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First published online September 3, 2004; 10.1104/pp.104.046417 Plant Physiology 136:2771-2781 (2004) © 2004 American Society of Plant Biologists The Elm1 (ZmHy2) Gene of Maize Encodes a Phytochromobilin Synthase1Boyce Thompson Institute, Cornell University, Ithaca, New York 14853 (R.J.H.S., T.D.-B., T.P.B.); School of Biological Sciences, University of Southampton, Southampton SO16 7PX, United Kingdom (P.J.L., M.J.T.); Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (J.F.G.-M.); and Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606–8502, Japan (P.J.L., T.K.)
The light insensitive maize (Zea mays) mutant elongated mesocotyl1 (elm1) has previously been shown to be deficient in the synthesis of the phytochrome chromophore 3E-phytochromobilin (P  B). To identify the Elm1 gene, a maize homolog of the Arabidopsis PB synthase gene AtHY2 was isolated and designated ZmHy2. ZmHy2 encodes a 297-amino acid protein of 34 kD that is 50% identical to AtHY2. ZmHY2 was predicted to be plastid localized and was targeted to chloroplasts following transient expression in tobacco (Nicotiana plumbaginifolia) leaves. Molecular mapping indicated that ZmHy2 is a single copy gene in maize that is genetically linked to the Elm1 locus. Sequence analysis revealed that the ZmHy2 gene of elm1 mutants contains a single G to A transition at the 3' splice junction of intron III resulting in missplicing and premature translational termination. However, flexibility in the splicing machinery allowed a small pool of in-frame ZmHy2 transcripts to accumulate in elm1 plants. In addition, multiple ZmHy2 transcript forms accumulated in both wild-type and elm1 mutant plants. ZmHy2 splice variants were expressed in Escherichia coli and products examined for activity using a coupled apophytochrome assembly assay. Only full-length ZmHY2 (as defined by homology to AtHY2) was found to exhibit PB synthase activity. Thus, the elm1 mutant of maize is deficient in phytochrome response due to a lesion in a gene encoding phytochromobilin synthase that severely compromises the PB pool.
The quality and quantity of incident light provides the plant with a rich source of information to monitor a constantly changing environment (Neff et al., 2000  ). Light signals may trigger immediate adaptive responses or may be integrated over long periods of time to regulate seasonal changes in growth. Despite the variety of light-mediated responses, genetic analyses have identified a relatively modest number of primary photoreceptors. Among these, the best characterized are the phytochromes, a family of photoreceptors responsive to changes in the flux of red (R) and far-red (FR) light (Smith, 2000; Quail, 2002).
In Arabidopsis, the phytochrome family consists of five genes: PHYA, PHYB, PHYC, PHYD, and PHYE (Clack et al., 1994
Spectrally active plant phytochromes are conjugates of PHY apoprotein covalently attached to the linear tetrapyrrole chromophore 3E-phytochromobilin (P
P
Characterization of the elm1 mutant strongly suggested that the mutant was deficient in a P
Isolation of the ZmHy2 Gene
A maize homolog of the Arabidopsis AtHY2 (P
The ZmHy2 gene is predicted to encode a 297 amino acid protein of 34 kD. Figure 1 shows an alignment of the predicted ZmHy2 protein sequence with the Arabidopsis AtHY2 protein, a putative AtHY2 ortholog from rice, and the PebB protein of Synechococcus sp. WH8020. The degree of conservation between these sequences (maize to rice 79% residue identity; maize to Arabidopsis 50% residue identity) is consistent with the identification of ZmHY2 as a putative P B synthase. Sequence analysis revealed a putative chloroplast transit peptide in the 30 residues of the ZmHY2 N-terminal region (Bannai et al., 2002).
