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Article

Sterol Profile in Leaves of Spring Oats (Avena sativa L.) Under Conditions of the Cryolithozone

by
Vasiliy V. Nokhsorov
1,*,
Valentina E. Sofronova
1,
Igor V. Sleptsov
1,
Svetlana V. Senik
2,
Lidia V. Petrova
3 and
Klim A. Petrov
1
1
Institute for Biological Problems of Cryolithozone, Siberian Branch of Russian Academy of Sciences, 41 Lenina Av., 677000 Yakutsk, Russia
2
Komarov Botanical Institute, Russian Academy of Sciences, 2 Professor Popov Str., 197376 St. Petersburg, Russia
3
Yakut Scientific Research Institute of Agriculture, Division of Federal Research Centre “The Yakut Scientific Centre of the Siberian Branch of the Russian Academy of Sciences”, 23/1 Bestuzhev-Marlinskogo Str., 677000 Yakutsk, Russia
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2024, 15(4), 1304-1320; https://doi.org/10.3390/ijpb15040090
Submission received: 13 November 2024 / Revised: 8 December 2024 / Accepted: 10 December 2024 / Published: 12 December 2024
(This article belongs to the Section Plant Physiology)
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Abstract

:
Plant sterols (STs) are essential for the regulation of fluidity and permeability of cell membranes, which have a wide structural diversity. The dynamics of changes in sterol molecular species in leaves of a valuable cereal crop, spring oat (Avena sativa L.), as a function of different sowing dates were studied. In particular, 11 molecular species of sterols (STs) and triterpenoids in A. sativa leaves were identified by GC-MS. Triterpenoids Ψ-taraxasterol, cyclolaudenol, and betulin were identified in A. sativa leaves for the first time, which may be related to adaptation to extreme climatic conditions of the cryolithozone. The dynamics of STs and triterpenoids changes were revealed during growth and development of the standard term and late summer sowing term during A. sativa hardening to low ambient temperatures. The ratio of β-sitosterol to campesterol was found to increase in response to low positive air temperatures, while the ratio of stigmasterol to β-sitosterol remained constant from mid-September to the end of October. Overall, leaves of standard-seeded A. sativa plants maintained higher levels of absolute STs and triterpenoids by 1.9-fold than leaves of late-seeded A. sativa plants. It is suggested that the ability of A. sativa plants to synthesize β-sitosterol and stigmasterol may be part of an evolutionary adaptation process to cope with wide temperature fluctuations and to maintain important membrane-bound metabolic processes.

1. Introduction

The problem of stress tolerance of agricultural crops to hypothermia has been one of the subjects of special attention by researchers in recent years. Spring oats are one of the most important cereal crops and are among the six cereals used for human consumption, livestock feed, forage, hay and silage [1]. In this context, it is important to identify adaptive defense mechanisms of A. sativa plants in order to improve their resistance to stress conditions. Secondary metabolites, in particular unusual fatty acids [2], isoprenoids, phenolics, and other compounds [3], play a special role in plant adaptation to stress conditions. The group of isoprenoids, one of the most diverse and numerous secondary metabolites, includes the triterpenes (C30) and STs (C18 − C29)—structurally diverse molecules that are practically inaccessible to chemical synthesis [4]. STs (isoprenoid derivatives) are structuring components of biological membranes [5], as well as precursors of the phytohormone brassinosteriods, which regulate plant growth and development [6].
The plant species are characterized by complex sterol compositions, in contrast to animals, which are dominated by cholesterol, and fungi, which are dominated by ergosterol [7]. β-sitosterol, stigmasterol (24-ethyl sterols), campesterol (24-methyl sterol) are the most abundant ∆5-sterols in plants, while avenasterol and cholesterol are present in small amounts in most plant species [8]. STs incorporated into membranes have different functions. It has previously been shown that β-sitosterol and campesterol, in contrast to stigmasterol, are the most effective compounds in limiting the mobility of fatty acids in phospholipids. In addition, unlike stigmasterol, β-sitosterol and campesterol reduce membrane permeability [5,9]. It has been suggested that the “stress” phytosterol stigmasterol may influence the distribution of other membrane lipids, metabolic processes in membranes, and signaling pathways that alter the expression of “stress” genes [10,11].
It should be noted that STs, being structural components of cell membranes, can affect the physical state of membranes under stress conditions not only through quantitative changes in the total STs, but also through changes in the ratio of their distinct components [10]. Also, the ratio of molecular species of STs can change during plant growth and development [12,13] and modulate plant signaling and defense responses [14,15].
The relationship between the sterol composition and plant resistance to low temperatures is not completely clear. At the same time, there is an increasing need to study the mechanisms of cold adaptation of plants growing in permafrost ecosystems. In addition to the fundamental interest, the study of the sterol profile of cultivated plants from this region has important applied significance, as sterols are widely used in so-called “functional nutrition” [16]. It is known that phytosterols are environmentally friendly feed additives that stimulate animal growth and positively influence the immune status of their organism [17].
In the natural conditions of Central and Northeast Yakutia, a significant part of wild fodder plants in cereal-sedge phytocenoses of shallow valley lands do not have enough time to complete the growth-development cycle; they are covered by snow while remaining green and form a valuable cryo-fodder for wild animals and livestock (the Yakut horse, reindeer, etc.) in conditions of prolonged and extremely cold winters [18,19]. Taking into account the above, spring oat plants of optimal and late sowing dates are considered the main crop for the production of green matter for silage or cryo-feed for livestock in the permafrost zone [18]. It can be assumed that spring oats can be considered as a source of phytosterols for additives in the fodder of northern animals in the autumn and winter period.
The purpose of this research is to study the structural diversity of STs in spring oat (A. sativa) leaves depending on different sowing dates and to identify the factors that determine the composition of these membrane components and their possible role in the process of adaptation in cold climate of the cryolithozone.

