ABA Biosynthetic and Catabolic Pathways - Abscisic Acid: Metabolism, Transport and Signaling - page 26

plumbaginifolia (Marin et al. 1996 ). The ZEP gene encodes a monooxygenase
with a FAD binding domain. Recombinant ZEP protein required additives exist-
ing in the stroma fraction for its conversion activity from zeaxanthin to violaxan-
thin (Marin et al. 1996 ). Then, it was revealed that the activity of ZEP depended
on a reduced ferredoxin (Bouvier et al. 1996 ). Tobacco Npaba2 , Arabidopsis
Ataba1, rice Osaba1, and tomato hp3 mutants impaired in ZEP genes show
wilty and non-dormant seed phenotypes, and accumulate high levels of zeaxan-
thin but not epoxycarotenoids such as violaxanthin and neoxanthin (Table 2.1 )
(Agrawal et al. 2001 ; Duckham et al. 1991 ; Galpaz et al. 2008 ; Marin et al. 1996 ;
Rock and Zeevaart 1991 ). VDE may not be responsible for ABA biosynthesis, as
the Arabidopsis npq1 mutant defective in the VDE gene accumulated significant
amounts of epoxycarotenoids such as violaxanthin and neoxanthin that are precur-
sors of ABA (Niyogi et al. 1998 ).
After the epoxidation step, all- trans -violaxanthin is converted to the 9- cis iso-
mer prior to oxidative cleavage of the epoxycarotenoids to form xanthoxin by
9- cis -epoxycarotenoid dioxygenase (NCED) (Fig. 2.2 ). There are two possible
substrates for NCED in plants, the 9- cis isomers of violaxanthin and neoxanthin
(Fig. 2.2 ). In Arabidopsis leaves, all- trans -violaxanthin and 9′- cis -neoxanthin
were found to be the two major epoxycarotenoids (40.2 and 51.8 %, respectively)
of the total epoxyxanthophyll content. All- trans -neoxanthin and 9- cis -violaxan-
thin represented only 5.4 and 2.6 %, respectively (North et al. 2007 ). In tomato
leaves, a similar composition of epoxyxanthophylls was observed (Parry and
Horgan 1991 ). Therefore, 9′- cis -neoxanthin was supposed to be the major sub-
strate of NCED in leaves. Two enzymatic steps are thought to be involved in the
synthesis of 9′- cis -neoxanthin from all- trans -violaxanthin, involving neoxanthin
synthase (NSY) and an unknown isomerase (Fig. 2.2 ). Three different types of
NSY have been reported to date (Table 2.1 ). The first NSYs were biochemically
isolated from tomato and potato as homologs of capsanthin capsorubin synthase or
lycopene ʲ -cyclase (Al-Babili et al. 2000 ; Bouvier et al. 2000 ). The tomato NSY
enzyme was then found to be identical to the B gene encoding a fruit-specific lyco-
pene ʲ -cyclase isoform (Ronen et al. 2000 ), suggesting that NSY might be capa-
ble of converting both lycopene to ʲ -carotene and violaxanthin to neoxanthin in
tomato. However, neoxanthin was still produced in the old - gold mutant, a loss of
function allele of the B gene (Hirschberg 2001 ; Ronen et al. 2000 ). Other genes
responsible for neoxanthin synthesis were identified in the Arabidopsis aba4 and
tomato neoxanthin-deficient 1 ( nxd1 ) mutants lacking both isomers of neoxan-
thin (Neuman et al. 2014 ; North et al. 2007 ). The mutated genes in each mutant
encoded different types of unknown proteins. The Arabidopsis aba4 mutant had
a defect in a gene encoding a functionally unknown chloroplastic protein with
four transmembrane domains. The loss of neoxanthin in the mutant resulted in
reduced levels of ABA, and the phenotype was obvious under dehydration condi-
tions. The ABA-deficient phenotypes of the aba4 mutant were milder than those
of aba1 since aba4 was able to produce a small amount of ABA. The existence
of 9- cis -violaxanthin in the aba4 mutant could account for the mild phenotypes
(North et al. 2007 ). Biochemical analysis of recombinant ABA4 protein had