Maize Genetics Cooperation Newsletter vol 86 2012

 

 

Data-mining the B73 genome sequence for carotenoid biosynthesis gene candidates.

 

            --Stinard, PS

 

            Many of the genes associated with classical carotenoid-deficient endosperm mutants of maize have been cloned and characterized (e.g. y1 (phytoene synthase; Buckner et al. 1990.  Plant Cell 2:867-876); vp5 (phytoene desaturase; Li et al. 1996.  Plant Molecular Biology 30:269-279); y9 (zeta-carotene isomerase; Li et al. 2007.  Plant Physiology 144:1181-1189); vp9 (zeta-carotene desaturase; Matthews et al. 2003.  J Exp Bot 54:2215-2230); ps1 (lycopene beta-cyclase; Singh et al. 2003.  Plant Cell 15:874-884); and vp2 (4-hydroxyphenylpyruvate dioxygenase; Matthews et al. 2003.  J Exp Bot 54:2215-2230).  However, to date, many carotenoid-deficient loci have eluded association with steps in the carotenoid biosynthetic pathway.  The list of uncharacterized genes includes lw1, lw2, lw3, lw4, w3, y8, y10, and cl1.  We report here the association of these loci (with reasonable confidence) to specific gene products.  Our technique was to identify characterized Arabidopsis orthologs of carotenoid biosynthetic genes and perform BLAST searches against the maize B73 genome (version 2) using the MaizeGDB genome browser tools.  The results are summarized in Figures 1 and 2, and Tables 1 and 2.

 

            With the exception of vp2, the characterized genes involve steps in the direct pathway leading from geranylgeranyl diphosphate to beta-carotene.  vp2, however, is implicated in the biosynthetic pathway for plastoquinone (Figure 1), an electron receptor involved in the desaturation steps between phytoene and lycopene.  We first examined steps in the plastoquinone biosynthetic pathway in Arabidopsis.  The PDS1 gene in Arabidopsis encodes 4-hydroxyphenylpyruvate dioxygenase, involved in the conversion of 4-hydroxyphenylpyruvate to homogentisic acid (Norris et al. 1995.  Plant Cell 7:2139-2149).  The Genbank sequence for PDS1 (NCBI Reference Sequence: NM_100536.3) was used to BLAST against the maize genome and picked up homology to gene model GRMZM2G088396 (Chr5:83859479..83861633), which is located on 5S near the estimated location of vp2 (Chr5:78386141..80842741), and which encodes a putative 4-hydroxyphenylpyruvate dioxygenase.  This is consistent with the data of Matthews et al. (2003).

 

            The PDS2 gene in Arabidopsis encodes homogentisate solanesyltransferase, involved the conversion of homogentisic acid to 2-demethyl-plastoquinol-9 (Tian et al. 2007.  Planta 226:1067-1073).  The Genbank sequence for PDS2 (NCBI Reference Sequence: NM_001161137.1) was used to BLAST against the maize genome and picked up homology to gene model GRMZM2G113476 (Chr2:206847694..206863769), which is located on 2L near the estimated location of w3 (Chr2:204481904..205710630), and which encodes a putative prenyltransferase/ zinc ion binding protein with high sequence homology to the Arabidopsis homogentisate solanesyltransferase gene.  Thus the maize w3 locus is an excellent candidate for the gene encoding maize homogentisate solanesyltransferase.  A UniformMu line (UFMu-02780) carrying an insert (mu1031674) in this gene model segregates for a white endosperm viviparous mutant allele of w3.  Although this result is suggestive, confirmation that the w3 locus encodes homogentisate solanesyltransferase will require molecular analysis.

 

            The remaining uncharacterized genes were placed in the biosynthetic pathway leading from 1-deoxy-D-xylulose-5-P (DOXP) to isopentenyl-diphosphate (IPP), part of the plastidial DOXP/MEP pathway (Figure 2; reviewed in Lichtenthaler 2004.  Proceedings of the 16th International Plant Lipid Symposium, Budapest, Hungary, pp. 11-24).  Whereas most of the reduced carotenoid mutations in genes involved in the later, purely plastidial parts of the carotenoid biosynthetic pathway exhibit vivipary due to reduced synthesis of ABA, mutants in genes of the MEP pathway might be expected to exhibit a less severe phenotype due to shuttling of intermediates from the alternative cytosolic MVA pathway (Rodr�guez-Concepci�n 2006.  Phytochemistry Reviews 5:1-15).  Thus, mutants in MEP pathway genes might be expected to produce low levels of endosperm carotenoids and exhibit dormancy, i.e. a �lemon white� phenotype.  Such mutants include lw1, lw2, lw3, lw4, cl1, and y10.

