Small kernel2 Encodes a Glutaminase in Vitamin B₆ Biosynthesis Essential for Maize Seed Development

A small kernel mutant in maize highlights vitamin B6 biosynthesis in embryogenesis and endosperm development.

Abstract

Vitamin B₆, an essential cofactor for a range of biochemical reactions and a potent antioxidant, plays important roles in plant growth, development, and stress tolerance. Vitamin B₆ deficiency causes embryo lethality in Arabidopsis (Arabidopsis thaliana), but the specific role of vitamin B₆ biosynthesis in endosperm development has not been fully addressed, especially in monocot crops, where endosperm constitutes the major portion of the grain. Through molecular characterization of a small kernel2 (smk2) mutant in maize, we reveal that vitamin B₆ has differential effects on embryogenesis and endosperm development in maize. The B₆ vitamer pyridoxal 5′-phosphate (PLP) is drastically reduced in both the smk2 embryo and the endosperm. However, whereas embryogenesis of the smk2 mutant is arrested at the transition stage, endosperm formation is nearly normal. Cloning reveals that Smk2 encodes the glutaminase subunit of the PLP synthase complex involved in vitamin B₆ biosynthesis de novo. Smk2 partially complements the Arabidopsis vitamin B₆-deficient mutant pdx2.1 and Saccharomyces cerevisiae pyridoxine auxotrophic mutant MML21. Smk2 is constitutively expressed in the maize plant, including developing embryos. Analysis of B₆ vitamers indicates that the endosperm accumulates a large amount of pyridoxamine 5′-phosphate (PMP). These results indicate that vitamin B₆ is essential to embryogenesis but has a reduced role in endosperm development in maize. The vitamin B₆ required for seed development is synthesized in the seed, and the endosperm accumulates PMP probably as a storage form of vitamin B₆.

The term vitamin B₆ refers to six water-soluble compounds, including pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their 5′-phosphorylated derivatives (Drewke and Leistner, 2001; Mooney et al., 2009). Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B₆, which serves as a cofactor for over 170 enzymes that are mostly involved in amino acid, lipid, and carbohydrate metabolism (Percudani and Peracchi, 2003). In addition, vitamin B₆ has also been identified as a potent antioxidant that quenches singlet oxygen as efficiently as do vitamins C and E (Ehrenshaft et al., 1999). This function has been implicated in conferring plant resistance to abiotic stresses (Shi et al., 2002; Shi and Zhu, 2002; Chen and Xiong, 2005; Titiz et al., 2006).

Vitamin B₆ is synthesized de novo in plants, fungi, archae, and most eubacteria, but not in animals, including humans, which have to obtain it from dietary sources. The biosynthesis of vitamin B₆ involves two de novo pathways and a salvage pathway. The seven-step DXP-dependent de novo pathway, which occurs exclusively in the γ-subdivision of proteobacteria, uses 1-deoxy-d-xylulose-5-phosphate (DXP) and 4-phosphohydroxy-l-Thr to synthesize pyridoxine 5′-phosphate (PNP; Kennedy et al., 1995; Zhao and Winkler, 1996; Cane et al., 1998; Laber et al., 1999). In contrast, plants, fungi, and most eubacteria use a DXP-independent pathway that synthesizes PLP from Gln, ribose 5-phosphate (or ribulose 5-phosphate), and glyceraldehyde 3-phosphate (or dihydroxyacetone phosphate; Tambasco-Studart et al., 2005). This pathway consists of two enzymes that form a Gln amidotransferase (PLP synthase) comprised of a synthase subunit (PDX1) and a glutaminase subunit (PDX2; Tambasco-Studart et al., 2005). PDX2 catalyzes release of an ammonia from Gln that is combined by PDX1 with ribose 5-phosphate and glyceraldehyde 3-phosphate to synthesize PLP (Burns et al., 2005; Tambasco-Studart et al., 2005). The PLP synthase complex has a dodecameric cogwheel structure that includes 12 PDX1-PDX2 heterodimers (Strohmeier et al., 2006). Channeling of ammonia between the active centers of PDX1 and PDX2 is mediated by a Met-rich hydrophobic tunnel (Strohmeier et al., 2006).

In addition to the de novo pathways, PLP can be derived through a salvage pathway. As illustrated in Supplemental Figure S1, B₆ vitamers are interconvertible. Interconversions of PN, PL, PM, and corresponding 5′-phosphate derivatives are mediated by kinase and phosphatase reactions (Yang et al., 1996, 1998). PL can be reduced to PN by a pyridoxal reductase (Guirard and Snell, 1988; Nakano et al., 1999), whereas PNP and PMP can be converted to PLP by a PMP/PNP oxidase (Zhao and Winkler, 1995). Salt-Overly-Sensitive4 (SOS4) encodes the PN/PL/PM kinase (Shi et al., 2002; Shi and Zhu, 2002) and PDX3 encodes the PNP/PMP oxidase (Sang et al., 2007), and PLR1 the PL reductase in Arabidopsis (Arabidopsis thaliana; Herrero et al., 2011). The salvage pathway is universally present in all kingdoms, including those that cannot synthesize vitamin B₆ de novo.

