Isolation And Functional Analysis Of ERECT PANICLE 2 (EP2) In Rice (Oryza Sativa, L.)

With the global explosion of human population, increasing grain yield will be a major challenge. Improvement of plant architecture is considered as a valuable approach to increase grain yield, because crop plants with desirable architecture are able to produce much higher yields. The most striking example is the use of semi-dwarf stature for rice and wheat in the late 1950s, which has greatly improved plant architecture and yield potential. Except for plant height, tiller, panicle and leaf morphology also plays important roles in shaping the crop high yield ideotype. The rice panicle architecture, which determines grain yield directly, is one of the most important agronomical traits. The rice panicle architecture is determined fundamentally by the number of primary branches (length of main axis) and the number of spikelets on each primary branch (length of the primary branch). Since 1980s, many high-yielding japonica rice varieties characterized erectness panicle have been released in China. EP-type varieties usually carry short-erect panicles and leaves, which are beneficial for improvement on ventilation and penetrating light, which show higher photosynthesis and material production capacity in their populations. Panicle erectness is considered as one perfect plant architecture in japonica rice. However, the molecular mechanism underlying domestication of erect panicle is largely unknown and all EP varieties are derived from japonica, no EP-type indica varieties have been released so far.Here, we identified two allelic erect-panicle mutants in indica rice, erect panicle2-1(ep2-1) and erect panicle2-2 (ep2-2), exhibiting the characteristic erectness panicle phenotype. We cloned the new erect panicle gene, ERECT PANICLE 2 (EP2), on chromosome 7 by map-based cloning. In present study, several techniques, including biochemical and molecular biological technique, have been used to explore the function of EP2 gene. The results indicated that:1. To clarify the inheritance of ep2, ep2-1 was crossed with a wild japonica rice cultivar Zhonghua 11. All the F1 plants showed wild-type phenotype, while the F2 plants were segregated into two groups with a 3:1 ratio. This result showed that ep2 is a single recessive nuclear gene. We cloned the new erect panicle gene, ERECT PANICLE 2 (EP2), on chromosome 7 by map-based cloning. Sequence analysis showed that the gene had different mutations in ep2 mutant alleles. And the genetic complementation experiments showed complementation of the ep2-1 phenotype. This result shows that we cloned the EP2 gene, which controlling the erectness panicle.2. The EP2 gene is a single-copy gene in rice genome and contains 10 exons, and encodes 1365 amino acid residues. By homologous sequence BLAST, we found that EP2 encodes a plant specific protein of unknown biochemical function. And the EP2 genes are highly homologous in gramineous crops.3. The EP2 gene plays pleiotropic roles in an array of plant architecture. In addition to affecting the panicle traits, but also affect other traits, such as seed size, leaf size and plant height. Anatomical investigations revealed that the ep2 mutants have more small vascular bundles and sclerenchyma cells, and thicker stem than wild-type plants, explaining the panicle erectness phenotype in ep2 mutants.4. The EP2 gene is mainly expressed in the stem, leaf and young panicle, with the highest expression in panicle by RT-PCR analysis. GUS reporter with a fusion of the EP2 promoter showed that EP2 is expressed.in vascular bundles of stem, leaf blade and leaf sheath. The the expression of GUS was also detected in tiller base, axillary bud, lamina joint and young panicle in pEP2:GUS transgenic plants. This results show that the expression of EP2 gene has a strong spatial and temporal nature.5. To ascertain the subcellular localization of EP2, GFP was fused to the C terminus of EP2 under the control of the 35S promoter, and the fusion gene was transformed into rice protoplast. We found that EP2-GFP has a reticulate fluorescence pattern, and co-localizated with the endoplasmic reticulum specific marker (ER-mCherry). Therefore, we concluded that EP2 is an endoplasmic reticulum-localized protein.6. The expression of GA-related genes in the mutant was showed by RT-PCR. We found that the expression of OsGA20ox3 and OsGA3ox1 are decreased significantly in the mutant. GA20ox and GA3ox play a key role in the GA synthesis pathway, especially in the formation of active GA molecule. This shows that the EP2 protein regulated the expression of OsGA20ox3 and OsGA3ox1, controlling the plants height.7. Analysis the plant traits at the mature stage, we found that ep2-2 mutant improves the rice plant architecture. The ep2-2 mutant had shorter plant height, thicker stem and erectness panicle, which increase the lodging resistance. In addition, the ep2-2 mutant showed more grain number per panicle than the wild-type.According to the results above, we concluded that we cloned a new EP2 on the chromosome 7. The ep2 mutants have more vascular bundles and sclerenchyma cells, and thicker stem than wild-type plants, resulting panicle erectness phenotype in ep2 mutants. The EP2 encodes a novel plant-specific protein, which localizes to the endoplasmic reticulum with unknown biochemical function. It shows that the EP2 could be used in breeding work as a new erectness panicle resource.