Elsevier

Plant Science

Volume 177, Issue 1, July 2009, Pages 68-80
Plant Science

Analysis of the metabolome and transcriptome of Brassica carinata seedlings after lithium chloride exposure

https://doi.org/10.1016/j.plantsci.2009.03.013Get rights and content

Abstract

Exposure of B. carinata seedlings to increasing concentrations of a non-physiological ion, lithium, showed significant effects on the germination rate, root length, chlorophyll content and fresh weight in brown-seeded and yellow-seeded near-isogenic lines. Metal content analysis and phytochemical profiling indicated that lithium hyper-accumulated and the lipid and phenolic composition dramatically changed in brown-seeded seedlings. Here, sinapic acid esters and chloroplast lipid were replaced by benzoate derivatives, resveratrol and oxylipins after lithium exposure. In contrast, the yellow-seeded plants maintained the same phenolic and lipid composition before and after exposure to lithium and did not tolerate the high metal concentrations tolerated by the brown-seeded line. Microarray analysis using a Brassica napus 15,000 expressed sequence tag (EST) array indicated a total of 89 genes in the brown-seeded line and 95 genes in the yellow-seeded line were differentially expressed more than 20-fold after treatment of B. carinata seedlings with lithium chloride and more than 1083 genes with expression changes greater than 2-fold. The putative functions of the differentially expressed genes included proteins involved in defense, primary metabolism, transcription, transportation, secondary metabolism, cytochrome P450, as well as proteins with unknown functions. Transcriptome changes between yellow-seeded and brown-seeded B. carinata seedlings after lithium chloride exposure indicated that the two lines responded differently to lithium treatment. The expression patterns generally supported the phytochemical data. From the results of this study, B. carinata brown-seeded germplasm showed an ability to survive under moderately high concentrations of lithium chloride (>150 mM). The ability to accumulate this metal ion suggests brown-seeded B. carinata has some potential in phytoremediation of lithium-contaminated water and soil.

Introduction

Metal salt contamination of soils is a serious environmental problem with potential harmful consequences to agriculture and human health. About 20% of the world's cultivated land and nearly half of all irrigated lands are affected by salinity [1]. The problem of salinity is increased further because of poor drainage and the use of poor quality water for irrigation. Metal salt contamination can arise from a variety of sources and activities, including mining of natural fertilizers and other minerals, manufacturing and disposal of engine additives and pharmaceutical use.

In nature, plant species possess a range of mechanisms involved in the detoxification of metals, allowing some to survive better than others under metal stress [2]. Salt tolerance in plants is not associated exclusively with cellular metal homeostasis, but also with adaptation to the secondary effects of salinity. These secondary effects include oxidative damage and changes to the levels and composition of fatty acids of the major glycerolipids in roots and leaves of a wide range of plant species [3]. Morphological and physiological processes can also be affected by salinity. Tobacco exposed to Li+ salt shows inhibition of the rhythmic movements of pulvini and petals, disrupted pollen development (inducing symmetrical mitoses in the microspores), and a block in pollen germination [4]. Zhu speculated that three interconnected activities are important for plants to achieve salt tolerance—prevention or alleviation of damage, re-establishment of homeostasis, and resumption of growth [1]. Since these processes are not clearly understood, the molecular basis of cellular ion homeostasis and damage control becomes very important. Understanding the mechanism will impact strongly on the success of developing salt tolerant crops [5].

Several species of the mustard Brassica family, e.g. B. juncea, are not only important sources of edible oil, but also are known to be tolerant to heavy metals and are potential candidates for phytoremediation [5], [6]. B. carinata (A.) Braun (Ethiopian mustard) is an allotraploid member of the cruciferous plant family and is composed of two genomes, one originating from B. nigra and one from B. oleracea [7]. B. carinata primarily has been grown in Ethiopia as a vegetable and oilseed crop [8]. Cultivars of yellow-seeded B. carinata are currently being developed in North America for the biodiesel and fish feed biorefinery markets. This species induces stress-related genes after exposure to copper [9], but broader defense responses to a range of metals at the genetic and molecular level are unknown for B. carinata.

