View of CAM (Crassulacean Acid Metabolism) Biology, Chlorophyll Fluorescence, and Leaf Optical Properties
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MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the dissertation of Nicolas Yebit Fondom Candidate for the Degree: Doctor of Philosophy Director Dr. Alfredo J. Huerta Reader Dr. David L. Gorchov Reader Dr. Nancy Smith-Huerta Reader Dr. Richard C. Moore Graduate School Representative Dr. Michael W. Crowder ABSTRACT PHYSIOLOGICAL AND BIOCHEMICAL ADAPTATIONS IN SOME CAM SPECIES UNDER NATURAL CONDITIONS: THE IMPORTANCE OF LEAF ANATOMY by Nicolas Y. Fondom This dissertation consists of an introduction and three independent research chapters. Chapter one (introduction) presents a review of CAM (Crassulacean Acid Metabolism) biology, chlorophyll fluorescence, and leaf optical properties. Chapter two addresses the physiology, anatomy, and biochemistry of two tropical Peperomia species in the field in the El Cielo Biosphere Reserve in Tamaulipas, Mexico. Chapters three and four address similar studies in two morphs of Agave striata (red vs. green leaves) under natural condition in the high plateau desert region of Miquihuana, Tamaulipas, Mexico. The summary for each of the research chapters is discussed. In chapter two, I tested, under field conditions, the hypotheses that relative thickness of the spongy mesophyll layer is an indication of CAM and that water distribution within the leaf tissues of the two Peperomia species studied changes differently in response to drought. The results showed that when well-watered, P. obtusifolia has typical C3-activity and P. macrostachya has typical CAM-activity, with spongy mesophyll layer thickness of 26% and 76% respectively. In addition, the results also showed that the distribution of water in the leaf tissue in P. obtusifolia changes in response to drought, from the hydrenchyma to the chlorenchyma, but this is not the case in P. macrostachya. In chapter three, I tested the hypothesis that leaf epidermal pigments screen light of particular wavelengths from reaching the photosynthetic machinery, reducing dependence on the xanthophyll cycle as an energy dissipation process. The results showed that Fo (dark level fluorescence), Fv/Fm (maximum PSII efficiency), and ΦPSII (quantum yield of PSII) were higher under sun in leaves of the red morph compared to the green morph. Leaf surface temperature, xanthophyll conversion, and qN (non-photochemical quenching) were significantly higher during the day for leaves of the green morph compared to the red morph. From these results, it is clear that the sub-epidermal anthocyanins serve as a photoprotective barrier in the leaves of the red morph. Finally, in chapter four, the first goal of this project was to test the hypothesis that leaf wax development is a delayed response to sunlight exposure following cutin development. The second goal of this project tested the hypothesis that during leaf ontogeny in the two morphs of A. striata, energy dissipation shifts from non-photochemical to photochemical quenching. The results showed that on both the adaxial and abaxial leaf surfaces of young and mature leaves of both morphs, leaf wax thickness increased from the basal end of the leaf whereas cutin thickness decreased from the basal end of the leaf. Thus, as leaf age in both morphs of A. striata, cutin deposition exceeds wax deposition at the early stages of development. Furthermore, chlorophyll fluorescence data supported the photoprotective function of anthocyanin, indicating that the deposition of anthocyanins may help to reduce dependence of the leaf on energy dissipation via the xanthophyll cycle and non-photochemical quenching during leaf ontogeny. PHYSIOLOGICAL AND BIOCHEMICAL ADAPTATIONS IN SOME CAM SPECIES UNDER NATURAL CONDITIONS: THE IMPORTANCE OF LEAF ANATOMY A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Botany by Nicolas Yebit Fondom Miami University Oxford, Ohio 2009 Dissertation Director: Dr. Alfredo J. Huerta TABLE OF CONTENTS 1. Chapter 1. Introduction……………………………………………………………………….1 1.1. What is CAM?……………………………………………………………………………2 1.1.1. Taxonomic distribution of CAM plants……………………………………..…….2 1.1.2. Regulation of CAM cycle…………………………………………………………2 1.1.3. Variability in CAM metabolism…………………………………………………..5 1.1.4. Carbon gain/balance in CAM plants………………………………………………6 1.2. Chlorophyll Fluorescence………………………………………………………………...8 1.2.1. The basis of chlorophyll fluorescence and its measurements……..………………9 1.2.2. Chlorophyll fluorescence during CAM-Phases…………………..……………...13 1.3. Leaf optical properties……………….………………………………………………….14 1.3.1. The link between leaf spectral characteristics and stress………..……………….15 1.3.2. Leaf optical properties and anthocyanin content…………………………..