A Comparative Study of Ethylene Emanation upon Nitrogen Deficiency in Natural Accessions of Arabidopsis thaliana

Abstract

An original approach to develop sustainable agriculture with less nitrogen fertilizer inputs is to tackle the cross-talk between nitrogen nutrition and plant growth regulators. In particular the gaseous hormone, ethylene, is a prime target for that purpose. The variation of ethylene production in natural accessions of the model species Arabidopsis thaliana was explored in response to the nitrate supply. Ethylene was measured with a laser-based photoacoustic detector. First, experimental conditions were established with Columbia-0 (Col-0) accession, which was grown in vitro on horizontal plates across a range of five nitrate concentrations (0.5, 1, 2.5, 5, or 10 mM). The concentrations of 1 and 10 mM nitrate were retained for further characterization. Along with a decrease of total dry biomass and higher biomass allocation to the roots, the ethylene production was 50% more important at 1 mM than at 10 mM nitrate. The total transcript levels of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASES (ACS) in roots and those of ACC OXIDASES (ACO) in shoots increased by 100% between the same treatments. This was mainly due to higher transcript levels of ACS6 and of ACO2 and ACO4 respectively. The assumption was that during nitrogen deficiency, the greater biomass allocation in favor of the roots was controlled by ethylene being released in the shoots after conversion of ACC originating from the roots. Second, biomass and ethylene productions were measured in 20 additional accessions. Across all accessions, the total dry biomass and ethylene production were correlated negatively at 1 mM but positively at 10 mM nitrate. Furthermore, polymorphism was surveyed in ACC and ethylene biosynthesis genes and gene products among accessions. Very few substitutions modifying the amino acids properties in conserved motifs of the enzymes were found in the accessions. Natural variation of ethylene production could be further explored to improve Nitrogen Use Efficiency (NUE), in particular by manipulating features like the biomass production and the timing of senescence upon nitrogen limitation.

Keywords: Arabidopsis; biomass; ethylene; natural variation; nitrogen.

Figure 1

Biomass production, nitrogen and carbon tissue concentration, pigment levels, and ethylene emanation in response to nitrate supply in Columbia-0 accession. Arabidopsis thaliana Col-0 seedlings were grown across a range of nitrate concentrations (0.5, 1, 2.5, 5, or 10 mM) and harvested 13 days after germination. (A) Representative pictures of petri plates containing 100 seedlings. Scale bar: 5 cm, (B) Total dry biomass, (C) Root to shoot dry biomass ratio, (D,E) Total nitrogen (D) and carbon (E) concentrations per dry weight of root (black columns) or shoot (white columns) tissues. (F–H) Pigment concentrations per fresh weight of shoot tissues: total chlorophyll (Chla+b) (F), carotenoids (G) and anthocyanins (H), (I) Ethylene production per hour and per dry weight of total seedlings. Mean of four samples (100 pooled seedlings) ± SD. Letters indicate significant differences (Tukey's HSD test, p < 0.1).

Figure 2

Transcript abundance of ACC SYNTHASE and ACC OXIDASE multigene family. Transcript levels of the most highly expressed ACS (A) and ACO (B) members are presented in root and shoot tissues of Col-0 seedlings grown at 1 or 10 mM nitrate, 13 days after germination. Data represent mean abundance with the abundance of the most highly expressed (normalized by ACT2 and UBQ10 levels) family member in one organ, set as one under 10 mM nitrate condition (ACS6 = 1 and ACO2 = 1, respectively for ACS and ACO families). Mean of two or three pools of 100 organs ± SD (each sample assessed by three technical replicates).

Figure 3

Biomass production and ethylene emanation in response to nitrate supply in Arabidopsis accessions. Accessions were grown at 1 or 10 mM nitrate supplies and harvested 13 days after germination. (A,B) Total dry biomass, (C,D) Root to shoot dry biomass ratio, (E,F) Ethylene emanated per hour and per hundred seedlings, (G,H) Ethylene emanated per hour and per dry weight. Parameters were measured at 1 mM (A,C,E,G) and 10 mM (B,D,F,H) nitrate. Mean of four samples (100 pooled seedlings) ± SD. Asterisks represent significant (p < 0.1, Kolmogorov-Smirnov two-sided test) differences between one accession and Col-0.

Figure 4

Correlation between biomass production and ethylene emanation in response to nitrate availability in Arabidopsis accessions. Hundred seedlings of each accession were grown at 1 mM (A) or 10 mM (B) nitrate supplies and harvested 13 days after germination. Total dry weight biomass and ethylene emanation normalized to hundred seedlings were measured in four replicates. Accession names indicate means of four replicates. Linear regression models indicated a significant negative association between the total dry biomass and ethylene emanation at 1 mM and a positive association at 10 mM (slopes different from zero: p < 0.001). Model details at 1 mM: p < 0.001, adjusted R² = 0.15, F-statistic = 14.01 on 1 DF, residual SE = 0.124 on 72 DF; and 10 mM: p < 0.001, adjusted R² = 0.15, F-statistic = 14.82 on 1 DF, residual SE = 0.146 on 76 DF.

Figure 5

Polymorphism density in DNA coding sequences of ACC SYNTHASES and ACC OXYDASES genes. Aivailable coding gene sequences of ACS (A) and ACO (B) for 21 accessions from the McKhann et al. (2004) core set were compared to the corresponding Col-0 sequences retrieved from 250k SNP data published in Atwell et al. (2010) and Li et al. (2010). The density of single nucleotide polymorphism, defined as the number of SNPs per base pair in a given exon, is indicated by the color scale.

Figure 6

Amino acid sequence alignment of ACC synthase and ACO oxydase proteins. Available protein sequences of 21 accessions from the McKhann et al. (2004) core set were compared to the corresponding Col-0 sequences retrieved from 250k SNP data published in Atwell et al. (2010) and Li et al. (2010). Amino acid substitutions are indicated for conserved domains and motifs of ACS and ACO protein families. (A) The sequences of 11 ACS isozymes present seven conserved domains marked as boxes 1–7. The S residue marked at the sequence end of ACS1-6, 8, 9, and 11 is a putative phosphorylation site. (B) In ACO proteins, the so-called “2-His-1-carboylate facial triad” (HxD…H) motif is involved in co-factor (Fe²⁺) binding pocket, while the RxS motif is critical for substrate (ascorbate) binding pocket. The presence of one or more synonymous (green) or non-synonymous (red) amino acid substitutions in a given item is indicated.