Chemical Composition of Volatile and Extractive Organic Compounds in the Inflorescence Litter of Five Species of Woody Plants

The decomposition of plant litter, most of which is found in forests, is an important element of the global carbon cycle, as a result of which carbon enters the atmosphere in the form of not only CO2 but also volatile organic compounds (VOCs). Although the formation of litter is associated with autumn cooling, in the spring, there is a very intense fall of faded inflorescences of woody plants. This study examined the chemical composition of the litter and VOCs emitted from decaying inflorescences of four species of forest-forming trees: silver birch, European hornbeam, black alder and aspen. All litter emissions consisted of 291 VOCs, mainly terpenes actively participating in atmospheric processes. The detection of a number of typical mushroom metabolites, such as 1-octen-3-ol, known as “mushroom alcohol”, and alkyl sulphides, suggests that inflorescence-derived VOCs are a mixture of components of plant and microbial origin. In methanol extracts of the fallen inflorescences of all types, 263 organic compounds were identified, the majority of which were related to carbohydrates. Their share in the extracts was 72–76%. In general, the composition of the extractive compounds indicates the easy availability of this material for assimilation by various types of destructors.


Introduction
The formation and further decomposition of plant litter encompass the main element of the natural cycle of carbon and nutrients in the biosphere, including in forest ecosystems.In the process of litter decomposition, controlled by a number of insufficiently studied physical and biological factors, various volatile organic compounds (VOCs) enter the atmosphere [1].As they are photochemically active, they have a significant effect on chemical processes in the atmospheric boundary layer.Although it has been established that the main biogenic source of VOCs is the living vegetation on the continents [2][3][4][5], in recent decades, increasing attention has been paid to other previously unexplored sources, including plant litter [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22].The motivation to study the VOCs produced in leaf litter was, on the one hand, the recognition of their important role as key participants in atmospheric chemical processes and, on the other hand, the high degree of uncertainty in existing estimates of biogenic emissions [23,24].This uncertainty may be explained by the presence of additional previously unaccounted for sources of biogenic VOCs.The determination of the total reactivity of the hydroxyl radical in boreal forests clearly indicates the existence of a previously unaccounted for source of VOCs active in photochemical processes [25].This makes it necessary to characterise all natural sources of reactive VOCs [26].
The ecological significance of studying forest plant litter (chemical composition and the dynamics of its changes during the decomposition process) is also associated with the fact that it serves as a habitat and a food resource for a variety of organisms, including many species of saprotrophic invertebrates.These organisms play an active role in the decomposition of litter [27][28][29][30][31], and their activity is also accompanied by the emission of VOCs contained in dead plant material, as well as the release of their own volatile components into the gas phase [1].
In temperate and boreal climate zones, the formation of litter is associated with the fall of leaves in autumn during the onset of cold weather.Therefore, the exchange of trace gases between the forest floor and the atmosphere during the summer-autumn transition period has received much attention [32][33][34][35][36].However, in forest ecosystems, plant litter is not only made up of dead leaves, and its formation is not limited to the autumn period.In the spring and in the case of some tree species, even before foliage blooms, flowering begins, during and at the end of which part of the inflorescences dies and falls to the ground.The composition of VOCs released by the living flowers of woody plants has received much attention not only because of their involvement in atmospheric processes but also because of their role in interspecies communication.The smell of a flower is specific to each plant species and can be used by pollinators as a signal to locate and recognise the source of nectar or pollen.A recent review [37] quantitatively summarised data on the relative composition and release rates of VOCs in the floral fragrances of 305 plant species from 66 families.These data indicate that the main components of flower-derived VOCs are terpene compounds and benzenoids, which play an important role in atmospheric chemistry.However, until now, there is yet to be a discussion on the chemical composition of faded inflorescences and their role in the processes occurring under the forest canopy.
We hypothesise that the release of volatiles from spent inflorescences continues even after they have fallen, and, thus, this process serves as a source of highly reactive VOCs during the spring-summer transition period.In addition, it can be assumed that the chemical composition of fallen inflorescences makes them an attractive food resource for saprotrophic organisms.In accordance with this, the purpose of this work was to study the chemical composition of the volatile emissions of the fallen inflorescences of forest-forming woody plants in the mid-latitude and boreal zones of the northern hemisphere and to determine the composition of the extractive compounds contained in them.