2. Materials and Methods

2.1. Characteristics of the A. sativa Species

Currently, out of 30 varieties of spring oats (A. sativa) included in the State Register of Breeding Achievements in the East Siberian region, 3 varieties are cultivated in Yakutia: Pokrovsky, Pokrovsky 9, and Vilensky. The Vilensky variety A. sativa was selected for the experiments. This variety was created at the Yakutsk Research Institute of Agriculture by hybridization of the local variety Pokrovsky 9 × 2154 (Wodan × Khibiny 2). The authors of the variety are Petrova L.V., Rozhin V.S., Danilova V.P. It is included in the State Register for the East Siberian (XI) region and is distributed in all agricultural zones of the Republic of Sakha (Yakutia). The weight of 1000 grains is 31–38 g. It is medium-early—the vegetation period is 65–70 days. Resistance to lodging is at the level of the standard Pokrovsky variety. The protein content is 10.2–11.9%, the grain weight is 460−580 g/L. It is moderately susceptible to dusty and hard parsha. Under field conditions it was poorly affected by bacterial disease, red-brown spot, and stem rust. The average yield in the East Siberian region was 21.6 centners/hectare. In the Republic of Sakha (Yakutia), the gain to the Pokrovsky standard was 2.5 centners/hectare with a yield of 25.7 centners/hectare. The maximum yield (61.2 centners/hectare) was obtained in 2014 in the Transbaikal Territory.

2.2. Experiments on Cultivation of the First Sowing and Late Summer (Second Term) Crops of A. sativa

Sown oats (Avena sativa L.), the Vilensky cultivar of local selection, were grown on an experimental plot without irrigation, located on the first supra-floodplain terrace of the Lena River (Yakutsk vicinity, 62°15′ N, 129°37′ E). Soils of the site are meadow-chernozem soils formed on light loam. Experiments were conducted in 2022 and 2023. A. sativa were sown in two terms (Table 1). In the first term, plants were sown at the optimal dates for the climatic region, and in the second term, at later dates. The scheme of the experiments was planned in such a way that the plants of late sowing did not have coarsening of shoots until the period of reaching average daily low-positive temperatures from 5 to 0 °C (late September–early October). Young plants of late sowing underwent a period of hardening average daily air temperatures from 10 to 0 °C for at least 4 weeks. The average daily and hourly temperatures and precipitation were monitored according to the data of the Yakutsk Department of Hydrometeorology and Environmental Monitoring of Roshydromet. Young formed green leaves were used as research material.
As a quantitative indicator reflecting the ratio of heat and moisture, G. T. Selyaninov’s HTK (hydrothermal coefficient) was used, which is determined by the formula:
HTK = P/0.1 × T > 10 °C,
where T > 10 °C is the sum of average daily air temperatures for the period with air temperatures above 10 °C; P is the amount of precipitation for the same period [20].
The dry weight of plant material was determined by drying parallel samples (50 mg, 3–4 repetitions) to constant weight in a desiccator (SHS-20-02 SPU, Smolensk, Russia) at 100 °C and the basic error of temperature stabilization ±2 °C.

2.3. Collection and Storage of Samples Prior to Analysis

The plant samples were collected in the Republic of Sakha (Yakitia), Central Yakutia, in the 2022–2023 season. They were collected in three locations, and from four to six samples were collected from each one. After three samples had been combined, they were collected in dependance from each growth period (Tillering, Start of entering the tube, Emergence into the tube, Tubing, Hatching, Milk ripening). The samples of A. sativa leaves were collected in the first half of the day (9:00–11:00). Flag leaves of A. sativa were selected for biochemical analysis. Leaf samples were collected from medium biomass (2–5 g) without root system which was 2 m2 plot in triplicate. They were immediately fixed in liquid nitrogen and transported in Dewar vessels to the laboratory.
Samples of A. sativa leaves were stored in a freezer at −80 °C (Panasonic, Tokyo, Japan) prior to analysis. For biochemical studies, the samples of leaves A. sativa fixed in liquid nitrogen were dried in the lyophilizer (VirTis, New York, NY, USA). The data on air temperature in the habitats of herbaceous plants were taken from the Internet resource (http://www.pogodaiklimat.ru/weather.php?id=24959 (accessed on 2 August 2024)). Weather conditions in the years of the experiment were typical for Central Yakutia (Table 1).