Vitamin B₆ plays important roles in plant growth, development, and stress responses. A complete deficiency of vitamin B₆ causes embryo lethality (Tambasco-Studart et al., 2005; Titiz et al., 2006), whereas attenuated vitamin B₆ biosynthesis negatively affects plant growth and development and reduces stress tolerance (Chen and Xiong, 2005; Wagner et al., 2006; Titiz et al., 2006). The Arabidopsis genome contains three PDX1 homologs (AtPDX1.1, AtPDX1.2, and AtPDX1.3) and one PDX2 (Tambasco-Studart et al., 2005). AtPDX1.1 and AtPDX1.3 have the PLP synthase activity (Titiz et al., 2006; Tambasco-Studart et al., 2007), and AtPDX1.2 functions by enhancing the catalytic activity of PLP synthase (Moccand et al., 2014). The Arabidopsis pdx2 knockout mutants and pdx1.1:pdx1.3 double mutants blocking the vitamin B₆ biosynthesis are embryo lethal (Tambasco-Studart et al., 2005; Titiz et al., 2006). The atpdx1.1 and atpdx1.3 single mutants have reduced growth (particularly root growth in pdx1.3) and are hypersensitive to oxidative stresses (Chen and Xiong, 2005; Wagner et al., 2006; Titiz et al., 2006). A recent study revealed that differential expression of AtPDX1.1 and AtPDX1.3 in developing roots affects homeostasis of auxin and ethylene (Boycheva et al., 2015). Biosynthetic pathways of both hormones involve PLP-dependent enzymes (Boycheva et al., 2015). The stress tolerance function is partially attributed to the antioxidant properties of vitamin B₆. Stress-induced reactive oxygen species can be quenched by vitamin B₆ (Ehrenshaft et al., 1999; Bilski et al., 2000, Mittler, 2002). In addition to total vitamin B₆ levels, proper balancing of B₆ vitamers by salvage reactions is essential for normal growth and development (Colinas et al., 2016). In the PMP/PNP oxidase-deficient pdx3 mutant of Arabidopsis, increased levels of PMP and reduced levels of PLP and PL are associated with impaired growth and development (Colinas et al., 2016).

Although vitamin B₆ biosynthesis is essential for embryogenesis in Arabidopsis (Tambasco-Studart et al., 2005), the roles of vitamin B₆ in embryogenesis and endosperm development in grasses has not been addressed. Whereas Arabidopsis seeds develop a transient endosperm that is quickly consumed by the developing embryo leaving a single layer of aleurone cells in mature seeds, endosperm is a major component of the grass seed. Here, we report a detailed characterization of small kernel2 (smk2), a vitamin B₆ biosynthetic mutant of maize. Molecular analysis indicates that Smk2 encodes the glutaminase subunit of the PLP synthase. Null mutations of Smk2 arrest embryogenesis at the transition stage, whereas endosperm development is only moderately affected. Vitamin B₆ levels are decreased dramatically in both the endosperm and embryo of the smk2 mutant, indicating that embryogenesis is more sensitive to reduced vitamin B₆ levels than endosperm development. Furthermore, our results also indicate that the developing endosperm accumulates vitamin B₆ in the form of PMP, probably as a storage form of the vitamin.

RESULTS

Phenotypic Characterization of smk2-1

The recessive embryo-lethal small kernel2-1 (smk2-1) mutant was isolated from the UniformMu transposon mutagenesis population (McCarty et al., 2005). Self-pollinated Smk2-1 heterozygous plants segregate smk and wild-type kernels in ratios that range from 1:3 to 1:6.5, depending on the season. PCR genotyping enabled by cloning of the gene indicated that the underrepresentation of mutant homozygote is caused by reduced transmission of the mutant allele through pollen, suggesting that environmental factors affect pollen transmission. The smk2-1 kernels were smaller than wild-type siblings throughout kernel development (Fig. 1, A and B). At maturity, the mutant kernels contained normal-appearing but slightly smaller endosperms, whereas the embryos were aborted (Fig. 1C). Histological sections showed that at 18 days after pollination (DAP), wild-type embryos had reached late stage embryogenesis as indicated by formation of scutellum (SC), leaf primordia, shoot apical meristem (SAM), and root apical meristem (Fig. 1, D and F). By contrast, the smk2-1 mutant embryos were arrested at an early transition stage (Fig. 1, E and G). Although morphologically normal, endosperms of smk2-1 mutant seeds contained fewer cells than in endosperms of wild-type siblings (Fig. 1, D and E). Development of basal endosperm transfer cells was not visibly affected in the mutant (Supplemental Fig. S2). Overall, these results suggest that the smk2-1 mutation arrests embryogenesis at the transition stage and causes a moderate reduction in endosperm size in maize. Consistent with the early block in embryogenesis, mature mutant seed invariably failed to germinate. In addition, attempts to rescue the smk2-1 mutants on Murashige and Skoog medium were not successful. Hence, the smk2-1 mutation was maintained as a heterozygote.

Figure 1.

Seed development is affected in the smk2-1 mutant. A, An ear segregating smk2-1 mutants (arrows). B, Germinal face of the wild-type and smk2-1 mutant kernels at 18 DAP. C, Section of mature wild-type and smk2-1 kernels. D to G, Cytological sections of wild-type (D and F) and smk2-1 mutant kernels (E and G) at 18 DAP. LP, Leaf primordia; RAM, root apical meristem; Em, embryo. Scale bars = 0.5 cm in A to C, 1 mm in D and E, 500 µm in F, and 100 µm in G.

Cloning of Smk2

Smk2 was cloned by transposon tagging based on the Southern-blot cosegregation analysis. A 5.1 kb EcoRI fragment that hybridized to the Mutator-Don Robertson (MuDR)-specific probe was found to cosegregate with the smk2-1 mutant in a 24-plant population (Fig. 2A). Further analysis in a 70-plant population did not recover any recombinations, indicating a tight linkage between the smk2 phenotype and the MuDR insertion. This 5.1 kb EcoRI fragment was cloned by screening a size-selected λ-phage library. Sequencing of the fragment revealed that the MuDR was inserted in GRMZM2G023528 (accession AY109859). This gene contains seven exons, and the MuDR element is inserted in the first intron (Fig. 2B). To confirm that this candidate is the causal gene for the smk2-1 phenotype, we isolated alleles of this gene from the Trait Utility System in Corn population (Bensen et al., 1995). Eleven independent alleles were identified, named from smk2-2 to smk2-12, respectively (Fig. 2B). Crosses of each allele to the smk2-1 allele showed noncomplementation of the smk phenotype in F1 seeds, thus confirming that GRMZM2G023528 is the causal gene for the smk2 mutation.

