Phenolic Compounds as Biomarkers of Interactions between the Endophyte Klebsiella oxytoca and the Common Duckweed, Lemna minor L. Belgrade

: the common duckweed ( Lemna minor L.) as a model organism is experiencing a form of a renaissance. In this study, our focus was on the interactions between duckweeds and a rhizosphere-associated bacterial strain Klebsiella oxytoca (Access. No. MK212915). Five distinct phenolic compounds were identified by liquid chromatography—mass spectrometry: luteolin 6,8-di-C-hexoside, p-hydroxybenzoic acid, caffeic acid, apigenin 6-C-(2′′-pentosyl)hexoside and p-coumaric acid. All of the identified compounds reflect the colonization of the plant by K. oxytoca . This paper is another call for all plant physiologists to focus their research on L. minor and to analyze different aspects of complex plant/bacterium interactions.


Introduction
The common duckweed (Lemna minor L.) is a cosmopolitan, miniature, fast-reproducing higher aquatic plant of simplified morphology. In the last decade, the common duckweed is experiencing a form of a renaissance, after decades of being largely substituted by Arabidopsis thaliana [1]. Its simple and small genome as well its high vegetative reproduction rates, low requirements for in vitro growth and ability to thrive even under unfavorable conditions makes the common duckweed as well as many other related species of the same family (Lemnaceae), an almost ideal model organism for a wide variety of studies. One of the important traits of the common duckweed is its ability to co-exist with a large number of various microorganisms in its natural habitat, which makes it particularly well-suited for the research of plant-microorganism interactions [1,2]. In this work, we analyzed phenolic compounds associated with the co-cultivation of an endophytic bacterium, K. oxytoca, and duckweed (L. minor). These compounds correspond to various stages of colonization of the plant by bacteria and antioxidative responses of the plant to bacterial presence. Therefore, we proposed that they can be used as biomarkers of interactions between these two species.

Bacterial strain Klebsiella oxytoca 14bg Access. No. MK212915 and Growth Conditions
K. oxytoca was isolated and identified in our previous studies and selected for the ensuing experiments due to its high resistance to phenol, ability to eliminate phenol and a positive effect on the multiplication rates of the duckweeds in vitro. This strain was also identified as an endophyte [4]. Bacterial monoculture containing K. oxytoca was grown and maintained on a Luria-Bertani (LB) medium, prepared according to the previously applied protocol [4].

Co-Cultivation of K. oxytoca and Duckweed
In our previous work, we observed a change in color of nutrition MS medium (from colorless to green-brownish) during experiments with multiplication rates of duckweeds. This change in color appeared only in duckweed specimens with K. oxytoca, and not with other bacteria, and was more pronounced in phenol-free MS media than in phenol-supplemented MS media (with 500 mg L −1 of phenol) [4]. This prompted further investigation. Briefly, an overnight culture of K. oxytoca in 5 mL of LB medium was used to inoculate sterile MS medium containing duckweeds. Overnight culture was briefly centrifuged, the supernatant discarded and the remaining debris resuspended in sterile MS medium (1 mL) which was then transferred to a flask containing sterile MS medium (100 mL) with 100-150 surface-sterilized duckweeds. Duckweeds and bacteria were then co-cultivated for 3 days. After 3 days, samples of this MS medium were taken for LC-MS analysis.

Liquid Chromatography Mass Spectrometry (LC-MS)
MS nutrition medium with duckweeds and K. oxytoca was analyzed using LC-MS. System used for LC-MS was: UHPLC Accela 6000-ESI-LTQ Orbitrap XL with column C18 with 1.7 microns diameter, of the manufacturer Thermo Fisher Sci, USA. Gradient elution with two elution buffers was performed. Elution buffers A and B were (A)-0.1% aqueous solution of formic acid (HCOOH) and (B)-acetonitrile.

Bibliographical Analysis
Bibliographical analysis was conducted using the Publish or Perish software [5] and the data was retrieved from Google Scholar database.

Change of Color of MS Medium
A change in color of MS medium used for co-cultivation of K. oxytoca and duckweeds (from colorless to green-brownish) was apparent already after 48 h of co-cultivation (Figure 1). The color became darker as the co-cultivation progressed.

Bibliographical Analysis
Bibliographical analysis of publications containing key words "duckweed phenolic compounds liquid chromatography mass spectrometry bacteria rhizosphere" showed that there are 489 research papers published between 1960 and 2021 corresponding to these search criteria. The majority of these papers (457) were published since 2001.
When keywords "duckweed phenolic compounds liquid chromatography mass spectrometry Klebsiella oxytoca" were used as search criteria, only 17 papers published between 2001 and 2021 were retrieved. Out of these, 10 articles were presented with an h index.

Discussion
The observed change in color of MS medium used for co-cultivation of K. oxytoca and L. minor is probably due to release of p-hydroxybenzoic, caffeic and p-coumaric acids, known for their brownish color. Luteolin 6,8-di-C-hexoside is abundant in many plants, where it has immunomodulatory effects [6]. p-Hydroxybenzoic acid is accumulated in plants that are under bacterial attack; it is also an intermediary compound in bacterial metabolism of some monoaromatic compounds [7]. Caffeic acid is an essential biomolecule of all plants, as it is a ligning precursor. However, it's an uncommon metabolyte in bacteria [8]. Apigenin 6-C-(2′′-pentosyl)hexoside is also a flavonoid, found in root exudates. Its synthesis is stimulated during the endophytic colonization of roots [9]. p-Coumaric acid is also associated with the endophytic colonization of roots and with plants' defense response [10]. Furthermore, it has antimicrobial properties [11]. Therefore, all detected biomolecules can be associated with the endophytic colonization of the duckweed root; with the plant-bacteria signalization; and with bacterial modulation of plants' antioxidative response. In our previous work [3] we also demonstrated the importance of bacterial modulation of antioxidative response of L. minor. Namely, the presence of bacteria H. paralvei modulated the expression of plants' peroxidases and alleviated phenolinduced stress on the plants. Similar processes might be happening in K. oxytoca/L. minor interactions as well and the afforementioned phenolic compounds might in turn function to regulate bacterial activity.
Bibliographical analysis revealed that this specific interaction between L. minor and K. oxytoca is insufficiently investigated. Since K. oxytoca strain 14bg used in this study [4] was identified as an endophyte with potential positive effect on multiplication rates of the duckweeds and an ability to remove phenol from MS medium, more analyses would contribute to their better application in bioremediation and/or agriculture. In the decade of re-discovery of L. minor as a model organism, the future of this research area looks more promising than ever [1].

Conclusions
Five phenolic compounds that reflect the interactions between endophytic K. oxytoca strain 14bg and L. minor, the common duckweed, were identified by LC-MS. According to the findings presented in this paper, interactions between the common duckweed and bacterium K. oxytoca are under-investigated and therefore, more research is needed.