2.4. Extraction of Lipids and Analysis of Sterols and Triterpenoids

For lipid extraction, lyophilically dried A. sativa leaves (30 mg) were extracted three times with a mixture of chloroform and methanol (2:1, by volume). The resulting chloroform extracts were evaporated on a UL-2000 rotary evaporator (Ulab, Shanghai, China). Re-dissolved in pyridine (15 μL), C24 hydrocarbon (Sigma-Aldrich, St. Louis, MO, USA) was added as an internal standard and converted to TMSi derivatives using BSTFA c 1% TMCS. To ensure sufficient completeness of the silylation reaction, the extracts with BSTFA were heated in a desiccator (SHS-20-02 SPU, Smolensk, Russia) at 100 °C for 15 min. The obtained silyl derivatives were analyzed by GC-MS using an Agilent Maestro instrument (Interlab, Moscow, Russia) with an Agilent 5975C mass-selective detector (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was performed on an Agilent HP-5ms (5%-phenyl)-methylpolysiloxane capillary column, 30 m long, 0.25 mm internal diameter and 0.25 μm thickness of the stationary phase film in the mode of linear temperature programming from 70 °C to 325 °C, at a rate of 6 °C/min, carrier gas—helium. The analysis was carried out in the mode of constant helium flow rate through the column (1 mL/min). The temperature of the evaporator was 300 °C, the temperature of the detector was 250 °C, the division ratio at sample input was 1:20. The mass spectrum was scanned from 50 to 1050 a.u.m. at a rate of 2 scans/s. Chromatograms of samples were recorded by total ion current. Data acquisition was performed using Agilent ChemStation E.02.02.1431 software (Agilent Technologies, Santa Clara, CA, USA).
Mass spectrometric data were processed and interpreted using the AMDIS program, NIST 2011 standard library, and the Christie Mass Spectra of Some Miscellaneous Lipids-Archive were used [21]. Retention indices (RI) of TMSi sterol derivatives were calculated using a calibration mixture of n-alkanes (Sigma, Deisenhofen, Germany) based on AMDIS capabilities. The relative retention time of hydrocarbon (C24) RRT (1.000). The quantitative processing was carried out using the specialized computer program UNICHROM (http://www.unichrom.com) (accessed on 12 August 2024).

2.5. Activity of Sterol Methyltransferase and Acyl-Lipid Desaturase Enzymes

The activity of the sterol methyltransferase (SMT) enzymes that catalyze the methylation reaction at the 24th carbon atom (C24) in the side chain was determined as the ratio 24-methyl sterols/24-ethyl sterols [22]. The activity of sterol C-22 desaturase was determined as the ratio of stigmasterol/β-sitosterol.

2.6. Data Analysis

The data were processed by one-factor analysis of variance with a significance level of 0.05 in Microsoft Excel 2010. The figures were plotted using OriginPro 2021 software package (OriginLab, Northampton, MA, USA) and Microsoft Excel 2010. The arithmetic mean values and their standard deviations are presented in the figures. Analysis of STs and triterpenoids was performed on three biological samples (three analytical replicates each).
Statistical processing of STs profiles was performed using the principal component method in the MetaboAnalyst resource (www.metaboanalyst.ca) (accessed on 8 July 2024). The data were normalized against the internal standard (hydrocarbon C24) and autoscaled.

3. Results

3.1. Growth and Development of A. sativa and the Temperature Course During the Period of Experiments

The experiments with A. sativa were carried out during the 2022–2023 season. The growth and development of A. sativa of the first sowing date was observed from 3 June to 26 August 2023. During this period, there was 117 mm of precipitation. Daylight hours decreased from 19.8 to 15.0 h. Mean day and night minimum air temperatures were 19.3 ± 3.4 and 12.3 ± 3.7 °C, respectively, until August 20 (Figure 1). From 21 August to 26 August, a gradual decrease in their mean daily values from 15 to 11 °C was observed with night cooling to 6.4 ± 3.3 °C. The growth processes and stages of plant development during the first sowing period took place at the optimal dates for the region (Figure 2A). Experiments with late sowing date plants were conducted from 27 July to 18 October 2022 (Figure 2B). The sum of liquid precipitation was 107.6 mm. The mean daily air temperature during this period decreased from 23.1 ± 1.8 to −2.6 ± 2.0 °C, and the duration of the photoperiod was reduced from 17.9 to 9.8 h. The beginning of weak frosts was observed from the end of the second decade of September, and a persistent decrease in the mean night temperature below 0 °C occurred after 27 September. Snow cover was established on 10 October, and by 18 October, the average height of the snow layer reached 4–6 cm. A. sativa plants of the late sowing date showed a slowdown in development rates due to an increase in the duration of interphases compared to plants of the first sowing date (Table 1).

3.2. GC-MS Sterols Profile A. sativa Leaves

To study the lipid-dependent mechanisms of cold acclimation in A. sativa cultivar adapted to cryolithozone conditions, plants were harvested and fixed for further extraction at different developmental stages and the air temperature in September and October, listed in Table 1. Late-sown plants that had ceased development were harvested at the onset of low positive temperatures and then when temperatures fell below freezing. As a result of GC-MS analysis, a series of 11 sterol and triterpenoid compounds were identified from A. sativa leaves (Figure 3, Table 2). The mass spectral data are given in Table 3. STs and triterpenoids were isolated and analyzed from total lipid extracts without prior TLC purification or solid phase extraction. Among the identified compounds, the triterpenoid betulin (more than 90% of the total triterpenoids) dominated in A. sativa leaves. The other two triterpenoids, cyclolaudenol and Ψ-taraxasterol, were found in lower amounts, 1.7 and 7%, respectively. These triterpenoids were identified in A. sativa leaves for the first time and their mass spectra are shown in Figure 4. Sterols were represented by Δ0-, Δ5-, and Δ7-components. Major sterols were β-sitosterol (5) (more than 60% of sum) and sitostanol (6) (up to 30%), and stigmasterol (4) (up to 17.1%), whereas cholesterol (1), campesterol (2), campestanol (3), sitostanol (6), Δ5-avenasterol (7), Δ7-avenasterol (8) were minor (less than 9% of the sum of STs).

