Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium

Abstract

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ(1)-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum×morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136-KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.

Keywords: 3′ RACE; 3′ rapid amplification of cDNA ends; AAAP; ABA; APC; ATF; Abiotic stresses; C-repeat binding factor; CAT; CBF; Chrysanthemum lavandulifolium; ClTUA; ESTs; Flowering and floral development; GSA; LHT; ORF; P5C; P5CDH; P5CR; P5CS; PCR; PDH; PRE; ProT; Proline; Proline dehydrogenase gene; Proline transporter gene; RT-PCR; RT-qPCR; abscisic acid; amino acid transporter family; amino acid-polyamine-choline; amino acid/auxin permease; cationic amino acid transporter; expressed sequence tags; glutamate-semialdehyde; lysine–histidine transporter; open reading frame; ornithine-δ-aminotransferase; polymerase chain reaction; proline dehydrogenase; proline transporter; proline- or hypoosmolarity-responsive element; real-time quantitative PCR; reverse transcription-polymerase chain reaction; Δ(1)-Pyrroline-5-carboxylate synthetase gene; Δ(1)-pyrroline-5-carboxylate; Δ(1)-pyrroline-5-carboxylate dehydrogenase; Δ(1)-pyrroline-5-carboxylate reductase; Δ(1)-pyrroline-5-carboxylate synthetase; α-tubulin gene in Chrysanthemum lavandulifolium; δOAT.