THE INTEGRATION of LIGHT and PLASTID SIGNALS by Michael E
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THE INTEGRATION OF LIGHT AND PLASTID SIGNALS By Michael E. Ruckle A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Biochemistry and Molecular Biology 2010 ABSTRACT THE INTEGRATION OF LIGHT AND PLASTID SIGNALS by Michael E. Ruckle Proper chloroplast biogenesis and function are essential for agriculture and life on earth because photosynthesis drives plant growth, development, and reproduction. Photosynthesis-related gene expression was previously reported to be induced by light signaling and repressed by plastid signaling. Although light signaling and plastid signaling were previously thought to independently regulate the expression of these genes, data indicating that the regulation of photosynthesis-related gene expression by light and plastid signals depends on common promoter elements led me to hypothesize that light signaling and plastid signaling might be interactive processes and that these interactions might be significant. I first tested this hypothesis by screening a group of Arabidopsis mutants with defects in plastid signaling for light signaling phenotypes. Based on results from these experiments, I conclude that the blue light receptor cryptochrome1 (cry1) contributes to both the light and the plastid signaling that regulates the expression of genes encoding the light-harvesting chlorophyll a/b-binding protein (Lhcb) of photosystem II. I provide evidence that plastid signaling broadly ³UHZLUHV´OLJKWVLJQDOLQJDQGWKDWLQWKHFDVHRIWKHFU\VLJQDOLQJWKDWUHJXODWHVLhcb H[SUHVVLRQWKLV³UHZLULQJ´LVODUJHO\FDXVHGE\ the conversion of long hypocotyl 5 (HY5) from a positive regulator to a negative regulator of Lhcb expression. HY5 is a bZIP-type transcription factor that acts downstream of cry1 and other photoreceptors. I found that cry1-dependent plastid signals are genetically distinct from GENOMES UNCOUPLED 1 (GUN1)-dependent plastid signals and that the interactions between light and plastid signals appear critical for proper chloroplast biogenesis. Addtionally, I found that plastid signals can broadly affect light-regulated development of Arabidopsis seedlings. Results from these developmental assays are consistent with cry1 and GUN1 helping integrate chloroplast function with light regulated development. Based on these findings, I hypothesized that the interactions between light and plastid signaling promote chloroplast biogenesis by optimizing the expression of chloroplast-related genes for particular light environments and that plastid signaling broadly regulates light signaling by affecting possibly numerous signaling factors that act downstream of photoreceptors. We tested these ideas with time-resolved- expression profiling. Results from the expression profiling are consistent with interactions between light and plastid signaling optimizing not only chloroplast biogenesis but also coordinating plant growth and development with chloroplast function. Results from the reverse genetic analyses of Arabidopsis mutants yielded mutant alleles that cause abnormal chloroplast biogenesis. These alleles have defects in eighteen genes that encode transcription factors, signaling factors, and proteins of no known function. These findings provide evidence that light and plastid signaling are interactive processes that not only promote chloroplast biogenesis and function but also affect diverse processes related to plant growth and development. ACKNOWLEDGEMENTS First and foremost I would like to thank Rob Larkin, for always having his door open for helpful discussions, for pushing me as hard as he could to help me reach my potential, for always providing me with support for my ideas, and for being an excellent mentor. I owe much of my success to him. I would also like to thank my committee members Drs. Beronda Montgomery, Robert Last, Sheng Yang He, and Christoph Benning, for their support and guidance. I would like to thank my family, especially my mom, for providing me with the strong support that I needed along the way. I would like to my friends at MSU, for making graduate school enjoyable, and for always offering a helping hand when I needed it. I would like to thank Neil Adhikari for putting up with me as his office partner and lab-mate. Much of the work presented here came at the aid of several amazing students. I would like to thank Andrea Stavoe, Chris Sinkler, Lauren Lawrence, and Stephanie Demarco for all of their help. iv TABLE OF CONTENTS LIST OF TABLES««««.