Ethylene-induced inhibition of root growth requires abscisic acid function in rice (Oryza sativa L.) seedlings

Abstract

Ethylene and abscisic acid (ABA) have a complicated interplay in many developmental processes. Their interaction in rice is largely unclear. Here, we characterized a rice ethylene-response mutant mhz4, which exhibited reduced ethylene-response in roots but enhanced ethylene-response in coleoptiles of etiolated seedlings. MHZ4 was identified through map-based cloning and encoded a chloroplast-localized membrane protein homologous to Arabidopsis thaliana (Arabidopsis) ABA4, which is responsible for a branch of ABA biosynthesis. MHZ4 mutation reduced ABA level, but promoted ethylene production. Ethylene induced MHZ4 expression and promoted ABA accumulation in roots. MHZ4 overexpression resulted in enhanced and reduced ethylene response in roots and coleoptiles, respectively. In root, MHZ4-dependent ABA pathway acts at or downstream of ethylene receptors and positively regulates root ethylene response. This ethylene-ABA interaction mode is different from that reported in Arabidopsis, where ethylene-mediated root inhibition is independent of ABA function. In coleoptile, MHZ4-dependent ABA pathway acts at or upstream of OsEIN2 to negatively regulate coleoptile ethylene response, possibly by affecting OsEIN2 expression. At mature stage, mhz4 mutation affects branching and adventitious root formation on stem nodes of higher positions, as well as yield-related traits. Together, our findings reveal a novel mode of interplay between ethylene and ABA in control of rice growth and development.

Figure 1. Ethylene responses in etiolated mhz4 seedlings.

Rice seedlings were grown in dark for 3 d in the presence of various concentrations of ethylene. (A) Ethylene-response phenotypes of WT (Nipponbare) and mhz4 seedlings grown in air or 10 ppm ethylene. Bar = 10 mm. (B) Ethylene dose-response curves for root length (top) and relative root length (bottom) in WT and mhz4 seedlings. Each point is average of 20 to 30 seedlings and bars indicate SD. (C) Ethylene dose-response curves for coleoptile length (top) and relative coleoptile length (bottom) of WT and mhz4 seedlings. Others are as in (B). (D) Expression of ethylene-inducible genes in both shoot and root of WT and mhz4 seedlings. Dark-grown 2 d-old seedlings were treated with or without 10 ppm ethylene for 8 h and the RNA was isolated for quantitative PCR. Data are the mean ± SD of four replicates. (E) Expression of ethylene-inducible genes in root (left) or shoot (right) of WT and mhz4 seedlings. Others are as in (D).

Figure 2. Map-based cloning of mhz4 locus.

(A) Fine mapping of MHZ4 gene. The locus was mapped to chromosome 1 within a 30 Kb region between Idl1–1.55 and Idl1–1.58 markers. Mutation site is indicated in schematic diagram of MHZ4 gene. Black boxes represent exons. (B) Schematic structure of the MHZ4 protein. The structure is predicted using the SMART software (http://smart.embl-heidelberg.de). Mutation site is shown. cTP indicates chloroplast transit peptide identified using the ChloroP 1.1 program (http://www.cbs.dtu.dk/services/ChloroP/). Blue columns represent transmembrane domains. (C) Confirmation of mhz4 mutation site in both genomic DNA and cDNA by PCR. M: marker. (D) Functional complementation of mhz4 mutant. MHZ4 genomic DNA (4213 bp) was transformed into mhz4 plants (MHZ4/mhz4), rescuing the enhanced coleoptile elongation and reduced root inhibition phenotypes of mhz4 in the presence of ethylene (10 ppm). Bottom is confirmation of the transgene by PCR using the genomic DNA as templates. Bar = 10 mm. (E) Phylogenetic analysis of MHZ4/OsABA4 and its homologous proteins from other plants. The phylogenetic tree is generated by Maximum Likelihood method. Accession numbers are as follows: Vitis vinifera, XP_002283875; Populus trichocarpa, XP_002305164; Ricinus communis, XP_002521222; Citrus sinensis, ADH82117; Glycine max, XP_003532342; Medicago truncatula, XP_003618901; Arabidopsis thaliana, NP_564889; Brachypodium distachyon, XP_003565288; Setaria italica, XP_004968073; Zeay mays, ACN29324; Physcomitrella patens, Pp1s108_75V6; Anabaena cylindrica, WP_015215835; Synechocystis, BAA18538.

Figure 3. MHZ4 expression and protein subcellular localization.