 

            The DXS gene in Arabidopsis encodes DOXP synthase, involved in the conversion of pyruvate and glyceraldehyde-3-P to 1-deoxy-D-xylulose-5-P (DOXP).  Vallabhaneni and Wurtzel (2009.  Plant Physiology 150:562-572) and Cordoba et al. (2011.  J Exp Bot 62:2023-2038) report three DXS genes in maize, dxs1, dxs2, and dxs3.  These correspond to maize gene models GRMZM2G137151 (Chr6:146378393..146382661), GRMZM2G493395 (Chr7:14077852..14081075), and GRMZM2G173641 (Chr9:20462059..20467072) respectively.  Cordoba et al. indicate that of these three DXS genes, dxs1 is expressed the most in leaves, and dsx2 and dsx3 are expressed the most in yellow endosperms, with dsx2 expressed more highly than dsx3.  The y8 gene is estimated to be at Chr7:14027268..14618739, which overlaps the dsx2 location and is therefore a candidate gene for dxs2.  Although y8 mutants are homozygous viable and therefore not traditional �lemon whites,� the expression pattern of the three DXS genes may explain how a knockout in dsx2 could result in the y8 mutant phenotype.  It is possible that a knockout of dsx2 might not be fully compensated for by dxs3 expression in the endosperm, leading to the pale yellow y8 mutant phenotype.  A fully functional dsx1 gene would allow normal carotenoid production in the rest of the plant (i.e. a fully viable green plant).  On the other hand, if only the dxs1 gene were knocked out, one would expect a yellow endosperm albino seedling mutant.  w14 (estimated to be at Chr6:148253633..148506034) is a possible classical maize gene candidate for the dxs1 locus.

 

            The DXR gene in Arabidopsis encodes 1-deoxy-D-xylulose 5-phosphate reductoisomerase, involved in the conversion of 1-deoxy-D-xylulose-5-P (DOXP) to 2-C-methyl-D-erythritol-4-P (MEP).  The Genbank sequence for DXR (NCBI Reference Sequence: NM_125674.2) was used to BLAST against the maize genome and picked up homology to gene models GRMZM2G056975 (Chr3:30226804..30233358) and GRMZM2G036290 (Chr8:8094442.. 8101055), both of which encode maize DXR protein and show high homology with each other and the Arabidopsis gene.  These are excellent candidates for the duplicate genes cl1 (Chr8:33707329.. 33742708) and Clm1 (chromosome 8S, location unknown).  Note that mutants at cl1 lead to a reduction in both endosperm and plant carotenoids.  Variants at the Clm1 locus are able to compensate for the reduction in plant carotenoids in cl1 mutants, but not for the reduction in endosperm carotenoids.  This could be due to tissue-specific differences in expression of the two DXR genes.

 

            The CDPMEK gene in Arabidopsis encodes 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, involved in the conversion of 4-diphosphocytidyl-2-C-methylerythritol to 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate.  The Genbank sequence for CDPMEK (NCBI Reference Sequence: NM_128250.3) was used to BLAST against the maize genome and picked up homology to gene model GRMZM5G859195 (Chr3:187922271..187927591), which is located on 3L and which encodes 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase with high sequence homology to the Arabidopsis gene.  The maize y10 locus is estimated to be at Chr3:205199570..205264647, which seems a little far from the location of GRMZM5G859195.  However, the genetic map of chromosome 3 places y10 close to na1 (Chr3:184214701..185318488).  Thus, the maize y10 locus is an excellent candidate for the gene encoding maize 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase.

 

            The ISPF gene in Arabidopsis encodes 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, involved in the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate.  The Genbank sequence for ISPF (NCBI Reference Sequence: NM_180640.2) was used to BLAST against the maize genome and picked up homology to gene models AC209374.4_FG002 (Chr5:196279295..196281037) and GRMZM5G835542 (Chr4:155830779..155832786), both of which encode maize 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and show high homology with each other and the Arabidopsis gene.  These are excellent candidates for the duplicate factor loci lw3 (Chr5:188462959..190607852) and lw4 (Chr4:155828832..155834753).

 

            The HDS gene in Arabidopsis encodes 4-hydroxy-3-methylbut-2-enyl diphosphate synthase, involved the conversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate to 4-hydroxy-3-methylbut-2-enyl diphosphate.  The Genbank sequence for HDS (NCBI Reference Sequence: NM_125453.6) was used to BLAST against the maize genome and picked up homology to gene model GRMZM2G137409 (Chr5:182124005..182130631), which is located on 5L near the estimated location of lw2 (Chr5:174149224..175478743), and which encodes 4-hydroxy-3-methylbut-2-enyl diphosphate synthase with high sequence homology to the Arabidopsis gene.  Thus, the maize lw2 locus is an excellent candidate for the gene encoding maize 4-hydroxy-3-methylbut-2-enyl diphosphate synthase.

 

            Finally, the HDR gene in Arabidopsis encodes 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, involved the conversion of 4-hydroxy-3-methylbut-2-enyl diphosphate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).  The Genbank sequence for HDR (NCBI Reference Sequence: NM_119600.3) was used to BLAST against the maize genome and picked up homology to gene model GRMZM2G027059 (Chr1:272936836 to 272940502), which is located on 1L near the estimated location of lw1 (Chr1:271108631..273434076), and which encodes 4-hydroxy-3-methylbut-2-enyl diphosphate reductase with high sequence homology to the Arabidopsis gene.  Thus the maize lw1 locus is an excellent candidate for the gene encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase.