Plant Material and Techniques for Field Experiments
This study examined the composition of organic compounds released into the gas phase from the inflorescence litter of five species of deciduous trees: silver birch (Betula pendula), European hornbeam (Carpinus betulus), Norway maple (Acer platanoides), aspen (Populus tremula) and crack willow (Salix fragilis).All these types of woody plants are characteristic of forests not only in Poland but also in the entire temperate and boreal zone of the European continent.Freshly fallen inflorescences of all plant species were handcollected from the Las Zwierzyniecki Nature Reserve near Bialystok, Poland, in early May 2022.The reserve's vegetation consists of a stand of trees in which the dominant species is hornbeam, with admixtures of English oak, ash, aspen, maple, silver birch, black alder and various species of willow (Salix spp.).In the groundcover layer, we could distinguish the following plants: yellow wood warbler, yellow wood anemone, wood anemone, greater chickweed and common ground elder.Some of the collected inflorescences were used in field experiments to study the composition of VOCs during decomposition under natural conditions.For incubation, litter bags (200 × 200 × 20 mm) with a terylene mesh bottom and a mesh size of 1.5 mm were used; each bag contained approximately 10 g of inflorescences.The litter bags were laid out on the natural layer of the previous year's leaf litter under the trees whose flower litter was studied.The bags were covered on top with a terylene mesh (mesh size 5 mm) to prevent wind drift and foreign materials from having an effect.The duration of this stage of the experiment was 14 days.

VOC Determination
The determination of the composition of the volatile components of the freshly collected inflorescence litter and that incubated for two weeks was carried out according to a previously described method using solid-phase microextraction combined with gas chromatography with mass spectral detection (HS-SPME/GC-MS) [38,39].According to the results of the experiments described in the cited papers, which were carried out to determine the composition of VOCs of various natural materials from the available range of sorption fibres, the choice was made to use DVB/CAR/PDMS fibre (Sigma-Aldrich, Poznan, Poland).
The inflorescence litter delivered to the laboratory was placed in a glass container with a volume of 0.25 L, and this was sealed using a lid with a port to introduce the sorption fibre through a silicone membrane.After standing for 0.5 h at room temperature, fibre preconditioned according to the producer's recommendations was introduced into the gas phase above the leaves.After exposing the fibre for 2 h (every 15-20 min, the contents of the container were shaken), it was injected for 15 min into the injection port of a gas chromatograph HP7890A with a 5975C VL MSD Triple-Axis Detector (Agilent Technologies, Santa Clara, CA, USA).The apparatus was fitted with an HP-5ms capillary column (30 m × 0.25 mm i. d., 0.25 µm film thickness), with electronic pressure control and a split/splitless injector.The latter was operated at 220 • C in splitless mode.The helium flow rate through the column was 1 mL min −1 in constant-flow mode.The initial column temperature was 40 • C, and it rose to 220 • C at a rate of 3 • C min −1 .After integration, the fraction of each component in the total ion current (TIC) was calculated.The precision of the method was studied by three replicate extractions and analyses.The peak areas of the extract components obtained by replicate analyses were used for the calculation of their relative standard deviation (RSD) values.On average, the RSD amounted to 2% for the main peaks (more than 10% of the TIC), 6% for medium peaks (more than 1% of the TIC) and 18% for peaks that amounted to ≤0.5% of the TIC.
To calculate the linear temperature-programmed retention indices (RIs) of the analytes, the SPME fibre was inserted for 2-3 s into the headspace of the vial with a mixture of C 6 -C 18 n-alkanes.Their separation was performed under the above conditions.The RI values of the separated components were calculated according to the following equation: where t x is the retention time of the analyte, t n is the retention time of the n-alkane eluting directly before the analyte, and t n+1 is the retention time of the n-alkane eluting directly after the analyte.

