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Isolation and Characterization of a Spotted Leaf 32 Mutant With Early Leaf Senescence and Enhanced Defense Response in Rice

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Isolation and Characterization of a Spotted Leaf 32 Mutant With Early Leaf Senescence and Enhanced Defense Response in Rice

Liting Sun et al. Sci Rep.

Abstract

Leaf senescence is a complex biological process and defense responses play vital role for rice development, their molecular mechanisms, however, remain elusive in rice. We herein reported a rice mutant spotted leaf 32 (spl32) derived from a rice cultivar 9311 by radiation. The spl32 plants displayed early leaf senescence, identified by disintegration of chloroplasts as cellular evidence, dramatically decreased contents of chlorophyll, up-regulation of superoxide dismutase enzyme activity and malondialdehyde, as physiological characteristic, and both up-regulation of senescence-induced STAY GREEN gene and senescence-associated transcription factors, and down-regulation of photosynthesis-associated genes, as molecular indicators. Positional cloning revealed that SPL32 encodes a ferredoxin-dependent glutamate synthase (Fd-GOGAT). Compared to wild type, enzyme activity of GOGAT was significantly decreased, and free amino acid contents, particularly for glutamate and glutamine, were altered in spl32 leaves. Moreover, the mutant was subjected to uncontrolled oxidative stress due to over-produced reactive oxygen species and damaged scavenging pathways, in accordance with decreased photorespiration rate. Besides, the mutant showed higher resistance to Xanthomonas oryzae pv. Oryzae than its wild type, coupled with up-regulation of four pathogenesis-related marker genes. Taken together, our results highlight Fd-GOGAT is associated with the regulation of leaf senescence and defense responses in rice.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Phenotypic comparison of the wild-type 9311 and spl32 plants.
(A,B,C) Phenotypes of 15 (A), 23 (B), 31 (C) days old wild-type 9311 and spl32 seedlings. The white arrows indicate the fourth real leaves of the wild-type 9311 and spl32 plants. The inserts show enlargement of the fourth real leaves of the wild-type 9311 and spl32 mutant (AC). (D) Phenotypic comparison of the wild-type 9311 and spl32 mutant at heading stage. The insert shows flag leaves of the wild-type 9311 and spl32 plants. (E) Comparison of the wild-type 9311 and spl32 seeds (dehulled). Bars = 5 cm in A, B and C, 15 cm in D and 5 mm in (E). (F) Determination of the chlorophyll contents of the fourth leaves of the wild-type 9311 and spl32 mutant at three different developmental stages: 15, 23, 31d after sowing. Chla, Chlorophyll a; Chlb, chlorophyll b; Car, total carotenoids. Data represent means ± SD of three independent measurements. Asterisks indicate the statistical significance levels according to Student’s t test: **P < 0.01 and *P < 0.05. FW, fresh weight.
Figure 2
Figure 2. Transmission electron microscopy analyses.
The wild-type 9311(A,C,E) and spl32 (B,D,F) mutant at 15d (A and B), 23d (C and D), 31d (E and F) after sowing. Bars = 5 μm. Thy: thylakoid lamellae; OB: osmiophilic body.
Figure 3
Figure 3. Gene expression analyses.
The expression analysis of two senescence-associated transcription factors (OsWRKY23 and OsWRKY72), two senescence-associated genes (Osl43 and Osl85) SGR (A) and photosynthesis-associated genes (B) at tillering stage. Data represent means ± SD of three independent measurements. Asterisks indicate the statistical significance levels according to Student’s t test: **P < 0.01 and *P < 0.05. The accession numbers were listed as follows: OsWRKY23 BAG98560.1; OsWRKY23 BAG98549.1; Osl43 AAK82986; Osl85 AAL65398; SGR AAW82954.1; rbcS AAR19268.1; lhcA AAB65793.1; lhcB (Arabidopsis thaliana), AAA32760; rbcL NP_039391.1; psaA AJC99402.1; psbA AJC99383.1; petD AJC99432.1; ndhA AJC99458.1; atpA AJC99399.1.
Figure 4
Figure 4. Cloning of SPL32 gene and phylogenetic analysis of Fd-GOGAT homologs.
(A) The SPL32 locus was mapped to the long arm of chromosome 7 between the markers RM118 and RM172. (B) Mapping of the SPL32 locus between markers zzy-21 and zzy-23 based on bacteriophage P1-derived artificial chromosome (PAC) or bacterial artificial chromosome (BAC) clone sequence (PAC1, P0047B07; BAC2, OJ1477_F01; PAC3, P0496C02). ‘n’ represents homozygous recessive individuals derived from an F2 population and an F2:3 population from the cross spl32 × 02428. The number of recombinants is indicated below the map. (C) the SPL32 locus was narrowed down to a 66-kb region; there are 8 putative open reading frames (ORFs). (D) The gene structure of 9311. The junction of the third exon and intron had a single base mutation (G into