|Alos, Enriqueta - VALENCIAN INST., SPAIN|
|Roca, Maria - INST. SCIENT. RSCH, SPAIN|
|Iglesias, Domingo Jose - VALENCIAN INST., SPAIN|
|Minguez-Mosquera, Maria Isabel - INST. SCIENT. RSCH, SPAIN|
|Borges-Damasceno, Cynthia Maria - CORNELL UNIVERSITY|
|Campbell-Rose, Jocelyn - CORNELL UNIVERSITY|
|Talon, Manuel - VALENCIAN INST., SPAIN|
|Cercos, Manuel - VALENCIAN, INST., SPAIN|
Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 8, 2008
Publication Date: July 1, 2008
Citation: Alos, E., Roca, M., Iglesias, D., Minguez-Mosquera, M., Borges-Damasceno, C., Thannhauser, T.W., Campbell-Rose, J., Talon, M., Cercos, M. 2008. An evaluation of the basis and consequences of a stay-green mutation in the navel negra (nan) citrus mutant using transcriptomic and proteomic profiling and metabolite analysis. Journal of Plant Physiology. 147:1300-1315. Interpretive Summary: A mutant, termed navel negra (nan), was identified among a population of orange trees (Citrus sinensis) whose fruit developed a dark brown color upon ripening. This change in coloration was found to be confined to the zest. In order to determine the molecular basis of the abnormal coloration the pigment levels in the zest of the mutant were compared with those of wild-type over a range of developmental and ripening stages. The wild-type and mutant showed no differences in the total chlorophylls and carotenoid levels in the immature and mature green stages. However, where as the abundance of chlorophyll decreased dramatically during ripening in the wild-type fruit, it remained unchanged in nan. Expression analysis for the genes associated with pigment biosynthesis and catabolism was carried out which revealed that expression was similar in wild-type and nan in terms of relative abundance and changes during development. Treatment with the plant hormone ethylene caused a substantial reduction in of total chlorophyll in wild-type while no significant change could be detected in nan. Significantly, the expression patterns of the pigment biosynthesis and catabolism genes remained similar in both suggesting that nan is ethylene responsive but that this response is upstream of the signaling pathway that induces chlorophyll degradation. In order to gain insight into the consequences of the nan mutation comparative transcriptome and proteome analysis were carried out in parallel. The gene and protein expression profiling analyses together with the metabolite analyses reveal that the nan mutant shows numerous hallmarks of oxidative stress in the form of genes or proteins with a defined role in senescence, or in providing protection against reactive oxygen species. It is notable that nan shows substantial changes in gene and protein expressions at the mature green stage, prior to the normal onset of chlorophyll degradation or any detectable difference in chlorophyll levels compared with wild-type. This observation, together with the metabolite data, suggests that nan represents a new class of stay-green mutant with a lesion in a regulatory pathway that is upstream of the signal that induces chlorophyll degradation and that the mutant exhibits symptoms of oxidative stress prior to the onset of the normal degreening process.
Technical Abstract: A Citrus sinensis spontaneous mutant, navel negra (nan), produces fruit with an abnormal brown colored flavedo during ripening. Analysis of pigment composition in the wild type (WT) and nan flavedo suggested that typical ripening-related chlorophyll (Chl) degradation, but not carotenoid biosynthesis, was impaired in the mutant, identifying nan as a Type C stay-green mutant. nan exhibited normal expression of Chl biosynthetic and catabolic genes and chlorophyllase activity, but no accumulation of dephytylated chlorophyll compounds during ripening, suggesting that the mutation is not related to a lesion in any of the principal enzymatic steps in Chl catabolism. Transcript profiling using a citrus microarray indicated that a citrus ortholog of a number of SGR (stay green) genes was expressed at substantially lower levels in nan, both prior to, and during, ripening. However, the pattern of catabolite accumulation and SGR sequence analysis suggested that the nan mutation is distinct from those in previously described stay-green mutants and is associated with an upstream regulatory step, rather than directly influencing a specific component of Chl catabolism. Transcriptomic and comparative proteomic profiling further indicated that the nan mutation resulted in the suppressed expression of numerous photosynthesis-related genes and in the induction of genes that are associated with oxidative stress. These data, in addition to metabolite analyses, suggest that nan fruit employ a number of molecular mechanisms to compensate for the elevated Chl levels and associated photooxidative stress.