To functionally test the predicted plastid localization of ZmHY2, green fluorescent protein (GFP) was fused to the C terminus of predicted full-length ZmHY2 protein and tobacco leaves infiltrated with an Agrobacterium culture containing the ZmHy2 expression construct (see "Materials and Methods"). A control plasmid carrying a 35S:GFP cassette resulted in predominantly cytosolic localization of GFP as shown by merged images of chlorophyll autofluorescence (magenta) and GFP fluorescence (green; Fig. 2, A–C). In contrast, the majority of ZmHY2:GFP protein was targeted to tobacco chloroplasts as shown by the overlay of chlorophyll (magenta) and GFP fluorescence images (yellow; Fig. 2, D–F). These data strongly suggest that ZmHY2 is plastid localized.
ZmHy2 Is Genetically Linked to the Elm1 Locus
To genetically map the ZmHy2 gene, a 1,000-bp genomic fragment internal to ZmHy2 was labeled and hybridized to DNA isolated from 4 maize inbred lines that served as parents in generating the IBM94 (Lee et al., 2002
DNA-blot analysis was also performed with elm1 mutants and near isogenic wild-type siblings to identify a potential lesion in the ZmHy2 gene of elm1 plants. Despite performing a number of restriction digests, no RFLPs were identified that distinguished ZmHy2 alleles from Elm1 and elm1 individuals. This indicated that the lesion in elm1 was not due to a large insertion or structural rearrangement of the ZmHy2 gene. However, small insertions or deletions in the ZmHy2 gene of elm1 plants would not be detectable using DNA-blot analysis. Therefore, to further investigate genetic linkage between elm1 and ZmHy2, an elm1 mutant [W22] was crossed to a wild-type plant from a different inbred background [H99]. The F1 plant was self-pollinated to generate a segregating F2 population. DNA was extracted from six phenotypically wild-type and six phenotypically mutant plants and digested with a restriction enzyme that distinguished the ZmHy2 alleles of W22 (ZmHy2-W22) and H99 (ZmHy2-H99). Filters were probed with the gene-specific ZmHy2 fragment, and complete linkage was observed between the W22 allele and the elm1 phenotype (data not shown), suggesting that the ZmHy2 gene was linked to the elm1 mutant phenotype.
To precisely define the lesion in the ZmHy2 gene of elm1 plants, RT-PCR was used to amplify ZmHy2 cDNA products from Elm1 and elm1 plants. It is important to note that elm1 was identified as a spontaneous mutant derived from a highly inbred [W22] line. Thus, near isogenic comparisons can be made between Elm1 and elm1 plants. Sequencing of full-length cDNA products from elm1 seedlings identified a single bp deletion immediately 3' to the junction of exons III and IV of the wild-type Elm1 transcript. The resulting frame shift creates an open reading frame of approximately 500 bp that diverges from the wild-type sequence and results in a premature stop codon. No other variations were detected between ZmHy2 sequences of Elm1 and elm1 siblings across the entire coding region of the gene.
To investigate the putative splice junction lesion of ZmHy2 in elm1 mutants, PCR was used to amplify across intron III from Elm1 and elm1 genomic DNA. Cloning and sequence analysis of ZmHy2 genomic PCR products revealed a G to A transition at the 3' splice acceptor site of intron III (Fig. 4A). The dinucleotide AG is normally essential to define the 3' splice site, and alterations to this guanidine have previously been reported to result in missplicing and the creation of frameshift mutations (Brown, 1996
A PCR-based assay was developed to confirm the association of this nucleotide change with the elm1 phenotype (Fig. 4B). PCR primers designed to exon III and exon IV were used to amplify ZmHy2 genomic sequence from wild-type and elm1 plants. The resulting products were digested with the enzyme NlaIV and analyzed by gel electrophoresis. An NlaIV site 110 bp into the fragment was ablated by the A to G transition mutation in the elm1 mutant allele, allowing the presence of the single nucleotide polymorphism to be easily assayed. A second NlaIV site 246 bp into the fragment provided a control for complete digestion. Figure 4B shows the results obtained using DNA extracted from two independent pools of wild-type Elm1 and mutant elm1 seedlings.
