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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes^1,[W]

Faculty of Biology, Center for Applied Biosciences, University of Freiburg, 79104 Freiburg, Germany

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5' untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1.

The plant carotenoid biosynthetic pathway is localized in the plastid and has been molecularly elucidated in recent years (for review, see DellaPenna and Pogson, 2006). It diverges from C₃ carbon metabolism by the action of the enzyme deoxyxylulose phosphate synthase, followed by a series of enzymes of the so-called nonmevalonate pathway (for review, see Hunter, 2007), yielding isopentenyl diphosphate (IPP; C₅), the building block of all isoprenoids. IPP and its isomer dimethylallyl diphosphate (DMAPP) are then converted into carotenes through chain-elongating condensation reactions catalyzed by geranylgeranyl diphosphate synthase (GGPPS) and phytoene synthase (PSY; Fig. 1 ). The triene chromophore of phytoene (C₄₀), the first carotene formed, is then extended to form the colored undecaene in lycopene catalyzed by phytoene desaturase (PDS) and -carotene desaturase (ZDS). Plant-type carotene desaturation utilizes specific poly-cis-configured isomers of intermediates (Bartley et al., 1999) involving two cis-trans isomerases, carotenoid isomerase (CrtIso; Isaacson et al., 2002, 2004; Park et al., 2002) and -carotene isomerase, which has come to light recently (Li et al., 2007a). Lycopene cyclization is accomplished by lycopene β-cyclase (B-LCY), which creates β-ionone rings to produce β-carotene, whereas lycopene -cyclase (E-LCY) creates one -ring. -Carotene (β,-carotene) is thought to be synthesized by both enzymes. The biosynthesis of oxygenated xanthophylls involves the hydroxylation at C-3 of - and β-carotene and the epoxidation of hydroxylated β-carotene derivatives. Hydroxylation of the β- and -rings is carried out by two nonheme diiron β-hydroxylases and by two P450-type -hydroxylases, respectively, to form zeaxanthin and lutein (Tian and DellaPenna, 2004; Kim and DellaPenna, 2006). Zeaxanthin epoxidase (ZEP) converts zeaxanthin into violaxanthin via antheraxanthin by introducing C-5,C-6-epoxy groups into the C-3-hydroxy-β-rings, a reaction that is reverted by violaxanthin deepoxidase; this constitutes the so-called xanthophyll cycle (for review, see Niyogi, 1999). Finally, the enzyme neoxanthin synthase catalyzes the conversion of the diepoxidated precursor violaxanthin into an allenic carotenoid, a reaction representing the classical final step in plant xanthophyll formation.

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. The carotenoid and ABA biosynthesis pathway. AAO-MoCo, Abscisic aldehyde oxidase-molybdenum cofactor; -HYD, -hydroxylase; β-HYD, β-hydroxylase; GGPP, geranylgeranyl diphosphate; NXS, neoxanthin synthase; SDR, short-chain dehydrogenase; VDE, violaxanthin deepoxidase; Z-ISO, -carotene isomerase.

Several reports have shown that ABA formation is regulated at the level of carotenoid cleavage, such as in avocado (Persea americana; Chernys and Zeevaart, 2000), tomato (Solanum lycopersicum; Thompson et al., 2000b), tobacco (Nicotiana tabacum; Qin and Zeevaart, 2002), and Arabidopsis (Iuchi et al., 2001; Wan and Li, 2006). However, roots, where carotenoids are scarce, are a major source of ABA, and the rate-limiting step in carotenogenesis may become regulatory.

In many, but not all (Diretto et al., 2007), nongreen tissues, PSY, catalyzing the first committed step in carotenogenesis, is thought to be rate-limiting. In ‘Golden Rice’ (Ye et al., 2000), for instance, the effectiveness of PSY (Paine et al., 2005), and not that of the bacterial carotene desaturase CrtI (Al-Babili et al., 2006), was decisive for increased carotenoid levels. Similarly, the overexpression of a bacterial PSY in canola (Brassica napus) seeds led to increased β-carotene formation (Shewmaker et al., 1999), while constructs harboring additional pathway genes affected product pattern only (Ravanello et al., 2003). Rate limitation by PSY was also proposed for ripening fruit of tomato (Fraser et al., 1994) and Arabidopsis seeds (Lindgren et al., 2003).

The central role of PSY is corroborated by findings in deetiolating tissues, where coordinated carotenoid biosynthesis is vitally important. Here, PSY was shown to be light (phytochrome) regulated, whereas GGPPS and PDS were not (von Lintig et al., 1997; Welsch et al., 2000). Consequently, the Arabidopsis PSY promoter region showed G-box-like elements mediating light regulation in addition to elements responsible for high basal promoter activity (Welsch et al., 2003, 2007).

Carotenoid pathway flux regulation is often shared by multiple PSYs. While there is only one PSY gene in Arabidopsis, there are two in tomato, where PSY1 is a fruit- and flower-specific isoform and PSY2 predominates in green tissues (Bartley and Scolnik, 1993; Fraser et al., 1999; Giorio et al., 2007). There are also two PSYs in cassava (Manihot esculenta; J. Arango, unpublished data), two in carrots (Daucus carota; Just et al., 2007), and three in rice, as we show here.

