PMC

Effect of the bmr6-3 mutation on the structure of SbCAD2. (A) G(X)GGV(L)G motif and its interaction with the phosphate backbone of NADP⁺. (B) SbCAD2 bmr6-3 variant at residue 191. The G191S mutation will affect the interaction between this flexible motif and phosphate groups.

Graphical comparison of active site residues interacting with the NADP⁺ cofactor of (A) AtCAD5 and (B) SbCAD2. Residue M60 and C95 change to A and V, respectively, whereas the other residues are conserved. This will potentially affect substrate binding and specificity.

Representation of the configurations of the secondary structural elements β11, α6, and β12 in (A) wild-type SbCAD2 and (B and C) the bmr6-27 variant of SbCAD2. B and C represent two possible configurations of the corresponding secondary structure. In B, significant repositioning of the α6-helix occurred. In C, a significant reduction of the secondary structure in all three structural elements occurred.

The bmr6-ref, but not the bmr6-3 mutation, causes downregulation of SbCAD2 expression in 2-week-old plants. (Top lane) Products from RT–PCR with primers specific for a fragment of the cDNA encoding SbCAD2. (A) 1–4, N3 cDNA; 5–8, bmr6-3 cDNA. (B) 1–6, BTx623 cDNA; 7–12, BTx623 bmr6-ref cDNA. Odd numbers correspond to cDNA obtained from shoots; even numbers cDNA obtained from roots. (Bottom lane) Products from RT–PCR with primers for Ubiquitin gene as a cDNA loading control. The size marker is Bioline Hyperladder II (cat. no. 33054).

Ribbon diagram representation for the structure of SbCAD2. The significant arginine mutations found in SbCAD2_27b are indicated in blue. The dimer contact positions for βD–F are highlighted in yellow. The three secondary structural elements, β11, β12, and α6 are not maintained in the CAD model of bmr6-27 mutant due to the degenerative hydrophobic interactions caused by the arginine residues highlighted in blue.

Molecular markers can distinguish between the wild-type and mutant SbCAD2 alleles. (A) CAPS marker specific for the bmr6-ref allele. Lanes 1 and 2 show the undigested 992-bp PCR product from N6 and bmr6-ref DNA, respectively. Lanes 3 and 4 show the respective PCR products after digestion with the restriction enzyme TaiI. The 363-bp diagnostic fragment is indicated with a solid triangle. (B) CAPS marker specific for the bmr6-3 allele. Lanes 1 and 2 show the undigested PCR product from N3 and bmr6-3 DNA, respectively. Lanes 3 and 4 show the respective PCR products after digestion with the restriction enzyme TauI. The 138-bp diagnostic fragment is indicated with a solid triangle.The size marker is Sigma PCR Marker [catalog (cat.) no. P9577].

Organization of the sorghum (A) and rice (B) CAD8 gene clusters. Block arrows represent the genes and their orientation. Arrows with the same color represent genes more similar to each other than to the other members of the cluster. The yellow arrows represent a gene downstream of the CAD8 cluster present in both the rice and sorghum genomes. The scale is approximate and based on the sorghum and rice physical map. The represented sorghum and rice fragments span ∼21.5 and 26 kb, respectively.

Sequence comparison of the translated sequences of Sorghum bicolor CAD family members, maize ZmCAD2 (Zea mays sequence AJ005702.1), and Arabidopsis AtCAD5 (Arabidopsis thaliana sequence At4g34230). The conserved amino acids identified by as part of the substrate binding pocket are indicated by asterisks above the sequences. The conserved glycine-rich motif G(X)GGV(L)G is indicated by a dotted line below the sequences. Italicized letters indicate completely conserved residues across all 14 sequences.

Phylogenic relationship between the sorghum CAD family and CAD proteins from other species. The branch lengths indicate the relative distance between sequences, with the exception of the putative sorghum sorbitol dehydrogenase, which is distantly related to the CAD family (accession no. Sb07g025220.1). The sequences used to build the tree were: Selaginella moellendorffii SmCAD1-1 (protein ID 450958 of the JGI Selanginella genome database); Picea abies PtCAD (O82035); Zea mays ZmCAD2 (CAA06687); Arabidopsis thaliana AtCAD1 (At1g72680), AtCAD2 (At2g21730), AtCAD3 (At2g21890), AtCAD4 (At3g19450), AtCAD5 (At4g34230), AtCAD6 (At4g37970), AtCAD7 (At4g37980), AtCAD8 (At4g37990), and AtCAD9 (At4g39330); Populus tremuloides PtSAD (AAK58693), PtCAD (AAF43140); Oryza sativa OsCAD1 (NP_001064283.1), OsCAD2 (NP_001046132.1), OsCAD3 (AAP53892), and OsCAD4 (NP_001068303.1). OsCAD5 (BK003971), OsCAD6 (CAD39907), OsCAD8 A-C (BK003972), and OsCAD9 (AAN05338). Sorghum sequences are those described in . The rice CAD gene nomenclature follows .