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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (6): 1401-1415.doi: 10.3724/SP.J.1006.2022.12032

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Mechanism of drought and salt tolerance of OsLPL2/PIR gene in rice

ZHOU Wen-Qi1,2(), QIANG Xiao-Xia3, WANG Sen4, JIANG Jing-Wen1, WEI Wan-Rong1,*()   

  1. 1Key Laboratory of Southwest China Wildlife Resources Conservation / College of Life Sciences, China West Normal University, Nanchong 637000, Sichuan, China
    2Crops Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
    3Lanzhou No. 4 High School, Lanzhou 730050, China
    4Shannxi Province Animal Husbandry Industry Experimental Demonstration Center, Xianyang 713702, Shaanxi, China
  • Received:2021-05-01 Accepted:2021-10-19 Online:2022-06-12 Published:2021-11-20
  • Contact: WEI Wan-Rong E-mail:zhouwenqi850202@163.com;weiwr18@126.com
  • Supported by:
    Young Scientific and Technological Talents Support Project of Gansu Association for Science and Technology in 2020 and the Agricultural Science and Technology Innovation Program of Gansu Academy of Agricultural Sciences(2020GAAS34)

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Abstract:

Drought threatens global agricultural production and limits the prospects for sustainable agricultural development. Plant leaf epidermis plays a vital role in the process of growth, development, and resistance to adversity stress, and water and gas exchange with the external environment. In this study, compared with the wild-type Zhonghua 11 (ZH11), we found that mutants less pronounced lobe epidermal cell 2-1 (lpl2-1) and less pronounced lobe epidermal cell 2-2 (lpl2-2) were more sensitive to drought and salt stress response, and the survival rate after rewatering was extremely significantly reduced, which was less than half of the control. Compared with ZH11, lpl2-1 and lpl2-2 had shorter plant height, shorter root length, significantly increased stomatal density and stomatal openings in the same phyllodes, and the serrated lobe of the epidermal cell margin becomes smoother, and the epidermal cell nesting was not tight, which might result in faster and more water loss of lpl2-1 and lpl2-2 than ZH11. The water loss experiment of separated leaves also proved that the water loss rate of lpl2-1 and lpl2-2 leaves was higher than that of the ZH11 in equal time. Overexpression of OsLPL2 was transferred into lpl2-1, and the OE-OsLPL2/lpl2-1 transgenic positive plants recovered the smooth epidermis of lpl2-1 and the sensitive phenotype to drought and salt stress. These results showed that OsLPL2 gene not only controlled the microfilament synthesis and morphogenesis of rice epidermal cells, but also played a key role in response to plant stress by regulating stomatal density, stomatal conductance, and root growth and development. This study provides a theoretical basis for revealing the molecular regulation mechanism of OsLPL2 in response to drought stress in rice.

Key words: rice, drought tolerance, salt resistance, SCAR/WAVE complex, plant leaf epidermis

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 酶切位点
Restriction enzyme cutting site
p1301-LPL2F
p1301-LPL2R		 GGTACCATGGCCATCCCCGTCGAGG
ACTAGTTCAAGTAGCTCTCTGTGGCAATG		 Kpn I /Spe I
RT-LPL2-F
RT-LPL2-R		 CCTGGAATCATCTCGTGTTATCC
GCTGGGCAGAGTCATTGTAAAG
TUB-F
TUB-R		 TTTCACTCTTGGTGTGAAGCAGAT
GACTTCCTTCACGATTTCATCGTAA

Table 2

Genetic code and function annotation of OsLPL2 in different species"

物种
Specie		 同源基因
Orthologous gene		 功能预测
Putative function
水稻Oryza sativa LOC_Os03g05020 PIR, putative, expressed
拟南芥Arabidopsis thaliana AT5G18410 Transcription activators
玉米Zea mays GRMZM2G113174 PIROGI; similar to KLK/PIR/PIR121/PIRP (KLUNKER, PIROGI)
玉米Zea mays GRMZM2G170567 PIROGI; similar to KLK/PIR/PIR121/PIRP (KLUNKER, PIROGI)
二穗短柄草Brachypodium distachyum Bradi1g75470 Protein PIR
高粱Sorghum bicolor Sb01g047340 Protein PIR
白杨树Populus trichocarpa POPTR_0013s04800 PIR121; transcription activator
葡萄Vitis vinifera GSVIVG00026355001 GSVIVG00026355001
人类Homo sapiens HsPIR121/CYFIP2 KLK/PIR/PIR121/PIRP (KLUNKER, PIROGI)

Fig. 1

Homology analysis of OsLPL2/PIR in different species AtPIR is a homologous protein of PIR in Arabidopsis thaliana, OsLPL2 is a homologous protein of PIR in rice, HsPIR121 is a homologous protein of PIR in human, and ZmBRK2 is a homologous protein of PIR in maize. Homologous sequence analysis showed that the amino acid similarity is 72.9%. OsLPL2 has particularly 93% similarity to maize BRK2 homologous proteins."