3.3. Dynamics of Changes in STs in A. sativa Leaves at Different Sowing Dates

All the STs and triterpenoids identified in the leaves of A. sativa plants of the first sowing period had different dynamics depending on the phenological phase and the ambient temperature. In plants of the summer sowing period (3 June), the maximum level of the sum of STs and triterpenoids content was observed in young outgrown leaves (4 July), when A. sativa plants were in the early tubule emergence phase, compared to other vegetation phases. During the summer period (mid-July and early August), when hot and dry weather prevailed and night and mean day air temperatures reached 14.7 ± 2.9 °C and 20.5 ± 6.2 °C, respectively, the levels of STs and triterpenoids were at the same level (about 11–12 mg/g DW). At the end of August, when A. sativa plants were in the milk ripeness phase, there was a 1.5-fold decrease in the level of STs content compared to June and July. The dynamics of individual triterpenoid and sterol components in the leaves of A. sativa plants of the first sowing is visualized in the form of a heat map (Figure 5). Hierarchical clustering allowed us to identify groups of components with similar dynamics during A. sativa development. The primary split in the hierarchical dendrogram (Figure 5) shows a group consisting of two compounds whose levels increase during the development of A. sativa–β-sitosterol and campesterol. Other components (all triterpenoids and three sterols–sitostanol, Δ7-avenasterol, Δ5-avenasterol) showed the opposite dynamics. A high level of the “stress” sterol stigmasterol was observed in young leaves when A. sativa plants were in the tube emergence phase, then the level of its content decreased 1.3 times in August (milk ripeness phase) compared to July (tube emergence phase). A similar dynamic was observed for campesterol and cholesterol.
In cryolithozone conditions, late summer sowing is carried out in the middle of the growing season (mid-July). The second sowing of A. sativa was carried out on 27 July, so that the plants would be preserved in a green state in the autumn under snow. In the leaves of the A. sativa of late summer sowing, the total content of STs and triterpenoids did not exceed 8 mg/g DW, which was about 1.5–2.0 times lower than in the leaves of the early sowing. As in A. sativa leaves of the I sowing, the three identified triterpenoids, together with sitostanol, changed synchronously during the development of A. sativa plants; their accumulation was observed in the trumpeting phase at the onset of low positive quenching air temperatures (1.3 ± 1.7 °C) from mid to late September (Figure 6). In October, a 1.5-fold decrease in Ψ-taraxasterol, betulin, and cyclolaudenol occurred under the influence of near-zero average daily and negative night air temperatures from −1 to −5 °C and a reduction in photoperiod (up to 9.8 h). The dynamics of the changes in sitostanol were similar for triterpenoids, with the content of this compound increasing in response to low positive temperatures and decreasing in October, when stable snow cover and negative ambient temperatures are established. Among the STs in the leaves of late summer A. sativa at the tillering stage, β-sitosterol dominated, its content decreased in late September and in October against the background of low air temperatures. The minor sterols Δ5-avenasterol and campesterol had similar dynamics. The “stress” sterol stigmasterol increased in early September. All other STs were present at low levels in A. sativa leaves.

3.4. Dynamics of Changes in STs Ratios and Activity of Some Enzymes in A. sativa Leaves at Different Sowing Dates

The sterol profile in leaves of different sowing dates of A. sativa was used to indirectly estimate the dynamics of activity of some enzymes involved in the biosynthesis of STs (Figure 7a,b). The ratio of β-sitosterol to campesterol decreased in the leaves of early sown A. sativa plants during growth and development. In the leaves of late sowing A. sativa plants (II sowing term), a different dynamic of changes in these parameters was observed. The ratio of β-sitosterol to campesterol was found to increase in response to chilling and freezing air temperatures. Thus, the activity of SMT in late-summer A. sativa leaves increases in response to low air temperatures and reduced photoperiod (Figure 7b). From mid-September to October the ratio of stigmasterol to β-sitosterol increases due to an increase in stigmasterol level. The level of sterol C-22 desaturase activity also increases compared to early September (Figure 7a).

3.5. PCA of Leaves of I and II Sowing Dates of A. sativa

For statistical analysis of the obtained data, a matrix including 27 observations for 11 identified compounds in leaves of summer and late summer A. sativa crops was created (Figure 8). The resulting data set was processed by Principal Component Analysis (PCA) with auto scaling performed with the publicly available resource MetaboAnalyst (www.metaboanalyst.ca). The variances for components 1 and 2 was 51.3% and 23.9%, respectively. The graph of estimations of PCA shows that biological replicates cluster well with each other, and the plants of I sowing form a trend coinciding with the development of plants under air temperature decreasing and photoperiod shortening, whereas samples of plants of II sowing do not form such an obvious trend. Apparently, this reflects the disturbance of normal development of late-sown plants under conditions of premature cold onset.