The presence of a 3' terminal AG is highly conserved in introns of many plant and animal nuclear genes. The elm1 mutation changes the authentic 3' splice sequence from AG:GG to AA:GG such that splicing is expected to occur at the new +1 AG dinucleotide (AAG:G) producing an out-of-frame mRNA. However, a number of reports have described splicing events at an AA dinucleotide, including at least one instance in maize (Aroian et al., 1993 In light of these data, an RT-PCR-based assay was used to investigate the possibility that in elm1 mutants a proportion of ZmHy2 transcripts may be spliced at the authentic position, using the AA as the 3' splice site and generating in-frame mRNAs. A primer (RS302) was designed that would only form a perfect complement to ZmHy2 transcripts that were spliced in-frame either in wild-type Elm1 transcripts or in elm1 transcripts using the AA as 3' splice site. The use of the +1 AG as a splice acceptor would delete a single G residue from the mature transcript and result in noncomplementarity at the 3' end of the primer (Fig. 5A). When used in conjunction with a primer designed to exon II (Pcb10), a 199-bp product was amplified from plasmid DNA containing in-frame product (Fig. 5B). However, very little product was obtained when using plasmid containing the mismatched base (+1 AG), indicating that this PCR assay can differentiate between these two splicing events.
To estimate the relative product abundance of ZmHy2 splice products in wild-type and elm1 plants, a semiquantitative PCR assay was developed. A primer designed to exon IV (Elm4) was used in conjunction with Pcb10 to assess differences in template concentration. RNA was extracted from wild-type and mutant seedling leaf tissue, and cDNA products were amplified by RT-PCR using Pcb10/RS302 and Pcb10/Elm4 primer pairs. Interestingly, primer pair Pcb10/RS302 amplified a 199-bp product from both wild-type and mutant cDNA (Fig. 5B), although the yield was much lower for elm1 plants. These data suggest that a small amount of ZmHy2 product is spliced using the AA dinucleotide as the 3' splice site in elm1 mutants. Primer pair Pcb10/Elm4 amplified similar yields of products from wild type and mutants, thus confirming an approximately equal concentration of template. In addition to the 199-bp product, a 283-bp product was amplified from mutant elm1 cDNA using primer pair Pcb10/RS302. This 283-bp product was sequenced and found to include intron III but not intron II of the unprocessed transcript. The absence of intron II in these products suggests that they are not amplified from contaminating genomic DNA but are derived from partially spliced transcripts.
In the analysis of ZmHy2 RT-PCR products, it became apparent that multiple transcripts of varying size were being amplified from Elm1 and elm1 cDNA. Comparisons of RT-PCR products revealed that all deleted regions began immediately downstream of an exon boundary and terminated with an AG dinucleotide. These observations suggested that multiple ZmHy2 transcripts were differentially spliced products of a single gene. The cloning and sequencing of 28 ZmHy2 RT-PCR products (14 each from wild-type and elm1 samples) identified multiple 3' splice acceptor sites in introns III and IV (Fig. 6).
All ZmHy2 mRNA species identified from wild-type seedlings utilized the same 3' splice acceptor in intron III (Fig. 6A). This event was designated type 1 splicing at the boundary of exon III and exon IV and the resulting mRNA denoted III/IV1. In elm1 mutants the AG dinucleotide defining this site is mutated, resulting in the creation of a novel splice acceptor site one nucleotide downstream and designated III/IV1E. In 10 of 14 sequences from elm1 seedlings, the type 1E pattern of splicing was observed (Fig. 6B). However, one sequence was found to contain a 45-bp deletion (relative to III/IV1), corresponding to the presence of an alternative 3' splice site AG present 45 nt downstream of the exon III/IV boundary. This event was designated type 2 splicing at the boundary of exon III and exon IV. In contrast to III/IV1E mRNAs, III/IV2 mRNAs remain in-frame with the predicted full-length wild-type ZmHy2, although they contain an internal deletion corresponding to the loss of 15 amino acid residues (Fig. 1). An additional AG dinucleotide is present 36 nt downstream of the type 1 splice site, but no PCR products were amplified corresponding to this transcript length. Two sequences from elm1 were amplified in which intron III was not spliced from the mature transcript, and a single product was amplified from elm1 that was spliced in-frame. These latter observations are consistent with the RT-PCR results shown in Figure 5, suggesting that some in-frame ZmHy2 transcripts are generated in the elm1 mutants.
