Analyses Of Rice Genome Sequences And Expressed Sequence Tags And Studies Of Glyoxylate Cycle In Submerged Seedlings In Rice

1. Sequence analysis of rice genomeRice is the principal food for over half of the population of the world. With itsgenome size of 430 megabase pairs (Mb), the cultivated rice Oryza sativa species,has been chosen as a model plant for genome research. A comprehensive clone-basedrice chromosome 4 physical map was constructed through an integrated approach.The finished sequence of rice chromosome 4 consists of 34.5Mb, covering about98.7% of this chromosome. Rice chromosome 4 is annotated by a combination ofautomatic predictions, homology search against full-length cDNAs and ESTs. In thisstudy, a chromosome 4 gene, designated as OsAI gene, was identified to encodehomologous amino acids sequence to putative alpha-amylase inhibitor (AI). A 15-nucleotides insertion, resulting in the insertion of a pentapeptide, was found in theOsAI genes from most detected cultivars of indica but japonica. The grouping ofcultivated rice species by 15-nt of insertion was very analogical to the subspeciesclassification of indica and japonica. The similar insertion was identified in somecommon wild rice. The insertion probabaly originated from the common wild species.2. Sequence analysis of mRNA polyadenylation signals of rice genes The formation of eukaryotic mRNAs involves the cleavage and polyadenylation ofpre-mRNAs. To investigate the sequence requirement of putative polyadenylationsignals (PASs), poly(A) sites and downstream elements (DEs) in 3'-end-processing inrice, we compared expressed sequences tags (ESTs) with poly(A) extremity to full-length cDNA sequences and constructed a database of 12969 pre-mRNA sequences in-40 to +40 nucleotides (nt) surrounding the poly(A) sites, which were from 9953genes. The alternative poly(A) sites were revealed in approximately 25% of mRNAs.Nearly 80% of pre-mRNAs showed stringent requirement of the YA (CA or UA) atpoly (A) sites for polyadenylation. About 7.9% had the AAUAAA signals on –40 to –1 nt upstream of the poly(A) sites. Over 60% of mRNAs probably used the one-ortwo-base variants of AAUAAA hexamers as their PASs in 3' fragments. The single-base variants of AAUGAA revealed the high frequency in 11.5% of 3' fragments. TheDEs were detected in 90% of pre-mRNAs, especially more than half of the pre-mRNAs with multi-base variants of AAUAAA had the DEs surrounding the poly(A)site. The location of DE is also important for defining the cleavage site. Althoughmost of the rice pre-mRNAs did not contain AAUAAA signal, the existence ofdownstream elements ensured the efficiency of cleavage-polyadenylation.3. Study of glyoxylate cycle in submerged rice seedlingsUnder anaerobic conditions, respiration is blocked and ethanolic fermentationsubstantially contributes to NAD+ regeneration. The ethanolic fermentation producedacetaldehyde and ethanol by catalysis of pyruvate decarboxylase (PDC) and alcoholdehydrogenase (ADH). With the continuance of oxygen deficiency, activation of theethanolic fermentation would lead to the accumulation of acetaldehyde. The ability ofmetabolism of acetaldehyde from the ethanolic fermentation is important to survivalduring anoxia. Increased transcripts of rice Aldh2a have been identified in low oxygencondition, suggesting that acetaldehyde might be metabolized in the routine ofacetaldehyde/acetate/acetyl-CoA. Glyoxylate cycle is a modified form of thetricarboxylic acid (TCA) cycle for converting C2 compounds into C4 dicarboxylicacids. Isocitrate lyase (ICL) and malate synthase (MS) are the two characteristicenzymes of the glyoxylate cycle, which enable the cell to use acetyl-CoA to generateincreased levels of TCA cycle intermediates for biosynthetic pathways such asgluconeogenesis. By the research of submerged rice seedlings, we revealed theactivation of the glyoxylate cycle by identifying increased transcripts of the genes forICL and MS, two characteristic enzymes of the glyoxylate cycle. Northern analysisshowed that the level of mRNAs for ICL and MS dramaticallyincreased 24 to 72 hours after submergence, while it decreased when the seedlingswere turned back to the aerobic condition. The Northern-blot analysis also indicatedthat the induction of identified rice Pdc1 and Adh1 genes' expressions dramaticallyincreased in the seedlings submerged for 12 h, indicating that the ethanolicfermentation was more rapidly activated in the submerged tissues. Similarly, theactivities of PDC and ICL in the tissues submerged for 72 h were the 8.8 folds and 3.5folds of increase over that in the un-submerged seedlings, respectively. The Northernanalysis and the activity assays showed that ICL and MS were steadily active in theseedling submerged from 12 to 72 h, suggesting that the glyoxylate cycle might play ametabolic role during absence of oxygen.4. Study of genes encoding acetyl-CoA synthetase in submerged rice seedlingsAcetyl-CoA synthetase (ACS) is thought to be involved in ethanol utilization. ACScatalyzes the transfer of acetate to acetyl-CoA to activate metabolism of acetate. Itwas revealed that two open reading frames were homologous to the Arabidopsis ACSsequence, which located on rice chromosome 2 (OsACS1) and 4 (OsACS2),respectively. In aerobic condition, the transcripts for OsACS1 and OsACS2 werehardly detectable. Northern-blot analysis showed that the level of OsACS2 mRNAsdramatically increased in the submerged tissues after 24 h. Comparably, transcripts ofOsACS1 were steadily weak during the submergent treatment. We used sodium [1-14C]acetate as the substrate to measure the ACS activity by radioactive concentrationin the products, containing [1-14C]acetyl-CoA. The results showed that ACS activityin the tissues submerged for 72 h reached a 3-fold increase over the initial level inaerobic condition, suggesting an induction in ACS activity in the course of anoxictreatments. Considerable evidence has indicated that rice Aldh in cytoplasm (Aldh1)and mitochondria (Aldh2) contributed to acetate formation. It was proposed thataccumulation of acetate might occur in the prolonged period of low oxygen andfunction of ACS was associated with ALDH. Activation of OsACS2 in anaerobiccondition probably lessened the accumulation of acetate from acetaldehyde in theanaerobic condition. The transcripts of genes employed in ethanolic fermentation,oxidation of acetaldehyde, and the glyoxylate cycle has been identified to increaseunder submerged condition. The activation of ACS might link the oxidation ofacetaldehyde and the glyoxylate cycle in the metabolism of acetate in low oxygencondition.