Antisense inhibition of the beta-carotene hydroxylase enzyme in Arabidopsis and the implications for carotenoid accumulation, photoprotection and antenna assembly.

The xanthophylls are oxygenated carotenoids and are important structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex apoproteins (LHC) and contribute to photoprotection via non-photochemical quenching of chlorophyll fluorescence (NPQ) in oxygenic photosynthetic organisms. Previously, mutations have been described that disrupt many steps in the xanthophyll biosynthetic pathway. However, there are no definitive reports of a lesion that effects the beta-hydroxylase enzyme, which catalyzes hydroxylation of the beta-rings of beta-carotene and alpha-carotene, and is thus necessary for synthesis of essentially all xanthophylls of higher plant chloroplasts. We have utilized an antisense approach to effectively reduce levels of beta-hydroxylase in Arabidopsis thaliana in order to examine how a reduction in this enzyme impacts carotenoid biosynthesis and plant viability. Expression of the antisense beta-hydroxylase transgene resulted in a maximal reduction in violaxanthin of 64% and a maximal reduction in neoxanthin of 41%. This reduction was reflected in a 22% increase in beta-carotene and a reduction in the total carotenoid pool, whereas lutein levels were relatively unaltered. Despite the reduction in violaxanthin and neoxanthin, the antisense beta-hydroxylase plants had a wild-type complement of chlorophylls and LHCs on a fresh weight basis. Under high light stress, the unconverted pool of violaxanthin was the same size as in wild type and thus there was an even greater proportional reduction in zeaxanthin of 75%. Despite this marked decrease in zeaxanthin, NPQ only declined by 16%.

Calcium/calmodulin-dependent protein kinase II and arrestin phosphorylation in Limulus eyes.

In rhabdomeral photoreceptors, light stimulates the phosphorylation of arrestin, a protein critical for quenching the photoresponse, by activating a calcium/calmodulin-dependent protein kinase (CaM PK). Here we present biochemical evidence that a CaM PK that phosphorylates arrestin in Limulus eyes is structurally similar to mammalian CaM PK II. In addition, cDNAs encoding proteins homologous to mammalian and Drosophila CaM PK II in the catalytic and regulatory domains were cloned and sequenced from a Limulus lateral eye cDNA library. The Limulus sequences are unique, however, in that they lack most of the association domain. The proteins encoded by these sequences may phosphorylate arrestin.

Genetic manipulation of carotenoid biosynthesis and photoprotection.

There are multiple complementary and redundant mechanisms to provide protection against photo-oxidative damage, including non-photochemical quenching (NPQ). NPQ dissipates excess excitation energy as heat by using xanthophylls in combination with changes to the light-harvesting complex (LHC) antenna. The xanthophylls are oxygenated carotenoids that in addition to contributing to NPQ can quench singlet or triplet chlorophyll and are necessary for the assembly and stability of the antenna. We have genetically manipulated the expression of the epsilon-cyclase and beta-carotene hydroxylase carotenoid biosynthetic enzymes in Arabidopsis thaliana. The epsilon-cyclase overexpression confirmed that lut2 (lutein deficient) is a mutation in the epsilon-cyclase gene and demonstrated that lutein content can be altered at the level of mRNA abundance with levels ranging from 0 to 180% of wild-type. Also, it is clear that lutein affects the induction and extent of NPQ. The deleterious effects of lutein deficiency on NPQ in Arabidopsis and Chlamydomonas are additive, no matter what the genetic background, whether npq1 (zeaxanthin deficient), aba1 or antisense beta-hydroxylase (xanthophyll cycle pool decreased). Additionally, increasing lutein content causes a marginal, but significant, increase in the rate of induction of NPQ despite a reduction in the xanthophyll cycle pool size.