Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme

Abstract

Background: Among the dietary essential amino acids, the most severely limiting in the cereals is lysine. Since cereals make up half of the human diet, lysine limitation has quality/nutritional consequences. The breakdown of lysine is controlled mainly by the catabolic bifunctional enzyme lysine ketoglutarate reductase - saccharopine dehydrogenase (LKR/SDH). The LKR/SDH gene has been reported to produce transcripts for the bifunctional enzyme and separate monofunctional transcripts. In addition to lysine metabolism, this gene has been implicated in a number of metabolic and developmental pathways, which along with its production of multiple transcript types and complex exon/intron structure suggest an important node in plant metabolism. Understanding more about the LKR/SDH gene is thus interesting both from applied standpoint and for basic plant metabolism.

Results: The current report describes a wheat genomic fragment containing an LKR/SDH gene and adjacent genes. The wheat LKR/SDH genomic segment was found to originate from the A-genome of wheat, and EST analysis indicates all three LKR/SDH genes in hexaploid wheat are transcriptionally active. A comparison of a set of plant LKR/SDH genes suggests regions of greater sequence conservation likely related to critical enzymatic functions and metabolic controls. Although most plants contain only a single LKR/SDH gene per genome, poplar contains at least two functional bifunctional genes in addition to a monofunctional LKR gene. Analysis of ESTs finds evidence for monofunctional LKR transcripts in switchgrass, and monofunctional SDH transcripts in wheat, Brachypodium, and poplar.

Conclusions: The analysis of a wheat LKR/SDH gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes show lineage-specific differences between monocots and dicots, and findings suggest variation in activity of LKR/SDH genes among plants. Although most plant genomes seem to contain a single conserved LKR/SDH gene per genome, poplar possesses multiple contiguous genes. A preponderance of SDH transcripts suggests the LKR region may be more rate-limiting. Only switchgrass has EST evidence for LKR monofunctional transcripts. Evidence for monofunctional SDH transcripts shows a novel intron in wheat, Brachypodium, and poplar.

Figure 1

Organization of an LKR/SDH gene segment of the wheat genome. (A) Diagram of the wheat LKR/SDH genomic region spanned by BAC clone 0006M07. Genes are identified by purple boxes and regions of transposable elements by white boxes. Sequences of non-coding and non-repetitive type are indicated by grey boxes. Arrows indicate direction of transcription. (B) The LKR/SDH gene region is expanded to show exon/intron organization. Exons are indicated by numbered boxes and introns by intervening lines. The LKR domain exon boxes are blue and the SDH boxes are red. Two yellow exons are the proposed interdomain coding region of the full-length LKR/SDH protein. Start and stop codon positions of the full-length coding region are marked.

Figure 2

LKR/SDH amino acid sequences. The derived amino acid sequence of a wheat LKR/SDH protein is shown and compared to sequences derived from DNA sequences of other plants. Amino acid positions with no more than one difference among all plants are shaded yellow. Amino acids unique to monocots are shaded blue. Red lines indicate exon boundaries. Arrowheads indicate predicted start positions of monofunctional SDH transcripts for wheat (above sequences) and Arabidopsis (below sequences). An asterisk marks the approximately position ending the monofunctional LKR transcript. Exon numbers above the sequences indicate monocot exon numbers - dicot numbering is one less since exons 1 and 2 are fused in dicots. The poplar sequence is derived from poplar gene 1 as described below.

Figure 3

Phylogenetic analysis of LKR/SDH proteins. LKR/SDH amino acid sequences were used to generate a phylogenetic tree using Clustal W and described in Methods. Length of branches represent the number of amino acid substitutions per site. The percentage of replicate trees in which the associated sequences clustered in the bootstrap test are shown next to the branch points. Clusters of monocot and dicot sequences are labeled.

Figure 4

Genome-specific LKR/SDH primers. Common priming sites for all three genomes are F3 and R3. Genome-specific priming sites were developed in intron 22 for the B- and D-genomes (BF3 and DF3, respectively) and in exon 23 for the A-genome (AF3). (B) PCR products generated with each genome-specific LKR/SDH primer plus common primer R3 from genomic DNA of hexaploid wheat cv Chinese Spring group 6 chromosome nullisomic-tetrasomic genetic lines (N6A = nulli6A, N6B = nulli6B, N6D = nulli6D). Two amounts of sample were loaded for each DNA.

