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First published online December 27, 2007; 10.1104/pp.107.111120 Plant Physiology 146:1333-1345 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
PSY3, a New Member of the Phytoene Synthase Gene Family Conserved in the Poaceae and Regulator of Abiotic Stress-Induced Root Carotenogenesis1,[W],[OA]Department of Biological Sciences, Lehman College, The City University of New York, Bronx, New York 10468; and The Graduate School and University Center, The City University of New York, New York, New York 10016–4309
Abscisic acid (ABA) plays a vital role in mediating abiotic stress responses in plants. De novo ABA biosynthesis involves cleavage of carotenoid precursors by 9-cis-epoxycarotenoid dioxygenase (NCED), which is rate controlling in leaves and roots; however, additional bottlenecks in roots must be overcome, such as biosynthesis of upstream carotenoid precursors. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis; with PSY3 described here, maize (Zea mays) and other members of the Poaceae have three paralogous genes, in contrast to only one in Arabidopsis thaliana. PSY gene duplication has led to subfunctionalization, with each paralog exhibiting differential gene expression. We showed that PSY3 encodes a functional enzyme for which maize transcript levels are regulated in response to abiotic stresses, drought, salt, and ABA. Drought-stressed roots showed elevated PSY3 transcripts and ABA, responses reversed by rehydration. By blocking root carotenoid biosynthesis with the maize y9 mutation, we demonstrated that PSY3 mRNA elevation correlates with carotenoid accumulation and that blocking carotenoid biosynthesis interferes with stress-induced ABA accumulation. In parallel, we observed elevated NCED transcripts and showed that, in contrast to dicots, root zeaxanthin epoxidase transcripts were unchanged. PSY3 was the only paralog for which transcripts were induced in roots and abiotic stress also affected leaf PSY2 transcript levels; PSY1 mRNA was not elevated in any tissues tested. Our results suggest that PSY3 expression influences root carotenogenesis and defines a potential bottleneck upstream of NCED; further examination of PSY3 in the grasses is of value for better understanding root-specific stress responses that impact plant yield.
Abiotic stresses, such as water deficit, salinity, and high or low temperatures, have profound negative effects on plant growth; such stresses are the primary causes of crop productivity losses (Bray et al., 2000  ). For example, during the flowering and silking stages of maize (Zea mays), 4 d of mild drought stress can cause up to a 50% decrease in yield productivity (Classen and Shaw, 1970). Plants have evolved various levels of adaptation in response to stress conditions, and best understood is mediation by the hormone abscisic acid (ABA; Xiong et al., 2002).
Given its important role in plant stress tolerance, regulation of ABA biosynthesis and accumulation is a focal point of research. In higher plants, ABA is derived from the 9-cis-epoxycarotenoids 9-cis-violaxanthin and 9-cis-neoxanthin; these C40 compounds are cleaved by 9-cis-epoxycarotenoid dioxygenase (NCED) to form xanthoxin, a C15 intermediate, which is subsequently converted to ABA in two steps of oxidation (see Fig. 1
; for review, see Nambara and Marion-Poll, 2005
In leaves of dicot and monocot plants, including maize, de novo biosynthesis of ABA is induced by water stress (Sindhu and Walton, 1987 ). The cloning and characterization of maize Viviparous14, which encodes NCED1, led to identification of NCED as a rate-controlling enzyme in stress-induced de novo biosynthesis of ABA, particularly in leaf tissue (Schwartz et al., 1997; Tan et al., 1997). Similar results were obtained from characterization of other orthologous genes, PvNCED1 of bean (Phaseolus vulgaris; Qin and Zeevaart, 1999), VuNCED1 of cowpea (Vigna unguiculata; Iuchi et al., 2000), LeNCED1 of tomato (Solanum lycopersicum; Thompson et al., 2000a), and AtNCED3 of Arabidopsis (Arabidopsis thaliana; Iuchi et al., 2001). Stress-induced elevation of NCED transcript levels was observed to precede ABA accumulation (Qin and Zeevaart, 1999), and up- or down-regulation of the gene affected both ABA levels and drought sensitivity (Thompson et al., 2000b; Iuchi et al., 2001). Conversion of the downstream xanthoxin to ABA is not limiting as evidenced by the invariant levels of a requisite cytosolic enzyme activity in comparing normal and water-stressed leaves (Sindhu and Walton, 1987). Similarly, the upstream carotenoid levels are not thought to limit flux to ABA in leaves; leaf epoxycarotenoids are abundant (Parry et al., 1990), and transcripts for zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, the precursor for epoxycarotenoids, were shown to be constant or deceased in leaves under drought stress (Audran et al., 1998; Iuchi et al., 2000; Thompson et al., 2000a).