The use of multiple splice acceptor sites was also observed at the junction of intron IV and exon V. RT-PCR products were amplified from wild-type and elm1 cDNAs corresponding to splicing at three different positions at the 3' end of ZmHy2 intron IV. These species were designated type 1, type 2, and type 3 splicing at the boundary of exon IV and exon V (Fig. 6C). All three splice sites were defined by the presence of an AG dinucleotide (Fig. 6A). The observed frequencies of these events are shown in Figure 6C. In both vertebrate and plant introns, the 3' splice site AG is found within the 5 nt consensus GCAG:G (highly conserved AG motif is underlined and the cleavage site is denoted by a colon; Kramer, 1996
To assay the potential function of multiple ZmHy2 mRNA species in Elm1 and elm1 plants, four ZmHY2 derivatives were expressed in E. coli as fusions to glutathione S-transferase (GST). The constructs designated ELMA-D are illustrated in Figure 7A. All four lacked the N-terminal 38 residues predicted to constitute the chloroplast transit peptide (as determined by homology to Arabidopsis AtHY2). ELMA encodes the predicted mature full-length ZmHY2 protein. ELMB encodes a truncated ZmHY2 protein predicted to be the product of the IV/V1 splicing event. ELMC encodes a protein containing an internal deletion predicted to be the product of III/IV2 splicing in elm1 mutants. ELMD encodes a truncated protein resulting from III/IV1E splicing in elm1 mutants.
A coupled holophytochrome assembly assay was used to determine whether ZmHY2 or derivatives possessed P B synthase activity. ELMA-D proteins were purified as GST-fusions by affinity chromatography and incubated in a reaction mixture containing BV IX and cyanobacterial apophytochrome (Cph1). The phytochrome difference spectrum obtained following incubation with ELMA had a maximum at 676 nm and a minimum at 724 nm, which is consistent with a PB-Cph1 adduct (Kohchi et al., 2001) and suggested that ZmHY2 possesses PB synthase activity (Fig. 8). Difference spectra obtained following incubation with ZmHY2 derivatives ELMB-D (Fig. 8) did not differ from a no protein control. These data indicate that ELMB-D are not active in vitro.
ZmHy2 is a maize homolog of the Arabidopsis HY2 gene, which encodes a P B synthase (Kohchi et al., 2001). PB synthase catalyses the ferredoxin-dependent reduction of BV IX to 3Z-PB, a precursor of the phytochrome chromophore (Kohchi et al., 2001; McDowell and Lagarias, 2001). Although highly divergent in the amino-terminal regions of the proteins, AtHY2 and ZmHY2 are both predicted to be targeted to the plastid and are localized to chloroplasts in transient expression assays (Kohchi et al., 2001). Comparison of AtHY2 and ZmHY2 mature protein sequences shows a degree of conservation that is consistent with common enzymatic activity. Unfortunately, little is known about the domain structure of AtHY2, making it difficult to speculate on the functional significance of divergence seen between AtHY2 and potential orthologs. Comparison of plant PB synthase proteins with cyanobacterial bilin reductases may identify certain residues essential to function (Fig. 1). However, as has been previously noted, conserved residues may be plesiomorphies shared by this large protein family and do not necessarily define regions essential to PB synthase activity per se (Kohchi et al., 2001). Recombinant AtHY2 and ZmHY2 both exhibit PB synthase activity in an in vitro coupled holophytochrome assembly assay (Kohchi et al., 2001).