 

            Thus, gene candidates can be assigned to nearly all of the loci associated with reduced endosperm carotenoids.  Mutants, many of which are derived from populations carrying active transposable elements, exist for all of these loci, so it should be a simple matter to determine whether these mutants are due to lesions at the candidate loci.  However, there are still genes in the carotenoid biosynthetic pathway for which mutants have not yet been identified.  One possible explanation is that some of these genes occur as duplicate loci in maize for which two or more genes would need to be knocked out in order to observe a mutant phenotype.  One such example is the genes homologous to the Arabidopsis gene ISPD (Figure 2; NCBI Reference Sequence: NM_126305.2), encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase.  The Arabidopsis gene picks up homology with maize gene models GRMZM5G856881 (Chr3:170115790..170118780) and GRMZM2G172032 (Chr8:164748939..164752371).  These genes encode a putative 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase with homology to each other and to the Arabidopsis gene.  We predict that if both genes were knocked out, a reduced endosperm carotenoid mutant phenotype would result.  This and other examples of predicted duplicate genes are summarized in Table 2.  Reverse genetics tools such as the UniformMu project may someday identify knockouts in these individual genes that may then be combined to test this hypothesis.

Figure 1.  Plastoquinone biosynthetic pathway.  Classical maize gene candidates are listed at the left of each step.  ? = uncharacterized duplicate factor loci.  Arabidopsis genes are in parentheses.

 

 

4-hydroxyphenylpyruvate

vp2                    4-hydroxyphenylpyruvate dioxygenase (PDS1)

homogentisic acid

w3                     homogentisate solanesyltransferase (PDS2)

2-methyl-6-solanyl-1,4-benzoquinol (2-demethyl-plastoquinol-9)

? ?                     2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (VTE3)

plastoquinol-9

Figure 2.  DOXP/MEP pathway.  Classical maize gene candidates are listed at the left of each step.  ? = uncharacterized duplicate factor loci.  Arabidopsis genes are in parentheses.

 

 

                                       pyruvate + glyceraldehyde-3-P

y8 w14 ?                                                            DOXP synthase (DXS)

                                     1-deoxy-D-xylulose-5-P (DOXP)

cl1 clm1                                                             DOXP reductase (DXR)

                                   2-C-methyl-D-erythritol-4-P (MEP)

? ?                                                                      CDP-ME synthase (ISPD)

                              4-diphosphocytidyl-2-C-methylerythritol (CDP-ME)

y10                                                                     CDP-ME kinase (CDPMEK)

                             4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate

lw3 lw4                                                              MEcPP-synthase (ISPF)

                                    2-C-methyl-D-erythritol-2,4-cyclodiphosphate

lw2                                                                      HMBPP-synthase (HDS)

                                    4-hydroxy-3-methylbut-2-enyl diphosphate

lw1                                                                      HMBPP reductase (HDR)

dimethylallyl-diphosphate (DMAPP)           isopentenyl-diphosphate (IPP)

Table 1.  Classical maize carotenoid genes and predicted gene models.

 

 

Classical Maize Gene

Location

Arabidopsis Gene Candidate

Orthologous Maize Gene Model

vp2

5S (5.04)

AT1G065701 (PDS1)

GRMZM2G088396

w3

2L (2.08)

AT3G11945 (PDS2)

GRMZM2G113476

y8

7S (7.01)

AT4G15560 (DXS)

GRMZM2G493395

w14

6L (6.05)

AT4G15560 (DXS)

GRMZM2G137151

cl1

3S (3.04)

AT5G62790 (DXR)

GRMZM2G056975

Clm1

8S

AT5G62790 (DXR)

GRMZM2G036290

y10

3L (3.07)

AT2G26930 (CDPMEK)

GRMZM5G859195

lw3

5L (5.06)

AT1G63970 (ISPF)

AC209374.4_FG002

lw4

4L (4.06)

AT1G63970 (ISPF)

GRMZM5G835542

lw2

5L (5.05)

AT5G60600 (HDS)

GRMZM2G137409

lw1

1L (1.10)

AT4G34350 (HDR)

GRMZM2G027059

 

1  TAIR locus name (from www.arabidopsis.org).

Table 2.  Predicted duplicate factor maize carotenoid genes and gene models.

 

 

Arabidopsis Gene

Orthologous Maize Gene Model

Location

AT3G634101 (VTE3)

GRMZM2G082998

1L

AT3G63410 (VTE3)

GRMZM2G099206 (pseudogene?)

3S

AT4G15560 (DXS)

GRMZM2G137151

6L

AT4G15560 (DXS)

GRMZM2G493395

7S

AT4G15560 (DXS)

GRMZM2G1736412

9S

AT2G02500 (ISPD)

GRMZM5G856881

3L

AT2G02500 (ISPD)

GRMZM2G172032

8L

 

1  TAIR locus name (from www.arabidopsis.org).

2  Data from Cordoba et al. 2011.  J Exp Bot 62:2023-2038.

 

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