Determination of the Composition of Extractive Substances
A total of 5 g of freshly harvested inflorescences was ground to a size of about 0.5 mm, placed in a conical flask, filled with 25 mL of methanol and placed on a magnetic stirrer.After extraction at room temperature with stirring for half an hour, the solvent was separated by filtration through a paper filter.Extraction with fresh portions of methanol was repeated twice.The combined extracts were dried by adding MgSO 4 and then evaporated to dryness on a rotary evaporator.Of the resulting material, 5-10 mg was transferred to a 2 mL vial and dissolved in 220 µL dry pyridine, and 80 µL BSTFA/TMCS was added.The reaction mixture was heated for 0.5 h at a temperature of 60 • C to obtain trimethylsilyl (TMS) derivatives.
The GC separation of TMS derivatives and their identification were carried out on the GC-MS apparatus mentioned in Section 2.4, equipped with the same HP-5ms capillary column.The helium flow rate through the column was 1 mL min −1 .One microliter of the sample was injected with the aid of an HP 7673 autosampler.The injector heated to 280 • C worked in split mode; the split ratio was 10:1.The initial column temperature was 50 • C, rising to 325 • C at 3 • C min −1 .Electron ionisation mass spectra were obtained at 70 eV of ionisation energy.Detection was performed in full-scan mode from 41 to 600 a. m. u.After integration, the fraction of each component in the total ion current (TIC) was calculated.
The separation of C 8 -C 40 n-alkanes in n-hexane was carried out under the above conditions, and the recorded retention times were used to calculate the linear temperatureprogrammed retention indices (RIs) of the separated compounds.

Component Identification
The separated components of the volatile emissions and the TMS derivatives of the compounds extracted from the fallen inflorescences were identified by their mass spectra using an automatic GC/MS processing system equipped with an National Institute of Standards and Technology NIST 14 electron ionisation mass spectra library.The calculated values of the retention indices were used as an independent analytical parameter.The mass spectrometric identification was considered reliable if its results were confirmed by the calculated RI values, that is, if their deviation did not exceed ±10 units from the standard RI values published in accessible databases [40][41][42].If the results of the mass spectrometric identification were not confirmed by the RI values due to their absence in the available databases, or if the discrepancy exceeded 10 u.i., the identification was considered tentative.