Fig. 2

lpl2-1 had shorter plants, shorter root length, and shorter root cell length at seedling stage A-D: lpl2-1 plants grown at 1, 2, 3, and 4 weeks, respectively; E, F: scanning images of root cells in ZH11 and lpl2-1 plants for growing one week, respectively; G, H: scanning images of root cells in ZH11 and lpl2-1 growing for 4 weeks; I: plant height was measured from 1 to 4 weeks, N = 30; J: root length was measured from 1 to 4 weeks, N = 30, 1, 2, 3, and 4 represent 1, 2, 3, and 4 weeks, respectively. A-D: Bar: 1 cm; E-H: Bar: 100 μm. *: P < 0.05; **: P < 0.01."

Fig. 3

Leaf epidermal morphology of mutant lpl2-2 A: mature stomata and pavement cells (PCs) with waved lobes were obviously observed in ZH11 (oblique black arrows); B: lpl2-2 showed epidermal PCs with gentler lobes (oblique black arrows), small black triangles indicate GMC-like cells; C: stomatal opening of young leaves, N = 100; D: amplified mature stomata is composed of two GCs and SCs in ZH11 (oblique black arrows); E: amplified mature stomatal structure of lpl2-2 mature leaves; F: stomatal opening of mature leaves, N = 100; G, H: stomatal density of the 4th, 5th, flag leaf and secondary flag leaf in ZH11 and lpl2-2, N > 3000; A-E: bar: 20 μm. **: P < 0.01 (Student's t-test). PC: pavement cells; SC: subsidiary cells; GC: guard cell; SP: stomatal opening; SL: stomatal length; SW: stomatal width."

Fig. 4

lpl2 is more sensitive to drought stress A, E: ZH11 and lpl2-1 before drought treatment; B, F: ZH11 and lpl2-1 after drought; C, G: ZH11 and lpl2-1 after rehydration; I: ZH11 and lpl2-2 before drought treatment; J: ZH11 and lpl2-2 after drought; K: ZH11 and lpl2-2 after rehydration; D, H, L: survival rate of ZH11, lpl2-1 and lpl2-2. A-D, I-L: plants of ZH11 and lpl2-1 were responsive to drought at seedling stage, bar: 10 cm; E-H, plants of ZH11 and lpl2-2 were responsive to at heading stage, Bar: 20 cm. Each experiment, 50 seedlings were selected and biological experiments were repeated for 3 times. D, H, L: N = 150. **: P < 0.01 (Student's t-test)."

Fig. 5

lpl2 is more sensitive to salt stress A: the seedlings of ZH11 and lpl2-1; B: the response of lpl2-1 was more sensitive than CK with different concentrations of NaCl; C: after recovery from watering, the surviving plants of lpl2-1 were significantly less than those of CK; D: the seedlings of ZH11 and lpl2-2; E: the response of lpl2-2 was more sensitive than CK with different concentrations of NaCl; F: after recovery from watering, the surviving plants of lpl2-2 were significantly less than those of CK; A-F: bar: 10 cm. In each experiment, 25 seedlings were selected and four biological replicates were performed. G, H: survival rate of lpl2-1, lpl2-2, and ZH11, N = 100, **: P < 0.01."

Fig. 6

Water loss rate of ZH11, lpl2-1, and lpl2-2 at seedling and heading stages A: comparison of water loss rate between ZH11 and lpl2-1 at seedling stage, the fourth and fifth leaves; B: comparison of water loss rate between ZH11 and lpl2-1 at heading stage, flag leaf and secondary flag leaf. N = 50; ** indicates significant difference with the control group at P < 0.01. There was no significant difference in stomatal density and water loss rate between lpl2-1 and lpl2-2, but the difference was extremely significant between lpl2 and CK."

Fig. 7

OE-OsLPL2/lpl2-1 restores the tolerance of lpl2-1 to drought and salt stress A: ZH11 and Com #3 before drought treatment at seedling stage; B: ZH11 and Com #3 after drought treatment; C: ZH11 and Com #3 after rehydration treatment; D: survival rate. N = 150, three biological replicates, 50 seedlings each experiment; E-H: plant phenotypes of ZH11 and Com #3 were treated with different concentrations of NaCl for 0, 6, 8, and 5 d after recovery, respectively. I: survival rate of Com #3 and ZH11, after watering, was not significantly different, bar: 10 cm, N = 100, four biological replicates were performed, 25 seedlings each time, NS: not significant."

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