Dynamics of alkylresorcinols during rye caryopsis germination and early seedling growth

1 Introduction

Saturated alkylresorcinols (ARs) and their unsaturated counterparts constitute a large family of essential non-isoprenoid phenolic compounds widely distributed in cereals and other plants [1, 2]. In particular, ARs accumulate to high levels in rye grains (Secale cereale L.) with levels ranging from 0.36 up to 3.2 g kg^–1 [3]. Resorcinolic lipids occur in the testa and pericarp of cereal caryopses [4], whereas the epicuticular wax layer contains only a small amount of these compounds. Due to their unique biological activities, ARs are often considered natural biofungicides [5, 6]. In fact, alkylresorcinols are potent antimicrobials significantly contributing to a critical defensive chemical barrier in cereal grains against numerous bacterial and fungal pathogens [1]. For example, ARs isolated from rye grains were found to inhibit growth of the plant pathogenic fungi Fusarium sp. and Rhizoctonia sp. [7]. Besides cereal grains, ARs have been shown to occur in developing coleoptiles [8] and in intracuticular waxes of rye leaves [9]. There is still substantial lack of an understanding of the biosynthesis process of these intriguing secondary metabolites. In order to fill this serious knowledge gap, in this study we explored the dynamics of AR metabolism during the germination of caryopses and early seedling growth of rye.

2 Materials and methods

3 Results and discussion

Rye caryopses contain large quantities of alkylresorcinols [1, 3]. In the present study, the total concentration of ARs in the tested intact rye caryopses was 581.9 mg kg^–1, which is within the lower content range previously reported in Swedish cereal cultivars (549–1022 mg kg^–1) [12]. Saturated homologues, such as 1,3-dihydroxy-5-n-heptadecylbenzene (AR C_17:0) and 1,3-dihydroxy-5-n-nonadecylbenzene (AR C_19:0) were the most predominant ARs in all samples tested in this study (Table 1). Similar results were reported in the earlier studies of Deszcz and Kozubek [8] and Kulawinek et al. [2] who showed that these constituents are the major secondary compounds in rye grains and seedlings, respectively. Existing differences in AR concentrations between various cultivars of the same cereal species have been well documented [3]. In fact, both quantitative and qualitative levels of ARs in plant organs and tissues can be affected by various, often hard-to-define, biotic and abiotic factors not only within distinct cultivars but also between individual plants of the same cultivar, even within the same ecological niche [10, 13–16]. During the germination of rye caryopses, significant changes in the abundance and/or composition of the ARs were observed. Compared to the AR content of whole ungerminated grains (about 582 mg kg^–1), the content in 1-day-old germinating caryopses was reduced to about 505 mg kg^–1, while the respective homologue profiles remained almost identical, indicating that the ARs present in the germinating grain originated from the original storage pool; yet it remains unclear whether the seedlings are able to utilize ARs accumulated in the seeds. Since light conditions favor the energy-dependent uptake of exogenous ARs, while limiting their accumulation in green seedlings [8], our experiments were performed in darkness, which not only accurately mimicked basic conditions of rye germination but also appeared to stimulate de novo production of resorcinolic lipids. Table 1 summarizes both the AR content and composition during germination and early seedling growth. The AR content in the coleoptiles of 3-day-old seedlings was 2.03 mg kg^–1 and increased during further seedling growth. Interestingly, AR homologue composition in the developing seedlings was clearly distinct from that observed in the grains. There was a slight, about 3%, decrease in the content of unsaturated AR homologues, but significant alterations were detected in the pattern of the saturated ones. In fact, the ratios of C_19:0/C_21:0 and C_21:0/C_23:0 changed from initial 2.7 and 4.1 to 1.9 and 1.8, respectively, during seedling development. Interestingly, the proportion of saturated AR homologues increased from 82% to about 92% during seedling growth, while that of the monounsaturated counterparts decreased. In addition, the proportions of short (≤C₁₇), long (C₁₉–C₂₁) and very long (≥C₂₃) AR homologues varied dynamically during germination and seedling development. The group of very long side chain homologues was minor in all analyzed samples, but their content significantly increased from 4.4% to 8.9% during seedling development. An accumulation mainly of these homologues in cuticular waxes of the leaves of 3-week-old rye plants was also noted by Ji and Jetter [9].

Table 1

Modification in content and homologue composition of ARs during germination of rye caryopses and early seedling development.

Sample	Content*, mgkg–1 DW	R#:  Homologue composition, %
		C15:0	C17:1	C17:0	C19:1	C19:0	C21:1	C21:0	C23:1	C23:0	C25:1	C25:0
Ungerminated caryopses	581.90±5.32a	3.50	7.40	39.60	7.40	26.30	2.30	9.10	0.60	2.10	0.30	1.40
1-day-old germinating caryopses	505.27±6.24b	3.00	6.61	37.14	7.61	27.63	2.70	10.31	0.40	2.50	0.20	1.90
Coleoptiles of 3-day-old seedlings	2.03±0.03c	7.43	5.45	37.13	6.44	20.79	2.97	10.89	0.00	5.94	0.00	2.97
Coleoptiles of 5-day-old seedlings	6.25±0.09d	9.62	1.76	21.64	3.85	29.33	1.92	24.04	0.64	5.93	0.00	1.28

DW, dry weight. ^*Mean values±SD of alkylresorcinol contents from n=3 experiments. Values followed by different letters (a-d) are significantly different at p=0.05 according to Duncan’s multiple range test. ^#R, C₁₅–C₂₅ saturated or monounsaturated side-chain.

Although the process of AR biosynthesis in plants still remains largely undescribed, some encouraging results have been published. It has been discovered that aliphatic carbon chains of fatty acids serve as precursors in the biosynthesis of ARs [17], which takes place in plastids [8], i.e., the same organelle in which fatty acids are synthesized in plant cells. Furthermore, the production of cuticular wax components also begins in plastids, from where they are exported to the plant cuticle via the endoplasmic reticulum and the plasma membrane [18]. Thus, any disorder or induced alterations in the functionality of these organelles leads to significant modifications of AR content and homologue patterns [19]. Our current study provides novel insight into the AR dynamics during rye caryopsis germination and early seedling growth.