Here, we address the question of how ABA biosynthesis is linked to carotenoid biosynthesis in rice roots. Aiming to assess the relative effectiveness of different PSYs in carotenoid biosynthesis in different versions of ‘Golden Rice’, we discovered a novel PSY paralog that adds to the two that were already known to occur in Poaceae (Gallagher et al., 2004). We document here that this PSY form is feedback regulated and plays a specialized role in abiotic stress-induced ABA formation.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Cloning and Phylogenetic Analysis of Phytoene Synthase Genes from Rice

PSY1 from rice (OsPSY1) was cloned from rice leaf RNA by reverse transcription (RT)-PCR using primers deduced from available cDNA sequence information (accession no. AJ715786; The Institute for Genomic Research [TIGR] rice genome annotation LOC_Os06g51290). A similar approach using sequence information for the OsPSY2 cDNA given by Gallagher et al. (2004; accession no. AY024351) was not successful, as the 5' end of this sequence up to position 330 was a cloning artifact and does not occur in the rice genome. Therefore, the 5' end was cloned using RACE and used to assemble the full-length OsPSY2 cDNA sequence (identical with accession no. AK073290; TIGR rice genome annotation LOC_Os12g43130).

A third gene, OsPSY3, was discovered in the databases (accession nos. EAZ10037, BAF25806, AK108154, and NM_001070427), and we used this information to clone the corresponding cDNA. However, the N terminus was approximately 40 amino acids shorter than that of OsPSY1 and other PSY sequences. ChloroP (Emanuelsson et al., 1999) failed to identify a plastid transit peptide, and so did in vitro chloroplast import. An annotation from TIGR rice gene models (LOC_Os09g38320) indicated an additional 20 N-terminal amino acids that, when included into the full-length cDNA, yielded successful ChloroP targeting and in vitro import (see below). The deduced amino acid sequence is given in Supplemental Figure S1.

A comparison of the three rice PSYs with PSY orthologs revealed that they grouped together with PSY from daffodil (Narcissus pseudonarcissus) in a separate branch of monocot PSYs (Fig. 2A ). This group is distinct from PSYs of dicot origin, including PSY1 and PSY2 from tomato. Because all rice PSYs are more closely related to each other than to PSY1 and PSY2 from tomato, it can be concluded that PSY gene multiplication in Solanaceae and Poaceae occurred independently.

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Phylogeny of rice PSYs and PSY gene structures. A, A phylogenetic tree of selected PSY amino acid sequences was calculated using the neighbor-joining algorithm (Saitou and Nei, 1987). Abbreviations and GenBank accession numbers are as follows: SlPSY1 (S. lycopersicum; EF534740), CaPSY (C. annuum; P37272), SlPSY2 (EF534738), HaPSY (Helianthus annuus; CAC19567), TePSY (Tagetes erecta; AAM45379), AtPSY (A. thaliana; NP_197225), CpPSY (Citrus x paradisi; AAD38051), OsPSY2, ZmPSY2 (Z. mays; AAX13807), OsPSY3, ZmPSY3 (ABD1761 [Li et al., 2007b]), NpPSY (N. pseudonarcissus; P53797), ZmPSY1 (AAR08445), and CmPSY1 (Cucumis melo; P49293). B, Gene structures of Arabidopsis and rice PSYs. Exon/intron structures of Arabidopsis PSY (AtPSY) and rice PSYs (OsPSY1–OsPSY3) were calculated by comparing transcript sequences with genomic regions of the corresponding genes. The following full-length cDNAs were used: AtPSY (NM_121729), OsPSY1 (NM_001065182), and OsPSY2 (AK073290). As annotations for OsPSY3 containing the 5' end are not available, transcript start prediction was performed using FGENESH. Numbers within exons indicate the number of the corresponding exon, and numbers above exons correspond to the exon size (in base pairs).

Chloroplast Import Properties of Rice PSYs

The newly identified OsPSY3 encodes a protein of 443 amino acids, compared with 420 and 398 amino acids for OsPSY1 and OsPSY2, respectively. As shown in Supplemental Figure S1, the overall amino acid identity of the three PSYs is 68% similarity and 58% identity, on average. The C-terminal two-thirds of the PSYs (starting at amino acid 126 in the OsPSY1 sequence) are highly conserved, with 94% similarity and 70% identity, on average. The large divergence in the N termini is partially due to the plastid transit peptides, which are known for low sequence conservation.

ChloroP predicted transit peptides of 21, 80, and 53 amino acids for OsPSY1, OsPSY2, and OsPSY3, respectively (Supplemental Table S1). Consistent with this, all three PSYs were imported into chloroplasts, as indicated by their protease resistance and decreased molecular masses after import (Fig. 3 ). The apparent molecular masses of the mature proteins were approximately 42 kD for OsPSY1, 40 kD for OsPSY2, and 43 kD for OsPSY3. Subfractionation of plastids following protein import revealed that all three PSY proteins were associated with the plastid membranes.

View larger version (59K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. In vitro import of OsPSYs into pea chloroplasts. OsPSY cDNAs were in vitro translated in the presence of [35S]Met and incubated with pea chloroplasts. SDS-PAGE and subsequent autoradiography showed that the precursor protein (TP) is truncated and protease resistant (thermolysin; Th). Subfractionation showed the localization in the membrane (mem) fraction and absence in the stroma (str). In contrast to predictions performed with ChloroP (Supplemental Table S1), the molecular masses of the processed rice PSYs are in a comparable range, with 42 kD for OsPSY1, 40 kD for OsPSY2, and 43 kD for OsPSY3.

