Summary of the glycolytic pathway. Reversible enzymes that involve PPi are shown in gray boxes. The solid gray arrow represents transcriptional activation of PPDK by the O2 transcription factor. AGPase, ADP-Glc pyrophosphorylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PEPC, PEP carboxylase; PFK, ATP-dependent phosphofructokinase; PK, pyruvate kinase.

PFPα and PFPα retrogene structure. A, Structure of the PFPα genomic sequence. Thick bars and thin line represent exons and introns, respectively, whose length corresponds to the scale bar. The gray exon is not present in the PFPα retrogene. B, Genomic sequence of PFPα showing exons 13, 14, and 15 (gray-shaded uppercase letters) and introns 13 and 14 (lowercase letters). The underlined 105-bp region, encoding 35 amino acids, is absent in the PFP retrogene. In the PFPα retrogene, the entire region between the terminal T of exon 13 and the first G of exon 15 (shown with white letters on black boxes) is absent. C, Amino acid sequences of PFPα retrogene (PFPα R; top) and PFPα (PFPα L; bottom), aligned to show polymorphisms and the 35-amino acid region not present in the retrogene product (black box). The underlined region shows the 14-amino acid region used for peptide antibody production.

Expression of PFPα in maize endosperm. A, RT-PCR of PFPα in W64A wild-type (WT), W64A o2, and K0326Y QPM developing endosperm RNA. Three biological replicate ears are shown for each genotype. Primers flank the 105-bp region absent in the retrogene transcript and are common to both PFPα (top band) and PFPα retrogene (bottom band). B, As for A but comparing CM105 wild type, CM105o2, and CM105 QPM. C, qRT-PCR of PFPα comparing expression in W64A wild type, W64A o2, K0326Y QPM, and K0326Y by W64Ao2 RILs with defined genotypes at chromosome 1, 7, and 9 QPM QTLs (Holding et al., 2011), as shown in D. The PCR product does not span the 105-bp polymorphism, and primer-binding sites are common between PFPα and PFPα retrogene. Differential expression is derived from full-length PFPα (Supplemental Fig. S1). The y axis shows relative expression (log₂ to the wild type using RBR1 as an internal control gene; Sabelli et al., 2005). Values are derived from three biological replicate ears, and sd values represent means of three technical repeats, as described previously (Holding et al., 2011). D, RT-PCR of same lines as in C with genotype at the three QTLs depicted (Holding et al., 2011). A refers to the K0326Y QPM allele, B refers to the W64Ao2 allele, and lines were selected for homozygosity at each of the chromosome 1, 7, and 9 QTLs. Thus, AA BB BB has the homozygous K0326Y QPM genotype only at the chromosome 1 QTL, BB AA BB has the homozygous K0326Y QPM genotype only at the chromosome 7 QTL, and BB BB AA has the homozygous K0326Y QPM genotype only at the chromosome 9 QTL. The chromosome (chr) number for each QTL genotype is shown below the genotype designation. E, Kernel vitreousness/opacity of the RILs shown in C and D using a light box. [See online article for color version of this figure.]

PFP subunit accumulation in endosperm and embryo. A, PFPα protein gel blot of 12- and 18-DAP CM105 wild-type (WT), CM105o2, and CM105 QPM endosperm extracts. B, PFPβ protein gel blot of 12- and 18-DAP CM105 wild-type, CM105o2, and CM105 QPM endosperm extracts. C, PFPα protein gel blot of 18-DAP CM105 wild-type, CM105o2, and CM105 QPM embryo extracts. D, PFPα protein gel blot of 18-DAP endosperm extracts of representative vitreous, semivitreous, and opaque RILs derived from the W64Ao2 × K0326Y QPM cross selected on the basis of phenotype rather than genotype. Kernel opacity phenotypes are shown below. Numbers below the panel refer to the RIL number whose genotype at chromosome 1, 7, and 9 QTLs (Holding et al., 2011) follows the same nomenclature described Figure 3D legend, as follows: 164, BB AA AB; 208, AA BA AA; 175, AA BA BB; 176, AA BA BB; 185, BB BB BB; 194, BB BB BB. [See online article for color version of this figure.]

Size-exclusion chromatography analysis of PFP complex formation in maize endosperm. The panels show protein gel blots of SDS-PAGE of eluted fractions from HPLC separation of native endosperm extracts. Elution times of protein standards are shown in the key at bottom. Genotypes are shown on the left, and the developmental stages and the antibodies used are shown above each panel. WT, Wild type.

Size-exclusion chromatography analysis of PFP complex formation in 18-DAP maize embryo. The panels show protein gel blots of SDS-PAGE of eluted fractions from HPLC separation of native embryo extracts. Elution times of protein standards are shown in the key at bottom. Genotypes are shown on the left, and the antibodies used are shown above each panel. WT, Wild type.

PFP and PPDK activity in 18-DAP W64A wild-type (WT), W64A o2, and K0326Y QPM endosperm. Activity units (y axes) are in μmol min⁻¹ mg⁻¹ protein. Values (means ± se) with the same letter (A) above the bar are not significantly different at P = 0.05, as determined by a two-tailed t test. Bars shown with a B above are significantly different at this level. Error bars show se.

PFP induction in root tissue in response to stress. Lanes show protein gel blots of W64A wild-type root proteins (10 μg) from plants grown in tissue culture boxes and treated to induce heat or cold shock and/or UPR stress as follows: lane 1, 25°C control; lane 2, 42°C for 12 h; lane 3, 42°C + 5 mm DTT for 12 h; lane 4, 25°C + 5 mm DTT for 12 h; lane 5, 4°C for 12 h; lane 6, 4°C + 5 mm DTT for 12 h. Antibodies used were HSP90, HSP101, HSP70 (BiP), PFPα, and PFPβ, which are indicated at left.