Multiple loss-of-function mutations of carotenoid cleavage dioxygenase 4 reveal its major role in both carotenoid level and apocarotenoid composition in flue-cured mature tobacco leaves.

Apocarotenoid volatiles contribute to the flavor of many agricultural products. In many flowering plants, carotenoid cleavage dioxygenase 4 (CCD4) is involved in the decomposition of carotenoids and resultant production of C13-apocarotenoids, such as ß-ionone derived from ß-carotene. To understand the possible role of tobacco CCD4 genes (NtCCD4-S, NtCCD4-T1 and NtCCD4-T2) in these processes, we analyzed loss-of-function phenotypes. RNA interference transgenic plants showed yellow color in mature (senescent) leaves. Mature leaves of chemically induced double-mutant plants showed a stronger yellow color, and those of triple-mutant plants showed a pronounced yellow color. Carotenoid analysis of the leaves from mutants showed that lutein and ß-carotene increased in line with the degree of color change compared to wild type, whereas there was little change in green color in their young leaves. This result indicates that CCD4s are important for the decomposition of carotenoids in the tobacco leaf maturation process. Analysis of apocarotenoids in flue-cured leaves of the multiple-mutant plants showed that many compounds, including megastigmatrienones, were decreased in comparison to wild type, whereas intriguingly ß-ionone and dihydroactinidiolide were increased. Our results suggest that CCD4s play a key role in both carotenoid level and apocarotenoid composition in flue-cured mature tobacco leaves.

Functional analysis of Arabidopsis CYP714A1 and CYP714A2 reveals that they are distinct gibberellin modification enzymes.

Endogenous levels of bioactive gibberellins (GAs) are controlled by both biosynthetic and inactivation processes, and some cytochrome P450s are involved in this control mechanism. We have previously reported that CYP714B1 and CYP714B2 encode the enzyme GA 13-oxidase, which is required for GA1 biosynthesis, and that CYP714D1 encodes GA 16α,17-epoxidase, which inactivates the non-13-hydroxy GAs in rice. Arabidopsis has two CYP714 members, CYP714A1 and CYP714A2. To clarify the possible role of these genes in GA metabolism, enzymatic activities of their recombinant proteins were analyzed using a yeast expression system. We found that the recombinant CYP714A1 protein catalyzes the conversion of GA12 to 16-carboxylated GA12 (16-carboxy-16ß,17-dihydro GA12), a previously unidentified GA metabolite. Bioassays of this GA product showed that CYP714A1 is an inactivation enzyme in Arabidopsis. This was confirmed by the extreme GA-deficient dwarf phenotype shown by CYP714A1-overexpressing plants. Intriguingly, the recombinant CYP714A2 protein catalyzed the conversion of ent-kaurenoic acid into steviol (ent-13-hydroxy kaurenoic acid). When GA12 was used as a substrate for CYP714A2, 12α-hydroxy GA12 (GA111) was produced as a major product and 13-hydroxy GA12 (GA53) as a minor product. Transgenic Arabidopsis plants overexpressing the CYP714A2 gene showed semi-dwarfism. GA analysis showed that the levels of non-13-hydroxy GAs, including GA4, were decreased, whereas those of 13-hydroxy GAs, including GA1 (which is less active than GA4), were increased in the transgenic plants. Our results suggest that the CYP714 family proteins contribute to the production of diverse GA compounds through various oxidations of C and D rings in both monocots and eudicots.

CYP714B1 and CYP714B2 encode gibberellin 13-oxidases that reduce gibberellin activity in rice.