Figure 2.

Cloning of Smk2. A, Southern hybridization of an F2 segregating family of smk2-1 mutants. DNAs were digested with EcoRI and probed with the 1.3-kb EcoRI-BamHI fragment of the MuDR element. A 5.1-kb fragment cosegregating with smk2-1 mutants is indicated by the arrow. The genotype N (nonsegregating, i.e. wild type) and S (segregating, i.e. heterozygote) was determined by phenotyping the selfed progeny of these individuals. B, Gene structure of Smk2 and Mu insertion sites. Boxes represent exons and lines introns. Triangles denote Mu insertions.

SMK2 Shows a High Similarity to the Glutaminase Subunit of PLP Synthase and Is Localized in Cytosol

BLAST analysis identified a single copy of the Smk2 gene in the B73 genome (RefGen_v3, Maize GDB; Schnable et al., 2009). Smk2 encodes a 255-amino acid protein that shows a high similarity to the glutaminase subunit of PLP synthases. The SMK2 protein sequence is 66% identical and 85% similar to AtPDX2 of Arabidopsis (Tambasco-Studart et al., 2007); 46% identical and 60% similar to YaaE of Bacillus subtilis (Raschle et al., 2005), and 38% identical and 53% similar to SNZ (snooze)-proximal open reading frame 1 (SNO1) of Saccharomyces cerevisiae (Rodríguez-Navarro et al., 2002; Dong et al., 2004). In addition, the highly conserved signature sequence of glutaminase subunits ([G/A]LI[L/I/V]PGGEST[S/T/A]; Zalkin and Smith, 1998), and the catalytic triad sites Glu-His-Cys (Bauer et al., 2004) are present in SMK2 (Fig. 3A). The two plant proteins, SMK2 and AtPDX2, are similar in size, whereas the bacterial and yeast, YaaE and SNO1, are slightly shorter in the C terminus. The sequence similarity indicates that Smk2 encodes the glutaminase subunit of PLP synthase in the vitamin B₆ biosynthesis pathway.

Figure 3.

Smk2 encodes a cytosolic protein that shows significant similarity to the glutaminase subunits of PLP synthases. A, Alignment of SMK2 with characterized glutaminase subunits from S. cerevisiae (SNO1), Arabidopsis (AtPDX2), and B. subtilis (YaaE). The blue box indicates the signature motif of glutaminase subunits ([G/A]LI[L/I/V]PGGEST[S/T/A]). The red boxes highlight the conserved catalytic triad sites (Glu-His-Cys). B, SMK2-GFP or GFP-SMK2 fusion protein was transiently expressed in tobacco leaf epidermal cells with ADH-RFP as a cytosolic marker. Fluorescent signals were observed under confocal laser-scanning microscopy at 36 h after infiltration. C, Western-blotting analysis of tobacco leaves transiently expressing GFP, SMK2-GFP, or GFP-SMK2 with anti-GFP antibody. Rubisco large subunit stained with Coomassie Blue serves as a loading control. DIC, differential interference contrast. Scale bars = 10 µm.

To determine the subcellular localization, SMK2 was fused with GFP at the N or C terminus and transiently expressed in tobacco leaves via Agrobacterium infiltration. The alcohol dehydrogenase-RFP fusion (ADH-RFP) was coexpressed as the cytosolic marker (Denyer et al., 1996; Heazlewood et al., 2004; Giegé et al., 2003). Confocal microscopy analysis indicated that the green signals from both SMK2-GFP and GFP-SMK2 were merged with the red signals from ADH-RFP (Fig. 3B), indicating that SMK2 is localized in the cytosol. To test whether SMK2 carries an N-terminal signal peptide that could be cleaved after localization, we compared the size of SMK2-GFP and GFP-SMK2 in these leaves by western blot. As shown in Figure 3C, the anti-GFP antibody detected a single 56-kD band that appeared to be indistinguishable in size between SMK2-GFP and GFP-SMK2, suggesting that SMK2 is unlikely to have a cleavable signal peptide.

Vitamin B₆ Deficiency Arrests Embryogenesis But Has Reduced Effects on Endosperm Development

As wild-type Smk2 transcripts were not detected in the embryo and endosperm of smk2-1 mutants by reverse transcription (RT)-PCR analyses (Supplemental Fig. S3), smk2-1 is probably a null allele. Hence, smk2-1 was subjected to further analyses. To test whether the smk phenotype is associated with a vitamin B₆ deficiency, we measured the vitamin B₆ contents in the embryo and endosperm of the wild-type and smk2-1 kernels by high-performance liquid chromatography (HPLC). The five B₆ vitamers were identified and quantified based on the standards (Supplemental Fig. S4). To further aid the identification and quantification of PMP and PLP, the embryo and endosperm extracts were treated with alkaline phosphatase, which converts PMP to PM and PLP to PL, respectively. As a result, the treatment caused a complete disappearance of the PMP and PLP peaks and increases in the PM and PL peaks (Supplemental Fig. S4). The decreased amounts of PMP and PLP were in a good agreement with the increased amount of PM and PL in both the embryo and endosperm. Noted in the standard injection is the much stronger emission signal of PL than that of PLP at 395 nm and such that the PL signal converted from PLP was amplified. The results showed that the content of total vitamin B₆ was drastically reduced in the smk2-1 embryo and endosperm in comparison to the wild type (Fig. 4A). This reduction was found in the smk2-1 mutants from multiple ears, confirming that the mutation in Smk2 causes the vitamin B₆ deficiency. In wild-type kernels, PMP and PLP are the major B₆ vitamers, together accounting for about 90% of total vitamin B₆ in both embryos and endosperms at 18 DAP (Fig. 4B). PMP accounted for ∼50% of vitamers in the embryo and 75% of vitamers in the endosperm (Fig. 4B). During seed development, PLP content increased in the embryo but remained relatively unchanged in the endosperm. In contrast, PMP levels increased dramatically in the endosperm from 6 to 18 DAP (Fig. 4B).