The functionality of OsPSY1 has been shown previously through overexpression in rice grains, which resulted in ‘Golden Rice 2’ (Paine et al., 2005). OsPSY2 was shown to be functional by overexpression in a bacterial carotenoid-producing system (Gallagher et al., 2004). To investigate the functionality of the newly identified OsPSY3 and to assess possible isomeric state differences of the phytoene formed, OsPSYs, truncated to remove the transit peptide (Fig. 4B ), were expressed in Escherichia coli as N-terminally 6xHis-tagged proteins. The recombinant proteins were chaotropically refolded, purified, and incubated with a recombinant purified GGPPS from Sinapis alba (Welsch et al., 2000), providing the GGPP substrate. The reaction was started with DMAPP and IPP followed by HPLC analysis on a C₃₀ reverse-phase column capable of separating geometric isomers. The retention time and spectra obtained (Fig. 4, A and C) confirmed that OsPSY3 encodes a functional PSY. The isomeric state of the phytoene produced by all rice PSYs was identical and corresponded to 15,15'-cis-phytoene.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. In vitro phytoene production with OsPSYs. OsPSYs truncated by the transit peptide were expressed as N-terminally 6xHis-tagged proteins in E. coli, purified, and incubated with a recombinant, purified 6xHis-GGPP synthase from S. alba in the presence of DMAPP and IPP. A, The HPLC traces shown give the result with OsPSY3 (+OsPSY3) as an example; an incubation with GGPPS alone is shown as a control (–OsPSY3). B, Coomassie blue-stained SDS-PAGE gel. C, Spectrum of the 15,15'-cis-phytoene formed, which was identical for all OsPSYs.

Real-time RT-PCR was used to investigate the expression levels of all three PSYs in different tissues (leaves, roots, and endosperm) and in developing and mature leaves (Fig. 5A ).

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Tissue specificity and light induction of rice PSYs. A, Expression levels of OsPSYs in roots (ro), developing leaves (dl), mature leaves (ml), and endosperm (en). Transcript levels normalized to 18S rRNA are expressed relative to root levels. B, Expression levels of OsPSYs and AtPSY during deetiolation. Seven-day-old etiolated rice seedlings (D) and 3-d-old etiolated Arabidopsis seedlings were illuminated for 24 h with red (R), far-red (FR), and white (WL) light. Expression levels of PSYs, normalized to 18S rRNA of the corresponding sample, are expressed relative to the levels detected in roots (as in A). AtPSY transcript levels are displayed relative to the levels in etiolated seedlings.

In photosynthetically active tissues, all three PSY mRNAs were present, albeit at different levels. Compared with roots, OsPSY1 showed 16 and 6 times higher transcript levels in developing leaves and mature leaves, respectively, whereas OsPSY2 transcript levels in developing leaves were only 3 times higher and declined in mature leaves to very low levels. This indicates that OsPSY1 represents the enzyme mainly responsible for carotenoid supply in chloroplasts. In contrast, OsPSY3 transcript levels were comparably low in both roots and leaves and remained unchanged in leaves at both developmental stages.

The coordinated supply of carotenoids is vitally important during the formation of the photosynthetic apparatus during deetiolation. In Arabidopsis, PSY represents the first light-induced step within the carotenoid pathway mediated mainly by the phytochrome system, involving phyA and phyB (von Lintig et al., 1997; Welsch et al., 2003). To investigate possible differences in their expression modes toward light, we determined the transcript levels of all rice PSYs in etiolated rice seedlings that had been illuminated with red, far-red, and white light for 24 h (Fig. 5B). Arabidopsis seedlings were treated and analyzed for AtPSY expression accordingly.

Red light raised OsPSY1 and OsPSY2 transcript levels approximately 6-fold over the dark control, and far-red light increased them in the 3- to 4-fold range. White light was most effective, yielding increased levels approximately 14-fold higher than in the dark control. In contrast, OsPSY3 was not light regulated. OsPSY1 and OsPSY2 behaved somewhat different compared with the only PSY of Arabidopsis, in which far-red light was more effective than red light, the former light quality giving an even stronger increase than white light.

OsPSY3 Plays a Specialized Role in Stress-Induced ABA Formation

While OsPSY1 and OsPSY2 play a predominant role in photosynthetic tissues, the almost identically low OsPSY3 transcript levels in leaves and roots as well as the photoinsensitivity of the expression profile pointed to a different role. Therefore, the possibility of an OsPSY3 involvement in the biosynthesis of ABA was investigated.

Three-week-old rice seedlings grown hydroponically in a mineral salt solution were transferred into a liquid medium supplemented with 250 mM NaCl, and the time course of OsPSY3 transcript accumulation was measured during the first 6 h, separately in roots and leaves. OsPSY3 transcript levels increased rapidly in roots, up to 15 times within the first hour following salt treatment (Fig. 6A ) and up to 22-fold after 2 h, followed by a decline after 6 h to levels approximately 5-fold higher than in untreated roots. In leaves of the same plants, OsPSY3 levels followed the same time course but reached only approximately half this induction level. In contrast, OsPSY2 responded only moderately (7-fold), and OsPSY1 levels were salt insensitive. Among the downstream carotenoid biosynthetic genes investigated (OsPDS, OsZDS, OsB-LCY, and OsZEP), only OsZEP showed a 2-fold increase after 2 h (Fig. 6B).

View larger version (32K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Expression levels of OsPSYs and OsZEP following salt and drought treatment. A to C, Salt treatment. Rice grown hydroponically in mineral solution for 2 weeks was subjected to salt stress with 250 mM NaCl for the times indicated (+). Control plants were transferred into fresh mineral solution and harvested at the same times (–). All expression levels were normalized to 18S rRNA levels of the corresponding samples and expressed relative to levels detected in the untreated sample (0 h) of the corresponding tissue (root or leaf). A, OsPSY3 expression levels in roots and leaves. B, Transcript levels of OsPSY1, OsPSY2, and OsZEP in roots. C, Carotenoid content and pattern of rice roots. D, Drought response of OsPSY3 in roots. For further explanation, see text.

