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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (7): 1832-1842.doi: 10.3724/SP.J.1006.2022.12028

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Physiological characters and gene mapping of a precocious leaf senescence mutant ospls7 in rice (Orzo sativa L.)

HUANG Fu-Deng1(), HUANG Yan2, JIN Ze-Yan2, HE Huan-Huan2, LI Chun-Shou1,*(), CHENG Fang-Min2, PAN Gang2   

  1. 1Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China
    2College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, China
  • Received:2021-04-20 Accepted:2021-10-20 Online:2022-07-12 Published:2021-11-15
  • Contact: LI Chun-Shou E-mail:pahfd@126.com;lichunshou@126.com
  • About author:First author contact:

    ** Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(31771688);National Natural Science Foundation of China(31971819);Major Projects of Rice Breeding in the 14th Five Year Plan of Zhejiang Province(2021C02063-1)

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Abstract:

Leaf senescence is the final stage of leaf development, however, premature aging of leaves, especially functional leaves, leads to reduction of yield and quality. Thus, it is very important for developing novel crop germplasms with delayed leaf-senescence characteristics through investigating the molecular and physiological mechanism of leaf senescence. In this study, a stable precocious leaf senescence mutant ospls7 was obtained from 60Co γ-radiated upland rice cultivar Monolaya, and its morphology, physiological characteristics of leaf senescence, cytological observation of internodes, genetic analysis and gene mapping were investigated. Under field conditions, leaf senescence was noticed as early as the 3-4-leaf seedling stage, featuring yellowing and browning at the edge of tip and the upper middle parts of old leaves and finally wilting. Due to shorter length of the parenchyma cells, panicle length and all internodes length of ospls7 were significantly shorter compared with wild type plants at the mature stage, resulting in dwarf phenotype in ospls7. Physiological analysis of leaf senescence indicated that compared to the wild type plants, the total chlorophyll contents, net photosynthetic rate, soluble protein content, and catalase (CAT) activity of the second and third leaves from top in ospls7 were significantly declined at the booting stage, which in turn resulting in the accumulation of H2O2 and a steady increase of malondialdehyde (MDA) contents in the mutant leaves. Moreover, due to significant up-regulation of ABA biosynthetic genes (OsNCED3 and OsAAO3) and significant down-regulation of the ABA catabolism genes (OsABA8ox2 and OsABA8ox3), the endogenous ABA levels in the leaves of ospls7 were significantly higher compared with the wild type at the booting stage. Genetic analysis and gene mapping showed that ospls7 was controlled by a single recessive nuclear gene, located in a region of 207 kb between SSR marker RM25040 and the InDel marker ID74-33/34 of chromosome 10. These results would further facilitate the cloning and functional analysis of OsPLS7 gene.

Key words: rice, ospls7, precocious leaf senescence, physiological analysis, gene mapping

Table S1

Primers for qRT-PCR analysis of ABA biosynthetic and metabolic genes"

基因
Gene		 基因号
Locus ID		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
OsZEP1 LOC_Os04g37619 GGTGCGATAACGTCGTTGATC GTATGGTCTATAAGTGGTAGC
OsNCED1 LOC_Os02g47510 ACCATGAAGTCCATGAGGCT TCTCGTAGTCTTGGTCTTGG
OsNCED2 LOC_Os12g24800 ATGGAAACGAGGATAGTGGT CTTATTGTTGTGCGAGAAGT
OsNCED3 LOC_Os03g44380 CTCCCAAACCATCCAAACCG TGAGCATATCCTGGCGTCGT
OsNCED4 LOC_Os07g05940 ATCTCCTTCTCCCTCCTCCCA TCGCACCCTGCTTGATCTTGC
OsNCED5 LOC_Os12g42280 TCCGAGCTCCTCGTCGTGAA AGGTGTTTTGGAATGAACCA
OsSDR LOC_Os03g59610 GACCTGACGAGACGATGTCC GCAACCTTGCTTTCCAACC
OsAAO1 LOC_Os07g18154 TTCGCCATTTGTTCGTAA CAGAGGAGGTTGCTCAAG
OsAAO2 LOC_Os07g18158 CCCTTGACGCCAACACTG CCGCTTTCGCCACTTATT
OsAAO3 LOC_Os07g07050 CGCCTGGTAAAGTGTCTA AATTGCTCCTTGAGTGGT
OsABA8ox1 LOC_Os02g47470 AAGCTGGCAAAACCAACATC CCGTGCTAATACGGAATCCA
OsABA8ox2 LOC_Os08g36860 CTACTGCTGATGGTGGCTGA CCCATGGCCTTTGCTTTAT
OsABA8ox3 LOC_Os09g28390 AGTACAGCCCATTCCCTGTG ACGCCTAATCAAACCATTGC

Fig. 1

Phenotypes of ospls7 and its wild-type (WT) plants at different growth stages A: seedling stage; B: booting stage; C: leaves at the booting stage, and F means flag leaf and 2-4 means 2nd to 4th leaf from top, respectively; D: panicle and the internodes at the mature stage, P means panicle, 1-6 means the 1st to 6th internode from top, respectively; E: the length of different internodes at the mature stage in 2020; F-I: longitudinal sections of the 2nd internode from top (F-G) and leaf (H-I) in wild-type plants (F, G) and ospls7 (G, I); Bar: 20 cm in A-E; Bar: 50 μm in F-G; Bar: 20 μm in H-I. ** : P < 0.01."