Figure 4.

HPLC analysis of vitamin B₆ content in embryo and endosperm of wild-type (WT) and smk2-1 kernels. A, Total vitamin B₆ content in embryo and endosperm of WT and smk2-1 kernels. Total vitamin B₆ value represents the sum of five B₆ vitamers. B, B₆ vitamer levels in embryo and endosperm of WT kernels at different developmental stages. C, B₆ vitamer levels in embryo and endosperm of 18 DAP smk2-1 kernels. Error bars represent the se of three independent experiments. For each vitamer, different letters indicate significant differences, according to the Waller-Duncan k-ratio t test with P = 0.05. Asterisks represent significant differences (P < 0.01). PLP, Pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate; PL, pyridoxal; PM, pyridoxamine; PN, pyridoxine.

In the mutant, the level of PLP was comparable in embryo and endosperm (Fig. 4C). As in wild type, PMP accumulated to higher levels than PLP in the endosperm, although the levels of each vitamer are drastically reduced. These results suggest that embryogenesis is more sensitive to vitamin B₆ deficiency than endosperm development in maize.

The Maize Smk2 Partially Complements the Arabidopsis pdx2.1 and the Yeast MML21 Mutant Phenotypes

The maize SMK2 shares a 66% amino acid identity with the Arabidopsis AtPDX2, which is implicated in vitamin B₆ biosynthesis (Tambasco-Studart et al., 2005). To address the functional relationship between these two genes, we tested whether Smk2 could complement the Arabidopsis pdx2 mutant. The Arabidopsis T-DNA insertion mutant pdx2.1 (SALK_072168) was obtained from the Arabidopsis Biological Resource Center. Development analysis indicated that embryogenesis of the pdx2.1 mutant was arrested at the globular stage, producing albino seeds (Supplemental Fig. S5; Tambasco-Studart et al., 2005). The mutant seeds could not germinate and were hence completely embryo lethal. When heterozygous pdx2.1 plants were transformed with the maize Smk2 driven by the CaMV 35S promoter, we were able to identify six viable plants that were homozygous for pdx2.1. All six plants were confirmed to contain a Smk2 transgene that was expressed (Supplemental Fig. S6, A and B). While these plants were viable, they grew slower than the wild type and the seeds produced showed a high percentage of lethality (Fig. 5, A–D). Phenotype analysis of developing transgenic embryos revealed varying degrees of rescue ranging from partial to complete complementation (Supplemental Fig. S6, C–E). Thus, Smk2 can only partially rescue the pdx2.1 mutant. HPLC analysis indicated that vitamin B₆ levels in Smk2 transgenic plants were about one-third of the wild-type level (Fig. 5I). We speculate that the partial rescue may be due to suboptimal function and/or expression of the Smk2 transgene in the heterologous system. Partial rescues are common in complementation tests, as the transgene cannot completely mimic the endogenous gene expression.

Figure 5.

Smk2 partially complements the embryo-lethal phenotype of Arabidopsis pdx2.1 mutant. A to D, Three-week-old plants of Col-0 (A) and pdx2.1 mutants carrying Smk2 transgene (B–D). Different transgenic lines show different extents of restored growth of pdx2.1 mutants. E to H, Three-week-old plants of Col-0 (E) and pdx2.1 mutants carrying Smk2 and ZmPDX1.1 transgene (F–H). I, The total vitamin B₆ level of wild-type, pdx2.1/Smk2-C, and pdx2.1/Smk2+ZmPDX1.1-C plants. Error bars represent the se of three independent experiments. Different letters indicate significant differences, according to the Waller-Duncan k-ratio t test with P = 0.05. C, Complementation.

We tested the possibility that partial rescue is due to incompatibility between maize SMK2 and Arabidopsis AtPDX1 subunits of the PLP synthase complex. PDX1 and PDX2 have to form a complex where PDX2 catalyzes the removal of the ammonia group from a Gln molecule and PDX1 transfers it to a specific substrate (Raschle et al., 2005). The activity requires proper channeling of the cell-damaging free ammonia from PDX2 to PDX1 without leakage; thus, accurate association between PDX1 and PDX2 is necessary. X-ray structural analysis of the PDX1:PDX2 complex showed that the N terminus of PDX1 interacts with PDX2 (Strohmeier et al., 2006), and interestingly the N-terminal sequences of maize PDX1s are divergent from the Arabidopsis PDX1 (Supplemental Fig. S7). For this reason, we created transgenic plants expressing ZmPDX1.1 (GRMZM2G120652) and Smk2 in the pdx2.1 mutant in Arabidopsis. The transgenic lines were verified by PCR genotyping (Supplemental Fig. S6G). The pdx2.1 plants coexpressing both maize genes showed a slight improvement in growth over the plants expressing only Smk2. However, growth was still slower than the wild type (Fig. 5, F–H). Consistent with the growth phenotype, HPLC analysis detected an increase in the total level of vitamin B₆ in these plants that was still lower than wild type (Fig. 5I). These results indicate that the cause of partial rescue of the pdx2.1 mutant likely involves transgene expression as well as subunit compatibility. The CaMV 35S promoter confers relatively low expression during the early stages of embryogenesis (Sunilkumar et al., 2002).

