“OsDHODH” encoding a putative cytosolic dihydroorotate dehydrogenase (DHODH) in rice. cytosolic dihydroorotate dehydrogenase is involved in plant stress response and that OsDHODH1 could be used in engineering crop plants with enhanced tolerance to salt and drought. 
As OsDHODH1 was induced in rice seedlings upon salt and drought stresses, it was of interest to investigate the function of OsDHODH1 in vivo. The tolerance of E. coli cells harboring the OsDHODH1 gene to salt and osmotic stresses was assayed; recombinant cells with OsDHODH1 significantly improved the salt and osmotic tolerance.
Two OsDHODH1-OX lines (S1 and S2) and two OsDHODH1-KD lines (A1 and A2) were analyzed for salinity and osmotic tolerance. There was no significant difference in the growth rates between WT and transgenic plants at the seedlings stage under normal conditions. However, in comparison to WT, when cultured on 1/2 MS medium supplied with 200 mM NaCl, OsDHODH1-OX lines showed improved tolerance to salt, and OsDHODH1-KD lines were more sensitive. The seedling heights of S1 and S2 were higher than those of WT, A1 and A2. The chlorophyll content, proline content and relative electrolyte leakage were also tested. Under normal conditions, the chlorophyll and proline contents and relative electrolyte leakage showed no significant difference between the WT and transgenic lines. After salt treatment, the chlorophyll and proline contents in OsDHODH1-OX were higher than in WT and OsDHODH1-KD. The relative electrolyte leakages in OsDHODH1-OX were lower than in WT and OsDHODH1-KD.
The transgenic rice plants with sense-OsDHODH1 and antisense-OsDHODH1 were obtained by Agrobacterium-mediated transformation. The expression of OsDHODH1 in transgenic lines and wild type (WT) were analyzed by semi-quantitative RT-PCR. The results showed that the expression levels of OsDHODH1 in three independent OsDHODH1-overexpression lines (OsDHODH1-OX lines S1, S2 and S3) were significantly higher than those in WT plants and three independent OsDHODH1-knock-down lines (OsDHODH1-KD lines A1, A2 and A3). Furthermore, we compared the DHODH activities among the WT and transgenic lines and found that the enzymatic activity of DHODH in the OsDHODH1-OX lines was 90% higher than those in the WT. However, the enzymatic activity of DHODH did not show a significant reduction from the OsDHODH1-KD lines.
The tissue-specific expression analysis suggested that OsDHODH1 is constitutively expressed in rice culms, leaves, roots and immature spikes at the adult stage, and shoots and roots at the seedling stage. To test whether OsDHODH1 exactly encodes a cytosolic protein, the OsDHODH1-GUS fusion gene is introduced into onion epidermal cells by an Agrobacterium-mediated transient expression. The OsDHODH1-GUS fusion protein is localized throughout the cells, indicating that OsDHODH1 is a cytosolic protein. The expression of OsDHODH1 in rice seedlings under NaCl, temperature, drought, abscisic acid (ABA) and H2O2 treatments by semi-quantitative RT-PCR showed that the expression of OsDHODH1 is induced by salt, drought and high-temperature stresses and ABA treatment, but not markedly affected by low temperature or H2O2 treatment.
The predicted protein of OsDHODH1 is composed of 414 amino acids with a molecular weight of 45.31 kDa and an isoelectric point (pI) of 6.29. There are no signal peptides or transmembrane domains in the OsDHODH1 sequence. A comparison with its genomic sequence revealed that OsDHODH1 comprises seven exons and six introns. A homology search against the GenBank database showed that OsDHODH1 is homologous to various cytosolic DHODH proteins in eukaryotes, with 84–87% amino acid similarities with the corresponding proteins from Arabidopsis thaliana, Pyrus pyrifolia and Lycopersicon esculentum. A multiple sequence alignment indicated that the DHO_dh domains are conserved among all known plant cytosolic DHODH proteins. To investigate the evolutionary relationships among dihydroorotate dehydrogenase proteins from different organisms, a phylogenetic tree is constructed using the neighbor-joining method. The tree could clearly divide into two big families: family 1 and family 2. The position of OsDHODH1 in the tree also supported that OsDHODH1 belongs to the family 1, the plant cytosolic DHODH proteins.
Labs working on this gene
1. State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
- Liu, W.-Y., Wang, M.-M., Huang, J., Tang, H.-J., Lan, H.-X. and Zhang, H.-S. (2009), The OsDHODH1 Gene is Involved in Salt and Drought Tolerance in Rice. Journal of Integrative Plant Biology, 51: 825–833. doi: 10.1111/j.1744-7909.2009.00853.x