Identification Of NYE1, NYE2, CRN1 And Their Roles In Chlorophyll Degradation In Arabidopsis

Chlorophyll(Ch1) loss during leaf senescence and fruit ripening represents the most splendid autumn scenery that attracts millions of people every year.A biochemical pathway of Ch1 degradation has been established in last decades.However,the genetic regulation of Ch1 catabolism is barely understood.This thesis will describe some new discoveries and discuss their significance in this process by using Arabidopsis as a model plant.A non-yellowing mutant,nye1-1,was identified from the M2 population of fast-neutron mutagenized Arabidopsis thaliana(Co1-0).The degradation of both Ch1 and Ch1 binding proteins were greatly restrained in nye1-1.Nevertheless,neither photosynthesis- nor senescence-associated process was significantly affected,suggesting that nye1-1 was a type-C cosmetic stay-green mutant.Enzymatic analysis revealed that the total Chlorophyllase activity was not obviously influenced while a significant reduction in PaO activity was detected in nye1-1.However,no detectable accumulation of either chlorophyllide a or pheophorbide a was observed based on HPLC analysis.Reciprocal crossings revealed that the mutant phenotype was caused by a monogenic semi-dominant nuclear mutation.We identified NYE1 by positional cloning and confirmed by genomic complementation test.nye1-1 was a null mutant with the L10＞stop mutation.Constitutive over-expression of NYE1 could result in either pale-yellow true leaves or even albino seedlings.Further analysis revealed the severity of the degreening in the NYE1 over-expressors was correlated with the expression level of NYE1.These results collectively indicated that NYE1 plays an important regulatory role in Ch1 degradation during leaf senescence.NYE1 was drastically induced by senescence signals and encodes a novel chloroplast protein,which was highly conserved among plant species.NYE2 is a putative paralog of NYE1 with an approximately 75%similarity in Arabidopsis.Whether NYE2 acts in the Ch1 degradation pathway remains unknown.A T-DNA insertion line of NYE2 wasn't stay-green during leaf senescence.However,by employing dexamethasone (DEX)-inducible over-expression system,we found over-expression of NYE2 could efficiently cause leaf yellowing phenotype,even in nye1-1 background.This result indicated that NYE2 may be also involved in Ch1 degradation,possibly through mimicking the function of NYE1.Further characterization of nye1-1/nye2-1 double mutant will facilitate elucidating of the role of NYE2 in Ch1 breakdown.Although highly conserved,domain sequences of NYE1/2 hardly provide any cues about its possible biochemical roles. Identification of additional stay-green genes will be exclusively important for the further understanding the regulatory mechanisms of Ch1 catabolic pathway and may also give inspiration to the function of NYE1/2.Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes share similar expression pattern with NYE1.Subsequently characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1(Co-regulated with NYE1).CRN1 encodes a putative chloroplast protein with a typical esterase/lipase domain(EC3.1.1),supposing it may have chlorophyllase/pheophytinase activity.The Ch1 breakdown was drastically restrained in crn1-1 under various leaf senescence conditions,which was comparable with that in acd1-20(a knockout mutant of PaO),but much severer than that in nye1-1. Surprisingly but interestingly,the photochemical efficiency of PSII(by evaluating the ratio of Fv/Fm) began to decrease more rapidly after two days dark-incubation of detached leaves in crn1-1,as compared with Co1-0 and nye1-1,supposing that CRN1 may also contribute to the maintaining of the residual capability of photosynthesis during leaf senescence.At the protein level,we found the large subunit of Rubisco degraded normally in crn1-1 during leaf senescence,while all the Ch1 binding proteins detected were more or less retained, suggesting the removal of Ch1 from pigment-protein complex appears to be a perquisite step for both Ch1 and Ch1 binding protein degradation,however,the mechanism seems to be distinct in different stay-green mutants.To explore the function of NYE1 and CRN1,we also employed yeast two hybrid system to study their possible interaction status with other Ch1 degradation proteins.Our preliminary result showed NYE1,CRN1,PaO and RCCR didn't interact with each other.Conclusively,the thesis mainly contributed to the molecular identification and functional characterization of three Ch1 degradation related genes:NYE1, NYE2 and CRN1 by using both forward and reverse genetic approaches in Arabidosis.