The elm1 mutant of maize has previously been characterized as severely deficient in active phytochrome pools (Sawers et al., 2002
Missplicing of intron III of ZmHy2 in elm1 plants leads to the accumulation of two species of ZmHy2 mRNA. The predominant mRNA species encodes a truncated protein (ELMD), while a minor form encodes a protein containing an internal deletion (ELMC). The potential accumulation of ELMC and ELMD offers a further explanation of the partial light responsiveness of elm1 mutant plants. However, when tested in a coupled holophytochrome assembly assay, neither ELMC nor ELMD exhibited measurable P
In both wild-type and elm1 backgrounds, intron IV of the ZmHy2 gene is spliced at multiple 3' splice sites. Splicing is a two step cleavage-ligation reaction catalyzed by the machinery of the spliceosome (Kramer, 1996
Despite the absence of a well-defined BPS and 3' splice junction at intron IV, ZmHy2 IV/V3 RT-PCR products were clearly the dominant form isolated from wild-type plants (chi-square test of observed against equal frequency at all sites;
The elm1 mutant is the first phytochrome-deficient mutant of maize to be characterized, and it has proven a useful resource in beginning to understand the role of phytochrome signaling in maize development and growth (e.g. Sawers et al., 2002
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
Segregating elm1 mutant stocks was as previously described (Sawers et al., 2002
Genomic sequence not present in database searches was amplified using the Genome Walker kit (CLONTECH, Palo Alto, CA) according to the manufacturer's protocol. The nested ZmHy2-specific primers PCB2.1 (5'-GTCAAGCTCCGAAGCAGTCTAATTTTGG-3') and PCB2.2 (5'-CATTGAGAACGGTGTTATCCTCATTTGC-3') were used for amplification. Sequence alignments and gene assemblies were performed with Sequencher (Gene Codes, Ann Arbor, MI) and DNAstar (DNAstar, Madison, WI) software.
RNA isolation and RT-PCR was as previously described (Sawers et al., 2002
A HindIII restriction site polymorphism was used to map ZmHy2 in the BNL96 population (Burr et al., 1988
Primers ELM5 and ELM4 (5'-ACTTTCGCGAACTGCTCCGTCC-3') were used to amplify a 549-bp genomic region spanning introns III, IV, and V. PCR products were digested with the restriction enzyme NlaIV, fractionated on 2% (w/v) agarose gels, and detected using ethidium bromide. The presence of the G to A transition at the 5' end of intron III was detected by the ablation of an NlaIV site. See Figure 4B for details.
To generate a translation protein fusion between ZmHY2 and GFP, the full-length ORF of ZmHY2 was amplified by PCR and introduced into the binary vector pGWB5 (a gift of Dr. Tsuyoshi Nakagawa, Shimane University, Japan) using the GATEWAY in vitro site-specific recombination (Invitrogen). This binary construct contains a cauliflower mosaic virus 35S promoter to confer constitutive expression of the ZmHY2:GFP introduced into Agrobacterium tumefaciens strain GV3101 (pMP90; Koncz et al., 1984
Vectors were constructed to express four species of ZmHy2 products. Transcripts encoding mature peptides without predicted chloroplast transit sequences were amplified using the primers ELMBGLIIFWD (5'-GGCAGATCTGGGTCCGGCTGCTCGTACCAG-3') and ELMSALIREV (5'-CTTGTCGACTATCCATCCACAGCGTCGCCGACGAA-3'), which contained BglII and SalI sites (underlined) respectively, and cloned into the pGEMT-EZ vector for sequence analysis. Cloned fragments representative of different species of ZmHy2 were excised using BglII and SalI and subcloned into the Escherichia coli expression vector pGEX-6-P1 (Amersham Pharmacia Biotech, Piscataway, NJ) using BamHI and SalI sites. The constructed vectors contained mature ZmHy2 sequence fused 3' to the GST gene of Schistosoma japonicum under the control of the Ptac promoter. Constructs are detailed in Figure 7.