The induction of OsPSY3 implies that it drives downstream carotenoid biosynthesis, in which case one would expect increased steady-state concentrations of carotenoids. Therefore, the carotenoid content in roots at 4 and 6 h after transfer into salt solution was determined (i.e. at times after maximal induction). Contrary to expectations, the carotenoid content decreased by almost 50% compared with that in the untreated control roots (Fig. 6C). This implied a decreased pathway flux toward carotenoid formation or an increased pathway flux toward ABA. To discriminate between both options, quantitative ABA measurements were carried out using liquid chromatography-mass spectrometry (LC-MS).

Three ABA peaks were resolved, peaks 1 and 2 representing trans- and cis-ABA, respectively, as revealed by retention time and the corresponding tandem mass spectrometry (MS²) fragmentation pattern; both were indistinguishable from the authentic reference (Fig. 7 ). The third peak had a trans-like MS² fragmentation pattern; this structurally unknown ABA population remained constant under all conditions examined and therefore is not considered further. Under salt stress, the concentration of the physiologically active form, cis-ABA, increased significantly, accompanied by small amounts of trans-ABA. Quantitative analysis using internal standardization revealed that the time course of ABA formation in rice roots upon salt stress mirrored the increase in OsPSY3 transcripts (Fig. 8A ). Compared with the untreated control, ABA content reached a maximum of approximately 3- to 4-fold levels after 4 h of salt treatment (2 h after the transcript levels peaked) and remained approximately constant at an elevated level within the measuring interval.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. LC-MS determination of ABA in rice roots. LC-MS revealed the occurrence of three ABA species that coeluted with trans-ABA (peak 1) and cis-ABA (peak 2). The identities were confirmed by the characteristic MS2 fragmentation pattern. The structures of the main fragments are given as determined by Ross et al. (2004). Peak 3 showed a fragmentation pattern like that of trans-ABA, but its structure was not resolved. This ABA species remained constant under all conditions applied and therefore was not considered further.

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. ABA content in rice roots after salt and drought treatment. Three-week-old rice seedlings were treated with 250 mM NaCl as described. Plants were harvested at the times indicated, and the ABA content was determined by LC-MS using internal standardization. Each bar represents the mean ABA content of 22 biological replicates. As controls, plants were transferred into fresh mineral solution, harvested at the same times, and subjected to ABA analysis (con). A, Salt treatment. B, Drought treatment. DW, Dry weight.

OsPSY3 Is under Positive ABA Feedback Control

The rapid induction of OsPSY3 in roots upon salt treatment can be interpreted as a result of osmosensing. It seems unlikely, however, that these mechanisms are responsible for the simultaneous induction of OsPSY3 in leaves (Fig. 6A). Since root-derived ABA is transported upward in the xylem, it is conceivable that increased apoplastic ABA levels induce OsPSY3 transcript amounts in remote target cells.

We tested the possibility of ABA-induced ABA synthesis by transferring rice seedlings grown in mineral solution into the same solution containing 100 µM ABA. Under these conditions, OsPSY3 showed a dramatic increase; there was also a low-level response of OsPSY1 and OsPSY2 transcripts, as well as for OsZEP (Fig. 9 ). However, in contrast to salt treatments, the mRNA levels showed no decrease, even after 6 h.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Expression levels of OsPSYs and OsZEP in roots following ABA application. Three-week-old rice seedlings were grown hydroponically as described and transferred to fresh mineral solution containing 100 µM ABA (+). RNA was isolated from roots at the times indicated, and the expression levels were determined by real-time RT-PCR. Control seedlings were transferred to fresh mineral solution and harvested at the same times (–). All expression levels were normalized to 18S rRNA levels of the corresponding samples and expressed relative to levels detected in the untreated sample (0 h).

NCEDs form a small gene family and have been reported to be rate-limiting in ABA formation (see introduction). We investigated the expression levels of OsNCED3 (accession no. AY838899), OsNCED4 (accession no. AK119780), and OsNCED5 (accession no. AY838901), the closest rice homologs of maize (Zea mays) vp14, in roots of salt-stressed rice seedlings, as above. All three genes were strongly induced by salt treatment following a time course that correlated with OsPSY3 and ABA induction (Fig. 10A ). These data can also explain the decreased carotenoid content observed at steady state after OsPSY3 and NCEDs induction. OsNCED4 showed by far the strongest response among these oxygenases. Just like OsPSY3, there was also an ABA-dependent response, indicating the presence of a positive feedback regulation loop (Fig. 10B). Again, OsNCED4 was shown to be most responsive under these conditions.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. Expression levels of OsNCEDs in roots, comparing salt treatment and ABA application. Three-week-old rice seedlings were treated with salt (A) and with 100 µM ABA (B) as described (+). RNA was isolated from roots at the times indicated, and the expression levels of OsNCED3, OsNCED4, and OsNCED5 were determined by real-time RT-PCR. Control seedlings were transferred to fresh mineral solution and harvested at the same times (–). All expression levels were normalized to 18S rRNA levels of the corresponding samples and expressed relative to levels detected in the untreated sample (0 h).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

PSY gene duplications are known, for instance, in tomato, where carotenoids are needed in chloroplasts and massively in fruit chromoplasts. The two tomato PSYs largely share these different tasks, thanks to their tissue-specific expression. However, rice does not develop chromoplasts; therefore, the roles of its three PSYs are expected to differ in some other aspect.