VOC Composition
The composition of volatile emissions was determined for freshly fallen inflorescences and those exposed to natural conditions for two weeks.The obtained chromatograms of the VOCs in both sets of experiments contained peaks of 291 compounds, whose individual contribution to the total ion current of the chromatograms was not lower than 0.01%.Of these, 246 compounds (84% TIC) were identified by their mass spectra and calculated retention indices; most of the unidentified peaks belonged to minor (0.01-0.05%TIC) VOC components.The chromatograms of the litter of the different species contained 51 to 119 peaks of organic C 1 -C 20 compounds of various classes.The largest number of peaks was recorded in the chromatograms of hornbeam (C.betulus) inflorescences and the smallest in the chromatograms of silver birch (B.pendula) inflorescences.
The components identified during the GC-MS analysis were divided into 12 groups, as shown in Table 1, together with the main representatives of each group (only compounds are given whose share in the TIC of at least one of the chromatograms was not less than 0.5%).The complete composition of the volatiles is shown in Table S1 in the Supplementary Information.Although representatives of all 12 VOC groups were present in the volatile emissions of the inflorescence litter of each studied plant species, their individual compositions turned out to be specific.Only 11 identified compounds were common to all samples: acetic acid, toluene, p-cymene, αand β-pinenes, 3-carene, limonene, α-copaene, β-caryophyllene, nonanal and 1-undecene.This kind of compositional specificity was also observed in the case of the fresh and exposed litter of each species.For example, of 146 hornbeam VOCs, only 48 (32%) were recorded in both chromatograms.The lowest similarity (only 16% of "common" components) was observed in the case of maple (A.platanoides) inflorescence fall.Terpene compounds, including 43 monoterpenes and 63 sesquiterpenes, formed the group with the most components in both the "fresh" and "not-fresh" (exposed for two weeks) litter.In the group of terpene compounds, α-pinene, 3-carene, limonene, αcopaene and β-caryophyllene accounted for the largest share.The second largest group (38 components) was formed by carbonyl C 3 -C 12 compounds (common to all samples, with aliphatic aldehyde nonanal present at the highest concentration).The next largest group (28 compounds) was formed by C 6 -C 16 alkanes and alkenes, which contributed 1.2-21% to the TIC of the different chromatograms.A significant contribution to the ion current of the chromatograms (from 2.5 to 28% TIC) was also made by 24 aromatic C 7 -C 10 compounds, which included hydrocarbons and their oxygenated derivatives, such as benzaldehyde, benzyl alcohol and 2-phenylethanol.Aliphatic alcohols, carboxylic acids and furans had a smaller but nevertheless significant contribution.
In all four cases, the chromatograms of the inflorescences exposed for two weeks showed a greater number of peaks than those of the freshly collected inflorescences of the same species (last row of Table 1).Both qualitative and quantitative changes in VOC composition were particularly noticeable in the case of sesquiterpenes.It can be assumed that "new" compounds formed as a result of various kinds of destructive processes, such as the hydrolysis of glycosides.This hypothesis is supported by the fact that, in plant tissues, most terpenoids are represented by various glycosides [43,44].
It is also impossible to exclude the participation of microorganisms-destructors both in the biochemical decomposition of plant glycosides and in the formation of volatile compounds, including terpenoids, as their inherent waste products [45,46].The participation of litter-degrading microorganisms in the emission of VOCs is supported by the presence of characteristic components such as C 8 alcohols and carbonyl compounds: unsaturated alcohol 1-octen-3-ol (known as "mushroom alcohol"), ketone 1-octen-3-one and octan-3one [47].The S-containing compounds found in the VOCs also had a microbiological origin; dimethyl sulphide and dimethyl disulphide have been found in the volatile secretions of fungal species, such as Aspergillus versicolor, Penicillium commune and Phialophora fastigiate, cultivated on different media [45].
In summary, the volatile emissions from the flower litter of the studied species of woody plants are a blend of low-molecular-weight metabolites of plant and microbial origin.Among them, terpene compounds predominate, unsaturated in their chemical nature.Their lifetime in the atmosphere is limited to a few minutes due to rapid gas-phase reactions with permanent components of the Earth's atmosphere such as OH and NO 3 radicals [48].These processes lead to the rapid formation of toxic photo-oxidants (ozone and organic peroxides), many of which exhibit phytotoxic effects and have a negative impact on plant communities, as well as human health [49].An increase in the tropospheric ozone concentration can also affect the climate by perturbing the Earth's radiation budget, as O 3 is the third-most important greenhouse gas [50].In addition, relatively low volatile compounds during gas-phase oxidation form products prone to nucleation and secondary aerosol formation, which also affect the radiative budget of the troposphere [51].
According to our observations, this source of VOCs during the transition from spring to summer is relatively short term, occupying a time interval of several weeks.This is due not only to the rapid microbiological decomposition of flower litter, which does not contain stable biopolymers, such as lignocellulose and lignin, but also to the activity of invertebrates for which flower litter serves as food.For example, insects, mainly Eurydema sp., were found in the litter bags covered with a mesh with large cells (mesh size of 5 mm).The nutritional value of flower litter for herbivorous insects can be judged by the composition of the extractive substances that it contains.