Bioactive gibberellins (GAs) control many aspects of growth and development in plants. GA(1) has been the most frequently found bioactive GA in various tissues of flowering plants, but the enzymes responsible for GA(1) biosynthesis have not been fully elucidated due to the enzymes catalyzing the 13-hydroxylation step not being identified. Because of the lack of mutants defective in this enzyme, biological significance of GA 13-hydroxylation has been unknown. Here, we report that two cytochrome P450 genes, CYP714B1 and CYP714B2, encode GA 13-oxidase in rice. Transgenic Arabidopsis plants that overexpress CYP714B1 or CYP714B2 show semidwarfism. There was a trend that the levels of 13-OH GAs including GA(1) were increased in these transgenic plants. Functional analysis using yeast or insect cells shows that recombinant CYP714B1 and CYP714B2 proteins can convert GA(12) into GA(53) (13-OH GA(12)) in vitro. Moreover, the levels of 13-OH GAs including GA(1) were decreased, whereas those of 13-H GAs including GA(4) (which is more active than GA(1)) were increased, in the rice cyp714b1 cyp714b2 double mutant. These results indicate that CYP714B1 and CYP714B2 play a predominant role in GA 13-hydroxylation in rice. The double mutant plants appear phenotypically normal until heading, but show elongated uppermost internode at the heading stage. Moreover, CYP714B1 and CYP714B2 expression was up-regulated by exogenous application of bioactive GAs. Our results suggest that GA 13-oxidases play a role in fine-tuning plant growth by decreasing GA bioactivity in rice and that they also participate in GA homeostasis.

Involvement of the CYP78A subfamily of cytochrome P450 monooxygenases in protonema growth and gametophore formation in the moss Physcomitrella patens.

CYP78 is a plant-specific family of cytochrome P450 monooxygenases, some members of which regulate the plastochron length and organ size in angiosperms. The CYP78 family appears to be highly conserved in land plants, but there have been no reports on the role of CYP78s in bryophytes. The moss, Physcomitrella patens, possesses two CYP78As, CYP78A27 and CYP78A28. We produced single and double mutants and overexpression lines for CYP78A27 and CYP78A28 by gene targeting to investigate the function of CYP78As in P. patens. Neither the cyp78a27 nor cyp78a28 single mutant showed any obvious phenotype, while the double mutant exhibited severely retarded protonemal growth and gametophore development. The endogenous levels of some plant hormones were also altered in the double mutant. Transgenic lines that overexpressed CYP78A27 or CYP78A28 showed delayed and reduced bud formation. Our results suggest that CYP78As participate in the synthesis of a critical growth regulator in P. patens.

Contribution of strigolactones to the inhibition of tiller bud outgrowth under phosphate deficiency in rice.

Strigolactones (SLs) or SL-derived metabolite(s) have recently been shown to act as endogenous inhibitors of axillary bud outgrowth. SLs released from roots induce hyphal branching of arbuscular mycorrhizal (AM) fungi that facilitate the uptake of inorganic nutrients, such as phosphate (Pi) and nitrate, by the host plants. Previous studies have shown that SL levels in root exudates are highly elevated by Pi starvation, which might contribute to successful symbiosis with AM fungi in the rhizosphere. However, how endogenous SL levels elevated by Pi starvation contribute to its hormonal action has been unknown. Here, we show that tiller bud outgrowth in wild-type rice seedlings is inhibited, while root 2'-epi-5-deoxystrigol (epi-5DS) levels are elevated, in response to decreasing Pi concentrations in the media. However, the suppression of tiller bud outgrowth under Pi deficiency does not occur in the SL-deficient and -insensitive mutants. We also show that the responsiveness to exogenous SL is slightly increased by Pi deficiency. When Pi-starved seedlings are transferred to Pi-sufficient media, tiller bud outgrowth is induced following a decrease in root epi-5DS levels. Taken together, these results suggest that elevated SL levels by Pi starvation contribute to the inhibition of tiller bud outgrowth in rice seedlings. We speculate that SL plays a dual role in the adaptation to Pi deficiency; one as a rhizosphere signal to maximize AM fungi symbiosis for improved Pi acquisition and the other as an endogenous hormone or its biosynthetic precursor to optimize shoot branching for efficient Pi utilization.

