Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase ␦ (Pol ␦) extends the upstream Okazaki fragment and displaces the 5-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase ␦ only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.During eukaryotic DNA replication, the lagging strand is replicated via synthesis and maturation of Okazaki fragments. These fragments are short stretches of DNA that are joined to generate a continuous strand (1). Each fragment is initiated when DNA polymerase ␣/primase (Pol ␣) 3 makes an RNA/ DNA primer, synthesizing ϳ10 nucleotides (nt) of RNA followed by 10 -20 nt of DNA (2). The primer is then extended by a complex of DNA polymerase ␦ (Pol ␦), the sliding clamp, proliferating cell nuclear antigen (PCNA), and the clamp loader, replication factor C (RFC). When Pol ␦ encounters the 5Ј-end of the downstream Okazaki fragment, it displaces it into a flap. Cleavage of the flap by nucleases generates a nick, which is subsequently sealed by DNA ligase I to form continuous double-stranded DNA (3, 4).One pathway for cleavage of the flap employs flap endonuclease 1 (FEN1). FEN1 is a single-strand, structure-specific endonuclease that enters the 5Ј-end of the flap and tracks to the base for cleavage (3,5,6). Following displacement of a short flap, less than about 12 nt, by Pol ␦, FEN1 cleaves leaving a nick, the substrate for DNA ligase I (7-9). Because the RNA initiating the Okazaki fragments is ϳ10 nt in length, short flaps composed entirely of RNA are first displaced by Pol ␦. This does not obstruct FEN1, which is active on RNA (10, 11). In addition, displacement and cleavage occurs mostly within the first 25 nt of the downstream fragment, sufficient to remove the entire RNA/DNA primer, which is ϳ20 -30 nt in length (11). Previous biochemical studies demonstrated that primarily short flaps are cleaved by FEN1. It is likely that in vivo a series of short successive displacements by Pol ␦ and cleavages by FEN1 are effective for removal of the entire init...

Improving the performance of rice () under drought stress has the potential to significantly affect rice productivity. Here, we report that the ERF family transcription factor OsLG3 positively regulates drought tolerance in rice. In our previous work, we found that has a positive effect on rice grain length without affecting grain quality. In this study, we found that was more strongly expressed in upland rice than in lowland rice under drought stress conditions. By performing candidate gene association analysis, we found that natural variation in the promoter of is associated with tolerance to osmotic stress in germinating rice seeds. Overexpression of significantly improved the tolerance of rice plants to simulated drought, whereas suppression of resulted in greater susceptibility. Phylogenetic analysis indicated that the tolerant allele of may improve drought tolerance in cultivated rice. Introgression lines and complementation transgenic lines containing the elite allele of showed increased drought tolerance, demonstrating that natural variation in contributes to drought tolerance in rice. Further investigation suggested that plays a positive role in drought stress tolerance in rice by inducing reactive oxygen species scavenging. Collectively, our findings reveal that natural variation in contributes to rice drought tolerance and that the elite allele of is a promising genetic resource for the development of drought-tolerant rice varieties.

BackgroundRice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level.MethodsGas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated.ResultsWe characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na+ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment.ConclusionsMetabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress.