Functional characterization of an ornithine cyclodeaminase-like protein of Arabidopsis thaliana.
BACKGROUND
In plants, proline synthesis occurs by two enzymatic steps starting from glutamate as a precursor. Some bacteria, including bacteria such as Agrobacterium rhizogenes have an Ornithine Cyclodeaminase (OCD) which can synthesize proline in a single step by deamination of ornithine. In A. rhizogenes, OCD is one of the genes transferred to the plant genome during the transformation process and plants expressing A. rhizogenes OCD have developmental phenotypes. One nuclear encoded gene of Arabidopsis thaliana has recently been annotated as an OCD (OCD-like; referred to here as AtOCD) but nothing is known of its function. As proline metabolism contributes to tolerance of low water potential during drought, it is of interest to determine if AtOCD affects proline accumulation or low water potential tolerance.


RESULTS
Expression of AtOCD was induced by low water potential stress and by exogenous proline, but not by the putative substrate ornithine. The AtOCD protein was plastid localized. T-DNA mutants of atocd and AtOCD RNAi plants had approximately 15% higher proline accumulation at low water potential while p5cs1-4/atocd double mutants had 40% higher proline than p5cs1 at low water potential but no change in proline metabolism gene expression which could directly explain the higher proline level. AtOCD overexpression did not affect proline accumulation. Enzymatic assays with bacterially expressed AtOCD or AtOCD purified from AtOCD:Flag transgenic plants did not detect any activity using ornithine, proline or several other amino acids as substrates. Moreover, AtOCD mutant or over-expression lines had normal morphology and no difference in root elongation or flowering time, in contrast to previous report of transgenic plants expressing A. rhizogenes OCD. Metabolite analysis found few differences between AtOCD mutants and overexpression lines.


CONCLUSIONS
The Arabidopsis OCD-like protein (AtOCD) may not catalyze ornithine to proline conversion and this is consistent with observation that three residues critical for activity of bacterial OCDs are not conserved in AtOCD. AtOCD was, however, stress and proline induced and lack of AtOCD expression increased proline accumulation by an unknown mechanism which did not require expression of P5CS1, the main enzyme responsible for stress-induced proline synthesis from glutamate. The results suggest that AtOCD may have function distinct from bacterial OCDs.
Sandeep
S
Sharma
Suhas
S
Shinde
Institute of Plant and Microbial Biology, Academia Sinica, No, 128 Sec, 2 Academia Rd, Nankang Dist,, Taipei 11529, Taiwan. paulv@gate.sinica.edu.tw.
Paul E
PE
Verslues
D000595:Amino Acid Sequence; D000642:Ammonia-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003681:Dehydration; D035264:Flowers / Q000187:drug effects / Q000502:physiology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005838:Genotype; D055442:Metabolome / Q000187:drug effects; D055432:Metabolomics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009952:Ornithine / Q000494:pharmacology; D017440:Photoperiod; D018087:Plastids / Q000187:drug effects / Q000201:enzymology; D011392:Proline / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D034622:RNA Interference; D011994:Recombinant Proteins / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D011392:Proline; D009952:Ornithine; D000642:Ammonia-Lyases; C021914:ornithine cyclodeaminase