Tissue Specificity, Leaf Development, and Light Regulation

In rice endosperm, the transcript levels for all three PSYs are at the detection limit of real-time RT-PCR. Consequently, for the development of ‘Golden Rice’, a PSY cDNA was needed to produce phytoene (Burkhardt et al., 1997) together with a bacterial carotene desaturase CrtI (Ye et al., 2000; Hoa et al., 2003; Paine et al., 2005), all substituting for the low expression/insufficient activity of the endogenous PSY/desaturase/isomerase system (Schaub et al., 2005). However, OsPSY3 was not known at that time and therefore was not included in these analyses and models. We show here that OsPSY3 has extremely low expression levels in the endosperm and, therefore, that carotenoids are below detection limits. One can argue, however, that some carotenoid biosynthesis must occur to supply the ABA needed to achieve seed dormancy. In all other tissues and developmental stages investigated, OsPSY3 transcript levels were constantly low and did not show significant differential expression (Fig. 5A).

It is not surprising that developing leaves show a stronger expression of both OsPSY1 and OsPSY2 than mature leaves, where steady-state turnover of carotenoids needs to be sustained. Here, OsPSY1 plays the predominant role. This is because OsPSY2 transcript levels decreased strongly during leaf development while OsPSY1 transcript levels remained at comparatively higher levels (Fig. 5). In this context, it is worth noting that OsPSY1 has been shown to be very efficient. Maize PSY1 cDNA was used to achieve elevated carotenoid levels in ‘Golden Rice 2’. OsPSY1 was about equally active and more efficient than PSYs from known carotenoid "superproducers," such as from pepper (Capsicum annuum), tomato, and daffodil (Paine et al., 2005). Maize PSY1 is also the subtype mainly responsible for carotenoid accumulation in grains, as shown by a largely reduced carotenoid content observed in a maize mutant (y1) containing a disrupted PSY1 gene, which led to a strong reduction of PSY1 transcripts in the endosperm (Buckner et al., 1996).

Chloroplast development is under phytochrome control, as are OsPSY1 and OsPSY2, which share similar expression patterns in response to phytochrome-targeting light qualities during deetiolation. It has been shown in Arabidopsis that PSY plays a fundamental role, effectively controlling the entry point into carotenoid biosynthesis during deetiolation (von Lintig et al., 1997; Welsch et al., 2003). This is similar in rice, because comparable light induction patterns were observed. Certain quantitative differences noted vis-a-vis the induction profile in Arabidopsis may be related to differences in phytochrome-mediated signaling between rice and Arabidopsis (for review, see Sawers et al., 2005). The strong effect exerted by white light on the mRNA expression of OsPSY1 and OsPSY2 may reflect the involvement of blue light signaling. This may be more pronounced in rice compared with Arabidopsis, where the investigation of phyA/phyB mutants (von Lintig et al., 1997) has shown the involvement of a blue light component in PSY regulation.

OsPSY3 and NCED Induction Correlates with Stress-Induced ABA Formation

OsPSY3 is strongly inducible in roots by high salt and drought. There is also some moderate participation of (the light-regulated) OsPSY2. The strong induction of OsPSY3 peaked after 2 h of salt treatment and correlated with the formation of mainly cis-ABA. Roots are known for their capability to form ABA, which can be exported to the shoot (Jeschke et al., 1997). However, ABA can be synthesized in all tissues that contain carotenoids, including epoxidated xanthophylls. In fact, leaves can be a source of ABA (Thompson et al., 2000b), which can be transported into the root (Hartung et al., 2002).

Because PSY is often rate-limiting in carotenogenesis, it appears reasonable to assume a similar role in rice roots, where stress-mediated induction of the specialized OsPSY3 mediates a "push" toward carotenogenesis. Along these lines of evidence, we did not find equivalent regulation patterns for PDS, ZDS, and B-LCY. A report on Arabidopsis seeds, where the overexpression of the only PSY gene was found to increase ABA formation (Lindgren et al., 2003), corroborates this view.

The stress-induced upregulation of carotenoid biosynthetic RNAs has been shown in tomato roots, where RNA levels for ZEP (Thompson et al., 2000a) and for a β-hydroxylase gene (Thompson et al., 2007) were up-regulated. Both enzymes are capable of shifting the xanthophyll pattern toward ABA precursors. OsZEP is also somewhat (3- to 4-fold) up-regulated in rice roots, although only to a minor extent compared with OsPSY3 (20-fold). The need for PSY induction in this process is novel and may be partially due to the fact that carotenoid levels in roots are very low.

Interestingly, OsPSY3 transcript levels decreased from a 20-fold induction level at 2 h to approximately 5-fold after 4 h, while the accompanying decline in ABA content was comparatively low (stabilizing at a 4- to 5-fold level) although salt stress continued. This seems to reflect a fine-tuning mechanism to attain adequate steady-state concentrations.

Drought conditions showed a temporally comparable but less pronounced expression course for OsPSY3 (11-fold after 2 h) that correlated with less pronounced changes of ABA levels.