Inhibition of shoot branching by new terpenoid plant hormones.

Shoot branching is a major determinant of plant architecture and is highly regulated by endogenous and environmental cues. Two classes of hormones, auxin and cytokinin, have long been known to have an important involvement in controlling shoot branching. Previous studies using a series of mutants with enhanced shoot branching suggested the existence of a third class of hormone(s) that is derived from carotenoids, but its chemical identity has been unknown. Here we show that levels of strigolactones, a group of terpenoid lactones, are significantly reduced in some of the branching mutants. Furthermore, application of strigolactones inhibits shoot branching in these mutants. Strigolactones were previously found in root exudates acting as communication chemicals with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Thus, we propose that strigolactones act as a new hormone class-or their biosynthetic precursors-in regulating above-ground plant architecture, and also have a function in underground communication with other neighbouring organisms.

The DDF1 transcriptional activator upregulates expression of a gibberellin-deactivating gene, GA2ox7, under high-salinity stress in Arabidopsis.

High-salinity stress affects plant growth and development. We have previously reported that overexpression of the salinity-responsive DWARF AND DELAYED FLOWERING 1 (DDF1) gene, encoding an AP2 transcription factor of the DREB1/CBF subfamily, causes dwarfism mainly by levels of reducing bioactive gibberellin (GA) in transgenic Arabidopsis. Here, we found that the GA 2-oxidase 7 gene (GA2ox7), which encodes a C20-GA deactivation enzyme, is strongly upregulated in DDF1-overexpressing transgenic plants. A loss-of-function mutation of GA2ox7 (ga2ox7-2) suppressed the dwarf phenotype of DDF1-overexpressing plants, indicating that their GA deficiency is due to overexpression of GA2ox7. Transient overexpression of DDF1 activated the promoter of GA2ox7 in Arabidopsis leaves. A gel shift assay showed that DDF1 binds DRE-like motifs (GCCGAC and ATCGAC) in the GA2ox7 promoter. In Arabidopsis under high-salinity stress, six GA2ox genes, including GA2ox7, were upregulated. Furthermore, the ga2ox7-2 mutant was less growth retarded than wild-type Col under high-salinity stress. These results demonstrate that, under salinity stress, Arabidopsis plants actively reduce endogenous GA levels via the induction of GA 2-oxidase, with the result that growth is repressed for stress adaptation.

dwarf and delayed-flowering 1, a novel Arabidopsis mutant deficient in gibberellin biosynthesis because of overexpression of a putative AP2 transcription factor.

A novel gibberellin (GA)-deficient mutant designated dwarf and delayed-flowering 1 (ddf1) was isolated from a library of activation-tagged Arabidopsis. This mutant showed dwarfism and late-flowering, but the phenotype was rescued by exogenous GA3 like known mutants defective in GA biosynthesis. The contents of bioactive GA4 and GA1 were in fact decreased in ddf1 at least partially through the repression of biosynthetic steps catalyzed by GA 20-oxidase (GA20ox). Genetic and molecular analyses revealed that the ddf1 phenotypes are caused by increased or ectopic expression of a putative AP2 transcription factor. Overexpression of DDF2, encoding another putative AP2 transcription factor closely related to DDF1, also conferred the ddf1-like phenotype. Among genes encoding (putative) AP2 transcription factors in Arabidopsis, DDFs are phylogenetically close to dehydration-responsive element binding protein (DREB1)/C-repeat binding factor (CBF) genes, which are known to be involved in stress responses. The ddf1 mutation upregulates a stress-related gene RD29A. DDF1 mRNA is strongly induced by high-salinity stress within 1 h. Moreover, transgenic plants overexpressing DDF1 showed increased tolerance to high-salinity stress. These results suggest that DDF1 is involved in the regulation of GA biosynthesis and stress tolerance. The possible relation between the contents of endogenous GAs and acquisition of stress protection is discussed.