As indicated in Figure 3A, SMK2 has a moderate similarity to SNO1 of S. cerevisiae, which encodes the glutaminase subunit of PLP synthase (Rodríguez-Navarro et al., 2002; Dong et al., 2004). To further explore the function of SMK2, we tested whether SMK2 could complement the SNO1 knockout mutant in S. cerevisiae. Yeast has three PDX2 genes, SNO1, SNO2, and SNO3, of which SNO1 plays a predominant role in PLP biosynthesis (Rodríguez-Navarro et al., 2002). The MML21 mutant strain, which was generated by disrupting SNO1 with the KanMX4 cassette, showed slow growth in medium lacking vitamin B₆ (Rodríguez-Navarro et al., 2002). To test whether Smk2 can rescue the sno1 mutant, we placed Smk2 into a yeast expression vector, pESC-HIS, which is under control of the GAL10 promoter. The SNO1 gene cloned in the same vector was used as a positive control, and the empty vector was used as a negative control. When cultured in medium supplemented with pyridoxine, all strains grew equally well (Fig. 6A). When cultured in medium deficient of pyridoxine, however, the MML21 mutant strains transformed with Smk2 grew faster than the strains with empty vector and slower than the strains with SNO1 (Fig. 6, A and B). These results indicate that Smk2 can partially rescue the pyridoxine auxotrophic phenotype of MML21 in yeast. Taken together, these results support the conclusion that SMK2 functions as the glutaminase subunit of PLP synthase in vitamin B₆ biosynthesis in maize.

Figure 6.

Smk2 partially restores the pyridoxine auxotrophy of the yeast mutant MML21. A, Growth of yeast strains on plates with or without pyridoxine. The strain MML21 is a SNO1 knockout mutant and shows slow growth in medium lacking vitamin B₆. MML21 was transformed with pESC-HIS-SNO1, pESC-HIS-Smk2, or empty pESC-HIS vector. The transformants were spotted on plates supplemented with or without pyridoxine. Each horizontal row represents a serial 10-fold dilution of each strain starting at an OD₆₀₀ of 0.5. B, Growth of yeast strains in liquid medium with or without pyridoxine. The strains grew in liquid medium starting at an OD₆₀₀ of 0.3. PN, pyridoxine. Values represent the mean OD values from three independent experiments. Error bars represent se.

Vitamin B₆ Rescues the smk2-1 Embryo-Lethal Phenotype

To further test whether the arrested embryogenesis is due to vitamin B₆ deficiency, we attempted to rescue the embryo-lethal smk2-1 mutants by applying vitamin B₆ to plants. Ears from self-pollinated Smk2-1 heterozygotes were sprayed with 2 mm vitamin B₆ (PN) daily after pollination. After 18 d, the unsprayed ears showed clear segregation of smk kernels as shown in Figure 1A, whereas the treated ears did not. Dissection indicated that mutant embryos from treated ears developed coleoptilar, SC, and SAM structures (Fig. 7B), whereas mutant embryos from untreated ears were uniformly blocked at the early transition stage (Fig. 7A). Notably, the structures of coleoptilar, SC, and SAM in the treated mutant embryos were not identical to those of wild-type siblings, suggesting that application of vitamin B₆ in plants can only partially rescue mutant embryogenesis. When the untreated mutant embryos were cultured on medium without vitamin B₆, they did not grow after 20 d in culture (Fig. 7C). By contrast, when the treated smk2-1 embryos (18 DAP) were cultured on medium containing vitamin B₆, they could grow and develop into seedlings (Fig. 7D). These rescued seedlings were confirmed to be homozygous for smk2-1 by PCR genotyping, and no expression of the wild-type Smk2 transcripts was detected by RT-PCR (Fig. 7, F and G). When the rescued smk2-1 seedlings were transplanted to soil and continuously watered with vitamin B₆, they grew slowly and died at the five-leaf stage. These results indicate that exogenous application of vitamin B₆ partially rescues the arrested embryogenesis of the smk2-1 mutant. However, watering the Smk2-1 heterozygous plants with vitamin B₆ did not rescue the smk kernels, suggesting that the capacity of transporting vitamin B₆ from maternal tissues to developing kernels is limited.

Figure 7.

Rescue of the homozygous smk2-1 mutant embryos by exogenous vitamin B₆ supplement. A and B, Histological section of smk2-1 mutant (A) and rescued smk2-1 (B) kernels at 18 DAP. C, The smk2-1 and wild-type (WT) embryos cultured on the medium without vitamin B₆ supplementation. Arrows indicate the WT embryos grown as a control. Squares indicate smk2-1 embryos. D, Rescued smk2-1 embryos grown on the medium with vitamin B₆ for 20 d. E, Gene structure of Smk2 and the location of MuDR insertion in the smk2-1 allele. The positions of primers for RT-PCR and genotype analysis are shown. F and G, The genotype (F) and expression levels (G) of Smk2 in WT and rescued plants. COL, coleoptile; Res, rescued smk2-1 plant. Scale bars = 200 µm in A and B.

Spatial and Temporal Expression Pattern of Smk2

To determine the temporal expression of Smk2, total RNA from different tissues was subjected to regular and quantitative RT-PCR (qRT-PCR) analyses. Smk2 expression was detected in all major plant organs (Fig. 8, A and B). Relatively high levels of expression were detected in root, stem, leaf, silk, and ear, while expression was low in developing kernels (Fig. 8, A and B). These results suggest that Smk2 is not seed specific but rather functions in many tissues. This conclusion is consistent with the basic cellular function of vitamin B₆.

Figure 8.

Spatial-temporal expression of Smk2 during plant growth and seed development. A, RT-PCR analysis of Smk2 expression in major tissues of maize and during seed development. ZmActin (GRMZM2G126010) was used as control. B, qRT-PCR determination of the expression levels in the tissues. ZmActin was used as an internal control to normalize RNA quantity, and the expression level of Smk2 in silk was set to 1. Data represents three biological replicates, and each was measured three times. Error bars represent se. C to H, RNA in situ hybridization of wild-type embryos at 5 (C), 7 (D), 9 (E), 15 (F and H), and 21 (G) DAP. The antisense probe of Smk2 was hybridized in C to G, and the sense probe in H (refer to ”Materials and Methods”). Scale bars = 500 µm.

