Study on Photosynthetic and Physiological Effects of a Novel Adaxially-Rolled Character in Rice（Oryza sativa L.）

Xiaoping GONG，Xiaoming KUANG，Ting LUO，Jiasheng Ll，Xubing ZHAO，Bingfeng ZHAO，Sheng ZENG，Xia YANG，Zhili ZHANGYudongnan Academy of Agricultural Sciences，Chongqing 408000，China

Study on Photosynthetic and Physiological Effects of a Novel Adaxially-Rolled Character in Rice（Oryza sativa L.）

Xiaoping GONG，Xiaoming KUANG，Ting LUO，Jiasheng Ll，Xubing ZHAO，Bingfeng ZHAO，Sheng ZENG，Xia YANG，Zhili ZHANG*
Yudongnan Academy of Agricultural Sciences，Chongqing 408000，China

［Objective］The main biological effects of leaf rolling of rice was studied to provide the theoretical basis for the application of RL（t）in the breeding of the rice.［Method］A rice mutant with adaxially-rolled leaf was found in breeding，temporarily named as RL（t）.The differences were researched in which light interception structure，light transmission photosynthetic efficiency and cellular structure comparison between RL（t）and its sister lines that had flat leaf.［Result］Both leaf rolling index and leaf erecting index of RL（t）were higher than those in 0731-3-1-1B.However，its basic leaf angle and leaf drooping angle were significantly lower than those in 0731-3-1-1B in upper three leaves，which caused a great raise in the photosynthetic rate on account of the light transmittance of RL（t）’s population were significantly higher than that of 0731-3-1-1B especially the upper and middle part.The stomata conductance and the intercellular CO2concentration of the upper three leaves and transpiration rate of the flag and the second leaf were also significantly higher than that of 0731-3-1-1B.The bulliform cells became smaller，leading to the rolling up of leaves.［Conclusion］The study provided a theoretical basis for the principle of highyielding breeding with the application of the adaxially-rolled leaf mutant in rice.

Rice；Leaf rolling index；Leaf erecting index；Photosynthetic rate；Light transmittance

Materials and methods

Materials and planting

The experiment was carried out in rice experimental field of Yudongnan Academy of Agricultural Sciences of Chongqing in 2012.Test materials were RL （t）and 0731-3-1-1B.The seeds were sowed at April 10th.The seedlings were transplanted at May 12th，which was single plant per hole. The row spacing×line spacing was 13.3 cm×25.0 cm.The experimental field was 10 m2，which was managed by conventional fertilizer and water management.Ultrastructure of leaf cells was observed in Rice Research Institute of Southwest University/Key Laboratory of security and control of transgenic plants in Chongqing.

Measurements and methods

Observation of phenotype of RL（t）and 0731-3-1-1B From the seedling stage to the mature period，leaf morphology were observed.

Ultrastructure of leaf cells in RL（t）and 0731-3-1-1B In the full heading stage，the fresh upper three leaves of RL（t）and 0731-3-1-1B were selected to fix by using FAA fixative which contained 90 ml of 70%alcohol，5 ml of 40%formaldehyde，5 ml glacial acetic acid，and stored at 4℃.Then treated by paraffin-section method and safranin-fast green staining，which the steps were fixation-dehydrationtransparency-waxdip-embedding-slicing-sticky piece-dewaxing-rehydrationsafranin-fast green staining-sealing. The thickness of slicing was 10 um. The slicing was sealed by Canada gum，and then observed and photographed by OLYMPUS BH type microscope［12］.

The determination of rolling index，erecting index，leaf base angle，drooping angle Each treatment had 10 plants.In full heading stage，blade length，leaf margin spacing of the widest part which was naturally rolled（Ln）and leaf width （Lw）of the upper three leaves of each plant was measured，and the leaf rolling index（LRI）was calculated.

Measured the angle between the leaf and stem，the line length from pulvinus to leaf apex of the natural rolling leaf（LNL）and the leaf erecting length，then calculated the leaf erecting index（LEI）.

The angle between the leaf and stem was leaf base angle.The angle between the line from pulvinus to leaf apex of the natural rolling leaf and the stem was drooping angle.

Determination of photosynthetic rate Each treatment had 10 plants. In grain filling stage，photosynthesis intensity of the upper three leaves of each plant was measured with LCA-4 portable photosynthesis system in the field.The measurement time was from 10:00 am to 12:00.When measuring，the leaf surface was directly illuminated，and the light intensity between 1 600 and 1 800 umol/（m2·s）.

Determination of light transmittance In the full heading stage，the light transmittance of RL（t）and 0731-3-1-1B was measured with ST-80 light meter（with linear probe）by Layered cut method.

Results and Analysis

The morphologic change of leaves of RL（t）and 0731-3-1-1B

The results showed that the maintainer line RL（t）was flat before the top third leaf sprouted，and the top three leaves showed moderate rolling.But that of 0731-3-1-1B showed flat in the whole growth period（Fig.1）.