Roots of maize and other plants also respond to osmotic or water stress through elevation of ABA, only some of which is due to increased translocation from other tissues (Rivier et al., 1983
To examine the nature of the root ABA bottleneck, we decided to look at maize, an important food crop worldwide, and model for translational genomics in the grass family (Poaceae; Lawrence and Walbot, 2007
The first enzyme in the plastid-localized carotenoid biosynthetic pathway, phytoene synthase (PSY), which is known to control flux to carotenoids in the seed (Gallagher et al., 2004 PSY3 cDNAs that we identified in the public databases were primarily associated with abiotic stress, suggesting that the PSY3 gene might play a role in regulating carotenoid flux in response to stress. We therefore tested this possibility and showed that, indeed, PSY3 gene expression represented at least one bottleneck in controlling flux to carotenoid precursors that are required for elevating ABA in maize roots.
Isolation of the Third PSY Gene Paralog from Maize, Rice, and Sorghum
We previously showed that PSY was encoded by two paralogs, PSY1 and PSY2, in 12 species across eight subfamilies of the grasses (Poaceae; Gallagher et al., 2004 To clone the maize PSY3 gene, we exploited synteny between maize and rice and used flanking markers to identify the maize syntenic region in maize bin 7.03 near umc1865 (Fig. 2 ); bacterial artificial chromosome (BAC) clones in this region were screened by PCR to identify one containing the maize PSY3 gene, which was sequenced by primer walking (GenBank accession no. DQ372936; see details in "Materials and Methods"). To facilitate gene annotation and later functional analyses, we used reverse transcription (RT)-PCR to clone full-length PSY3 cDNAs for maize and rice (as described in "Materials and Methods"). The cDNA clones containing the sorghum PSY1 (GenBank accession no. CD234165) and PSY3 (GenBank accession no. BG46454) genes were requested from the Comparative Grass Genomics Center. Analysis of genomic and cDNA sequences revealed conserved gene structure between PSY3 as compared to PSY1 and PSY2 in the grass family, as well as with Arabidopsis PSY (Fig. 3 ). All PSY genes possess six exons and five introns, except sorghum PSY3, whose third and fourth exons are fused. All of these PSY genes have long first exons and a very short second exon. With the exception of the sorghum-fused PSY3 exons, the sizes of the second, third, fourth, and fifth exons of all three groups of PSY genes in the Poaceae and Arabidopsis are identical with sizes of 51, 173, 236, and 193 bp, respectively. In addition, all PSY1s have a small first intron (approximately 100 bp) and a large second intron (>600 bp), but all PSY2s have a large first intron (approximately 400 bp) and a small second intron (approximately 100 bp; Fig. 3).
The deduced protein sequences for all three PSY genes of maize, rice, and sorghum were determined and used for phylogenetic analysis (Fig. 4A ). This analysis showed that all PSY3 proteins belong to a novel group, whereas the PSY1 group in monocot species in the grass family is most closely related to the PSY in the dicot plant Arabidopsis. PSY3 proteins possess a distinct domain found at the carboxyl terminal, R(H/R)XS(S/T)LT, a motif that separates these proteins from the members of the PSY1 group with the SLRNXQ(T/K) motif and PSY2 with the ARAAVAS(S/P) motif (Fig. 4B); H/R are charged amino acids, and S/T are polar but uncharged amino acids. All PSY3 proteins possess transit peptides for chloroplast targeting as predicted by the ChloroP 1.1 server (Emanuelsson et al., 1999 ). Maize PSY3 was predicted to be 47.3 kD (426 residues), having a 51-residue transit peptide and processed to a 41.9-kD (375 residues) mature plastid protein; rice PSY3 was predicted as 49.3 kD (444 residues) and having a 54-residue transit peptide and processed to a 43.5-kD (390 residues) mature plastid protein. Sorghum PSY3 was predicted as 48.9 kD (441 residues) with a 57-residue transit peptide and processed to a 42.7-kD (384 residues) mature protein. When comparing the processed and plastid-localized PSY proteins, the 41.9-kD maize PSY3 is expected to be larger by 2 kD at the N terminus as compared to maize PSY1 (39.8 kD) and PSY2 (39.4 kD). The unconserved first exon of the PSY genes encodes the PSY transit peptide and the N-terminal 2-kD extension in the case of PSY3 proteins; the last gene exon, which also lacks conservation among PSY genes, encodes the distinguishing C-terminal PSY motif (Fig. 4B).
