Identification And Gene Mapping Of An Early Senescent Leaf Mutant Esl6 In Oryza Sativa L.

Leaf, the main organ for photosynthesis of plant, generally senesces at the last stage of plant growth and development. Besides of the observed phenotypes of chlorosis and exsciccation, the senescence of leaf blades is a highly programmed process influenced by genetic factors and external factors（such as sunshine, diseases, high temperature, low temperature, water logging, drought and so on）, therefore, the researches of senescence were paid wildly attentions（Yoshida, 2003）. Earlier senescent plant could directly reduce rice yield and quality. For example, if the life of functional leaves postponed one day at the maturity stage, the rice yield would increase 2% in theory and 1% as a matter of fact（Liu, 1983）. In the process of leaf senescence, cell structure, metabolism, gene expression and regulation and other biological characters will be changed. To study the regulatory mechanism of senescence, cloning senescence gene based on the molecular and genetic standard is an efficient way.A mutant, tentatively named senescence early leaf 6（esl6）, with early senescence phenotype has been screened out in the progeny of an excellent indica restorer line Jinhui10 with seeds treated by EMS（ethyl methane sulfonate）. Jinhui10 has been used as wild type in order to analysis the morphological characteristics and agronomic trait of mutant esl6 in this study. By using transmission electron microscope and other equipment and technologies, we did some cytological observation. We prepare the mapping population of 1A/esl6 and take esl6 as a gene mapping. Phenotype analysis, agronomic characters analysis, photosynthetic pigment content analysis, cytological observation. The main results are listed as follows: 1. Phenotype identification of the esl6 MutantBefore the fourth leaf stage, no significant differences are observed between the esl6 and wild type. After that, the tip of fully expand leaves turns yellow while the base remains normal green in the esl6 mutant, and the phenotype sustains to the florescence. Subsequently, the whole leaf of esl6 turned yellow and the wild-type still kept green. The main agronomic traits were analyzed and the results showed that the plant height of esl6 was extremely lower than that of the wild type. In detail, the internode of inverted 1 and 2 decreased extremely significantly while the internodes of inverted 3 and 4 increased extremely. In addition, the length of the first, second and third leaf blades were extremely significantly shorter than those of the wild type. Except of flag leaf, the width of second and third leaf blades in the esl6 were significantly wider than those of the wild type. Further analysis indicated that the esl6 reduced in the traits of panicle length, primary and secondary branch number and filled grain number per panicle, while kept normal for the number of seed setting rate and 1000-grain weight. 2. Measurement of Photosynthetic Parameters and Photosynthetic Pigments ContentThe photosynthetic rate（Pn）, stomatal conductance（Gs） and transpiration rate（Tr） of the senescent leaf tip in the esl6 were all significantly lower than those of the wild type. While, the values of these photosynthetic parameters were significantly higher in the based leaf blade. The contents of photosynthetic pigments of first leaf from the top were significantly higher in the esl6 than those of the wild type at the third-leaf and fifth-leaf stages. Compared with those of the wild type, the contents of photosynthetic pigments declined significantly in the esl6, in detailly, the Chla, Chlb, total Chl and Car content were reduced by 68.74%, 63.73%, 67.99%and 57.63%, respectively. Interestingly, the photosynthetic pigments content of esl6’s leaf base were significantly increased, the Chla, Chlb, total Chl and Car contents increasing 16.04%, 36.96%, 19.32% and 18.82%, respectively. 3. The Determination of Active Oxygen Content and Protective Enzyme SystemThe H₂O₂, O^2- and ·OH contents in the leaf-tip and –base of esl6 increased significantly comparison to those of the wild type. In the senescent leaf tip, H₂O₂ was increased by 99.67%, O^2- was increased by 56.2% and H₂O₂ was increased by 119.33%. At the bottom green part, H₂O₂ was increased by 37.54%, O^2- was increased by 34.17% and H₂O₂ was increased by 46.45%. Compared with those of the wild type, the activities of CAT, SOD and POD are increased by 50.28%, 199.83% and 54.48%, respectively, in the leaf tip of esl6. In the leaf base, the activities of CAT and POD were decreased by 17.5% and 45.57%, while SOD kept no significant changing in comparison to those of the wild type. 4. The Ultra microstructure ObservationObservation by transmission electron microscope suggested no significant changing at the green leaf base and seriously damaged cell structures with bigger vacuole, broken cytomembrane and incomplete organelle at the senescent leaf tip of esl6. Compared with those of the wild type, the chloroplast of esl6 has abnormal structure with the broken thylakoid, incomplete grana lamella and more starch grains. 5. Analysis of Sugar ContentStarch staining demonstrated no obvious changing between the wild type and esl6 at the third-leaf stage. At the fifth-leaf stage, the starch content of esl6 was higher than that of the wild type while no changing for the new leaf blade. Sugar content measurement indicated that the contents of starches, glucose and cane sugar were increased by 239.45%, 40.03% and 53.42%, respectively in the esl6, leading to the significant different level compared with the wild type. And the fructose content had no differences between the wild type and esl6. 6. ESL6 Gene MappingGenetic analysis showed that the esl6 mutant was regulated by a single nuclear recessive gene. By using the F2 recessive genetic mapping population, esl6 was finally restricted between Indel marker Sind09-4 and Sind09-4 on chromosome 9 with the physical distance of 203 Kb.