Figure 5

ESTs matching BAC mTERF gene. Wheat and barley ESTs matching the BAC mTERF gene are aligned with the mTERF coding sequence. Start and stop codons are boxed. Sequence differences to the BAC are shaded in yellow and blue.

Figure 6

Compare LKR/SDH genome regions. LKR/SDH genomic regions of eight plants are compared for the relative location of three genes, i.e., LKR/SDH, mTERF, and PE. LKR/SDH genes = white boxes. mTERF genes = grey boxes. PE genes = black boxes. The numbers at the left indicated the total number of PE genes found 5' to the LKR/SDH genes in available genomic sequences. No additional wheat genomic sequence is available as indicated by the question mark. Gene lengths and spacing are drawn to scale. Gene lengths are the sum of exons plus introns. The three poplar genes are labeled LKR (monofunctional LKR) and genes 1 and 2 for the two full-length LKR/SDH genes. The poplar LKR/SDH genes are from BAC AC209229 and the PE genes from the genome assembly.

Figure 7

Wheat LKR/SDH EST alignment. The full-length LKR/SDH coding region derived from BAC 006M07 was used to align wheat ESTs matching with a BLASTn of e^-7 or lower. Red arrows indicate 5' reading ESTs initiating in the LKR or interdomain regions. For the remainder of the ESTs, black arrows show forward 5' reads and blue arrows show reverse 3' reads. Red vertical lines indicate the initial start codon of the LKR domain, the proposed start codon for a monofunctional SDH mRNA, and the common stop codon for both the bifunctional and SDH monofunctional mRNAs. Above the alignment are the numbered exons. Blue boxes indicate the LKR domain and red boxes the SDH domain. Yellow boxes are exons within the interdomain region. The black box represents the short sequence from intron 14 that is found at the 5' end of presumptive monofunctional SDH transcripts and not found in the red-arrowed ESTs. Three 5' and 3' EST pairs from the same clones are labeled as follows: 1, CJ882974 + CJ894783; 2, CJ881957 + CJ893808; 3, CJ883733 + CJ895693.

Figure 8

Switchgrass EST alignment. Switchgrass LKR/SDH ESTs are aligned to the maize coding sequence given in base pairs. Arrows indicate length of the ESTs and their direction of transcription. Black arrows are 5' reads and blue arrows are 3' reads. Regions of the LKR and SDH enzymatic domains are shown as boxes above the alignment. Paired 5' and 3' reads off the same cDNA clones are indicated either by ESTs on the same line or bracketed to the right of EST names. Asterisks indicated LKR ESTs with poly-A ends.

Figure 9

Switchgrass LKR ESTs. Five switchgrass ESTs that contain intron 12 sequence and a polyA tail are shown aligned to the maize LKR/SDH coding region. Sequences are given in black except for intron 12 which is in blue. The first stop codon for the reading frame into intron 12 is boxed. Differences among sequences are given in red and green for one or two differences to other sequences, respectively. PolyA tails of ESTs are shown in magenta.

Figure 10

Monofunctional wheat SDH ESTs. (A) Wheat ESTs are aligned to the wheat consensus full-length LKR/SDH coding sequence. Intron 14 sequences are shown in blue and green. The sequences of the first seven ESTs have intron 14 spliced out (dashes indicate absent intron sequences). The rest of the ESTs begin with sequence from the middle of intron 14 (bases in blue) to form an exon that is spliced directly to exon 15 in those ESTs. Bases in green are the presumptive transcription initiation signal region. Base differences in red are assumed to indicate homoeologue LKR/SDH sequences of hexaploid wheat. (B) Diagram of exon/intron organization of LKR/SDH bifunctional and SDH monofunctional transcripts. Exons are shown by boxes. The SDH first exon sequence is within the LKR/SDH intron 14 and is shown by the blue box. The green box is the non-transcribed sequence indicated in frame A. Exon numbers are given above (LKR/SDH) and below (SDH) exon boxes.