PSY3 Encodes a Functional PSY
We used heterologous functional complementation to verify whether the novel PSY3 proteins were functional (Gallagher et al., 2003
Maize PSY3 Is Mainly Expressed in Root and Embryo Tissue
To assess the role of maize PSY3, its tissue specificity was investigated with quantitative RT-PCR using endosperm and leaf tissues, where carotenoids generally accumulate to visible levels, and root and embryos, where carotenoids are barely detectable (Fig. 6
). In leaf and endosperm tissue, maize PSY3 mRNA levels were 4- to 5-fold lower than those of PSY2 and 10- to 15-fold lower than those of PSY1, which is consistent with the semiquantitative data we previously reported (Gallagher et al., 2004
Maize PSY2 and PSY3 mRNA Levels Up-Regulated by Drought We used E-northern from the National Center for Biotechnology Information (NCBI) to reveal possible factors that might influence PSY transcript levels as indicated by the source of abundant ESTs. We identified only six PSY3 ESTs from rice, but none from maize; as described in "Materials and Methods," five were related to the ABA pathway or associated with plants subjected to drought conditions. E-northern data suggested that PSY3 in the grass family may be involved in drought stress or in regulation of ABA biosynthesis under abiotic stresses. To test these hypotheses, we subjected maize seedlings to various abiotic stresses and measured PSY3 transcript levels in comparison to transcript levels for PSY1 and PSY2. To test for the effect of drought stress on PSY transcript levels, maize seedlings were subjected to drought conditions at the five-leaf stage. After 4 d of water deprivation, leaves began to wilt and ABA levels in leaf and root tissue began to increase (Fig. 7, A and B ); in parallel, mRNA level increases were observed for both PSY2 and PSY3 in leaves (Fig. 7A) and only PSY3 in roots (Fig. 7B). Transcript levels of PSY2 and PSY3 reached their highest levels in seedlings subjected to continuous drought stress for 8 d, showing a 16- and 17-fold increase in leaves, respectively; in leaves, PSY2 encoded the most abundant (3.4-fold compared to PSY3) transcript at 8 d, the time point after which water was restored. In contrast to leaves, at 4 d, roots showed a 38-fold increase of PSY3 transcripts and, by 8 d, roots showed 50-fold induction of PSY3 transcript levels as compared to predrought levels. Upon rewatering at 8 d, both ABA and mRNA levels of PSY2 and PSY3 dropped to normal levels within 4 h in both tissues. The rapid disappearance of PSY2 and PSY3 transcripts is suggestive of tight control of mRNA stability and/or gene transcription rate. In contrast, maize PSY1 mRNA levels were only slightly altered by drought stress in both tissues.
Response of Maize PSY Transcript Levels to Salt and ABA Treatment
For plants, the responses to drought and salt are closely related and their mechanisms overlap (Zhu, 2002 When maize inbred B73 seedlings were subjected to salt stress, PSY3 transcript levels were barely altered in leaves (Fig. 8A ), but increased in roots within 30 min; within 2 h of salt stress, PSY3 transcripts peaked 6-fold, compared to untreated controls, and then dropped to lower levels by 5 h (Fig. 8B). In contrast, PSY2 transcripts only slightly increased in salt-stressed root tissue. In leaves, PSY1 transcript levels decreased rapidly within 30 min and then remained at a low level, whereas transcript levels of PSY2 and PSY3 did not show significant changes (Fig. 8A).
To test PSY responses to ABA treatment, maize B73 seedlings were subjected to 100 mM ABA. In leaves, PSY2 transcript levels increased about 8-fold within 15 min, then dropped down to normal levels within 4 h (Fig. 9A ). In contrast, PSY1 mRNA levels steadily decreased upon ABA application, whereas PSY3 transcripts remained at a low level for the entire 4-h period. In roots, within 15 min of treatment, both PSY2 and PSY3 mRNA levels increased in response to ABA, with distinguishable temporal patterns; the PSY3 response was higher and peaked earlier at 15 min compared to 1 h for PSY2; PSY3 mRNA levels increased about 7-fold in 15 min and PSY2 mRNA levels increased 4.5-fold in 1 h, and then dropped after 2 h (Fig. 9B).
