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The Pennsylvania State University The Pennsylvania State University The Graduate School Department of Biology CHARACTERIZATION AND CLONING OF CAMOUFLAGE1, A MAIZE GENE FUNCTIONING IN THE TETRAPYRROLE SYNTHESIS PATHWAY A Dissertation in Biology by Mingshu Huang 2009 Mingshu Huang Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2009 ii The dissertation of Mingshu Huang was reviewed and approved* by the following: Sarah Assmann Waller Professor of Biology Chair of Committee David Braun Associate Professor of Biology Dissertation Advisor Paula McSteen Associate Professor of Biology Surinder Chopra Associate Professor of Crop and Soid Sciences Douglas Cavener Professor and Head of Biology *Signatures are on file in the Graduate School iii ABSTRACT During maize leaf development, polarized cell divisions result in clonal cell lineages arrayed along the long axis of the leaf. Utilizing this stereotypical division pattern, a collection of mutants that form chloroplast pigmentation sectors that violate the clonal cell lineages have been identified. Here, we describe the characterization of camouflage1 (cf1), a mutant which develops nonclonal, yellow-green sectors in its leaves. The cf1 gene was cloned by transposon tagging and found to encode porphobilinogen deaminase (PBGD), an enzyme that functions early in chlorophyll and heme biosynthesis. Although PBGD has been characterized biochemically, no viable mutations in this gene had been reported in plants. To investigate the in vivo function of PBGD, we characterized the cf1 mutant. Histological analyses revealed that cf1 yellow sectors display the novel phenotype of bundle sheath (BS) cell-specific death. Constant light suppressed cf1 sector formation, indicating that a dark/light transition is required to induce yellow sectors. Furthermore, sectors form only during a limited time in leaf development. Biochemical experiments determined that cf1 mutant leaves have decreased PBGD activity and increased levels of the enzyme substrate in both green and yellow regions. Moreover, the cf1 yellow regions displayed a reduction in catalase activity. A threshold model is proposed to explain cf1 variegation that incorporates photosynthetic cell differentiation, reactive oxygen species (ROS) scavenging and PBGD function. The restricted BS cell death in cf1 mutants resembles that observed in programmed cell death (PCD). PCD is commonly seen during plant development and in response to biotic and abiotic stress. Lesion mimic22 (Les22), another maize mutant in the tetrapyrrole biosynthesis pathway, displays a cell death pattern similar to PCD observed in the hypersensitive response (HR) during pathogen attack. Genetic analysis investigating whether cell death in the cf1 and Les22 mutants is mediated by the same mechanism showed that the two mutants initiate cell iv death independently. Moreover, accumulation of hydrogen peroxide (H2O2), a type of ROS involved in many PCD processes, is found in Les22 lesions but not in cf1 yellow or green sectors. Lack of a ROS burst, combined with a much reduced heme level in cf1 yellow sectors, supports our model that BS cell death in cf1 mutants is caused by a deficiency in ROS scavenging capability. Additionally, we found that functional chloroplasts are required for BS cell death in cf1 mutants, in contrast with the case of Les22. Therefore, the data suggest that PCD in cf1 is mediated by a different mechanism than in HR-related cell death. Gene duplication can increase functional diversity and specificity of genes. necrotic3 (nec3), a null mutation of cf1, is lethal if grown in a regular day/night cycle. When grown in a constant light environment, the cf1-nec3 phenotype is largely alleviated as evidenced by the seedling’s ability to make chlorophyll. These data indicate the presence of a functional duplicate gene of cf1. We probed a maize bacterial artificial chromosome (BAC) library and found a duplicate gene, camouflage2 (cf2). CF1 and CF2 share 97.5% identity in their protein sequence. Phylogenetic analysis revealed that cf1 and cf2 likely resulted from a gene duplication event. RT- PCR experiments determined that cf2 is expressed in all tissues examined, with the highest expression level detected in photosynthetic tissues. Similar to CF1, the CF2 protein also has a predicted plastid-target signal peptide in its N terminus, and a CF2 red fluorescent protein (CF2- RFP) fusion protein localizes to chloroplasts. The similarities in sequence, expression pattern and localization between CF1 and CF2 suggest that they have related functions in producing heme and chlorophyll. cf1 is the first viable mutant in PBGD in plants. Studies on cf1 mutants provide insight into the complicated regulatory network of tetrapyrrole biosynthesis, a pathway conserved from bacteria to higher eukaryotes including humans. Additionally, cf1 is the first mutant reported that display BS-specific cell death. This reveals a sensitivity difference of BS compared with mesophyll cells, to disturbance of an important metabolic pathway, probably due to an imbalance v between ROS generation and scavenging capability. Studies of a null mutation in cf1 uncovered the presence of a PBGD duplicate gene. Gene duplication of PBGD appears to be uncommon in plants, and this is the first case reported in the grass family, which may reflect the high degree of redundancy in the maize genome. vi TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................. viii LIST OF TABLES ................................................................................................................... x LIST OF ABBREVIATIONS .................................................................................................. xi ACKNOWLEDGEMENTS ..................................................................................................... xii Chapter 1 Introduction ............................................................................................................ 1 1.1 Maize leaf development and cell differentiation ........................................................ 1 1.2 Functional differentiation of C4 photosynthetic cells ................................................ 4 1.3 Chlorophyll and heme synthesis ................................................................................ 7 1.4 Cell death in plants ..................................................................................................... 13 1.5 Objectives of the research .......................................................................................... 15 Chapter 2 Phenotypic characterization and molecular cloning of camouflage1 ..................... 17 2.1 INTRODUCTION ..................................................................................................... 18 2.2 MATERIALS AND METHODS ............................................................................... 20 2.2.1 Growth conditions and genetic stocks ............................................................. 20 2.2.2 Starch staining and pigment analyses .............................................................. 20 2.2.3 Light and electron microscopy ........................................................................ 21 2.2.4 Photosynthesis and gas exchange measurements ............................................ 21 2.2.5 Cloning cf1 ...................................................................................................... 22 2.2.6 RT- and 5’ RACE-PCR ................................................................................... 22 2.2.7 Protein gel analyses and western blotting ....................................................... 24 2.2.8 PBGD sequence alignment .............................................................................. 25 2.2.9 PBGD subcellular localization ........................................................................ 25 2.2.10 PBG quantification, PBGD and catalase activity assays ............................... 26 2.3 RESULTS .................................................................................................................. 27 2.3.1 cf1 mutants develop nonclonal, yellow leaf sectors ........................................ 27 2.3.2 BS cell-specific death in cf1 yellow sectors .................................................... 29 2.3.3 Altered cell-specific RNA and protein accumulation ..................................... 38 2.3.4 Light/dark cycling is required for cf1 sector formation ................................... 40 2.3.5 Molecular characterization of cf1 .................................................................... 41 2.3.6 cf1 RNA expression ........................................................................................ 49 2.3.7 PBGD subcellular location .............................................................................. 50 2.3.8 PBGD activity ................................................................................................. 50 2.3.9 Diminished ROS scavenging capacity in cf1 yellow sectors .......................... 53 2.4 DISCUSSION ............................................................................................................ 53 Chapter 3 Bundle sheath cell death in cf1 requires functional chloroplasts............................ 62 3.1 INTRODUCTION ..................................................................................................... 62 3.2 MATERIALS AND METHODS ..............................................................................
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