4. Discussion

A. sativa belongs to the group of long-day plants and early spring crops with increased sensitivity to moisture deficiency. The growth processes and developmental stages of A. sativa plants during the first sowing period took place at the optimal time for the region due to the long daylight hours in the summer months, sufficient moisture availability (SST = 0.75 units), and optimal average daily air temperatures (18.8 ± 3.7 °C) [23,24]. In the case of the late sowing, after having passed the first developmental stages (tillering) with relatively optimal average daily temperatures and sufficient moisture availability (hydrothermal coefficient = 0.69 units) until the end of August, the plants experienced during the first three weeks of September the complex effect of a rapid decrease in average daily temperatures from 8.5 ± 2.0 °C to about 0 °C—a shortening of the photoperiod, and the first autumn frosts. The combination of the above stress factors resulted in a strong slowdown and eventually to a complete termination of A. sativa development in the tubing phase. It should be noted that the biological minimum for the formation of A. sativa generative organs is 10–12 °C. Despite the fact that spring oat practically stops growth processes at temperatures below 5 °C, they survive well at low positive temperatures [25]. In our study, night frosts of −5, −7 °C lasting three to five hours during 4 days in the middle of the first week of October resulted in plant death. The study of Rizza et al. [26] on different A. sativa cultivars showed that most cultivars do not survive at subzero temperatures. During the evolutionary process, spring cereals have not developed a genetically determined mechanism for resistance to subzero temperatures by extracellular ice formation.
It is worth noting that in the leaves of A. sativa of late summer sowing, the total STs content was about two times lower than in the leaves of early sowing. Studies on rice have shown that the concentration of membrane sterols increases during cold acclimation, and this effect is more pronounced in resistant rye cultivars [27].
The molecular diversity of STs in plants is associated with an attached lifestyle and is directed toward an optimal membrane structure [28]. In general, about five major molecular species of STs have been found in cell membranes of various plants. STs in plant cells are considered to be localized predominantly in the plasma membrane. STs are also present in small amounts in the endoplasmic reticulum [9], tonoplast [29] and mitochondrial membranes (up to 12%) [30]. A relatively small amount of STs is known to be present in chloroplast membranes [9]. A. sativa, in addition to high summer and low negative autumn air temperatures, is exposed to moisture deficit in summer and reduced photoperiod in autumn; all these limiting factors characterize the climatic conditions of permafrost ecosystems where this valuable cereal species grows [31]. Perhaps, the presence of 11 molecular species in the profile of A. sativa STs indicates the ability of this cereal to adapt to a wide range of abiotic factors in the cryolithozone of Yakutia. The mechanisms of adaptation of A. sativa to cold were previously studied using metabolomics methods. Acclimation to suboptimal temperatures in autumn has been shown to result in biochemical (accumulation of mono- and disaccharides and decrease in fatty acids and polyols), physiological and biophysical changes (decrease in leaf photochemical reflectance index and chlorophyll a fluorescence) [31,32]. The results obtained in the present study indicate that leaves of A. sativa of the standard sowing date maintained higher absolute concentrations of STs and triterpenes by 1.9 times than those of the late sowing date A. sativa. This seems to be related to the decrease in the functional activity of the photosynthetic apparatus in the autumn period [31], and consequently the decrease in the biosynthesis of lipid components of intracellular structures. It may also be associated with structural and functional changes in the mesophyll cells of leaves, including membrane remodeling.
According to our data, A. sativa synthesize Δ5-, Δ7- and Δ0-STs in leaves. The dominant molecular species of sterols were Δ5 sterols (β-sitosterol and stigmasterol) and Δ0 sterol (sitostanol). Previously, A. sativa leaves were shown to contain other sterols among the free sterols: Δ7-cholestenol, lophenol, and Δ7-stigmastenol, with β-sitosterol being the dominant sterol (more than 37%) [33]. Differences in sterol composition may be explained by random varietal characteristics of A. sativa or may be related to the cold acclimation of the cultivar. Under controlled conditions of short-term (1 h) cold stress (+4 °C), the total content of STs in the roots of wheat seedlings increased significantly compared to the control [34]. In the experiments with the late-summer A. sativa in natural conditions of the cryolithozone, +4 °C came in mid-September and also led to a significant increase in STs content in leaves by 1.5 times compared to early September. It should be noted that lower negative air temperatures in October (below 0 °C) lead to a decrease in the number of STs in A. sativa leaves, apparently this is due to damage in the membrane system of the mesophyll cells, leading to the death of the plants, which we observed on 18 October.
It is known that the functional state of the membrane depends on the balanced levels of sterols, mainly campesterol, β-sitosterol, and stigmasterol due to their ability to modulate membrane order [35,36]. Late spring oat (II sowing date) experienced wide fluctuations in ambient temperatures in September and October. At this time, the ratio of β-sitosterol to campesterol was found to increase in response to low positive air temperatures (Figure 7b). This is reasonable because campesterol is the best rigidifier of phospholipid bilayers compared to β-sitosterol and stigmasterol due to the least bulky side chain, and adaptation to low temperatures requires increased membrane fluidity.
It is known that the ratio of ST components, in particular stigmasterol to β-sitosterol, may influence the physicochemical properties of ordered microdomains, referred to as “lipid rafts” enriched with glycoceramides and STs [29,37]. Changes in lipid rafts can modulate plant signaling and defense responses. During the development and adaptation to the cold of plants sown in early summer, the ratio of stigmasterol to β-sitosterol gradually decreases during the period of vegetation. On the contrary, in plants sown in late summer, this parameter increases sharply in August and does not change from September until the snow falls. Both stigmasterol and β-sitosterol have 24-ethyl groups on the side chain but differ in the double bond at the C22 position initiated by the sterol C-22 desaturase. The trans-22 double bond reduces the ability of stigmasterol to order fluid phospholipid bilayers, leading to bilayer thinning and looser packing of lipids [38,39]. The conversion of β-sitosterol to stigmasterol has been shown to influence the response of plants to various biotic and abiotic stresses, including low and high temperatures [10,40], resistance to bacteria [41,42], nematode attack [43], and mechanical wounding [44], for which stigmasterol has been termed a “stress sterol” [11]. In contrast to animals, the unique ability of plants to synthesize 24-ethylsterols (β-sitosterol and stigmasterol) may be part of an evolutionary adaptation process to cope with wide temperature fluctuations and maintain important membrane-bound metabolic processes [45].
In addition to being involved in membrane adaptation to changes in environmental temperature, plant sterols can influence various developmental processes such as seed germination, plant phenotype, senescence, flowering time, and seed yield [36]. In particular, campesterol can influence plant growth and development as a precursor for brassinosteroids [46]. Artificial disruption of campesterol biosynthesis due to overexpression of the SMT2 gene leads to impaired development of Arabidopsis plants [47]. In the present study, the relationship between campesterol and development is observed by comparing the dynamics of sterol composition in plants of different sowing dates. Campesterol increased at the stage of emergence into the tube and accumulated at the late stage of development of A. sativa plants when sown in early summer. However, when the sowing date is late, cold disrupts the normal development of plants, and they do not have time to pass all stages of vegetation, and campesterol accumulation in the process of development is not registered in these plants. Similarly, stigmasterol dynamics were different in early and late sown plants. Stigmasterol plays a minor role in development compared with stress tolerance, but fluctuations in stigmasterol and C22-desaturase gene expression during developmental processes in different plants suggest that stigmasterol is involved in plant development [48].
A remarkable feature of A. sativa plants in the phase of emergence into the tube was an increase in the proportion of sitostanol. In plants of early sowing, its amount reached the level of β-sitosterol. Sitostanol, or other stanols (sterols with a saturated steroid nucleus), have not been previously observed in the composition of oats sterols [39,49] and have not been known as a developmental regulator in other plant species. Stanols have been found in some taxonomic groups of salt-tolerant plants. Their ability to synthesize stanols is thought to be an adaptive mechanism to cope with salinity [8]. The finding of the correlation of sitostanol levels with early development of A. sativa plants requires further study.
Studies on different cereal species have shown that late sowing of wheat increases lodging resistance by improving the biosynthesis and accumulation of lignin and cellulose, under Central European (Poland) conditions [50]. Late sowing of spring oats (1 September) produced the best forage value but the lowest dry matter yield for the three different cultivars studied [51]. The A. sativa cultivar used in our experiments was Vilensky, which was developed by hybridization of the locally released cultivar Pokrovsky 9 with ×2154 (Wodan × Khibiny 2). According to the data obtained, this A. sativa cultivar synthesizes triterpenes–Ψ-taraxasterol, betulin, and cyclolaudenol in leaves. Ψ-taraxasterol is a natural pentacyclic triterpene mainly extracted from dandelion (Taraxacum officinale F.H. Wigg.) and has anti-inflammatory, antioxidant, and anticarcinogenic activities [52]. Taraxasterol has demonstrated significant preventive and therapeutic effects in animal or cellular models of several diseases, including hepatic damage, gastritis, arthritis, lung inflammation, cancer, and immune system disorders [53,54]. Cyclolaudenol has previously been isolated from several plant species, including Coniogramme Japonica (Thunb.) [55], but the physiological role of this plant metabolite remains unclear. The significant amount of betulin in A. sativa leaves may be a consequence of adaptation to the low-temperature stress of cryolithozone. Betulin was first discovered and isolated from birch bark by T.E. Lovitz in 1788; this substance has a number of useful properties, including antioxidant activity [56]. β-sitosterol is also known to have high antioxidant activity. It has previously been shown that Arabidopsis mutants with high levels of β-sitosterol have greater resistance to oxidative stress compared to the wild type [57]. Experiments have demonstrated that a decrease in betulin and the sum of STs occurs in response to a temperature decline at the end of October. The decrease in STs leads to the accumulation of oxidative forms of lipids, which disrupts the integrity and functioning of cell membranes, which in turn can lead to a reduction in plant cell resistance.