NCEDs initiate ABA biosynthesis by cleaving 9'-cis-neoxanthin (and possibly also 9-cis-vioaxanthin) to form xanthoxin. Members of this small gene family represent rate-limiting steps in ABA biosynthesis and are involved in both drought (Iuchi et al., 2001) and salt stress (Barrero et al., 2006) responses. A rate-limiting function of NCEDs can also be confirmed for ABA synthesis in rice roots, because transcript levels of three NCED members (OsNCED3, OsNCED4, and OsNCED5) are strongly induced and correlated in time with stress-induced ABA formation (Fig. 10A). The relative induction rates were very high, in the range of OsPSY3 for OsNCED5 and vastly higher for OsNCED3 and OsNCED4. Consistently, this strong induction of xanthophyll cleavage led to decreased carotenoid contents. As shown by carotenoid analysis, the xanthophyll pool recovers and is replenished during the ongoing stress. This strongly suggests that the primary function of OsPSY3 is in the recovery of the xanthophyll pool rather than pushing ABA synthesis.

The Different Induction Characteristics of Rice PSYs Correlate with Promoter Architecture

OsPSY1 and OsPSY2 are light inducible, while OsPSY3 shows no light response but is stress regulated (with some participation of OsPSY2). These differences are mirrored at the level of cis-acting promoter elements (Fig. 11A ). In silico analysis shows that the light-regulated PSY genes (OsPSY1 and OsPSY2, AtPSY) contain one box I element (TTTCAAA) and three box IV elements (TAATTAAT), both of which are involved in the light regulation of Phe ammonia-lyase (Yamada et al., 1994). Furthermore, they contain the AE-box (AGAAACAA), which mediates the light regulation of glyceraldehyde 3-P dehydrogenase (Park et al., 1996). These cis-acting elements are absent from the promoter region of OsPSY3, which explains the lack of response to light.

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 11. cis-Acting elements within the AtPSY and OsPSY promoter regions. A, cis-Acting elements of 1-kb genomic regions upstream of the transcriptional start of the corresponding genes were predicted with PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html; Lescot et al., 2002). B, ABREs (in OsPSY1, OsPSY2, and OsPSY3) and CEs (in AtPSY and OsPSY3) in PSY promoters, as predicted by PlantCARE. The core sequences of ABREs and CEs, which are common in the majority of abiotic stress-regulated genes (Zhang et al., 2005), are shown at bottom.

Indeed, as reviewed by Yamaguchi-Shinozaki and Shinozaki (2005), a G-box also represents the core motif of the ABA-response element (ABRE), which is accompanied by a coupling element (CE). The ABRE-CE combination represents a crucial regulatory DNA structure in abiotic stress-dependent gene expression. In a genome-wide approach, Zhang et al. (2005) defined common ABRE-CE motifs and showed that they mediated the responses to both abiotic stimuli and the application of ABA.

Interestingly, both motifs find an almost perfect match in the OsPSY3 promoter (Fig. 11B). Therefore, the putative ABRE-CE found in the OsPSY3 promoter might be involved in the activation by both salt and drought stress as well as by ABA. ABRE-related motifs present in the promoters of OsPSY1 and OsPSY2 are only marginally related to the sequences provided by Zhang et al. (2005). In addition, a CE was not found in the promoters of OsPSY1 and OsPSY2, whereas in the AtPSY promoter only a CE was identifiable but an ABRE was missing.

Positive Feedback Regulation through ABA

This possible dual role of ABRE-CE elements may explain why the induction of OsPSY3 in leaves of salt-stressed plants occurs almost synchronously with roots, albeit to a somewhat lesser extent (Fig. 6A). While the induction in roots may be directly coupled to osmosensing, the induction in leaves may be due to root-to-shoot ABA transport. This dual response was observed also for OsNCED3, OsNCED4, and OsNCED5 in roots and indicates the occurrence of a positive feedback regulation mediated by ABA, as reported for several other genes involved in the biosynthesis of ABA (Xiong and Zhu, 2003; Barrero et al., 2006). Therefore, OsPSY3 may be a primary and a secondary responder to stresses resulting in enhanced carotenoid biosynthesis. In the first instance, OsPSY3 would support enhanced ABA biosynthesis in response to osmosensing; secondly, it would function as a mediator of ABA-enhanced ABA biosynthesis, thereby modulating the local response as well as mediating the long-range signaling response observed in leaves.

Evolution of Three Rice PSYs

In terms of the similarities of the exon/intron structures, the 5' UTR, and the promoter organization, OsPSY1 resembles the only PSY of Arabidopsis. Therefore, it can be concluded that both AtPSY and OsPSY1 represent descendants of an ancient PSY constituting a common ancestor of monocots and dicots. As the 5' UTR lengths of OsPSY2 and OsPSY3 are comparable with each other, but different from that of OsPSY1, it can be concluded that OsPSY3 evolved by a further gene duplication of OsPSY2 or vice versa.

While this article was in preparation, a publication by Li et al. (2007b) appeared, reporting on the identification of a third PSY in maize that is involved in stress-related processes. Orthologs were identified in silico in sorghum (Sorghum bicolor; accession no. AAW28997) and rice, the latter representing the PSY3 version investigated in detail here.

There are similarities but also differences in the responses of the maize and rice PSY3 orthologs upon salt and drought stress. While the time courses for PSY3 induction following salt treatment are equivalent in maize and rice, the response of ZmPSY3 appeared less pronounced compared with that of OsPSY3. This relates both to the induction rates in roots (20-fold for OsPSY3 versus 6-fold for ZmPSY3) and to the response of PSY3 in leaves, where ZmPSY3 was nonresponsive while OsPSY3 was responsive. In addition, the strong induction observed for OsPSY3 following ABA treatment is in contrast to a relatively weak response of ZmPSY3.

