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Environmental Control of Plant Growth and Development and its Relation to Plant Hormones

Environmental factors such as photoperiod, light quality, temperature, and water-deficit (drought) have pronounced effects on plant growth and development. Plant growth substances often play a role as intermediaries between the perception of an environmental factor and the ultimate morphological expression. Our aim is to elucidate how environmental factors regulate the metabolism and action of these plant growth substances.

One line of research is concerned with the regulation of abscisic acid (ABA) biosynthesis. Wilting (=loss of turgor) increases the ABA content of leaves many times, whereas in ripening fruits ABA biosynthesis appears to be developmentally regulated. Our evidence obtained in 18O2 labeling studies indicates that there is one universal pathway of ABA biosynthesis, although its regulation is different in various organs of the plant. ABA (C15) is derived by oxidative cleavage from larger molecules, the xanthophylls (C40). The cleavage enzyme that catalyzes this step has been obtained as a fusion protein of maize (VP14) and specifically cleaves 9-cis-epoxycarotenoids (Fig. 1). Because the cleavage of xanthophylls is thought to be the regulatory step in ABA biosynthesis, further work is directed toward understanding how the cleavage enzyme is regulated. Genes encoding the cleavage enzyme for ABA biosynthesis have also been isolated from bean leaves and ripening avocado fruits, and in each case the cleavage reaction appears to be a limiting step in ABA biosynthesis.

Figure 1. Pathway of abscisic acid biosynthesis in higher plants. The metabolic blocks in the vp14mutant of maize and the aba2 and aba3 mutants of Arabidopsis are indicated.

In another project we are investigating how stem growth in long-day rosette plants is regulated by gibberellins (GAs). In spinach, long days (LD) are required for stimulating GA biosynthesis that leads to accumulation of the active gibberellin, GA1. Thus, this gibberellin is produced in amounts sufficient for stem growth only when spinach is grown under long photoperiods (Fig. 2).

Figure 2. Spinach plants exposed to 0, 4, 11, and 18 long days (left to right). The plants first respond to long days with petiole elongation, followed by stem elongation (bolting) and flowering.

Our objective is to find out how long photoperiods control the activity of enzymes involved in GA biosynthesis, with emphasis on the steps late in the pathway, gibberellin 20-oxidase, 3ß-hydroxylase, and 2-oxidase. We have found that expression of the gene for 20-oxidase is greatly enhanced when spinach plants are transferred from short- to long-day conditions. This multifunctional enzyme catalyzes the oxidation and elimination of carbon-20 of C20-GAs (Fig. 3). By contrast, expression of the genes encoding 3ß-hydroxylase and 2-oxidase is only slightly affected by the length of day.

Figure 3. The steps catalyzed by gibberellin 20-oxidase in the conversion of C20- to C19- gibberellins.

In spinach, bioactive GA1, and its precursor GA20, are deactivated by 2-oxidation. In addition, the precursor GA53, a C20-GA, can be 2ß-hydroxylated to GA97. In fact, the most abundant GA present in spinach is GA97. A gene encoding a 2-oxidase (So2ox3), which specifically oxidizes C20-GAs (GA53 to GA97) was isolated from spinach and ectopically expressed in the rosette plant Nicotiana sylvestris. In SD, the transgenic plants have smaller leaves, but the dwarfed phenotype is much more striking under LD conditions (Fig. 4). Under these conditions, bolting and flowering are much delayed in comparison with wild type, and the final stem height is reduced by over 50%. Thus, stem growth and flowering in rosette plants can be promoted or delayed by overexpression of GA 20-oxidase or GA 2-oxidase, respectively.

Figure 4. Effect of ectopic expression of So2ox3 in Nicotiana sylvestris. Left: Wild-type plant with flower buds. Right: Plant transformed with So2ox3 gene, still in rosette stage. Both plants were grown in long- day conditions following germination.

Selected Publications

Schwartz SH, Qin X, Zeevaart JAD (2003) Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol 131: 1591-1601

Schomburg FM, Bizzell CM, Lee DJ, Zeevaart JAD, Amasino RM (2003) Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants. Plant Cell 15: 151-163

Schwartz SH, Qin X, Zeevaart JAD (2003) Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol 131: 1591-1601

Lee DJ, JAD Zeevaart (2002) Differential regulation of RNA levels of gibberellin dioxygenases by photoperiod in spinach. Plant Physiol 130: 2085-2094

Qin X, Zeevaart JAD (2002) Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance. Plant Physiol 128: 544-551

Schwartz SH, Qin X, Zeevaart JAD (2001) Characterization of a novel carotenoid cleavage dioxygenase from plants. J Biol Chem 276: 25208-25211

Liu J, Yu J, McIntosh L, Kende H, Zeevaart JAD(2001) Isolation of a CONSTANS ortholog from Pharbitis niland its role in flowering. Plant Physiol 125: 1821-1830

Seo M, Peeters AJM, Koiwai H, Oritani T, Marion-Poll A, Zeevaart JAD, Koornneef M, Kamiya Y, Koshiba T (2000) The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves. Proc Natl Acad Sci USA 97:12908-12913

Chernys JT, Zeevaart JAD (2000) Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado. Plant Physiol 124:343-353

Xu Y-L, Li L, Gage DA, Zeevaart JAD (1999) Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis. Plant Cell 11: 927-935

Qin X, Zeevaart JAD (1999) The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean. Proc Natl Acad Sci USA 96: 15354-15361

Zeevaart JAD (1999) Abscisic acid metabolism and its regulation. In: PPJ Hooykaas, MA Hall and KR Libbenga, eds Biochemistry and Molecular Biology of Plant Hormones. Elsevier Science, Amsterdam, pp 189-207

Helliwell CA, Sheldon CC, Olive MR, Walker AR, Zeevaart JAD, Peacock WJ, Dennis ES (1998) Cloning of the Arabidopsis ent-kaurene oxidase gene GA3. Proc Natl Acad Sci USA 95: 9019-9024

Koornneef M, Léon-Kloosterziel KM, Schwartz SH, Zeevaart JAD (1998) The genetic and molecular dissection of abscisic acid biosynthesis and signal transduction in Arabidopsis. Plant Physiol Biochem 36: 83-89

Kende H, Zeevaart JAD (1997) The five "classical" plant hormones. Plant Cell 9: 1197-1210

Schwartz SH, Léon-Kloosterziel KM, Koornneef M, Zeevaart JAD (1997) Biochemical characterization of the aba2 and aba3 mutants in Arabidopsis thaliana. Plant Physiol 114: 161-166           

Schwartz SH, Tan BC, Gage DA, Zeevaart JAD, McCarty DR (1997) Specific oxidative cleavage of carotenoids by VP14 of maize. Science 276: 1872-1874

Tan BC, Schwartz SH, Zeevaart JAD, McCarty DR (1997) Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA 94: 12235-12240

Xu Y-L, Gage DA, Zeevaart JAD (1997) Gibberellins and stem growth in Arabidopsis thaliana. Effects of photoperiod on expression of the GA4 and GA5 loci. Plant Physiol 114:1471-1476

Wu K, Li L, Gage DA, Zeevaart JAD (1996) Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach. Plant Physiol 110: 547-554 (1996).

Xu Y-L., Li L, Wu K, Peeters AJM, Gage DA, Zeevaart JAD (1995) The GA5  locus ofArabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression. Proc Natl Acad Sci USA 92: 6640-6644

Zeevaart JAD, Gage DA, Talon M (1993) Gibberellin A1 is required for stem elongation in spinach. Proc Natl Acad Sci USA 90: 7401-7405