5. Conclusions

Eight molecular species of STs and three molecular species of triterpenoids: Ψ-taraxasterol, cyclolaudenol, and betulin were identified by GC-MS in oat (A. sativa) leaves for the first time. The betulin, which is more than 90% of the total triterpenoids, dominated in oat leaves.
The group consisting of two compounds, the level of which increases during the development of first-sown oat plants, β-sitosterol and campesterol, was revealed. The other components (all triterpenoids and three sterols-sitostanol, Δ7-avenasterol, Δ5-avenasterol) showed the opposite dynamics.
In the leaves of the late date sowing, the total STs and triterpenoids content was about 1.5–2.0 times lower than in the leaves of the first sowing. Among STs, β-sitosterol was dominant throughout the entire autumn period and, unlike plants of the first sowing, did not depend on phenological phases of development. The relative content of the “stress” phytosterol stigmasterol in the leaves of late-sown oats increased by 1.6 times compared to early September, when night temperatures in the second half of September dropped from 4.6 ± 2.7 to near-zero temperatures.
The relative contents of three molecular species of triterpenoids in oat plants grown in the fall at chilling temperatures did not differ from those in plants grown in the summer. Their concentrations were practically constant in all experiments and did not depend either on the ambient temperature or on the phenological phases of oat plant development.
The results of the complex analysis of the composition and content of STs and triterpenes of oat plants of optimal and late summer sowing dates indicate the involvement of these metabolites in response to environmental changes (high and autumn low positive and negative temperatures, reduction in photoperiod).