Up-Regulation of PSY3 Expression Correlates with the Increase of Carotenoid Flux under Salt Treatment
The up-regulation of maize PSY3 in roots in response to drought, salt, and exogenous ABA strongly suggested that PSY3 plays a role in stress-induced carotenogenesis required for ABA and other apocarotenoids. Because roots do not ordinarily accumulate carotenoids, it is difficult to assess the correlation between increased PSY3 transcripts and increased carotenoid accumulation. However, it is possible to block the pathway using mutants that condition accumulation of pathway intermediates (Wurtzel, 2004
PSY3 Is a Key Regulator of Carotenoid Biosynthesis in Roots under Stress
We showed that PSY3 transcript levels are increased in response to drought, salt, and ABA treatment; elevated PSY3 transcripts were also shown to precede carotenoid and ABA accumulation in y9 roots. It has been previously shown in dicots that ZEP and HYD transcripts are elevated in roots subjected to drought stress (Audran et al., 1998
Reversal of drought stress in roots was observed within 2 h of rehydration, when PSY3 transcript levels decreased and ABA levels plummeted (Fig. 7B). One explanation for the rapid loss of ABA, besides loss of PSY3 transcripts, is that rehydration likely induced transcript levels of the ABA degradative enzyme ABA8ox; in Arabidopsis and bean, ABA8ox genes were up-regulated in response to drought stress and rehydration caused additional increases in transcript levels associated with reduced ABA (Kushiro et al., 2004 ; Yang and Zeevaart, 2006). To test whether reduced ABA was due to induction of the degradative enzyme ABA8ox, we measured corresponding maize transcripts. We identified maize orthologs of the recently identified rice gene (Saika et al., 2007) and examined two maize orthologs (ABA8ox1a and ABA8ox1b) that were most abundantly expressed in maize roots (R. Vallabhaneni and E.T. Wurtzel, unpublished data). Transcript levels of these root-abundant paralogs were measured in response to drought stress and rehydration; both genes showed increased transcript levels upon drought stress (Fig. 11, E and F). ABA8ox1a, having the most abundant root transcripts, showed a more rapid response to drought stress in comparison with ABA8ox1b; ABA8ox1a levels peaked at 4 d of drought stress compared to ABA8ox1b transcripts peaking at 6 d. ABA8ox1a, but not ABA8ox1b, also showed further elevation in response to rehydration within 2 h of watering at day 8 (Fig. 11E). These results explain the rapid loss of ABA upon watering and are consistent with previous reports of ABA8ox gene responses to drought stress in other plants (Kushiro et al., 2004; Yang and Zeevaart, 2006).
The role of carotenogenesis in plants is multifaceted, including functions in photosynthesis and photoprotection, precursors to ABA and to other apocarotenoids that function as signals in development, and in communication between plants and their biotic environment. Yet, in a species such as Arabidopsis, where a rate-controlling carotenoid biosynthetic enzyme, PSY, is encoded by a single-copy gene, responses leading to altered flux are limited to control of that single-copy gene. In contrast, we showed that maize and other members of the Poaceae have three paralogs; gene duplication has provided an opportunity for subfunctionalization, whereby gene family members vary in tissue specificity of expression and in responses to abiotic stress. Database searching led to the fortuitous discovery of PSY3 in the grasses; syntenic comparisons between the published rice genome and the available maize physical map facilitated isolation of the maize gene. We showed that PSY3 is present in maize, sorghum, and rice, three species that span two subfamilies in the Poaceae. Enzymes encoded by each of the three paralogs were shown to be functional, as demonstrated in a commonly used E. coli platform. Each enzyme is also predicted to be chloroplast-localized and marked by a paralog-specific C-terminal domain, whereas PSY3 is predicted to have an extended N terminus as compared to PSY1 and PSY2. The fact that PSY3 sequences across three species are more closely related than they are to PSY1 and PSY2 within a species suggests that gene family members may share paralog-specific roles in the plant either in terms of gene regulation or with regard to metabolon assembly and/or membrane-specific localization within different plastids. We discovered PSY3 among sorghum EST sequences, but we could not find any evidence that the gene was expressed in maize, suggesting that specific tissues or conditions were needed to elicit expression, which later proved to be true. In maize endosperm and leaves, tissues high in carotenoids, low levels of PSY3 transcripts were observed. In contrast, the gene is expressed in minimally carotenogenic tissues; further analysis of roots revealed that the absence of accumulated carotenoids is likely due to carotenoid cleavage because carotenoid accumulation can be observed if further conversions are blocked by mutations affecting carotenoid biosynthetic enzymes.
