The Pea Gene NA Encodesent-Kaurenoic Acid Oxidase

Isolation of Two CYP88A Genes from Pea

Two genes encoding cytochrome P450s of the subfamily CYP88A were isolated by library screening using a maize D3-like EST from soybean as probe. A partial cDNA of PsKAO1 was obtained from a pea cv Alaska apical bud cDNA library. The longest clone (500 bp) was extended by 3′- and 5-′ RACE using cDNA prepared from wild-typeNA (WL1769) as template (Frohman et al., 1988). A full-length cDNA, PsKAO2, was obtained from a pea cv Torsdag seed library.

PsKAO1 (CYP88A6, GenBank accession no.AF537321 sequenced from NA WL1769) and PsKAO2(CYP88A7, GenBank accession no. AF537322) showed close homology at the nucleotide level (60–100-bit score, 82%–93% identities) to AtKAO1 and AtKAO2 (BLASTN;Altschul et al., 1997). At the amino acid level, the full-length putative proteins PsKAO1 and PsKAO2 are similar to AtKAO1, AtKAO2, and CmKAO1 (644–661-bit score, 63%–65% identities, 79%–81% positives; National Center for Biotechnology Information Blast 2 sequences). Over the full-lengthPsKAO1 is similar to PsKAO2 at the nucleotide (462-bit score, 78% identity) and protein level (756-bit score, 74% identity, 86% positive; National Center for Biotechnology Information Blast 2 sequences). This is comparable with the Arabidopsis KAO putative proteins, where AtKAO1 is 76% identical to AtKAO2 (Fig. 3).

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Fig. 3.

Inferred phylogenetic relationship of KAOs [CYP88A] and representatives of related cytochrome P450 enzymes. Numbers shown represent the bootstrap support values (%). The phylogram was generated by PAUP 4.8b8 (Swofford, 1999) using putative amino acids of full-length genes (excluding gaps) with CYP701A as the outgroup. KAO proteins used in addition to the pea PsKAO1 and PsKAO2 were from Arabidopsis (AtKAO1 and AtKAO2,Helliwell et al., 2001), pumpkin (CmKAO1,Helliwell et al., 2000), rice (Oryza sativa; OsKAO1, GenBank accession no. AP000616), maize (D3, ZmKAO1, Winkler and Helentjaris, 1995), and barley (Grd5, HvKAO1, Helliwell et al., 2001). The related brassinosteroid biosynthetic enzymes used include Arabidopsis CPD (CYP90A1, Szekeres et al., 1996) and DWF4 (CYP90B1, Choe et al., 1998) and tomato (Lycopersicon esculentum)DWARF (CYP85A1, Bishop et al., 1999). The outgroup consisted of the cytochrome P450 monooxygenaseent-kaurene oxidases (CYP701A) from pumpkin CmKO1 (Helliwell et al., 2000) and Arabidopsis GA3(AtKO1, Helliwell et al., 1998) of the GA biosynthetic pathway.

Northern-Blot Expression Studies

The members of the pea kaurenoic acid oxidase (KAO) gene family are differentially expressed. PsKAO1 is expressed in the stem and to a lesser extent in the leaf, root, apical bud, pod, and seed, whereas PsKAO2 is only expressed in the seed (Fig.4A). PsKAO2 is expressed most strongly around the time of contact point when the embryo just fills the testa and the liquid endosperm is all consumed (Fig. 4B). This coincides with the rapid buildup of GA levels in maturing seeds (Frydman et al., 1974; Swain et al., 1993).

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Fig. 4.

A, PsKAO1 and PsKAO2transcript levels in various parts of wild-type pea (L107). Five micrograms of total RNA from the apical bud (all material above the uppermost fully expanded leaf), leaf (the uppermost fully expanded leaf), stem (internode immediately below the uppermost fully expanded leaf, the internode was 80%–100% fully expanded), and root (50 mm off the end of the tap and lateral roots) of 19-d-old seedlings; also, 5 μg of total RNA from seeds (3 d after contact point) and pods (that originally contained these seeds) from mature plants were loaded on the gel. B, PsKAO1 and PsKAO2 transcript levels in wild-type pea (L107) seeds at various developmental ages. Total RNA was extracted from whole seeds and their pods between 11 and 21 d after anthesis. Contact point (CP, the 1st d that no liquid endosperm remained in seeds) occurred at 17 d after anthesis. C,PsKAO1 transcript levels in wild-type NA (WL1769) and mutants na-1 (WL1766) and na-2 (L81) from the apical bud (all tissue above the uppermost fully expanded leaf) or fully expanded stem tissue of 18-d-old pea seedlings. The ethidium bromide (EtBr)-stained total RNA is included as a loading control for each northern analysis.

PsKAO1 Is Mutated in the na-1 andna-2 Mutants

The PsKAO1 cDNA from the na-1 line WL1766 contained a five-base deletion when compared with NA(WL1769). This would change the reading frame for the encoded protein leading to a premature stop codon. The predicted protein would be 194 amino acids long, which is much smaller than the expected 485-amino acid length of the putative PsKAO1 enzyme. The predicted protein would be truncated before the catalytic domains (Kalb and Loper, 1988), including the active haem-binding site common to all cytochrome P450 enzymes. There was markedly less PsKAO1 mRNA measured in the na-1 mutant tissue than the isogenic wild-type NA (Fig. 4C). This may be because of the instability of mRNA with a premature stop codon (Gutierrez et al., 1999).

The PsKAO1 from the na-2 line L81 also is altered compared with NA (WL1769). Initially, PCR of the cDNA obtained from RNA of na-2 stems produced three bands (Fig.5). When gel purified, the largest band (W) was found to have 48 bp incorrectly spliced out and the smallest band (Z) was found to have 364 bp incorrectly spliced out (Fig. 5). The band Y could not be gel purified. However, if the products W and Z were combined, melted, and annealed, the original three-band PCR pattern reappeared. This suggests that the Y band represents a duplex between the W and Z bands (Fig. 5). Genomic DNA sequence data was then used to further define the PsKAO1 na-2 mutation. The genomic sequence of na-2 revealed a 25-bp deletion (5 bp from the 3′ end of a large intron and 20 bp of exon sequence) compared with the wild type. The AG that is required for the positioning of splicing (Brown, 1996) was lost from the intron. Hence, splicing did not occur in the same place as in the wild type. Therefore, this mutation leads to aberrant processing of the resultant pre-RNA. The second and 18th AG after the deletion were used as 3′-splicing points for the RNA that produced the W and Z PCR bands, respectively. Splicing of the following intron was not affected (Fig.5).

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Fig. 5.

A, The cDNA PCR products of PsKAO1 from wild-type NA (WL1769) and mutants na-1 (WL1766) and na-2 (L81) using the same specific primers run on 1% (w/v) agarose/Tris-acetate EDTA gel containing EtBr at 80 V for 45 min. The na-2 bands have been labeled W, Y, and Z. B, Lanes W and Z contain previously gel-purified PCR product bands W and Z from na-2 mutant cDNA (see A). Lane W + Z is the product formed when the gel-purified products W and Z were combined, melted, and annealed (three cycles of melting at 95°C and annealing at 55°C then a 70°C extension). Lane na-2 is the PCR product of the na-2 mutant cDNA as seen in A and lane M is the SPPI-EcoRI size marker run on 11% (w/v) agarose/Tris-acetate EDTA gel containing EtBr. C, Schematic diagram explanations of the cDNA bands W, Z, and Y of A from sequence and experimental data (B). D, Schematic diagram of genomic DNA of PsKAO1 from sequence data. The 25-bp deletion site as well as the W and Z cryptic splice sites of the na-2mutation are indicated.

The Mutation in na-1 Cosegregates with thena Mutant Phenotype

DNA was extracted from individual plants from four segregating F₉ families from a cross between NA(WL1769) and na-1 (WL1766) plants (Fig. 2). The hexachloro fluorescein-labeled PCR products (243 bp in the wild type) identified the five-base deletion of the na-1 mutant gene when run on denaturing gel. The five-base deletion of PsKAO1cosegregates with the dwarf phenotype of na plants (data not shown). Of the 50 individuals in the four families, 12 were homozygous tall (NA NA), 26 were heterozygous tall (NA na), and 12 were homozygous dwarf (na na) in agreement with expected (χ² ₂ = 0.08;P > 0.9). Therefore, the na-1 phenotype cosegregates with the mutation in the PsKAO1 gene.

Yeast (Saccharomyces cerevisiae) Expression

Yeast strains WAT11 and WAT21 were transformed withPsKAO1 and PsKAO2 expression constructs and yeast strains expressing PsKAO1 and PsKAO2 were identified by RNA gel blots. Both yeast strains expressing PsKAO1 and PsKAO2 converted KA through to GA₁₂ at a greater rate than yeast expressing the Arabidopsis AtKAO1 (TableI). The intermediatesent-7α-hydroxykaurenoic acid and GA₁₂-aldehyde, as well as the final product GA₁₂, were detected (Table I) and confirmed to be authentic by comparison to known standards (Table II). The PsKAO2 construct expressed in both yeast strains appeared more effective than the PsKAO1 and converted all the KA substrate provided. Direct comparison may not be possible because the PsKAO1 construct may have four extra amino acids in the 5′-untranslated region because there were two possible start codons in the PsKAO1 sequence. GA₅₃ and GA₁₄ were not detected in any of the samples (Table I). WAT21 yeast transformed with PsKAO1 and PsKAO2 expression constructs fed with intermediatesent-7α hydroxy kaurenoic acid or GA₁₂-aldehyde converted these substrates to GA₁₂ although at a lower rate than expected from the KA feed data (data not shown). A substrate earlier in the GA biosynthetic pathway, ent-kaurene, was not metabolized by yeast expressing PsKAO1 or PsKAO2 (data not shown). Untransformed wild-type yeast did not metabolize KA to intermediates in the GA biosynthetic pathway (Table I).

The compound 7β-hydroxy-kaurenolide was detected and confirmed against authentic standard (Table II) in the samples with PsKAO1 and PsKAO2 activity after KA feeds but not when the intermediatesent-7α-hydroxykaurenoic acid or GA₁₂-aldehyde were used as substrates.ent-6α,7α-dihydroxy-kaurenoic acid was detected (TableI) and tentatively identified based on comparison with published spectra (Gaskin and MacMillan, 1991; Table II) in yeast with PsKAO1, PsKAO2, or AtKAO1 activity when fed with KA. However, this product was also present after feeds ofent-7α-hydroxykaurenoic acid (data not shown). Further conversion to fujenoic acid was not observed. Neither 7β-hydroxy-kaurenolide norent-6α,7α-dihydroxy-kaurenoic acid were detected in the wild-type untransformed yeast samples and appeared to be a result of the KAO activity. However, in the wild-type untransformed yeast and yeast expressing AtKAO1 and PsKAO1 samples, the C/D ring-rearranged compounds, stachenoic acid and trachylobanic acid, were present in significant amounts. However, neither compound was present in the more active PsKAO2-expressing samples that appeared to metabolize these compounds to the ent-7α hydroxy andent-6α,7α-dihydroxy derivatives and also through to GA₁₂-like derivatives (TableIII).

PsKAO1 and PsKAO2 Encode CYP88A Cytochrome P450 Monooxygenases

Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases from the subfamily CYP88A were identified in pea. They have high similarity to the recently cloned genes AtKAO1 and AtKAO2 of Arabidopsis (Helliwell et al., 2001) and CmKAO1 of pumpkin (Helliwell et al., 2000). They are grouped with the GA biosynthetic KAO enzymes in the subfamily CYP88A as defined by the maize D3 enzyme (Winkler and Helentjaris, 1995). The KAOs from the dicotelydons (pea, Arabidopsis, and pumpkin) are grouped separately from those of the monocotyledons (rice, maize, and barley; Fig. 3). In addition, the two enzymes of pea are grouped, as are those of Arabidopsis, suggesting that gene duplication occurred late in the evolutionary process. The KAO-deduced proteins contain the four catalytic domains (A–D) common to eukaryotic cytochrome P450s (Kalb and Loper, 1988). However, a critical conserved Thr of the A domain that was shown in the crystal structure of P450cam to form a hydrogen bond with Gly to produce an oxygen-binding pocket (Poulos et al., 1985), and may also be involved in oxygen transfer (Imai et al., 1989), is replaced by Ser in all the KAOs sequenced so far. In P450cam, this Thr could be replaced by Ser in site-directed mutagenesis without altering the monooxygenase activity, whereas replacement with amino acids without the hydroxyl group uncoupled the oxygen consumption of the enzyme (Imai et al., 1989).

Pea NA Encodes PsKAO1

The evidence shows that the pea NA gene encodes PsKAO1. First, PsKAO1 from both the na-1 andna-2 GA-responsive dwarf mutants had altered sequences. The mutations na-1 and na-2 are allelic and produced by independent mutational events (Reid et al., 1983). Furthermore, the five-base deletion in PsKAO1 associated with the na-1 mutation cosegregated with the dwarf phenotype. PsKAO1 is expressed in the tissues where thena mutant phenotype is expressed. The mutant naplant has dwarfed stature, reduced taproot length, and reduced leaflet area (Reid et al., 1983; Reid and Ross, 1993; Yaxley et al., 2001). The GA₁ levels are reduced in the shoot, roots, leaves, and pods (Potts and Reid, 1983; Ingram et al., 1984; Potts, 1986; Yaxley et al., 2001). PsKAO1 was expressed in stems, roots, leaves, apical buds, pods, and seeds (Fig. 4). However, the other pea CYP88A gene, PsKAO2, was only expressed in developing seeds and not any of the other tissues tested (Fig. 4). Because PsKAO2 was expressed in seeds, KAO activity could be expected in the seeds of thena mutant. This would explain the observation that mutantna plants have normal seed development and the same GA content in their seeds as wild-type pea seeds (Potts and Reid, 1983). In contrast, the two Arabidopsis genes have similar expression patterns. Probably because of this redundancy, no mutants have been found in Arabidopsis (Helliwell et al., 2001).

The na mutants are dwarfs with extreme reduction in internode lengths. The differences in internode lengths between WL1766 and L81 (Fig. 2) arise from differences in their genetic background (Reid et al., 1983). The putative proteins from the mutants (na-1 and na-2) are severely altered from wild-type PsKAO1. However, the double recessive with other GA biosynthetic mutants (na lh and na ls) are shorter than the single na mutant (Reid, 1986b). This could be because of limited activity by other KAO enzymes in pea shoots because extra bands were observed on genomic southern blots at low stringency (data not shown). Alternatively, there may be movement of intermediates from other tissues where thePsKAO2 gene is expressed. The latter is possible because the effect of the NA (PsKAO1) gene is graft transmissible (Reid et al., 1983; Proebsting et al., 1992) and mature pea seeds at sowing contain GA₂₀ (Ross et al., 1993). The other KAO gene (PsKAO2) is expressed in the seed (Fig. 4, A and B) and the seeds of na mutant plants develop normally (Potts and Reid, 1983; Potts, 1986). This may provide a limited amount of GA precursors in the stem of the mutant na seedlings allowing the epicotyl to develop almost normally before the extreme dwarfism sets in (Reid et al., 1983). Furthermore, the phenotype of the double mutant seedlings appears to reflect the accumulation of GAs in the seed (na sln, Reid et al., 1992;Ross et al., 1995; na le, Reid et al., 1983; Lester et al., 1999a; and na lsand na lh, Swain et al., 1995).

PsKAO1 and PsKAO2 Catalyze KA to GA₁₂

PsKAO1 and PsKAO2, when expressed in yeast, catalyzed the three steps from KA to ent-7α-hydroxykaurenoic acid to GA₁₂-aldehyde to GA₁₂(Table I; Fig. 1). This was observed previously with AtKAO1and AtKAO2 from Arabidopsis and HvKAO1 of barley (Helliwell et al., 2001). Many of the enzymes in the GA biosynthetic pathway are multifunctional (Hedden, 1997). This occurs normally where the enzyme catalyzes multiple oxidations at the same carbon position (Phillips et al., 1995;Xu et al., 1995; Helliwell et al., 1999) as occurs with all three oxidations from KA to GA₁₂ at the C-7 position (Hedden, 1997; MacMillan, 1997; Helliwell et al., 2001).

