial weight of wild type and  glossy    B. rapa cotyledons after severance,with standard error. Leaves were severed at the base of the petiole and allowed to air dry, estimatedtemperature: 22°C, estimated humidity: 69%. Bioassays conducted 7/8, 7/15, and 7/22 on plants planted6/23, 6/30, and 7/7, respectively, at Tufts University, Medford, MA. 3  020406080100050100150200250300350 Time (min.)    A  v  e  r  a  g  e   %   i  n   i   t   i  a   l   W  e   i  g   h   t Wild type 1st leavesGlossy 1st leaves Figure 2. The average percent initial weight of wild type and  glossy    B. rapa 1 st leaves after severance, withstandard error. Leaves were severed at the base of the petiole and allowed to air dry, estimated temperature:22°C, estimated humidity: 69%. Bioassays conducted 7/8, 7/15, and 7/22 on plants planted 6/23, 6/30, and7/7, respectively, at Tufts University, Medford, MA. 020406080100050100150200250300350 Time (min.)    A  v  e  r  a  g  e   %   i  n   i   t   i  a   l   W  e   i  g   h   t Wild type 2nd leavesGlossy 2nd leaves Figure 3. The average percent initial weight of wild type and  glossy    B. rapa 2 nd leaves after severance, withstandard error. Leaves were severed at the base of the petiole and allowed to air dry, estimated temperature:22°C, estimated humidity: 69%. Bioassays conducted 7/8, 7/15, and 7/22 on plants planted 6/23, 6/30, and7/7, respectively, at Tufts University, Medford, MA. 4  Fall data Overall, all leaves tended to lose water at a rapid but decreasing rate for the firstinitial 45-60 min before settling into a gentler, steady rate of water loss for the remainder of the assay (Figure 4).On average, standard cotyledons lost water at a faster rate than  glossy cotyledonsfor the duration of the assay. Standard cotyledons finished the assay with a smaller average percent initial weight than  glossy cotyledons on average (standard: 47.65%,  glossy : 56.23%).On average, standard 1 st leaves lost water at a faster rate than  glossy 1 st leaves for the duration of the assay. Standard cotyledons finished the assay with a smaller average percent initial weight than  glossy cotyledons on average (standard: 46.05%,  glossy :51.33%).On average, standard 2 nd leaves lost water at a faster rate than  glossy 2 nd leaves for the duration of the assay. Standard cotyledons finished the assay with a smaller average percent initial weight than  glossy cotyledons on average (standard: 43.53%,  glossy :48.45%). 020406080100050100150200250300350 Time (min.)    A  v  e  r  a  g  e   %   i  n   i   t   i  a   l   W  e   i  g   h   t Wild type cotyledonsGlossy cotyledons Figure 4. The average percent initial weight of wild type and  glossy    B. rapa cotyledons after severance,with standard error. Leaves were severed at the base of the petiole and allowed to air dry, estimatedtemperature: 24°C, estimated humidity: 27% Bioassays conducted on 10/10, 10/24, and 11/2 on  B. rapa  planted 9/22, 10/8, and 10/17 at Tufts University, Medford, MA. 5
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