To gain further insights of Smk2 expression during embryogenesis, we performed RNA in situ hybridization on sections of wild-type embryos at different developmental stages. Smk2 antisense and sense probes were used (Fig. 8, C–H). Smk2 mRNA signals were detected in 5 DAP early transition stage embryos (Fig. 8C). Expression was highest at the late transition stage (7 DAP; Fig. 8D) and then declined at the early coleoptile stage (9 DAP; Fig. 8E). At the leaf stage 1 (15 DAP), the signal appeared to spread over the whole embryo, and strong expression occurred in the SAM and in the epidermis of the SC (Fig. 8F). In 21 DAP embryos, expression was restricted to the top region of leaf primordia (Fig. 8G). As a negative control, the sense probe did not yield significant signals (Fig. 8H). These results suggest that embryogenesis requires high level of vitamin B₆ during the late transition stage, conforming to the observation that embryogenesis in smk2-1 mutant is arrested at the early transition stage.

DISCUSSION

In this work, we isolated and characterized the maize smk2 mutant obtained by transposon tagging. Multiple pieces of evidence indicate that Smk2 encodes the glutaminase subunit of the PLP synthase complex in vitamin B₆ biosynthesis. The evidence includes (1) the smk2 mutants are vitamin B₆–deficient, and exogenous application of vitamin B₆ could partially rescue the embryo-lethal phenotype of the smk2 mutant kernels (Fig. 7); (2) SMK2 shows significant similarity to PDX2 in Arabidopsis and SNO1 in yeast, which were previously characterized as glutaminase subunits (Fig. 3A); (3) the maize Smk2 partially complemented the Arabidopsis pdx2.1 and yeast MML21 mutants (Figs. 5 and 6). Furthermore, the SMK2 protein was localized to cytosol, indicating the compartment of vitamin B₆ biosynthesis (Fig. 3, B and C). Further biochemical and developmental analyses allowed us to dissect the differential roles of vitamin B₆ in embryogenesis and endosperm development in maize, an important monocot model crop.

Vitamin B₆ Biosynthesis Is Essential to Embryogenesis But Less So to Endosperm Development

Loss of Smk2 results in embryo lethality in maize, suggesting that vitamin B₆ is essential to embryogenesis. This essentiality is likely conserved in flowering plants, considering the cofactor function of vitamin B₆. In Arabidopsis, loss of PDX2 reduces vitamin B₆ content and causes embryo lethality as well (Tambasco-Studart et al., 2005; Titiz et al., 2006). The maize smk2-1 embryo is partially rescued by direct application of vitamin B₆ to developing ears. Continuous culture on vitamin B₆-rich medium allowed the partially rescued embryos to develop into seedlings. The biotin-deficient bio2 mutant of Arabidopsis can be rescued by watering heterozygous plants with biotin (Patton et al., 1998). But this is not the case for vitamin B₆ mutants of Arabidopsis and maize. This observation implies (1) the translocation efficiency of vitamin B₆ from maternal tissues or neighboring seeds to developing mutant seeds is limited; (2) embryogenesis requires endogenous biosynthesis of vitamin B₆.

In contrast to the strong block during the transition stage of embryogenesis, the smk2 mutation has less of an effect on endosperm development. The smk2 mutant kernel can form an endosperm filled with starch, suggesting that vitamin B₆ is not as essential to the endosperm development as it is to embryogenesis. One possibility is that endosperm development may not rely on high levels of vitamin B₆ for its cofactor function. The Enzyme Commission (http://www.chem.qmul.ac.uk/iubmb/enzyme/) has cataloged over 170 PLP-dependent activities mainly involving in amino acid metabolism. Endosperm, as the storage organ mainly for starch and proteins, may be less dependent on these activities since amino acids and Suc are supplied maternally. Starch biosynthesis appears to not involve PLP-dependent enzymes. Starch biosynthesis requires a concerted action of ADP-Glc pyrophosphorylase, starch synthase, and starch-branching and debranching enzymes (Sabelli and Larkins, 2009). Based on the vitamin B₆ database (http://bioinformatics.unipr.it/cgi-bin/bioinformatics/B6db/home.pl; Percudani and Peracchi, 2003), these four enzymes are not PLP dependent. An alternative possibility is that the endosperm has priority access to maternal PLP over the embryo because the maize endosperm is positioned between the maternal vasculature and the embryo. Indeed, a 7% of wild-type level PLP was detected in the smk2 mutant endosperms (Fig. 4), which may be near the threshold for normal endosperm metabolism. In that scenario, it is possible that the maternal vitamin B₆ is sufficient to maintain a basal level of amino acid metabolism in the smk2 endosperm.

During the seed development, the endosperm maintains a roughly constant level of PLP but accumulates significantly high levels of PMP, reaching more than three times the amount of PLP at 18 DAP (Fig. 4B). PMP serves as a cofactor for only a few enzymes (Agnihotri and Liu, 2001; Adrover et al., 2009). And no evidence indicates that the enzymes involved in starch biosynthesis are PMP dependent (http://bioinformatics.unipr.it/cgi-bin/bioinformatics/B6db/home.pl). PMP can be oxidized by the PMP/PNP oxidase (PDX3) to form PLP. Thus, it is reasonable to propose that the accumulated PMP in endosperms is a storage form that can be converted to PLP during seed germination to nurture the seedling growth.

