Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated.
Chlorophyll is a central player in harvesting light energy for photosynthesis, yet the rate-limiting steps of chlorophyll catabolism and the regulation of the catabolic enzymes remain unresolved. To study the role and regulation of chlorophyllase (Chlase), the first enzyme of the chlorophyll catabolic pathway, we expressed precursor and mature versions of citrus (Citrus sinensis) Chlase in two heterologous plant systems: (1) squash (Cucurbita pepo) plants using a viral vector expression system; and (2) transiently transformed tobacco (Nicotiana tabacum) protoplasts. Expression of full-length citrus Chlase resulted in limited chlorophyll breakdown in protoplasts and no visible leaf phenotype in whole plants, whereas expression of a Chlase version lacking the N-terminal 21 amino acids (ChlaseDeltaN), which corresponds to the mature protein, led to extensive chlorophyll breakdown in both tobacco protoplasts and squash leaves. ChlaseDeltaN-expressing squash leaves displayed a dramatic chlorotic phenotype in plants grown under low-intensity light, whereas under natural light a lesion-mimic phenotype occurred, which was demonstrated to follow the accumulation of chlorophyllide, a photodynamic chlorophyll breakdown product. Full-length and mature citrus Chlase versions were localized to the chloroplast membrane fraction in expressing tobacco protoplasts, where processing of the N-terminal 21 amino acids appears to occur. Results obtained in both plant systems suggest that Chlase functions as a rate-limiting enzyme in chlorophyll catabolism controlled via posttranslational regulation.
Harpaz-Saad
Smadar
S
Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, Bet-Dagan 50250, Israel.
Azoulay
Tamar
T
Arazi
Tzahi
T
Ben-Yaakov
Eran
E
Mett
Anahit
A
Shiboleth
Yoel M
YM
Hörtensteiner
Stefan
S
Gidoni
David
D
Gal-On
Amit
A
Goldschmidt
Eliezer E
EE
Eyal
Yoram
Y
D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D002734:Chlorophyll / Q000737:chemistry / Q000378:metabolism*; D032084:Citrus sinensis / Q000201:enzymology*; D028464:Cucurbita / Q000235:genetics* / Q000528:radiation effects; D015870:Gene Expression / Q000528:radiation effects; D015971:Gene Expression Regulation, Enzymologic / Q000528:radiation effects; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects; D005822:Genetic Vectors; D007425:Intracellular Membranes / Q000201:enzymology / Q000528:radiation effects; D008027:Light; D050505:Mutant Proteins / Q000378:metabolism; D010641:Phenotype; D030821:Plants, Genetically Modified; D011523:Protoplasts / Q000378:metabolism / Q000528:radiation effects; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D014026:Nicotiana / Q000235:genetics* / Q000528:radiation effects
D050505:Mutant Proteins; D011994:Recombinant Proteins; D002734:Chlorophyll; D012694:Serine; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase