Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado

Abstract

Avocado (Persea americana Mill. cv Lula) is a climacteric fruit that exhibits a rise in ethylene as the fruit ripens. This rise in ethylene is followed by an increase in abscisic acid (ABA), with the highest level occurring just after the peak in ethylene production. ABA is synthesized from the cleavage of carotenoid precursors. The cleavage of carotenoid precursors produces xanthoxin, which can subsequently be converted into ABA via ABA-aldehyde. Indirect evidence indicates that the cleavage reaction, catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED), is the regulatory step in ABA synthesis. Three genes encoding NCED cleavage-like enzymes were cloned from avocado fruit. Two genes, PaNCED1 and PaNCED3, were strongly induced as the fruit ripened. The other gene, PaNCED2, was constitutively expressed during fruit ripening, as well as in leaves. This gene lacks a predicted chloroplast transit peptide. It is therefore unlikely to be involved in ABA biosynthesis. PaNCED1 was induced by water stress, but expression of PaNCED3 was not detectable in dehydrated leaves. Recombinant PaNCED1 and PaNCED3 were capable of in vitro cleavage of 9-cis-xanthophylls into xanthoxin and C(25)-apocarotenoids, but PaNCED2 was not. Taken together, the results indicate that ABA biosynthesis in avocado is regulated at the level of carotenoid cleavage.

Figure 1

The cleavage reaction in ABA biosynthesis. Both 9-cis-violaxanthin and 9′-cis-neoxanthin can be cleaved to xanthoxin, which can subsequently be converted into ABA.

Figure 2

Alignment of the deduced amino acid sequences of PaNCED1, PaNCED2, and PaNCED3 from avocado with VP14 from maize. Amino acid residues identical in at least three of the sequences are indicated by black boxes. The arrows indicate the regions that were used in the design of the degenerate primers.

Figure 3

Dot blot demonstrating the specificity of the PaNCED probes. In vitro transcribed mRNA of PaNCED1, PaNCED2, and PaNCED3 (0.2 ng or 2 ng) were applied in duplicate to the membrane and hybridized with gene-specific probes against PaNCED1, PaNCED2, or PaNCED3.

Figure 4

Changes in ABA and ethylene levels, and in PaNCED1, PaNCED2, and PaNCED3 transcript accumulation during the course of avocado fruit ripening. A, Analysis of ABA and ethylene levels plotted as a function of days of ripening. B, Northern analysis of NCED gene expression in the same fruits. Total RNA (30 μg per lane) was isolated from fruit, separated by gel electrophoresis, and blotted onto nylon membranes. The same blot used for analysis of PaNCED1 was stripped and reprobed with probes against PaNCED2 and subsequently PaNCED3 and 17S rDNA. The specific probes used for the PaNCED genes are described in “Materials and Methods.” The 17S rDNA probe was used as a loading control.

Figure 5

Accumulation of ABA (A), and of PaNCED1, PaNCED2, and PaNCED3 transcripts (B) in response to wilting of avocado leaves. RNA-blot hybridizations were carried out with total RNA (30 μg per lane) isolated from leaves that had been wilted to increasing percentages of their fresh weights. The leaves were wilted using a pressure chamber for approximately 15, 30, and 50 min to achieve water losses of 5%, 12%, and 20%, respectively. After this time, the leaves were incubated in the dark for 4 h, and then frozen in liquid N₂. The specific probes used for the NCED genes and the probe (17S rDNA) used as a control are described in “Materials and Methods.”

Figure 6

Enzyme activities of the NCED1 and NCED3 proteins. A, Increase in xanthoxin formed from either 9′-cis-neoxanthin (▴) or 9-cis-violaxanthin (●) as a function of PaNCED1 (---) or PaNCED3 (—) protein concentrations. Assays contained 6 nmol of substrate. B, Xanthoxin formed by PaNCED1 (●) and PaNCED3 (▪) as a function of 9-cis-violaxanthin concentrations. The xanthoxin and C₂₅-apocarotenoids produced in the in vitro reaction were analyzed by HPLC and identified by mass spectrometry.