ABA Biosynthetic and Catabolic Pathways - Abscisic Acid: Metabolism, Transport and Signaling - page 30

difficulty showing the NSY activity since the protein tended to be insoluble (North
et al. 2007 ). The tomato nxd1 mutant had a defect in an unknown protein with-
out an obvious chloroplast targeting sequence. Although the tomato nxd1 mutant
did not produce either isomer of neoxanthin, it did not show any ABA-deficient
phenotypes. The existence of 9- cis -violaxanthin could compensate for the loss
of neoxanthin in the nxd1 mutant (Neuman et al. 2014 ). Arabidopsis NXD1 pro-
tein expressed in E. coli also failed to show NSY activity even when it was coex-
pressed with ABA4 (Neuman et al. 2014 ). Further analysis will be necessary to
reveal how these genes contribute to neoxanthin synthesis. The results from the
analyses of neoxanthin-deficient mutants indicate that the preferred substrate of
NCED could differ according to plant species and both 9- cis -violaxanthin and
9′- cis -neoxanthin could be the in vivo substrates of NCED. In fact, the parasitic
plant Cuscuta reflexa does not have neoxanthin but still accumulates ABA under
dehydration (Qin et al. 2008 ).
All- trans -epoxycarotenoids, violaxanthin and neoxanthin, are then converted
into 9- cis isomers by an unknown isomerase (Fig. 2.2 ). It has been a long-stand-
ing question that gene encodes the 9- cis -epoxycarotenoid-forming isomerase in
ABA biosynthesis. Recently, Alder et al. ( 2012 ) (Alder et al. 2012 ) found that the
rice strigolactone biosynthetic enzyme D27 had isomerase activity converting all-
trans - ʲ -carotene into 9- cis - ʲ -carotene and that the conversion activity was revers-
ible. The structural similarity between ʲ -carotene and epoxycarotenoids evokes the
possible involvement of D27 in ABA biosynthesis. Rice has two additional genes
homologous to D27, and Arabidopsis has three genes showing similarity to D27 .
Further analyzes of these genes and the corresponding mutants will help to dis-
cover the isomerase. Identification of the isomerase is the final piece for establish-
ing the main framework of the ABA biosynthetic pathway in plants.
9- cis isomers of violaxanthin and neoxanthin are then cleaved into C15 and
C25 compounds to produce the C15 xanthoxin, the direct precursor of ABA
(Fig. 2.2 ). The first discovery of 9- cis -epoxycarotenoid dioxygenase came in
the study of the viviparous, ABA-deficient, maize vp14 mutant (Schwartz et al.
1997b ; Tan et al. 1997 ). VP14 encodes a non-heme iron (II)-dependent dioxyge-
nase. Recombinant VP14 recognized the 9- cis configuration of C40 carotenoids
and was able to cleave 9- cis -violaxanthin, 9′- cis -neoxanthin, and 9- cis -zeaxanthin
at the 11-12 position but not all- trans isomers (Schwartz et al. 1997b ). After the
cleavage reaction in vitro, C25 compounds such as epoxy-apo-aldehyde or allenic-
apo-aldehyde were generated as by-products with C15 xanthoxin. Whereas xan-
thoxin was detectable in plants, neither of these two C25 by-products could be
detected (Parry and Horgan 1991 ; Schwartz and Zeevaart 2010 ). After the clon-
ing of VP14 , NCED genes were isolated from various plant species (Table 2.1 ).
Overexpression of NCED genes resulted in higher accumulation of ABA in the
transgenic plants, indicating that NCED catalyzes the rate-limiting step of ABA
biosynthesis. NCED genes form a small gene family in various plant species. Most
plants have a drought-inducible NCED gene whose expression levels are corre-
lated with the accumulation of ABA in response to drought. Carotenoids includ-
ing the substrate of NCED are abundantly localized at the thylakoid membrane in