Composition of Extractive Compounds
The chromatograms of the methanol extracts of the flower litter contained 263 peaks of organic compounds.Of these, 156 compounds (59%) were positively identified.Another 72 peaks were assigned to a specific class of compounds based on mass spectral data (a set of characteristic m/z values and the overall pattern of mass spectral fragmentation).Most belonged to the group of carbohydrates, totalling many thousands of individual compounds poorly represented in the available spectral and chromatographic databases (this circumstance makes unambiguous identification by GC-MS at the level of a chemical compound almost impossible).A characteristic of this group is the presence in the mass spectra of the TMS derivatives of a set of ions with m/z 204, 217, 361, 147 and 103 [42].
The identified compounds were roughly divided into seven groups, as shown in Table 2, along with the main representatives of each group.The eighth group consisted of compounds whose mass spectra did not allow them to be reliably assigned to any specific group of organic compounds.The full list of identified components is given in Table S2 in the Supplementary Information.The largest number of peaks (133) was recorded in the chromatogram of the maple inflorescence litter and the smallest number in the case of willow (67 peaks, last line in Table 2).The group with the most extractive compounds and making the greatest contribution to the TIC of all chromatograms was formed by monosaccharides and related compounds, such as sugar alcohols and acids, as well as some amino sugars.Of these, the main ones were glucose (18-21% TIC) and fructose (11-14% TIC), each of which was represented by three different anomers.The second most important group was formed by di-and trisaccharides, as well as glycosides, which accounted for 14 to 33% of the TIC.This group contained the largest quantities of sucrose, of which the relative contribution to the TIC was 2-13%.The aglycone components of glycosides were phenols (pyrocatechol, hydroquinone and 2-methoxyhydroquinone), phenolcarboxylic acids (salicylic and vanillic acids) and other phenolic compounds, such as sinapyl alcohol and tyrosol (4-hydroxyphenylethanol), as well as many flavonoids.The latter included catechin and epi-catechin, kaempferol, naringenin and quercetin.Phenolic compounds were also present in the extracts in a free state, forming the third largest group of extractive components.Their content in the extracts was 4-7% of the TIC.The contribution to the total ion current of the chromatograms of aliphatic C 2 -C 22 acids was approximately at the same level.In addition, the extracts of the aspen and maple flower litter contained a noticeable amount (7.41 and 2.95%, respectively) of free amino acids, including non-proteinogenic γ-aminobutyric acid.Interestingly, these components were almost completely absent from the flower litter of hornbeam and birch, which belong to the Betulaceae family.
Thus, this study indicates a high content of easily digestible organic compounds in the studied tree plant litter, which makes it an important seasonal resource for various inhabitants of forest soil, both microbes and other organisms of different levels of organisation.These microbial-zoological interactions in turn stimulate the intensification of abiotic processes, such as the physical evaporation of volatiles from degraded litter [1].

Conclusions
One of the results of this research is the confirmation of the hypothesis that the litter of woody plant inflorescences releases a range of VOCs into the atmosphere, with predominant participation in the emission of terpene compounds.During the spring-summer transition period, this source contributes to the pool of highly reactive components in the air of forests, the scale and significance of which require further study.The importance of this is explained by the participation of litter-derived VOCs in atmospheric processes leading to the formation of secondary pollution affecting the health of people and ecosystems.
A study of the composition of extractive compounds in flower litter showed a high content of easily biodegradable and easily metabolised compounds.This determines their accessibility to communities of microbes and saprotrophs, which play an important role in the life of forests and contribute to the emission of environmentally important VOCs into the atmosphere.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants13131829/s1,Table S1.Chemical composition (% of TIC) of VOCs emitted by fallen inflorescences of some forest-forming deciduous trees of the boreal and
* not found; ** below 0.01% of TIC; *** the identification of the corresponding compound is considered preliminary.

Table 2 .
Cont. not found; ** below 0.01% of TIC; *** the identification of the corresponding compound is considered preliminary. *