«««««««««««««««««««««««««Yii LIST OF ),*85(6««««««««««««««««««««««««««««YLLL CHAPTER 1: Introduction ,QWURGXFWLRQ««««««««««................................................................2 $GDSWLQJ WR WKH OLJKW«««««....................................................................14 Light-UHJXODWHG VLJQDO WUDQVGXFWLRQ QHWZRUNV«««««..............................12 Chloroplast Development.............................................................................22 3ODVWLG 6LJQDOLQJ««««...............................................................34 Integration of light and plastLG VLJQDOV«««««««««««««««42 )LJXUHV««««««««««««««««««««««««««««44 5HIHUHQFHV««««««««««««««««««««««««««1 CHAPTER 2: Plastid signals remodel light signaling networks and are essential for efficient chloroplast biogenesis in Arabidopsis Abstract.....................................................................................................71 Introduction...............................................................................................72 Results......................................................................................................77 Discussion.................................................................................................95 Materials and methods............................................................................105 $FNQRZOHGJHPHQWV«««««««««««««««««««««««10 )LJXUHV««««««««««««««««««««««««««««11 5HIHUHQFHV«««««««««««««««««««««««««««8 CHAPTER 3: Plastid signals that affect photomorphogenesis in Arabidopsis thaliana are dependent on GENOMES UNCOUPLED 1 and cryptochrome 1 Abstract...................................................................................................144 Introduction.............................................................................................145 Results....................................................................................................148 Discussion...............................................................................................157 Materials and methods............................................................................164 AcknowledgementV««««««««««««««««««««««8 )LJXUHV«««««««««««««««««««««««««««9 References««««««««««««««««««««««««««2 CHAPTER 4: Characterization of transcriptomes and signaling factors that contribute to chloroplast biogenesis in Arabidopsis Abstract...................................................................................................199 Introduction.............................................................................................200 Results....................................................................................................206 Discussion...............................................................................................231 Materials and methods............................................................................248 v $FNQRZOHGJPHQWV««««««««««««««««««««««««««3 )LJXUHV««««««««««««««««««««««««««««4 ReferencHV«««««««««««««««««««««««««««5 vi LIST OF TABLES -2 -1 Table 2.1 Segregation of the long hypocotyl phenotype in 25 ȝmol m s blue light inF seedlings 6 3 «««««««««««««««««««««««««««««« Table 4.1 Genes that exhibit enhanced light-induced expression in lincomycin-treated seedlings and their publicly available T-DNA alleles««««««««««««««3 Table 4.2 Genes that exhibit enhanced light-induced exprssion in lincomycin-treated seedlings and that do not have publicly available T-'1$DOOHOHV«««««««««7 Table 4.3 Alleles of genes that exhibit similar light-induced expression in lincomycin- treated and untreated seedlings that cause end phenotypes««««««««««8 Table 4.4 The diverse regulators of genes that exhibit enhanced light-induced expression in lincomycin-treated seedlings««««««««««««««««««2 vii LIST OF FIGURES Figure 1.1 The affect of light quality and quantity on plant growth and development««««««««««««««««««««««««««««««««4 Figure 1.2 3ODQWSKRWRUHFHSWRUV«««««««««««««««««««««««6 Figure 1.3 A simplified model of the known components of light signaling during phRWRPRUSKRJHQHVLV«««««««««««««««««««««««««47 Figure 1.4 The known light signaling network«««««««««««««««««8 Figure 1.5 Agriculturally important plastid types««««««««««««««««9 Figure 1.6 Examples the chloroplast adapWLQJWRWKHOLJKW««««««««««««1 Figure 1.7 Examples of mutations in chloroplast development««««««««««3 Figure 1.8 The balance of light and plastid signals during chloroplast development...54 Figure 1.9 Plastid signaling during chloroplast development«««««««««««6 Figure 1.10 Strategy IRUREWDLQLQJPXWDQWVWKDWUHJXODWHFKORURSODVWGHYHORSPHQW«8 Figure 2.1 Allelism of new gun mutants and cry1 mutants«««««««««««1 Figure 2.2 Expression of Lhcb and Rbcs in gun1 and cry mutants after chloroplast biogenesis was blocked««««««««««««««««««««««««««3 Figure 2.3 Expression of Lhcb and Rbcs in gun1 and cry1 after chloroplast biogenesis was blocked with various inhibitors of chloroplast biogenesis««««««««««4 Figure 2.4 Expression of Lhcb and Rbcs in cop1-4 and hy5 mutants