The rationale for investigating stress-induced regulation was based on prevalence of rice PSY3 ESTs associated with abiotic stress. Plant responses to drought and salt show overlapping mechanisms (Zhu, 2002
In nonstressed leaves, the mRNA levels of maize PSY2 were 3.4-fold higher than that of PSY3, suggesting that PSY2 is the primary gene responding to drought stress in leaves. However, the response mechanism may not involve increased flux to ABA because leaf ABA biosynthesis is limited not by the carotenoid precursor pool, but by NCED-mediated conversion of the xanthophyll precursors to ABA (Parry et al., 1990
In roots, transgenic overexpression of NCED previously suggested that there were other factors that were bottlenecks to ABA (Thompson et al., 2007
In summary, PSY3 expression plays a role in controlling flux to carotenoids in roots in response to drought stress; changes in PSY3 transcripts were accompanied by induced levels of carotenoid intermediates, elevation of HYD and NCED transcripts, and followed by accumulation of ABA. PSY3, which exists in multiple species within two subfamilies of the Poaceae, is a new target to consider for enhancing tolerance to drought and salt stress. Stress tolerance is an important factor affecting plant yield that could contribute to increasing the food supply or to improved biofuel production from grass species of the Poaceae.
Plant Materials Maize (Zea mays) inbred line B73 and mutant y9 (X07C; Maize Genetics Cooperation Stock Center, University of Illinois) and rice (Oryza sativa) ‘indica’ var. IR36 were propagated as follows. Maize B73 and y9 mutant were grown in a greenhouse with a photoperiod of 16 h supplemented with artificial lighting at 25°C with appropriate watering prior to drought, salt, or ABA treatment. Rice was grown under the same conditions and leaves were collected for cDNA isolation and gene cloning. The endosperm and embryo tissues of maize B73 were dissected at 20 DAP from field-grown plants and stored at –80°C until analysis.
Maize PSY1 cDNA sequence (GenBank accession no. ZMU32636) was used in BLAST analysis to identify a putative homolog from sorghum (Sorghum bicolor; GenBank accession no. BG46454; Altschul et al., 1997
cDNA sequences and corresponding protein sequences of PSYs of Arabidopsis (Arabidopsis thaliana), rice, sorghum, and maize were obtained from NCBI GenBank, some of which were deposited as a result of this work (DQ372936, DQ356431, AY705389, AY705390, DQ356430): Arabidopsis (AtPSY AAA32836), rice (OsPSY1, AAS18307; OsPSY2, AAK07735; OsPSY3, DQ356431), sorghum (SbPSY1, AY705389; SbPSY2, AW679367; SbPSY3, AY705390), maize (ZmPSY1, P49085; ZmPSY2, AAQ91837; ZmPSY3, DQ356430). Amino acid sequences were aligned using ClustalW and a neighbor-joining tree was constructed with 500 bootstrap replication support using MEGA3 software (Kumar et al., 2001 The genomic DNA sequences of PSYs of Arabidopsis, rice, sorghum, and maize were obtained from NCBI, Gramene, and PlantGDB for gene structure analysis: maize PSY1 (ZmPSY1; GenBank AY324431); rice PSY1 (OsPSY1; GenBank AP005750); sorghum PSY1 (SbPSY1; PlantGDB SbGSStuc11-12-04.5154.1); maize PSY2 (ZmPSY2; GenBank AY325302); rice PSY2 (OsPSY2; GenBank AL831803); sorghum PSY2 (SbPSY2; PlantGDB SbGSStuc11-12-04.12062.1); maize PSY3 (ZmPSY3; GenBank DQ372936; described in this article); rice PSY3 (OsPSY3; Gramene LOC_Os09g38320); sorghum PSY3 (SbPSY3; PlantGDB SbGSStuc11-12-04.766.1); Arabidopsis PSY (AtPSY; GenBank AB005238). The PSY gene structures were analyzed using Vector NTI Suite Version 9.0 (InforMax).