(A) MHZ4 expression in different rice organs detected by RT-PCR. Actin1 was used as an internal control. (B) Tissue-specific expression of MHZ4 revealed by promoter-GUS analysis. Transgenic plants expressing MHZ4pro::GUS were used for analysis. Rice organs were stained for GUS for two days. At least 10 samples for each organ were observed and representative ones are presented. (a–c) 1 d- to 4 d-old etiolated seedlings. (d) GUS signals in adventitious roots and lateral roots of 4 d-old seedlings. (e) GUS staining in vascular tissues of root tips. (f) GUS staining in quiescent center (arrow head) and root caps of root tips. (g, h) GUS staining in segments of young (g) and mature (h) leaf blades. (i) GUS staining in young stem nodes and the base of axillary buds. (j) GUS staining in adventitious roots derived from nodes. (k) GUS staining in the anthers and pistils of young flowers. (l) GUS staining in the lemma of flowers. (m) Staining in the top and bottom of an ovary. (n) GUS staining in a developing grain. Bars are 1 mm except for those indicated. (C) Subcellular localization of MHZ4 in chloroplasts of tobacco glandular hairs as revealed by GFP-fusion protein. The constructs were transiently expressed in tobacco leaf cells by microprojectile bombardment. GFP fluorescence was detected using confocal microscopy. Red fluorescence indicates chlorophyll. Yellow color indicates co-localization of MHZ4 with chloroplasts. Bar = 10 µm.

Figure 4. Ethylene-induced root inhibition is largely mediated through MHZ4-dependent ABA accumulation.

(A) ABA levels in WT and mhz4 seedlings in the absence or presence of ethylene. Two-day-old etiolated seedlings were treated with or without 100 ppm of ethylene for 48 h. Data are the mean ± SD of three replicates. * and ** indicate significant difference between the compared two samples at P<0.05 and P<0.01, respectively. (B) Expression of ABA-responsive gene OsMFT2 (LOC_Os01g02120). Upper, OsMFT2 transcripts in 3 d-old etiolated seedlings of WT in response to ABA (100 µM, 6 h). Bottom, OsMFT2 transcripts detected in mhz4 and WT etiolated seedlings. Actin1 was used as an internal control. (C) Rescue of the root length of mhz4 by ABA. Rice seedlings were grown in the dark for 2.5 days in the presence or absence (Mock) of 0.04 µM ABA. Each column is average of 40 seedlings and bars indicate SD. Different letters above each column indicate significant difference between the compared pairs (P<0.01). (D) Rescue of the reduced ethylene sensitivity of mhz4 roots by ABA. WT and mhz4 seedlings were grown in the dark for 2.5 d in the absence or presence of 10 µM ACC, with or without supplementation of 0.1 µM ABA. Bar = 10 mm. (E) Quantification of root inhibition in (D). Each column is average of 40 seedlings and bars indicate SD. ** indicates significant difference between the linked two samples at P<0.01. (F) Quantitative PCR analysis of MHZ4 expression in response to ethylene. WT seedlings were grown in the dark for 2.5 d and then treated with 10 ppm ethylene for 0–6 h. Data are the mean ± SD of four replicates. ** indicate significant difference compared to 0 h at P<0.01. (G) Ethylene-induced GUS activity in roots of transgenic plants harboring MHZ4pro::GUS construct. One-day-old etiolated seedlings were treated with or without 10 ppm ethylene for 24 h. The roots were cut off and stained for GUS activity for two days. Twenty to thirty roots were observed for each treatment and representative samples are presented. Bar = 1 mm. (H) Quantitative PCR analysis of OsIAA20 expression in response to ABA or ethylene+NDGA. Dark-grown 2 d-old WT seedlings were treated with 100 µM ABA for 6 h, or treated with 10 ppm ethylene for 8 h in the presence or absence (Mock) of 100 µM NDGA. The RNA from roots was isolated for quantitative PCR. Data are the mean ± SD of four replicates.

Figure 5. Enhanced ethylene-response in mhz4 coleoptiles is rescued by ABA and MHZ4 mutation leads to ethylene overproduction.