However, the reverse is observed under drought conditions, where maize PSY3 showed a stronger induction than rice PSY3. Although the regimes applied are not directly comparable, these differences might reflect adaptations to their respective natural habitats. Drought, for instance, is a major challenge for maize as opposed to rice, which usually faces wet conditions during seedling development.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Growth and Treatment

Rice (Oryza sativa var TP309) seeds were germinated in vermiculite under a 12-h light/dark cycle at 28°C. After 7 d, the seedlings were transferred to Yoshida solution (Yoshida et al., 1976) for growth for an additional 14 d under the same conditions. For salt stress, 21-d-old seedlings were transferred to fresh Yoshida solution containing 250 mM NaCl and incubated under otherwise identical conditions for 0, 2, 4, and 6 h.

For ABA treatment 21-d-old plants were transferred to fresh Yoshida solution containing 100 µM (±)-ABA (Sigma) and incubated for 0, 1, 2, and 6 h. For drought stress, the roots of 21-d-old rice plants were placed on paper towels for 0, 2, 4, and 6 h. For deetiolation, rice seeds were germinated for 7 d in darkness followed by a 24-h illumination period using the conditions given by Welsch et al. (2003). Arabidopsis (Arabidopsis thaliana) seeds (ecotype Wassilewskija) were etiolated for 3 d on moist paper and illuminated accordingly.

PSY Cloning

RNA was isolated as described for the real-time RT-PCR assays; all PCR fragments were cloned into the pCR2.1 vector (Invitrogen). For the cloning of OsPSY1, cDNA synthesis was carried out using Omniscript RT (Qiagen) and an oligo(dT) primer. The OsPSY1 cDNA was amplified with the primers 5'-CCCATCCAGTATAATAATGGCGG-3' (sense) and 5'-CCCGCCTCCTACTTCTGGCTATT-3' (antisense) using PWO polymerase (Peqlab).

For OsPSY2, cDNA was synthesized with SuperScript RT (Invitrogen) and an oligo(dT) primer. For the amplification of the 5' truncated cDNA fragment, the primers 5'-CGAGTACGCCAAGACCTTCT-3' (sense) and 5'-CTTGAACTGTGGGGCTTACC-3' (antisense) and Taq DNA polymerase (Eppendorf) were used. For the cloning of the 5' end of OsPSY2, a nested PCR was performed with the 5'/3' RACE kit (Roche Applied Science), using 5'-GGTCAAGCCTCATTCCTTCA-3' as the first primer and 5'-CGAGAGGGCTGCATCATAC-3' as the second primer. The full-length OsPSY2 cDNA was obtained by joining both fragments by overlap extension PCR.

For OsPSY3, a 5' truncated cDNA of OsPSY3 was amplified using primers 5'-GGTCGAGTACGCGAAGATG-3' (sense) and 5'-AACAACAATACCACAAGCTGATCA-3' (antisense) and AccuPrime GC-rich DNA polymerase (Invitrogen). For the amplification of the 5' end, we used genomic DNA and the primers 5'-TGATGTCCACCACCACCAC-3' (sense) and 5'-GTAGAATGTCTTGGCGTACT-3' (antisense) with AccuPrime GC-rich DNA polymerase. The full-length OsPSY3 cDNA was obtained by joining both fragments by overlap extension PCR.

TaqMan Real-Time RT-PCR Assay

RNA from rice endosperm was isolated as described (Schaub et al., 2005). RNA from roots and leaves was isolated using the plant RNA purification reagent (Invitrogen). RNA purification and on-column DNaseI digestion were performed using the Qiagen RNeasy mini kit. After first-strand cDNA synthesis, Taq-Man real-time RT-PCR assays were carried out with an ABI Prism 7000 (Applied Biosystems) using 18S rRNA levels for normalization. The relative quantity of the transcripts was calculated using the comparative threshold cycle method (Livak, 1997). Data were first normalized to the corresponding 18S rRNA levels and then calculated relative to a specific sample. Data represent averages of two biological replicates.

Primers and 6 FAM-labeled Taq-Man probes (Supplemental Table S2) were designed using Primer Express software (Applied Biosystems). For 18S rRNA quantification, the eukaryotic 18S rRNA endogenous control kit (Applied Biosystems) was used.

Enzymatic Assessment of Phytoene Synthases

Expression and Purification of Recombinant OsPSY Proteins
OsPSY cDNAs were truncated by the sequence encoding the transit peptide using PCR amplification with mutagenized primers. PCR products were subcloned into the vector pCOLDI (Takara-Clontech) to yield N-terminal 6xHis tag fusions. After transformation and induction, cell pellets from 1 L of culture were resuspended in 20 mL of lysis buffer (50 mM Na₂HPO₄, pH 7.6, 1 mM MgCl₂, and 5 mM β-mercaptoethanol) and lysed for 30 min on ice after adding 10 units of benzonase (Sigma) and lysozyme. After sonication and centrifugation (16,000g, 40 min), the resulting pellet was washed twice with water, once with 1% (v/v) Triton X-100, and once with 2 M urea.

Inclusion bodies obtained were solubilized in 10 mL of 6 M GuHCl containing 5 mM β-mercaptoethanol, and the solution was added dropwise to 450 mL of refolding buffer (50 mM HEPES-KOH, pH 7.6, 5 mM β-mercaptoethanol, 0.2% [w/v] N,N-dimethyldodecylamine N-oxide [LDAO], 1 mM MgCl₂, and 150 mM NaCl) and centrifuged for 20 min at 10,000g. Two milliliters of Talon resin (Clontech) was added to the supernatant, incubated for 20 min, and centrifuged for 10 min at 700g. The resin was washed (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM β-mercaptoethanol, 1 mM MgCl₂, and 0.035% [w/v] LDAO) and incubated with 3 mL of elution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol, 0.035% [w/v] LDAO, and 100 mM EDTA) for 20 min. After centrifugation, the supernatant was dialyzed against 500 mL of dialysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol, 1 mM MgCl₂, and 0.035% [w/v] LDAO) for 3 h.