The na Mutation Blocks GA Biosynthesis before GA₁₂-Aldehyde

To see if the three-step oxidation of KA observed in the yeast expression studies is demonstrated in the plant, we can look at the corresponding mutants. The pea na-1 and na-2mutants, which have a defective PsKAO1 gene, do not metabolize [³H] kaurenoic acid to substances co-eluting with GA₂₀, GA₁, or GA₈, even though NA plants do carry out this conversion (Ingram and Reid, 1987). This supports the yeast expression data (Table I) and the finding that the barley mutant (grd5) accumulates KA in its seed (Helliwell et al., 2001). However, metabolism studies show that na plants can convert [²H] GA₁₂-aldehyde to C19-GAs such as GA₂₀, GA₂₉, GA₁, and GA₈. In addition, application studies show that although na plants do not respond to precursors before GA₁₂-aldehyde, including KA and ent-7α-hydroxy-kaurenoic acid, they do respond to GA₁₂-aldehyde and readily convert labeled GA₁₂-aldehyde to GA₁ (Ingram and Reid, 1987). Thena mutation, therefore, appears to block the first two biosynthetic steps but not the final GA₁₂-aldehyde to GA₁₂ step in the plant. This could occur if the nature of the namutation altered the specificity of the PsKAO1 enzyme for the substrates. However, this is not likely because na-1(WL1766) is a null mutation.

Alternatively, there may be other specific or nonspecific enzymes present in the plant that can catalyze the last step but not the earlier steps catalyzed by PsKAO1 and PsKAO2 when expressed in yeast. A GA 7-oxidase dioxygenase has been found in pumpkin (Lange, 1997) in addition to the monooxygenase 7-oxygenase activity (Hedden et al., 1984) but has not yet been found in other species (Hedden and Phillips, 2000). Perhaps the most likely explanation is that nonspecific activity may be involved because multigene families of aldehyde oxidases have been cloned from maize and Arabidopsis (Sekimoto et al., 1997,1998) and some of these oxidize a wide range of aldehydes (Seo et al., 1998).

PsKAO1 and PsKAO2, when expressed in yeast, did not produce GA₅₃ from any of the precursors provided, although the early 13-hydroxylation GA biosynthetic pathway predominates in pea (Ingram et al., 1986; Reid and Ross, 1993; Poole et al., 1995). In immature pea and barley embryos, the formation of GA₅₃from GA₁₂ was associated with the microsomal fraction and required NADPH and oxygen (Kamiya and Graebe, 1983; Groβelindemann et al., 1992), suggesting that there may be another membrane-bound cytochrome P450 monooxygenase in pea catalyzing GA₁₂ to GA₅₃.

Additional Products of PsKAO Activity

In addition to ent-7α hydroxykaurenoic acid, GA₁₂-aldehyde, and GA₁₂, several side products of PsKAO1 and PsKAO2 activity were identified in the yeast expression studies. After either KA orent-7α-hydroxykaurenoic acid feeds, the byproductent-6α,7α-dihydroxykaurenoic acid was detected (Fig. 1). We also detected this compound as a product of AtKAO1 activity. The compound, ent-6α,7α-dihydroxykaurenoic acid, was noted in pumpkin (Hedden, 1997; MacMillan, 1997) and related products were previously detected in pea (Ingram and Reid, 1987). The product 7β-hydroxykaurenolide was detected after KA (but notent-7α-hydroxykaurenoic acid) feeds in yeast expressing PsKAOs and is presumably a side product of the formation of the double bond and epoxide from KA via ent-kauradienoic acid (Hedden, 1997). The P450–1 enzyme of Gibberella fujikuroi that catalyzes the four steps from KA to GA₁₄ also produced 7β-hydroxykaurenolide andent-6α,7α-dihydroxykaurenoic acid (Rojas et al., 2001). Fungi and higher plants appear to have evolved their GA biosynthetic pathway independently and P450–1 belongs to a different subfamily (CYP68) with low sequence homology to higher plant KAOs of subfamily CYP88A (Hedden et al., 2002). Because the additional products are common to both enzymes, they may be inevitable consequences of the reactions rather than specific products of the respective enzymes. In line with the expected difference between the fungi and higher plant KAO enzymes, the compound GA₁₄ was not detected from PsKAO activity. It was interesting to note, however, that the C/D ring-rearranged stachenoic acid and trachylobanic acid can act as substrates for the PsKAO activity.