Overexpression of Elm-GST constructs was performed in E. coli BL21 DE3 cells containing a plasmid encoding the trxA gene for E. coli thioredoxin (Yasukawa et al., 1995
Phytochromobilin synthesizing capacity for each purified protein fraction was determined in 1-mL assay mixes as described in Kohchi et al. (2001) Sequence data from this article have been deposited with the GenBank data library under accession numbers AY560384 and AI691542. Database accession numbers used in sequence alignments were BAB33374 (AtHY2), AK101395 (OsHY2), and Q02190 (PebB).
We thank Drs. J.W.S. Brown and C.G. Simpson (Scottish Crop Research Institute, Invergowrie, Dundee, Scotland) for discussions and comments on splicing of the ZmHy2 gene and Ms. Katya Anufrikova (Boyce Thompson Institute, Ithaca, NY) and Ms. Ling Bai (Cornell University, Ithaca, NY) for excellent technical assistance. We also thank Drs. Michael Edgerton (Monsanto, St. Louis), Paul Chomet (Monsanto/Mystic Research, Mystic, CT), and Ms. Moira Sheehan (Cornell University, Ithaca, NY) for many helpful discussions and Dr. Ben Burr (Brookhaven National Laboratories, Upton, NY) for contributing the mapping data. Received May 13, 2004; returned for revision July 6, 2004; accepted July 13, 2004.
1 This work was supported by the National Science Foundation (grant no. IBN 0110297 to T.P.B.) and in part by the Biotechnology and Biological Sciences Research Council International Scientific Interchange Scheme (award no. ISIS 982 to M.J.T. and T.K.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046417. * Corresponding author; e-mail tpb8{at}cornell.edu; fax 607–254–1242.
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RESULTS
ELMC).
) results in the ablation of the first NlaIV site. Bottom, Ethidium bromide stained agarose gel of digested products from a representative assay. DNA from two samples of three pooled wild-type (Elm1) and mutant (elm1) plants. Products were amplified with primers Elm4 and Elm5 and digested with NlaIV. Two NlaIV cut sites in wild-type fragments produced fragments of 303 bp, 136 bp, and 110 bp, whereas a single NlaIV cut site in elm1 mutants resulted in fragments of 303 bp and 246 bp.
, A single type 1 event was identified from elm1 mutants resulting in an in-frame transcript. Bottom, mRNAs arising from type 1 and type 2 splicing. The predicted protein sequence is shown below. C, Splicing of intron IV. Top, Genomic sequence of ZmHy2. Exons are shown boxed above sequence. Type 1, type 2, and type 3 cleavage sites are indicated by black arrows. Bars and numbers above splice junctions indicate the observed frequencies of these events. Bottom, mRNAs formed by type 1, type 2, or type 3 splicing. The predicted protein sequence is shown below.
2 = 46, P < 0.005). However, it should be noted that the assay used does not distinguish between differences in splicing efficiencies and differential stability of the resulting mRNA products. Both ZmHy2 IV/V1 and IV/V2 mRNAs are potential targets of the nonsense-mediated decay pathway, a system characterized in yeast, invertebrates, mammals, and plants that functions to rapidly degrade mRNAs containing premature stop codons (Pulak and Anderson, 1993
-galactoside. Cultures were incubated for a further 6 h at 18°C and then lysed by sonication in buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.05% [v/v] Triton X-100, 1 mM dithiothreitol (DTT), Complete Protease Inhibitor; F. Hoffman La Roche, Basel) using six 20-s pulses. Lysate was cleared at 45,000gmax for 60 min. Protein purification was performed using columns with a 1.5-mL bed of Glutathione Sepharose 4B resin (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's protocol. Removal of the GST protein domain with site-specific protease was not performed due to low expression level. 