…….15 1.4. General overview of the dissertation....………………………………………………....16 References……………………………………………………………………………….20 2. Chapter 2. Seasonal variation in photosynthesis and diel carbon balance under natural conditions in two Peperomia species that differ with respect to leaf anatomy.......................29 2.1. Abstract………………………………………………………………………………….30 2.2. Introduction……………………………………………………………………………...31 2.3. Materials and Methods………………………………………………………………….33 2.4. Results…………………………………………………………………………………...37 2.5. Discussion…………………………………………………………………………….....40 2.6. Conclusions………………………………………………………………………..….....44 References….....................................................................................................................45 3. Chapter 3. Field assessment of sub-epidermal leaf anthocyanin, PSII photochemistry, and the xanthophyll-cycle as photoprotective mechanisms in two morphs of Agave striata.........57 3.1. Abstract…...……………………………………………………………………………..58 3.2. Introduction…………………………………………………………………………...…59 ii 3.3. Materials and Methods……………………………………………………………….…61 3.4. Results…………………………………………………………………………………...66 3.5. Discussion……………………………………………………………………………….69 3.6. Conclusions……………………………………………………………………………...71 References……………………………………………………………………………….73 4. Chapter 4. Photoprotective mechanisms during leaf ontogeny: Cuticular development and anthocyanin deposition in two morphs of Agave striata that differ in leaf coloration…........86 4.1. Abstract………………………………………………………………………………….87 4.2. Introduction……………………………………………………………………………...88 4.3. Materials and Methods………………………………………………………………….90 4.4. Results………………………………………………………………………………...…94 4.5. Discussion…………………………………………………………………………….....97 4.6. Conclusions…………………………………………………………………………….101 References……………………………………………………………………………...103 5. Chapter 5. Conclusions and Future Prospects……………..………….……………...........117 References……………………………………………………………………………...123 iii LIST OF TABLES Chapter 2 Table 1: Thickness of the hydrenchyma, chlorenchyma, lower spongy mesophyll layer ………49 Table 2: Net CO2 gained and respiratory CO2 recycling of two Peperomia………………….....50 Table 3: RWC, ΨTotal, ΨPressure, and ΨSolute of two Peperomia species collected ………………..51 Chapter 3 Table 1: RWC, ΨTotal, ΨPressure, and ΨSolute of two morphs of A. striata collected at …….……..78 Chapter 4 Table 1: Adaxial leaf surface reflectance (%) in leaves of both morphs in all…………………108 iv LIST OF FIGURES Chapter 1 Figure 1: A generalized carbon fluxes and key CAM regulatory enzymes ……………………....4 Figure 2: A typical chlorophyll fluorescence kinetics or Kautsky curve ……………………........9 Figure 3: Cross-section photographs of both the green and the red morphs of A. striata……….16 Figure 4: Photographs oriented with adaxial epidermis up for both Peperomia species………..18 Chapter 2 Figure 1: Net CO2 exchange in two Peperomia species that differ with ………………………..52 Figure 2: Instantaneous water use efficiency in leaves of two Peperomia ……………………...53 Figure 3: Stomatal conductance and transpiration rate in two Peperomia………........................54 Figure 4: Total titratable acidity measured in leaves of two Peperomia …………………..……55 Figure 5: Chlorophyll fluorescence induction kinetics; Fo, Fv/Fm, ETR, ΦPSII, qP and qN …....56 Chapter 3 Figure 1: Ambient temperature and light intensity within experimental plots………………..…79 Figure 2: Total titratable acidity in leaves of both morphs measured during a 24 h period..........80 Figure 3: Chlorophyll fluorescence induction kinetics under full sunlight and shade; …………81 Figure 4: Percent adaxial surface reflectance, epidermal and sub-epidermal……………………82 Figure 5: Percent reflectance and change in reflectance in leaves of both morphs ......................83 Figure 6: Percent change in photochemical reflectance index (∆PRI), an indication of ……......84 Figure 7: Internal and surface leaf temperature in leaves of both the red and green ……………85 Chapter 4 Figure 1: A schematic structural arrangement of leaves in all three leaf-age groups……..……109 Figure 2: Average epidermal wax and cutin thickness in leaves of both morphs ………...........110 Figure 3: Percentage epidermis/sub-epidermis containing anthocyanin. ……………………...111 Figure 4: Percentage reflectance of adaxial surface in leaves of both morphs…………………112 Figure 5: NDVI (chlorophyll content) index, RRed:RGreen (anthocyanin content) ……………...113 v Figure 6: Chlorophyll fluorescence induction kinetics; Fo, FvFm, ΦPSII, and qN ……………..114 Figure 7: Surface leaf temperature in leaves of both the red and green morphs……………….115 Figure 8. Total titratable acidity measured in leaves of both the red and green morphs………116 vi DEDICATION This dissertation is dedicated to my mother, Mrs. Magdaline Ambit Fondom, for giving me every opportunity