(A) ABA rescue of enhanced ethylene-response phenotype in mhz4 coleoptiles. WT and mhz4 seedlings were grown in the dark for 2.5 d in the absence or presence of 10 ppm ethylene, with or without supplementation of 0.1 µM ABA. Bar = 10 mm. (B) Quantification of coleoptile growth with treatments in (A). Each column is average of 30 seedlings and bars indicate SD. ** indicates significant difference between the linked two samples at P<0.01. (C) Ethylene production in WT and mhz4 mutant. Data are the mean ± SD of three replicates. ** indicate significant difference compared to WT at P<0.01. (D) Expression of ethylene biosynthetic genes in 3 d-old etiolated seedlings of WT and mhz4. (E) Ethylene dose-response curves for coleoptile elongation in WT and mhz4 seedlings in the presence or absence of 5 µM of ethylene biosynthesis inhibitor AVG. Dark grown seedlings were treated with various concentrations of ethylene for 2.5 d. Each point is average of 25 to 30 seedlings and bars indicate SD. (F) Coleoptile elongation phenotypes of WT and mhz4 in the presence or absence of 5 µM of AVG. The ethylene concentration was 10 ppm. Others are as in (E). Bar = 10 mm. (G) OsEIN2 gene expression in WT and mhz4 etiolated seedlings detected by semiquantitative RT-PCR. Rice seedlings were grown in the dark for 3 days and RNA was isolated from the shoots and roots. Actin1 was used as an internal control.

Figure 6. MHZ4 overexpression confers enhanced and reduced ethylene responses in roots and coleoptiles, respectively.

(A) Ethylene response phenotypes in WT and MHZ4-overexpressing (MHZ4-OX) lines. Rice seedlings were grown in dark for 2.5 d in the presence or absence of 1 ppm ethylene. Bar = 10 mm. (B) Ethylene dose-response curves for root length (top) and relative root length (bottom) in WT and MHZ4-OX lines. (C) Ethylene dose-response curves for coleoptile length (top) and relative coleoptile length (bottom) in WT and MHZ4-OX lines. Others are as in (B). (D) Expressions of ethylene-inducible genes in roots (top) and shoots (bottom) of WT and MHZ4-OX lines. Dark-grown 2 d-old seedlings were treated with or without 10 ppm of ethylene for 8 h and the RNA was isolated for quantitative RT-PCR. Data are the mean ± SD of four replicates. (E) OsEIN2 gene expression in shoots of WT and MHZ4-OX lines detected by semi-quantitative PCR. Rice seedlings were grown in dark for 3 days and RNA was isolated from shoots. Actin1 was used as an internal control.

Figure 7. Genetic interactions of MHZ4 with ethylene receptor gene and MHZ7/OsEIN2 gene.

(A) Comparison of ethylene-response phenotypes of mhz4 Osers1 double mutant and the single mutants. Nipponbare (Nip) and Dongjin (DJ) are wild types. Double mutant between mhz4 and ethylene receptor OsERS1 mutant Osers1 (Dongjin background) were generated by crossing. Rice seedlings were grown in the dark for 3 d in the presence or absence of 1 ppm ethylene. (B) Quantification of root length (left) and relative root length (right) of the mutants in (A). Each column is average of 50 seedlings and bars indicate SD. ** indicate significant difference compared to air control at P<0.01. (C) Ethylene response phenotypes of double mutant mhz4 Osein2 and the single mutants. Osein2/mhz7-1 is in Nipponbare background. Rice seedlings were grown in dark for 3 d in the presence or absence of 10 ppm ethylene. (D) Ethylene dose-response curves for coleoptile length in various mutants. Each point is average of 20 seedlings and bars indicate SD. (E) Ethylene dose-response curves for root length (left) and relative root length in various mutants (right). Others are as in (D). Bars = 10 mm.

Figure 8. Phenotypic comparison of field-grown plants.

(A) Phenotypes of Five-week-old seedlings of WT and mhz4. (B) Plant phenotypes after heading. Please note the spots on some mhz4 leaves but not WT leaves. (C) Chlorophyll contents in the fourth leaves of plants. Chlorophyll was extracted with 95% ethanol from the fourth leaves from the top at heading stage. Each column is average of three measurements and bars indicate SD. ‘**’ indicate significant difference compared to WT (P<0.01). (D) Formation of branches and nodal adventitious roots in the main tiller of WT and mhz4 plants. (E) Branch length at each node from the main tillers of WT and mhz4. Each column is average of 30 to 35 plants and bars indicate SD. ‘**’ indicate significant difference compared to WT (P<0.01). (F) Percentage of nodal adventitious roots in the main tillers. 30 to 35 plants were investigated for WT and mhz4. (G) Effects of OsEIN2 mutation on branching and adventitious root formation of mhz4. Representative plants of WT, mhz4, Osein2 and mhz4 Osein2 double mutant were compared. (H) Quantification of branch length at each node in main tillers from 10–20 plants in (G). (I) Percentage of adventitious root formation at each node in main tillers from 10–20 plants in (G).