Author Contributions

Conceptualization, V.V.N. and V.E.S.; methodology, V.V.N., I.V.S., S.V.S. and L.V.P.; software, V.V.N. and I.V.S.; validation, V.V.N., I.V.S. and S.V.S.; formal analysis, V.V.N.; investigation, V.V.N., V.E.S. and S.V.S.; resources, V.V.N., I.V.S. and L.V.P.; data curation, V.V.N. and S.V.S.; writing—original draft preparation, V.V.N.; writing—review and editing, V.E.S., I.V.S. and S.V.S.; visualization, V.V.N. and I.V.S.; supervision, V.E.S. and S.V.S.; project administration, V.V.N.; funding acquisition, V.V.N., V.E.S., L.V.P. and K.A.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the State Assignment of the Ministry of Science and Higher Education of the Russian Federation (project No. AAAA-A21-121012190034-2) and FWRS-2022-0006.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data used to support the findings of this study can be made available by the corresponding author upon request.

Acknowledgments

We are grateful to Svetlana Petrova for the linguistic corrections and improvements.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. The air temperature course during the period of experiments.
Figure 1. The air temperature course during the period of experiments.
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Figure 2. A. sativa cultivar Vilensky, (A) standard sowing date (photo taken on 5 July) and (B) late sowing date (photo taken on 18 October).
Figure 2. A. sativa cultivar Vilensky, (A) standard sowing date (photo taken on 5 July) and (B) late sowing date (photo taken on 18 October).
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Figure 3. Representative GC-MS chromatogram of silylated free sterols and triterpens from A. sativa (4 July, leaves of the first sowing). Peaks: cholesterol (1), campesterol (2), campestanol (3), stigmasterol (4), β-sitosterol (5), sitostanol (6), Δ5-avenasterol (7), Δ7-avenasterol (8), Ψ-taraxasterol (9), cyclolaudenol (10), betulin (11).
Figure 3. Representative GC-MS chromatogram of silylated free sterols and triterpens from A. sativa (4 July, leaves of the first sowing). Peaks: cholesterol (1), campesterol (2), campestanol (3), stigmasterol (4), β-sitosterol (5), sitostanol (6), Δ5-avenasterol (7), Δ7-avenasterol (8), Ψ-taraxasterol (9), cyclolaudenol (10), betulin (11).
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Figure 4. Mass spectra and structural formulas of the first identified triterpenoid compounds (betulin-TMS ether (a), cyclolaudenol-TMS ether (b), Ψ-taraxasterol-TMS ether (c)) in spring oat (A. sativa) leaves.
Figure 4. Mass spectra and structural formulas of the first identified triterpenoid compounds (betulin-TMS ether (a), cyclolaudenol-TMS ether (b), Ψ-taraxasterol-TMS ether (c)) in spring oat (A. sativa) leaves.
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Figure 5. Hierarchical clustering heatmap of STs and triterpenoids in the leaves of A. sativa (I sowing date, 3 June) performed on the normalized data (using the Euclidean distance matrix with Ward’s method). Colors represent different concentrations indicated by the color bar.
Figure 5. Hierarchical clustering heatmap of STs and triterpenoids in the leaves of A. sativa (I sowing date, 3 June) performed on the normalized data (using the Euclidean distance matrix with Ward’s method). Colors represent different concentrations indicated by the color bar.
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Figure 6. Hierarchical clustering heatmap of STs and triterpenoids in leaves of A. sativa (II sowing date, 27 July) performed on the normalized data (using the Euclidean distance matrix with Ward’s method). Colors represent different concentrations indicated by the color bar.
Figure 6. Hierarchical clustering heatmap of STs and triterpenoids in leaves of A. sativa (II sowing date, 27 July) performed on the normalized data (using the Euclidean distance matrix with Ward’s method). Colors represent different concentrations indicated by the color bar.
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Figure 7. Dynamics of sterol C22-desaturase enzymes (a) and sterol molecular species ratio and activity of sterol methyltransferase (SMT) (b) in leaves of A. sativa. Means ± SDs. The experiments were performed with at least three biological replicates (n = 3); the different letters indicate significant differences (p ≤ 0.05).
Figure 7. Dynamics of sterol C22-desaturase enzymes (a) and sterol molecular species ratio and activity of sterol methyltransferase (SMT) (b) in leaves of A. sativa. Means ± SDs. The experiments were performed with at least three biological replicates (n = 3); the different letters indicate significant differences (p ≤ 0.05).