Table 1

Main agronomic traits of ospls7 and its wild-type (WT) plants"

性状
Trait		 2019 2020
野生型WT 突变体ospls7 野生型WT 突变体ospls7
株高 Plant height (cm) 92.27±3.13 74.23±4.65** 89.63±1.67 74.08±2.92**
穗长 Panicle length (cm) 24.87±1.04 19.34±1.15* 23.81±0.95 18.97±1.13*
有效穗数 Effective panicle number 7.75±0.96 5.23±0.45* 9.67±0.58 8.52±0.58*
每穗粒数 Grain number per panicle 176.61±15.83 112.63±11.76** 176.68±9.07 100.24±10.51**
结实率 Seed-setting rate (%) 82.81±5.83 69.84±3.97** 81.76±5.76 72.14±6.93**
千粒重 1000-grain weight (g) 19.57±0.94 15.98±0.59** 18.94±0.53 15.88±0.71**
单株产量 Yield per plant (g) 21.91±2.13 7.09±1.04** 27.38±3.78 8.84±1.37**

Fig. 2

Photosynthetic characteristics of leaves in the ospls7 and its wild-type (WT) plants at the booting stage A-D: Chl a (A), Chl b (B), the total Chl contents (C), and Chl a/b ratio (D) of leaves in ospls7 and wild-type plants; E-F: net photosynthesis rate (E) and Fv/Fm ratio (F) of leaves in ospls7 and wild-type plants. 1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top. *: P < 0.05; **: P < 0.01."

Fig. 3

O2?and H2O2 levels, and CAT and SOD activities in the leaves of ospls7 and its wild-type (WT) plants at the booting stage ?and H2O2 levels, and CAT and SOD activities in the leaves of ospls7 and its wild-type (WT) plants at the booting stage 1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top. *: P < 0.05; **: P < 0.01."

Fig. 4

MDA and soluble protein contents in the leaves of ospls7 and its wild-type (WT) plants at the booting stage 1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top. *: P < 0.05; **: P < 0.01."

Fig. 5

Phenotypes of ospls7 and its wild type (WT) seedlings in response to exogenous ABA A: the phenotypes of ospls7 and its wild-type (WT) plants under different exogenous ABA levels for seven days; B-C: shoot and root length of ospls7 and its WT plants under different ABA levels for seven days. Bar: 20 cm. *: P < 0.05."

Fig. 6

Endogenous ABA levels and qRT-PCR analysis of ABA biosynthetic and metabolic genes in leaves of ospls7 and its wild-type (WT) plants at the booting stage 1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top. *: P < 0.05; ** : P < 0.01."

Table S2

Molecular markers for OsPLS7 gene mapping"

标记
Marker		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
RM25000 CATTGAAGCAGGAGAAGGAGTTGC GATGCATCTGCTCCATCAATTCG
RM25011 AAGCTGCTGCTTCCACTTCACTTCG GTGGCCTCCTCGAGATCGAACG
RM25020 AATCCCTCTCGGCCCATCTCC CGAAGACGACGGCGATGACG
RM25030 GTGATGACGTGGACAAATCTCG GGGTAATCACTACTCACAGAGTTTGG
RM25034 TGTCATGTGGCAATATGAGAGC GACCTTTACCAAGCACATAGTCC
RM25038 CTTTAGAGGTTGCCGAACTGG GAGCGTTTGTAGGAAGTCTTATGG
RM25040 GGCTGGACTTCACTTGACTTTGG CCACACGACCATCTAAGTGAACAGG
ID74-31/32 TCATCTGGTGTTTTGTACCC GGAATGCCTTGGTTAGGTAT
ID74-33/34 GAAAATTGGAGGAGGAGGTA ACCCAAGGAGATAGCAAGTC
ID74-35/36 AGGTCTACCCAAAACAGGAG GTAAGGCCTGACTGGAAGTT

Table 2

Gene names and their functional annotations in the target interval"

基因号
Locus ID		 功能注释
Function annotation
LOC_Os10g07150 转座子蛋白 Transposon protein
LOC_Os10g07160 反转座子 Retrotransposon
LOC_Os10g07200 Hsp20/α晶体家族蛋白 Hsp20/alpha crystallin family protein
LOC_Os10g07210 Hsp20/α晶体家族蛋白 Hsp20/alpha crystallin family protein, putative, expressed
LOC_Os10g07229 脱氢酶 Dehydrogenase
LOC_Os10g07248 转座子蛋白 Transposon protein
LOC_Os10g07270 泛素羧基末端水解酶5 Ubiquitin carboxyl-terminal hydrolase 5
LOC_Os10g07280 表达蛋白 Expressed protein
LOC_Os10g07290 糖苷水解酶家族17 Glycosyl hydrolases family 17
LOC_Os10g07320 假定蛋白 Hypothetical protein
LOC_Os10g07340 表达蛋白 Expressed protein
LOC_Os10g07370 表达蛋白 Expressed protein
基因号
Locus ID		 功能注释
Function annotation
LOC_Os10g07380 表达蛋白 Expressed protein
LOC_Os10g07390 假定蛋白 Hypothetical protein
LOC_Os10g07400 类RPP13抗病蛋白1 Disease resistance RPP13-like protein 1
LOC_Os10g07420 表达蛋白 Expressed protein
LOC_Os10g07430 表达蛋白 Expressed protein
LOC_Os10g07440 表达蛋白 Expressed protein
LOC_Os10g07450 表达蛋白 Expressed protein

Fig. 7

Molecular mapping of OsPLS7 gene on chromosome 10"

Fig. 8

qRT-PCR analysis of genes expressed in the leaves in the target interval"

Fig. S1

Heatmap of gene expression in the target interval"

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