Biosynthesis of Vitamin B₆ in Developing Seeds and Transportation from Maternal Tissues

Although the disruptive Mu insertions occur in various positions of the Smk2 gene, the mutants are not completely vitamin B₆ deficient. A residual 7% of the wild-type vitamin B₆ level was consistently detected in the smk2-1 mutants (Fig. 4). This low level of vitamin B₆ raises the question on how developing mutant kernels acquire essential vitamin B₆. This result invokes two possibilities: (1) The developing mutant kernels may still retain a limited vitamin B₆ biosynthesis capability, and/or 2) the mutant kernels can obtain vitamin B₆ from maternal tissues or neighboring wild-type siblings. In both cases, the amount of vitamin B₆ synthesized or transported is limited (Fig. 4) and not sufficient to support normal embryogenesis. The first possibility could be realized by either a leaky mutation in smk2-1 or redundancy for PDX2; however, the evidence does not favor either scenario. First, the possibility that smk2-1 is leaky is not supported by the data. We analyzed multiple independent smk2 alleles that contain Mu insertions in different regions. In these alleles, the SMK2 proteins are predicted to be truncated to various lengths. All of the alleles show a typical smk phenotype. These results led us to rule out the possibility of leaky mutations. Secondly, we explored whether a Smk2 homologous gene might code for glutaminase activity in the maize. BLAST analysis did not uncover a homolog with significant similarity in the maize B73 RefGen_v3 draft, rendering this possibility unlikely. The second possibility for residual vitamin B₆ in smk2-1 mutant kernels is that a small amount of vitamin B₆ can be transported to developing kernels from maternal tissues. Although no vascular system exists between maternal tissues and developing kernels, small molecules such as vitamins (Patton et al., 1998), amino acids (Tegeder et al., 2000), hormones (Frey et al., 2004), and Suc (Weschke et al., 2000) are capable of being transported into filial tissue. In maize and several other tropical cereals, assimilates are delivered to the phloem terminals in the pedicel and then diffuse to endosperm transfer cells (Kladnik et al., 2004). The transport efficiency of different molecules varies. In maize kernels, the efficiency of movement of the vitamins thiamine and nicotinic acid is significantly lower than that of Suc (Shimamoto and Nelson, 1981). Thus, import of vitamin B₆ from maternal tissue to mutant smk2 kernels is likely insufficient to support embryo development. This notion is supported by our experiment where exogenous application of vitamin B₆ only caused a partial rescue of the embryo-lethal phenotype (Fig. 7B). This implies that the vitamin B₆ required for embryogenesis is synthesized within the developing embryo, whereas vitamin B₆ transport from maternal tissues to the embryo is minimal.

MATERIALS AND METHODS

Plant Material and Growth Conditions

The smk2-1 allele was isolated from the UniformMu population (McCarty et al., 2005), and other alleles were identified from the Trait Utility System in Corn population by PCR with gene-specific primers (Bensen et al., 1995). The maize plants were grown under natural conditions. The pdx2.1 T-DNA insertion line (SALK_072168) was obtained from the Arabidopsis Biological Resource Center. Arabidopsis (Arabidopsis thaliana) plants were grown under 16 h light/8 h darkness conditions at 22°C.

Complementation of Arabidopsis pdx2.1 Mutant

The full-length Smk2 CDS was amplified by using primers SMK2-BglII-F and SMK2-BstEII-R, which introduced BglII and BstEII restriction sites. The resulting PCR product was cloned into pCAMBIA3301 downstream of the CaMV 35S promoter. ZmPDX1.1 was introduced into pGWB2 vector. The constructs were introduced into Agrobacterium strain EHA105. The PDX2.1/pdx2.1 heterozygous plants were transformed by using the floral-dip method (Clough and Bent, 1998). T1 transgenic plants with Smk2 transgene were selected on BASTA-containing medium, and plants with both Smk2 and ZmPDX1.1 transgenes were selected on BASTA- and hygromycin-containing medium. Homozygous pdx2.1 plants were identified by PCR with primers pdx2.1-F1 and pdx2.1-R1. Transgenic plants containing Smk2 transgene were verified by PCR with primers SMK2-RTF2 and SMK2-RTR2, and those containing ZmPDX1.1 transgene were verified by PCR with primers ZmPDX1.1-F1 and GWB2-R1. Information of all primers used in this study is listed in Supplemental Table S1.

Yeast Complementation

Saccharomyces cerevisiae MML21, a knockout of the yeast SNO1 gene, was obtained from Dr. Jose E. Perez-Ortin (University of Valencia, Spain). For the complementation analysis, the full-length Smk2 CDS was amplified and ligated into the yeast expression vector pESC-HIS, which places Smk2 expression under the control of the GAL10 promoter. Transformation and screening of transformants were performed according to the protocol of Clontech (Takara). The transformants were cultured in synthetic dropout medium without vitamin B₆ for 30 h. And then, the starter culture was spotted onto solid medium or grown in liquid medium with optical density (OD₆₀₀) of 0.5 and 0.3, respectively.

Rescue of smk2-1 Kernels

The ears of self-pollinated Smk2-1 heterozygous plants were sprayed daily with 2mM pyridoxine from the first DAP. At 18 DAP, the embryos were isolated from sprayed kernels and cultured on an enriched-vitamin solid medium, composed of Murashige and Skoog salts, 3% Suc, 100 mg/L myoinositol, 500 mg/L MES, 0.9% agar, 0.1 mg/L 1-naphthylacetic acid, 1 mg/L 6-benzylaminopurine, 1 mg/L biotin, 1 mg/L nicotinic acid, 1 mg/L thiamine, and 100 µm pyridoxine, adjusted to pH 5.7 with NaOH. As a control, smk2-1 and wild-type embryos were isolated from unsprayed kernels and placed in a medium without pyridoxine. Each embryo cultured on enriched-vitamin solid medium was dripped with enriched-vitamin liquid medium every third day. After 20 d of embryo culture, rescued plants were transferred to soil and watered daily with 2 mm pyridoxine.