The yellow-seeded trait of all Brassica species in the triangle of U, with the exception of B. carinata, segregates as two or three recessive Mendelian alleles. After more than two decades of research, plant breeders have finally developed a yellow-seeded B. napus rapeseed (canola) line YN01-429 with improved meal quality and increased oil content [8]. In contrast, the yellow-seeded trait of B. carinata segregates as a single partially dominant Mendelian allele [8], but the basis of gene regulation for this trait is unknown except that the dihydroflavonol reductase gene is down-regulated in early stages of seed development [10]. In Arabidopsis, more than 20 yellow-seeded transparent testa mutants have been characterized [11], [12], but dominant yellow phenotypes have not been described.

Lithium is a non-physiological metal ion present in batteries and is a powerful mood-stabilizing therapeutic used in the treatment of bipolar depression. Our interest in lithium arose from its documented presence in surface and drinking waters [13]. Consequently, there is potential for B. carinata oilseed crops to come into contact with this metal and pose difficulties in their end use.

In the present study, we conducted an analysis of seedling growth and survival, element content, and phytochemical composition on B. carinata seedlings exposed to a range of lithium concentrations. Differential changes to the metabolome of brown-seeded and yellow-seeded B. carinata lines correlated with differential ability to accumulate lithium. An oligo-nucleotide array representing 15,000 genes from B. napus was used to investigate changes in the transcriptome of lithium-treated B. carinata seedlings [14]. Our analysis led to the discovery of 89 seedling genes in brown-seeded line and 95 genes in a yellow-seeded line that were differentially expressed more than 20-fold after treatment with lithium chloride, in addition to many other genes expressed at a weaker level. The results highlighted a broader spectrum of defense proteins than previously reported from B. carinata [9] and with a greater proportion of genes more strongly induced in the brown-seeded line than in the yellow-seeded line. We also discovered three compounds not reported previously from B. carinata, one of which has not been reported from any metal-stressed or salt-stressed plants.

Section snippets

Plant material and LiCl treatments

B. carinata seeds (S1, accession no. PGRC/E 21164, segregating for yellow- and brown-seeded phenotypes, were developed into third-generation near-isogenic lines (S3) by single seed descent. RFLP and RAPD gel analysis showed very little variation between the genomes of the two lines suggesting that they are very nearly isogenic [15]. Lines were grown in a soilless mix (Redi-Earth, Grace & Co., Ajax, ON) in a controlled environment chamber (Conviron, Winnipeg, Canada) under fluorescent and

Growth regulation, survival, and Li+ accumulation in yellow-seeded and brown-seeded lines

B. carinata grown in excess Li experienced a significantly decreased germination rate above a threshold concentration (Fig. 1). Fresh weight, root length and pigmentation showed almost no changes up to 30 mM Li exposure in either yellow-seeded or brown-seeded plants, although in general, brown-seeded seedlings initially were more stimulated in growth and more robust than yellow-seeded seedlings at low Li concentration. Growth decreased quickly after 30 mM exposure (Fig. 2A and B), such that more

Conclusion

Excess lithium chloride supplied to B. carinata led to dramatic effects on plant growth, secondary metabolite production and gene expression profiles. Germination rate, root length and fresh weight remained normal (or were stimulated in the brown-seeded line) when seedlings were exposed to less than 60 mM LiCl, but these growth determinants decreased quickly after seedlings were exposed to more concentrated LiCl. Under high Li+ concentration (>120 mM), brown-seeded B. carinata seedlings remained

Acknowledgements

X. Li and P. Gao were recipients of Visiting Fellowships to a Government of Canada Laboratory. This work was supported by the Canadian Crop Genomic Initiative and the Matching Investments Initiative of Agriculture and Agri-Food Canada in conjunction with a collaborative project with Forage Genetic International, Wisconsin, USA.

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