A) in ORF4 of spl32 mutant. (E) Complementation analysis of the spl32 mutant. The wild-type 9311 plant and homozygous recessive mutant derived from the cross spl32 × Nipponbare transformed with pFd-GOGAT show normal green leaves, whereas the spl32 mutant and homozygous recessive mutant of F2 population transformed with pΔFd-GOGAT show necrotic spots in leaves. (F) PCR analysis with primers Zf-1 showed the difference in the Fd-GOGAT cDNA in the wild-type 9311, spl32 mutant, ΔFd-GOGAT transgenic plants and Fd-GOGAT transgenic plants. Alternative splicing occurs in the coding region of Fd-GOGAT in spl32 mutant. (G) Phylogenetic analysis of Fd-GOGAT. Fd-GOGAT is most closely homologous to maize and sorghum Fd-GOGATs. The accession numbers were listed as follows: SiFd-GOGAT (Solanum lycopersicum), XP_004234830.1; StFd-GOGAT (Solanum tuberosum), XP_006363768.1; RcFd-GOGAT (Ricinus communis), XP_002526914.1; GmFd-GOGAT (Giycine max), XP_006576787.1; CsFd-GOGAT (Cucumis sativus), XP_004136778.1; OsFd-GOGAT (Oryza sativa), NP_001060520.1; ZmFd-GOGAT (Zea mays), NP_001105693.1; SbFd-GOGAT (Sorghumbicolor), XP_002463318.1; AtGLU1 (Arabidopsis thaliana), NP_850763.1; AtGLU2 (Arabidopsis thaliana), NP_181655.1.
Figure 5
Figure 5. Expression analysis and subcellular localization of Fd-GOGAT protein.
(A) The expression of Fd-GOGAT in different tissues. Leaf was selected from seedling of 30 days after sowing of the wild-type 9311; root, flag leaf, panicle, and leaf sheath were selected from the wild-type 9311 at heading stage. (B) The expression of Fd-GOGAT at three different developmental stages. The fourth leaves of 15, 23, 31 days after sowing in wild-type 9311 and spl32 mutant were selected. Data are means ± SD (n = 5). Asterisks indicate the statistical significance levels according to Student’s t test: **P < 0.01 and *P < 0.05. (C) GFP signals of the Fd-GOGAT-GFP fusion protein located in the chloroplasts of rice protoplasts. (D) GFP signals were dispersed in rice protoplasts. Bars = 10 μm.
Figure 6
Figure 6. Analysis of GOGAT and GS activities, determination of photorespiration rate, contents of ammonia (NH4+) and glutathione (GSH).
(A and B) Enzyme activity of GOGAT and GS in wild-type 9311 and spl32 mutant, non-spotted, spot-appearing and serious-spotted leaves of spl32 mutant and the corresponding sections of leaves in the wild-type 9311 at tillering stage. (C) Determination of NH4+ content in the wild-type 9311 and spl32 mutant at tillering stage. (D) Determination of net photosynthesis rate in atmospheric condition and hypoxia conditions using portable photosynthesis system LI-6400XTOPEN6.1. At tillering stage, new-fully expanded leaves of wild-type 9311 and spl32 mutant were chosen and net photosynthesis rate of mid-blade was measured before 12 o’clock. (E) Determination of GSH content in the wild-type 9311 and spl32 mutant at tillering stage. Data represent means ± SD of three independent measurements. Asterisks indicate the statistical significance levels according to Student’s t test: **P < 0.01 and *P < 0.05. FW, fresh weight.
Figure 7
Figure 7. Dying experiments and the determination of enzyme activities involved in scavenging and generating mechanism of ROS.
(A) non-spotted leaves and serious spotted leaves of spl32 mutant and the corresponding sections of leaves in the wild-type 9311 at tillering stage were stained by trypan blue and 3, 3-diaminobenzidine (DAB) respectively. Controls were not stained and decolorized directly by alcohol. (B) The contents of hydrogen peroxide (H2O2) and malonaldehyde (MDA) in the wild-type 9311 and spl32 mutant at tillering stage. FW, fresh weight. (C) The enzyme activities of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (POD), and peroxidase (APX) in wild-type 9311 and spl32 mutant at tillering stage. FW, fresh weight. (D) The enzyme activities of NADPH oxidase and polyamine oxidase (PAO) in wild-type 9311 and spl32 mutant at tillering stage. FW, fresh weight. Data represent means ± SD of three independent measurements. Asterisks indicate the statistical significance levels according to Student’s t test: **P < 0.01 and *P < 0.05. FW, fresh weight.
Figure 8
Figure 8. Detection of bacterial blight pathogen resistance and expression of resistance related genes.
(A) Lesion lengths were determined after plant leaves inoculated by bacterial blight pathogen zhe173. Bars represent ± SD of six replicates. (B) The expression of pathogenesis-related (PR) marker genes at tillering stage. Bars represent ± SD of three measurements. The gene numbers of PR genes are as follows: PR1a, AJ278436; PR1b, B109D03; PR5, X68197; PR10, D38170. Asterisks indicate the statistical significance levels according to Student’s t test: **P < 0.01 and *P < 0.05.

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