Subcellular Localization of SMK2

Full-length Smk2 CDS without stop codon was amplified by primers Smk2-F1 and Smk2-R1 from the maize inbred line B73 and cloned into the binary vector pGWB5 (for SMK2-GFP) by the GATEWAY in vitro site-specific recombination methodology (Invitrogen). Full-length AtADH CDS without stop codon was amplified by primers ADH-F1 and ADH-R1 and cloned into pH7RWG2 to generate the ADH-RFP construct (Karimi et al., 2002). To transiently express GFP-SMK2, full-length Smk2 CDS was introduced into pGWB6 vector. The binary constructs were introduced into Agrobacterium strain EHA105. Leaves of 4-week-old tobacco were coinfiltrated with the strains carrying ADH-RFP and SMK2-GFP or GFP-SMK2, respectively. Fluorescent signals were detected with an Olympus FV1000-IX81 confocal microscope at 36 h after infiltration.

For western-blotting analysis of the SMK2-GFP and GFP-SMK2 protein, total protein was extracted from leaves at 36 h after infiltration, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis,and analyzed by western blotting with an anti-GFP antibody (Sigma).

Light Microscopy of Cytological Sections

Wild-type and smk2-1 kernels were harvested from the same ear of a self-crossed heterozygous plant at 18 DAP. The kernels were cut along the longitudinal axis in three equal parts. The central slice containing the embryo was fixed for 1 d at 4°C in 4% paraformaldehyde. The fixed materials were dehydrated in a graded ethanol series (30%, 50%, 75%, 95%, and 100%), infiltrated, and embedded in paraffin. The samples were sectioned at 8 to 10 μm with a microtome (Jung Biocut 2035, Leica), stained with Johansen’s Safranin O, and observed with a Nikon ECLIPSE 80i microscope.

RNA Extraction, RT-PCR, and qRT-PCR

Total RNA was isolated by using the Qiagen Plant RNeasy kit (Qiagen) according to the manufacturer’s instructions and then treated with DNase I (New England BioLabs) to eliminate genomic DNA contamination. Complete removal of DNA was verified by performing PCR analysis on RNA samples before converting to cDNA. Reverse transcription reactions were performed using 1 µg of total RNA with random hexamers by SuperScript II Reverse Transcriptase (Invitrogen). qRT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad). Relative quantification was carried out using the 2^−ΔΔCT method. ZmActin was used as an internal control for RNA normalization. The expression level of Smk2 in silk was set to 1. The RT-PCR was performed in three technical replicates. The Smk2 cDNA was amplified with primers SMK2-CF1 and SMK2-CR1 from RNAs extracted from W22 leaves.

DNA Extraction, Southern Analysis, and Construction, and Screening of Genomic Library

Genomic DNA extraction and Southern hybridization analysis were performed as described previously (Tan et al., 2011). To reduce the high active Mu copy number, smk2-1 mutant was backcrossed twice with W22 and then self-pollinated to create an F2 segregating family. As homozygous smk2-1 mutants are embryo lethal, only heterozygotes and wild-type seeds can germinate. The genotype of each F2 plant was determined by scoring the selfed ear for segregating (S) or nonsegregating (N) the smk2-1 mutants. DNAs of the F2 plants were hybridized with different Mu-specific probes (Tan et al., 2011). Construction and screening of genomic libraries were carried out according to the method described previously (Tan et al., 1997).

In Situ Hybridization

In situ hybridization was performed as previously described (Shen et al., 2013). The DIG-labeled sense and antisense probes were synthesized by T7 and SP6 polymerases, respectively. The probe used to detect Smk2 transcripts corresponds to a 210-bp fragment from +29 to +238 bp of the Smk2 cDNA.

Vitamin B₆ Analysis by HPLC

Vitamin B₆ was extracted from maize embryos and endosperms and Arabidopsis rosette leaf tissue from pdx2.1/Smk2-OX, pdx2.1/Smk2+ZmPDX1.1-OX in Col-0 ecotype. To determine vitamin B₆ content, three independent maize kernel samples were obtained from three different cobs, and three independent Arabidopsis lines were sampled using six leaves per line. Vitamin B₆ was extracted by 5% (w/v) trichloroacetic acid in darkness (González et al., 2007). HPLC analysis of B₆ vitamer levels was performed as described previously (Valls et al., 2001). For alkaline phosphatase treatment, the embryo and endosperm were ground into dry powder under liquid nitrogen. One-half milliliter of 0.05 m phosphate buffer (pH 7.5) was added to 0.625 g of dry powder and followed by 100 U of alkaline phosphatase. The mixture was incubated at 37°C for 1 h. Then 5% (w/v) trichloroacetic acid was added and the sample was mixed and analyzed. Standards PMP, PLP, pyridoxal hydrochloride, pyridoxine hydrochloride, and pyridoxamine dihydrochloride were purchased from Sigma. PNP is not available commercially.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers SMK2 (AY109859), SNO1 (NP_013813), AtPDX2 (AT5G60540), YaaE (AKL87126), AtADH (AT1G77120), ZmPDX1.1 (NM_001147185), and ZmActin (NM_001155179).

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Scheme of de novo and salvage vitamin B₆ biosynthetic pathways.

• Supplemental Figure S2. The development of transfer cell layer at 18 DAP.

• Supplemental Figure S3. The expression of Smk2 transcript in embryo and endosperm of wild type and smk2-1 mutant.

• Supplemental Figure S4. Representative B₆ vitamer profiles of maize embryo and endosperm.

• Supplemental Figure S5. Characterization of Arabidopsis pdx2.1 mutants.

• Supplemental Figure S6. Characterization of transgenic plants.

• Supplemental Figure S7. Alignment of ZmPDX1s and AtPDX1s.

• Supplemental Table S1. Primer information.

Glossary

DAP

days after pollination