Sorghum PSY-containing ESTs, CD234165, AW679367, and BG46454, were requested and verified by further sequencing. Both CD234165 and BG46454 contained full-length sorghum PSY1 and PSY3 cDNAs, respectively. The maize PSY3 (sequence deposited as DQ356430) and rice PSY3 (sequence deposited as DQ356431) full-length cDNAs were amplified from cDNAs prepared from leaf tissue of the corresponding plant species using RT-PCR primers designed based on genomic DNA sequences of maize BAC clone b031205 and rice LOC_Os09g38320, respectively. The rice PSY3 (DQ356431) was subcloned into the pGEMT-vector (Promega) and renamed pGEMT-RPSY3 prior to sequencing of both strands and use in phylogenetic analysis. The maize PSY3 cDNA (DQ356430) was inserted into the pET23b (+) vector between EcoRI and XhoI, named pETb-MPSY3. Sorghum PSY1 from CD234165 and PSY3 from BG46454 were inserted between the EcoRI and HindIII sites of the pET23a (+) vector, designated as pETa-SPSY1 and pETa-SPSY3, respectively.
To test the function of PSY gene products, a heterozygous complementation assay was carried out as previously described (Gallagher et al., 2004
RNA isolation and cDNA synthesis were carried out as described (Gallagher et al., 2004
To carry out the drought stress experiment, maize B73 seedlings at the five-leaf stage (about 3 weeks) were deprived of water for 8 d and then rewatered. Leaves began wilting 4 d after withholding water. Therefore, leaves and roots were collected at 0, 4, 6, and 8 d after water was withheld and 2, 4, and 24 h after rewatering.
For high salt and ABA treatments, maize B73 seedlings were carefully removed from soil to avoid injury, rinsed with water, and then hydroponically grown in solutions that contained either 250 mM NaCl or 100 mM ABA [(±)-ABA; catalog A1049; Sigma]. For an effective concentration of 100 mM ABA, 200 mM of the enantiomeric mixture was used. Leaves and roots were collected after 0, 0.25, 0.5, 1, 2, 4, 5, 10, and 20 h of salt treatment, and after 0, 0.25, 0.5, 1, 2, and 4 h of ABA treatment. All plant materials were stored at –80°C until analysis. For the salt treatment of y9 mutants, y9 seedlings were treated with 250 mM NaCl and roots were collected for HPLC analysis to measure carotenoid content, which was performed and quantified as previously described (Li et al., 2007
ABA extraction was carried out according to the method described by Xiong et al. (2001) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AY705389, AY705390, DQ356430, DQ356431, and DQ372936.
The following materials are available in the online version of this article.
We are grateful to Rena Quinlan for critical reading of the manuscript, Christina Murillo for technical support, and Dr. Edward Kennelly and Dr. Bei Jiang for advice on HPLC methodology. Received October 17, 2007; accepted December 23, 2007; published December 27, 2007.
1 This work was supported by the National Institutes of Health (grant nos. S06–GM08225 and 1SC1GM081160–01), the Professional Staff Congress of The City University of New York, and New York State.  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: Eleanore T. Wurtzel (wurtzel{at}lehman.cuny.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.111120 * Corresponding author; e-mail wurtzel{at}lehman.cuny.edu.
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ABSTRACT
RESULTS
-cyclase; CYP97A, P450 carotene β-ring hydroxylase; VDE, violaxanthin de-epoxidase; ABA2, short-chain alcohol dehydrogenase; AAO3, ABA oxidase.
crtB, which carries a bacterial carotenoid gene cluster missing the bacterial PSY gene crtB (Misawa et al., 1990
-carotene by 
max) showing carotenoid content of y9 mutant seedling roots (the mean of N = 5) subjected to salt stress for hours indicated. B, ABA content of roots from maize y9 mutant seedlings and normal B73 seedlings subjected to salt stress for hours indicated. C, Quantitative RT-PCR of salt-treated roots for analysis of genes listed. Transcript levels were normalized to levels of β-actin transcripts measured in the same sample and are shown relative to PSY1 transcript levels in root at 0 h. Values represent the mean of three RT-PCR replicates ±SD from five pooled plants. Other genes are abbreviated as in 
