Fine mapping of the qHTB1-1QTL, which confers heat tolerance at the booting stage, using an Oryza rufipogon Griff. introgression line.

KEY MESSAGE: The qHTB1-1 QTL, controlling heat tolerance at the booting stage in rice, was fine mapped to a 47.1 kb region containing eight candidate genes. Two positional candidate genes showed significant changes in expression levels under heat stress. High-temperature stress at the booting stage has the potential to significantly limit rice production. An interspecific advanced backcrossed population between the Oryza sativa L. cultivar R53 and the wild Oryza rufipogon Griff accession HHT4 was used as the source material to develop a set of chromosome segment introgression lines to elucidate the genetic mechanism of the qHTB1-1 QTL in heat tolerance. A single-chromosome-segment introgression line, IL01-15, was used to develop secondary populations for the mapping of qHTB1-1 on chromosome 1 for heat tolerance at the booting stage. Using the BC5F2, BC5F3, and BC5F4 populations, we first confirmed qHTB1-1 and validated the phenotypic effect. The qHTB1-1 QTL explained 13.1%, 16.9%, and 17.8% of the phenotypic variance observed in the BC5F2, BC5F3, and BC5F4 generations, respectively. Using homozygous recombinants screened from larger BC6F2 and BC6F3 populations, qHTB1-1 was fine mapped within a 47.1 kb region between markers RM11633 and RM11642. Eight putative predicted genes were annotated in the region, and six genes were predicted to encode expressed proteins. The expression patterns of these six genes demonstrated that LOC_Os01g53160 and LOC_Os01g53220 were highly induced by heat stress in IL01-15 compared to R53. Sequence comparison of the gene-coding regions of LOC_Os01g53160 and LOC_Os01g53220 between R53 and IL01-15 revealed one synonymous and two nonsynonymous SNPs in exons, respectively. Our results provide a basis for identifying the genes underlying qHTB1-1 and indicate that markers linked to the qHTB1-1 locus can be used to improve the heat tolerance of rice at the booting stage by marker-assisted selection.

Overexpression of OsZHD1, a zinc finger homeodomain class homeobox transcription factor, induces abaxially curled and drooping leaf in rice.

Leaf rolling is receiving considerable attention as an important agronomic trait in rice (Oryza sativa L.). However, little has been known on the molecular mechanism of rice leaf rolling, especially the abaxial rolling. We identified a novel abaxially curled and drooping leaf-dominant mutant from a T1 transgenic rice line. The abaxially curled leaf phenotypes, co-segregating with the inserted transferred DNA, were caused by overexpression of a zinc finger homeodomain class homeobox transcription factor (OsZHD1). OsZHD1 exhibited a constitutive expression pattern in wild-type plants and accumulated in the developing leaves and panicles. Artificial overexpression of OsZHD1 or its closest homolog OsZHD2 induced the abaxial leaf curling. Histological analysis indicated that both the increased number and the abnormal arrangement of bulliform cells in leaf were responsible for the abaxially curled leaves. We herein reported OsZHD1 with key roles in rice morphogenesis, especially in the modulating of leaf rolling, which provided a novel insight into the molecular mechanism of leaf development in rice.

OsLOX2, a rice type I lipoxygenase, confers opposite effects on seed germination and longevity.

Rice production and seed storage are confronted with grain deterioration and loss of seed viability. Some members of the lipoxygenase (LOX) family function in degradation of storage lipids during the seed germination, but little is known about their influence on seed longevity during storage. We characterized the role of rice OsLOX2 gene in seed germination and longevity via over-expression and knock-down approaches. Abundant expression of OsLOX2 was detected in panicles, roots, and stems, but not in leaves. Moreover, OsLOX2 was highly induced during germination. OsLOX2 protein, located in the cytoplasm, showed a wide range of temperature adaptation (20-50 °C) and a substrate preference to linoleic acid. Lines over-expressing OsLOX2 showed accelerated seed germination under normal condition and lower seed viability after accelerated aging. RNA interference (RNAi) of OsLOX2 caused delayed germination and enhanced seed longevity. RNAi lines with strongly repressed OsLOX2 activity completely lost the capability of germination after accelerated aging. More lipid hydroperoxide were found in OE15 than the control, but less in RNAi lines than in the WT Nipponbare. Therefore, OsLOX2 acts in opposite directions during seed germination and longevity during storage. Appropriate repression of the OsLOX2 gene may delay the aging process during the storage without compromising germination under normal conditions.

Effects of external Mn2+ activities on OsNRAMP5 expression level and Cd accumulation in indica rice.

Manganese (Mn) transporter OsNRAMP5 was widely reported to regulate cadmium (Cd) uptake in rice. However, the relationship between OsNRAMP5 expression level and Cd accumulation, impacts of external ion activities on OsNRAMP5 expression level and Cd accumulation are still unclear. Investigations of the relationship between OsNRAMP5 expression level and Cd accumulation in three indica rice genotypes were conducted under various external Mn2+ activities ranging from Mn deficiency to toxicity in EGTA-buffered nutrient solution. Results in this work indicated that OsNRAMP5 expression level in roots significantly up-regulated at Mn phytotoxicity compared to that at Mn deficiency, which may stimulate by the increasing uptake of Mn. Our work also demonstrated that root Cd concentration of all the tested rice decreased notably when external Mn2+ activity reached the level of toxicity. This may explain by the increasing competition between the excess Mn2+ and Cd2+ as well as the disorder of element absorption caused by root damage at Mn toxicity. Our work also revealed that the relationship between OsNRAMP5 expression level in roots and Cd accumulation in roots was insignificant for all the tested genotypes. Besides, OsNRAMP5 expression level in roots seemed more related to root Mn accumulation. The fact that function of OsNRAMP5 mainly focuses on Mn uptake, together with the fact that many transporter genes involved in Cd uptake might result in the insignificant correlation between OsNRAMP5 expression level and Cd accumulation in roots. At last, multi-level regulating and processing of the process from gene expression to protein translation might account for the inconsistent relationship between root OsNRAMP5 expression level and Cd accumulation in roots.