The cells ultrastructure of leaves of RL（t）and 0731-3-1-1B

The results of paraffin section showed that compared with 0731-3-1-1B，the characteristic of vascular bundles and the characteristic cell layer of adaxial and abaxial side of thick-walled cells had not changed，but the bulliform cell of the adaxially-rolled leaf became smaller significantly.Meanwhile，stomatal complex area of leaves of RL（t）was smaller than that of 0731-3-1-1B（Fig.2）.

The determination of rolling index，erecting index，leaf base angle and drooping angle

The results showed that both leaf rolling index and leaf erecting index of the upper three leaves of RL（t）were higher than those in 0731-3-1-1B.The leaf rolling index of the upper three functional leaves of RL（t）was up to 54.4%-66.4%，and the leaf erecting index was 92.8%-99.7%.The leaf rolling index of the upper three functional leaves of 0731-3-1-1B was only 5.4%-8.6%，and the leaf erecting index was 68.3%-96.3%.Especially the difference between the third leaf of RL（t）and 0731-3-1-1B was great.The erecting index of the third leaf of RL（t）was up to 92.8%，which was only 68.3%of 0731-3-1-1B.

The leaf base angle and drooping angle of the upper three functional leaves of RL（t）were significantly lower than those in 0731-3-1-1B at 0.05 lev-el.The leaf base angle of the upper three functional leaves of RL（t）was 13.6°-15.6°，which was 18.6°-27.8°of 0731-3-1-1B.Especially the difference between the third leaf of RL（t）and 0731-3-1-1B was great.The leaf drooping angle of the upper three functional leaves of RL（t）was 6.6°-2.2°，which was12.4°-48.8°of 0731-3-1-1B（Table 1）.

The performance of photosynthetic rate

The results showed that the photosynthetic rate of the second leaf of RL（t）was significantly higher than that of 0731-3-1-1B at 0.01 level，the photosynthetic rate of the flag leaf was higher than that of 0731-3-1-1B，and the photosynthetic rate of the second leaf was similar to that of 0731-3-1-1B. The stomatal conductance and the intercellular CO2concentration of the upper three functional leaves of RL（t）and transpiration rate of the flag and the second leaf were also significantly higher than that of 0731-3-1-1B at 0.01 level，which was no significant difference between the second leaf of RL（t）and that of 0731-3-1-1B（Table 2）.

Table 2 The determination of photosynthetic rate of 0731-3-1-1B and RL（t）leaves

Table 3 The light transmittance of 0731-3-1-1B and RL（t）groups

Groups light transmittance

The results showed that the light transmittance of RL（t）was higher than 0731-3-1-1B，respectively 5.48%，16.31%，35.42%，39.19%，30.43%at the distance of 15 cm，30 cm，60 cm，90 cm and the part near the earth’s surface from the spike tip.It means that the difference of light transmittance of RL（t）and 0731-3-1-1B was gradually increased from the spike tip to bottom，but the difference was reduced in the stem base（Table 3）.

Conclusion and Discussion

The research indicated that both leaf rolling index and leaf erecting index of RL（t）were higher than those in 0731-3-1-1B.However，its basic leaf angle and leaf drooping angle were significantly lower than those in 0731-3-1-1B in upper three leaves，which caused a great raise in the photosynthetic rate on account of the light transmittance of RL（t）’s population were significantly higher than that of 0731-3-1-1B especially the upper and middle part.The difference of morphologic change of leaves was gradually increased from the flag leaf to the third leaf.The stomata conductance and the intercellular CO2concentration of the upper three leaves and transpiration rate of the flag and the second leaf were also significantly higher than that of 0731-3-1-1B at 0.05 level.Because the leaf morphology and plant type of two test materials which were flat at early growth stage were similar，and the difference of the light transmittance in the part near the earth surface was significantly reduced.Therefore，RL（t）mainly affected the leaf morphology and light transmittance of the upper and middle part of the groups，and less affected the lower part of the groups.The effect on simple leaf photosynthesis was to improve the photosynthetic rate of the upper two leaves especially the second leaf.The results of cell biology research showed that partial bulliform cells of leaf rolling maintaining line were atrophy and diminished，which led to the leaf rolled inward.Meanwhile，the stomata complex area was significantly smaller than flat leaf maintaining line，which changed the movement of stomatal guard cells and improved the transpiration.

It has been reported that leaf roll genes in rice showed rolling in the whole growth period，which led to limit the light utilization of rice in early growth stage，weak growth potential，decrease the photosynthetic rate of single leaf and limited the use value in the production［13-16］.Lu Jiangfeng［15］did the research from dry matter accumulation aspects，which indicated that dry matter production would affected the groups with moderate rolling degree in the early growth stage，the growth trend was clearly later，and the ultimate biological yield was similar to the groups with flat leaves.Chen zong-xiang et al.［16］found that leaf rolling group showed different performance in different stage.In early growth stage，it showed disadvantaged growth，such as the indexes of leaf age in seedling stage，stem tillers number，pseudostem diameter and seedling height were all lower than those of the control groups.In this study，the number of stem tillers increased slower，and the peak seedlings was less.Meanwhile，because the leaf was excessively rolled，the area which affected by light was significantly reduced，dry matter production was severely affected in the earlier stage，although it was significantly grew in later period but the dry matter accumulation was difficult to achieve the level of normal flat leaves. The obtained rice material RL（t）was flat in early growth stage and adaxiallyrolled in late growth stage.The leaf rolling gene could not only overcame the disadvantaged growth in early growth stage to some extent，but also gave full play to the advantages of the use of luminous energy in late growth stage.Therefore，the separation of the gene that controlled the leaf flat in early growth stage and adaxially-rolled in late growth stage has an important use value in high-yielding breeding.However，the ability of high-density planting，heterosis，the separation of the leaf rolling gene，functional verification and the application in breeding needed further study.

