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. 1998 Nov;118(3):1021-8.
doi: 10.1104/pp.118.3.1021.

Expression studies of the zeaxanthin epoxidase gene in nicotiana plumbaginifolia

C Audran  1 , C Borel, A Frey, B Sotta, C Meyer, T Simonneau, A Marion-Poll
Affiliations

Expression studies of the zeaxanthin epoxidase gene in nicotiana plumbaginifolia

C Audran et al. Plant Physiol. 1998 Nov.

Abstract

Abscisic acid (ABA) is a plant hormone involved in the control of a wide range of physiological processes, including adaptation to environmental stress and seed development. In higher plants ABA is a breakdown product of xanthophyll carotenoids (C40) via the C15 intermediate xanthoxin. The ABA2 gene of Nicotiana plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes the conversion of zeaxanthin to violaxanthin. In this study we analyzed steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in all plant organs, but transcript levels were found to be higher in aerial parts (stems and leaves) than in roots and seeds. In leaves ABA2 mRNA accumulation displayed a day/night cycle; however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the ABA2 mRNA level peaked around the middle of development, when ABA content has been shown to increase in many species. In conditions of drought stress, ABA levels increased in both leaves and roots. A concomitant accumulation of ABA2 mRNA was observed in roots but not in leaves. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.

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Figures

Figure 1
Figure 1
ABA2 mRNA accumulation in N. plumbaginifolia plants. Total RNA from roots (R), stems (St), rosette leaves (rL), cauline leaves (cL), and flowers (a, 3 mm; b, 5 mm; c, 8 mm; d, 15 mm; e, 20 mm; and f, 50 mm in length) were hybridized with an ABA2 probe and then with a 25S rRNA probe as a loading control.
Figure 2
Figure 2
Steady-state ABA2 mRNA levels in developing seeds of wild-type plants. RNA was extracted from seeds harvested from 3 to 22 DAP when seed capsules were opening. Ethidium bromide staining of 25S RNA is shown as a control.
Figure 3
Figure 3
ABA levels in response to dehydration in leaves of the wild type (WT) and of the mutant aba2-s1. ABA was quantified before (not stressed, NS) and after (stressed, S) detached leaves were placed for 1 h under a laminar flow hood. Similar results were obtained in four independent experiments. Only one is presented here. DW, Dry weight.
Figure 4
Figure 4
Immunoblot of leaf extracts from the mutant aba2-s1 and from the wild type (WT). Nine wild-type extracts were tested during a period of 24 h. Because the mutant aba2-s1 produced no detectable ABA2 protein, only one extract was loaded. Mutant leaves were collected 3.5 h after the beginning of the light period. Proteins were visualized with an antiserum against the ABA2 protein.
Figure 5
Figure 5
Diurnal rhythm in ABA2 mRNA abundance in N. plumbaginifolia leaves. Leaf total RNA was extracted at different intervals during 24 h and hybridized with an ABA2 probe. Ethidium bromide staining of 25S RNA is shown as a control.
Figure 6
Figure 6
ABA accumulation and ABA2 transcript levels in dehydrated leaves and roots. Entire N. plumbaginifolia plants were dehydrated under a laminar flow hood for 0 h (white bars), 4 h (checkered bars), or 8 h (black bars). Three independent experiments are presented here (1, 2, and 3). Relative abundance of ABA was measured in leaves (A) and in roots (B). Hormonal content was determined five times for each sample. Total RNA was extracted from leaves and roots and used for northern analysis using ABA2 and 25S rRNA probes. Relative ABA2 mRNA expression levels were determined using 25S rRNA as a standard in leaves (C) and in roots (D). Relative abundance of ABA was calculated by giving the value 1 to the ABA level observed at 0 h of dehydration, and relative abundance of ABA2 mRNA was calculated by giving the value 1 to the mRNA level observed at the same time in roots as in leaves, even if their absolute values were different.
Figure 7
Figure 7
ABA2 transcript levels in roots after an imposed dehydration. N. plumbaginifolia plants were cultivated in a hydroponic device. Roots were cut and divided into two batches. One was immediately sampled (100% RWC) and another was rapidly dehydrated in a stream of dry air until its weight was at 60% RWC. Samples were incubated from 30 min to 4 h. Total RNA from roots was extracted, and relative ABA2 mRNA expression levels were determined using 25S rRNA as a standard.
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