In Vitro Phytoene Production
Five micrograms of recombinant, purified 6xHis-OsPSY and 7.5 µg of purified 6xHis-SaGGPPS (Welsch et al., 2000) were incubated in 800 µL of reaction buffer (100 mM Tris-HCl, pH 7.6, 2 mM MnCl₂, 1 mM Tris[2-carboxyethyl]phosphine hydrochloride, 0.08% [v/v] Tween 80, 10 mM MgCl₂, and 20% [v/v] glycerol). The reaction was initiated by the addition of 40 µM DMAPP and 60 µM IPP (Isoprenoids Lc) and incubated for 1 h at room temperature. Assays were extracted (Welsch et al., 2000) and analyzed by HPLC (Hoa et al., 2003). In vitro protein import into isolated pea (Pisum sativum) chloroplasts was performed as described (Bonk et al., 1997).

Carotenoid Extraction and Quantification

Lipophilic compounds of 100 mg of root material were extracted three times with 2 mL of acetone followed by sonication. A total of 100 µL of -tocopherolacetate (2 mg mL^–1 in acetone; Sigma) was added as an internal standard. After centrifugation (3,000g, 5 min), the acetone phases were combined. Two milliliters of petroleum ether:diethyl ether (2:1, v/v) was added and filled up to a volume of 14 mL with 1% (w/v) NaCl. Samples were mixed and centrifuged. The petroleum ether:diethyl ether step was repeated, and the combined epiphases were dried and dissolved in 30 µL of chloroform. Ten microliters of each sample was injected into the HPLC system equipped with a C₃₀ column (YMC Europe) using a gradient system (Hoa et al., 2003). Peaks were normalized relative to the internal standard as described (Schaub et al., 2005).

ABA Extraction and Quantitative Analysis

Plant tissues were ground to a fine powder under liquid nitrogen and lyophilized. An amount not exceeding 200 mg was extracted at 1 h at 50°C using 3 mL of unbuffered 50 mM Tris. Samples were spiked with 50 ng of (±)-2-cis,4-trans-ABA-d6 (Icon Isotopes). After centrifugation (5,000g, 10 min), the supernatant was filtered and transferred into new tubes. Samples were acidified with 3 N HCl and partitioned three times against ethyl acetate. The combined epiphases were dried and redissolved in 200 µL of water, out of which 10 µL was subjected to LC-MS analysis.

Separation was carried out using a Finnigan Surveyor Plus HPLC system (Thermo Electron) equipped with a 3-µm Hypersil Gold C₁₈ reverse-phase column (150 x 4.6 mm; Thermo Electron). A gradient system was adopted from Ross et al. (2004) consisting of solvent A (0.05% [v/v] aqueous acetic acid) and solvent B (0.05% [v/v] acetic acid in acetonitrile). The gradient consisted of linear segments from 100% A to 50% A in 10 min and to 0% A in an additional 5 min at a flow rate of 0.5 mL min^–1. Final conditions were maintained for 15 min.

The Thermo Finnigan LTQ MS detector used was equipped with an electron spray ionization ion source and operated in the negative ion mode. Spectra were recorded using a source voltage of 4 kV and a capillary voltage of –100 V. The capillary temperature was set at 350°C. Nitrogen sheath gas flow, auxiliary gas flow, and sweep gas flow were set to 40, 10, and 15 arbitrary units, respectively. To record collision-induced MS² spectra, the normalized collision energy was set to 24%. Internal standard-based quantification was done using the MS data and the quantification software available in the Xcalibur 2.0 software package. Retention times and MS² fragmentation patterns were used for identification with the help of authentic reference standards; trans-ABA was from OlChemIm and (±)-ABA was from Sigma.

Sequence Analysis/Bioinformatics

Transit peptide predictions were done with ChloroP (Emanuelsson et al., 1999). Phylogenetic trees were calculated with the Vector NTI advance 10.0.1 software (Invitrogen). Transcriptional start prediction for OsPSY3 was performed using FGENESH (http://mendel.cs.rhul.ac.uk/mendel.php?topic=fgen), and cis-acting elements were analyzed using PlantCARE (Lescot et al., 2002).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NM_121729 (AtPSY), NM_001065182 (OsPSY1), AK073290 (OsPSY2), AY838899 (OsNCED3), AK119780 (OsNCED4), and AY838901 (OsNCED5).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Multiple alignment of PSYs from rice.

Supplemental Table S1. Predicted and apparent sizes of rice PSYs.

Supplemental Table S2. Sequences of primers and probes used for real-time RT-PCR.

	ACKNOWLEDGMENTS

 
We thank Jorge E. Mayer for valuable discussions.

Received January 30, 2008; accepted March 3, 2008; published March 7, 2008.

	FOOTNOTES

 
¹ This work was supported by the HarvestPlus research consortium and the Grand Challenges in Global Health initiative of the Bill and Melinda Gates Foundation.

² These authors contributed equally to the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Peter Beyer (peter.beyer{at}biologie.uni-freiburg.de).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.117028

^* Corresponding author; e-mail peter.beyer{at}biologie.uni-freiburg.de.

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