References

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一個新的水稻內(nèi)卷葉突變體的光合生理效應(yīng)

龔曉平，況曉明，羅 挺，李加勝，趙許兵，趙冰峰，曾 勝，楊 霞，張致力*（重慶市渝東南農(nóng)業(yè)科學(xué)院，重慶 408000）

［目的］明確水稻葉片前平后卷的主要生物學(xué)效應(yīng)，為RL（t）在水稻育種中的應(yīng)用提供理論依據(jù)。［方法］在水稻雜交后代材料中發(fā)現(xiàn)一個新的內(nèi)卷葉突變材料，暫時命名為RL（t），研究了RL（t）與平展葉姊妹系0731-3-1-1B在葉片受光姿態(tài)、光合效率及細(xì)胞組織結(jié)構(gòu)的差異。［結(jié)果］RL（t）較平展葉姊妹保持系三片功能葉明顯表現(xiàn)出卷曲度大、挺直度高、葉基角小、披垂角小，相應(yīng)地，提高了卷葉保持系群體各層尤其上部和中部的透光率；三片功能葉的氣孔導(dǎo)度、胞間CO2濃度和劍葉、倒二葉的蒸騰速率顯著高于平展葉姊妹保持系，從而提高了RL（t）劍葉、倒二葉的光合速率；由于卷葉保持系部分泡狀細(xì)胞適度萎縮變小，導(dǎo)致葉片內(nèi)向卷曲。［結(jié)論］該研究為內(nèi)卷葉性狀應(yīng)用于水稻高產(chǎn)育種提供了理論依據(jù)。

水稻；葉片卷曲度；挺直度；光合速率；透光率

R ice leaf is an important structure where the organisms carry out photosynthesis and form photosynthetic products，and it is an important factor to constitute plant type，so it has been the focus of attention in rice genetics and breeding［1-5］. Since Donald［6］proposed the concept of ideal plant，there has been a number of rice breeders proposed ideal plant model，such as the plant type model of leaf morphology was short shoot，upright leaf，big and erect panicle［7］，the upper three functional leaves model was long，straight，narrow，concave，thick［3］，the heavy panicle model，the adaxially-rolled，upright leaf model［8］and so on.These models are related with leaf erecting and leaf rolling. Moderate leaf rolling is conducive to maintaining leaf erecting，increasing light transmittance of the middle and lower part of plants，increasing？light utility efficiency，thereby improved the yield and quality of rice.In conclusion，leaf rolling is a valuable genetic resources.If leaf rolling trait was introduced into breed improvement，crop yield potential could be achieve a breakthrough［3，8-10］.

Germplasm resources of leaf rolling which could be stably inherited is rich in rice［11］.From the leaf rolling aspect，there are adaxially-rolled leaf that the both sides along the leaf veins rolled inward，and reverse rolled leaf that the both sides along the leaf veins rolled outwards.From the degree of roll in a single leaf，there are high degree of rolling，moderate rolling andslight rolling.From the period of leaf rolling，there are leaf rolling in the whole growth period，the leaf rolled in the seedling stage and unfolded in late growth stage，and the leaf was flat in the seedling stage and then rolled gradually［11］.In winter of 2003，rice breeders of Yudongnan Academy of Agricultural sciences of Chongqing found a rice mutant which the upper three leaves was adaxially-rolled after flatting in the earlier stage in Lingshui breeding base of Hainan province.By hybridizing this mutant with 0119-12-1-IB，the research group obtained a three-line hybrid rice maintainer line with adaxially-rolled leaf，which was tentatively named RL（t）.In this study，the main biological characteristics of RL（t）and its sister lines that had flat leaf 0731-3-1-1B was contrasted in order to clear and definite the main biological effect of adaxially-rolled leaf in rice.The study provided a theoretical basis for the principle of high-yielding breeding with the application of RL（t）in rice.

重慶市 “十二五”重大科技專項（CSTC，2012GGB8005）；重慶市科委科技攻關(guān)項目（CSTC，2011AB1080）。

龔曉平（1980-），男，重慶人，助理研究員，博士，從事水稻遺傳育種、分子生物學(xué)等方面的研究。*通訊作者，研究員，從事水稻遺傳育種方面的研究。

2014-12-20

2015-03-06

Supported by a grant from the Major Science and Technology Project of Chongqing during the 12thFive-Year Plan Period（No.CSTC，2012GGB8005）；Supported by a grant from the Science and Technology Key Project of Science and Technology Committee in Chongqing（No.CSTC，2011AB1080）.

.E-mail:zzllbn7513@sina.com

December 20，2014Accepted:February 6，2015