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Figure 8. The graph of estimations of PCA obtained on the basis of the analysis of the main components of the sterol profile of A. sativa leaves of I (blue arrow) and II crops (green arrow) in different phenological phases.
Figure 8. The graph of estimations of PCA obtained on the basis of the analysis of the main components of the sterol profile of A. sativa leaves of I (blue arrow) and II crops (green arrow) in different phenological phases.
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Table 1. Phenological phases of spring oat (A. sativa) development at different sowing dates (2022–2023 season).
Table 1. Phenological phases of spring oat (A. sativa) development at different sowing dates (2022–2023 season).
Timeframe for Collecting SamplesPhases of DevelopmentAir Temperature (°C) *Amount of Precipitation (mm) **Photoperiod (h)
T AverageT Night
I sowing date (3 June), summer period
4 JulyStart of entering the tube25.4 ± 4.320.1 ± 1.913.019.5
14 JulyEmergence into the tube14.7 ± 2.911.5 ± 0.97.618.9
5 August Hatching20.5 ± 6.214.2 ± 3.132.017.1
26 AugustMilk ripening11.2 ± 6.45.6 ± 3.226.615.0
II sowing date (27 July), autumn period
3 SeptemberTillering6.9 ± 1.46.1 ± 0.915.514.2
16 SeptemberStart of entering the tube 6.8 ± 4.14.6 ± 2.7 23.213.0
27 SeptemberEmergence into the tube1.3 ± 1.70.4 ± 1.219.411.9
10 OctoberEmergence into the tube−0.3 ± 1.1−1.1 ± 0.510.910.6
18 OctoberTubing−2.2 ± 2.3−2.6 ± 2.060 ***9.8
Note. *—24 h before taking samples; **—15 days before taking samples; data presented by the Yakutsk Department of Hydrometeorology and Environmental Monitoring of Roshydromet (http://www.pogodaiklimat.ru/weather.php?id=24959 (accessed on 17 September 2024)). ***—snow cover height. Snow cover was established on 10 October.
Table 2. Content of sterols and triterpenes in A. sativa leaves, % of the sum.
Table 2. Content of sterols and triterpenes in A. sativa leaves, % of the sum.
Sowing DateSterols,
% of Sum		Triterpenoids,
% of Sum
CholesterolCampesterolCampestanolStigmasterolβ-SitosterolSitostanolΔ5-AvenasterolΔ7-AvenasterolΨ-TaraxasterolCyclolaudenolBetulin
I (4 July)8.84.31.617.134.429.71.82.47.31.791.0
II (3 September)4.213.31.16.663.99.51.30.17.11.391.6
Table 3. Mass spectral data for trimethylsilylsterols and triterpenoid esters in spring oat (A. sativa) leaves.
Table 3. Mass spectral data for trimethylsilylsterols and triterpenoid esters in spring oat (A. sativa) leaves.
No.CompoundsRT, minRRTRIMain m/z Ratio
1Cholesterol38.8061.30203173458, 443, 368, 353, 329, 255, 213, 329, 129
2Campesterol39.9001.33873227472, 382, 367,343, 255, 213, 129
3Campestanol40.0021.34213282474, 384, 369, 305, 257, 215, 75
4Stigmasterol40.1981.34873301484, 469, 394, 379, 355, 351, 255, 213, 129, 83
5β-sitosterol40.7951.36873353486, 396, 381, 357, 303, 255, 213, 129
6Sitostanol40.9061.37243363488, 473, 431, 398, 383, 305, 257, 215, 75
7Δ5-avenasterol40.961 1.37433368484, 469, 394, 386, 371, 355, 343, 296, 281, 257, 255, 253, 213, 211, 129
8Δ7-avenasterol41.2031.38243420484, 469, 386, 371, 343, 281, 255, 253, 213
9Ψ-taraxasterol41.3371.38693398498, 483, 369, 297, 207, 189, 109, 72
10Cyclolaudenol41.9341.40693445512, 440, 422, 407, 379, 353, 300, 203, 175, 161, 147, 135, 121, 107, 95, 81, 73, 55
11Betulin42.8741.43843520586, 496, 483, 442, 393, 293, 279, 203, 189, 135, 109, 95, 72
Notes. The fragment corresponding to the molecular mass (M + TMS) is marked in bold. RRT is relative retention time based on C24 = 1.000. RI is a relative index based on interpolation of retention times of C8–C40 Alkanes Calibration Standard.
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Nokhsorov, V.V.; Sofronova, V.E.; Sleptsov, I.V.; Senik, S.V.; Petrova, L.V.; Petrov, K.A. Sterol Profile in Leaves of Spring Oats (Avena sativa L.) Under Conditions of the Cryolithozone. Int. J. Plant Biol. 2024, 15, 1304-1320. https://doi.org/10.3390/ijpb15040090

AMA Style

Nokhsorov VV, Sofronova VE, Sleptsov IV, Senik SV, Petrova LV, Petrov KA. Sterol Profile in Leaves of Spring Oats (Avena sativa L.) Under Conditions of the Cryolithozone. International Journal of Plant Biology. 2024; 15(4):1304-1320. https://doi.org/10.3390/ijpb15040090

Chicago/Turabian Style

Nokhsorov, Vasiliy V., Valentina E. Sofronova, Igor V. Sleptsov, Svetlana V. Senik, Lidia V. Petrova, and Klim A. Petrov. 2024. "Sterol Profile in Leaves of Spring Oats (Avena sativa L.) Under Conditions of the Cryolithozone" International Journal of Plant Biology 15, no. 4: 1304-1320. https://doi.org/10.3390/ijpb15040090

APA Style

Nokhsorov, V. V., Sofronova, V. E., Sleptsov, I. V., Senik, S. V., Petrova, L. V., & Petrov, K. A. (2024). Sterol Profile in Leaves of Spring Oats (Avena sativa L.) Under Conditions of the Cryolithozone. International Journal of Plant Biology, 15(4), 1304-1320. https://doi.org/10.3390/ijpb15040090

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