Structural Elucidation of Three Novel Kaempferol O-tri-Glycosides that Are Involved in the Defense Response of Hybrid Ornithogalum to Pectobacterium carotovorum

Ornithogalum is an ornamental flowering species that grows from a bulb and is highly susceptible to soft-rot disease caused by Pectobacterium carotovorum (Pc). Interspecific hybridization between O. thyrsoides and O. dubium yielded hybrids with enhanced resistance to that pathogen. The hybrids displayed distinct phenolic-compound profiles with several peaks that were specifically heightened following Pc infection. Three of these compounds were isolated and identified as novel kaempferol O-tri-glycosides. The structures of these compounds were elucidated using reversed phase high-performance liquid chromatography (RP-LC), RP-LC coupled to high-resolution mass spectrometry (RP-LC-MS), and nuclear magnetic resonance (NMR) (1D 1H and 13C, DEPT, HMQC, HMBC, COSY, and NOE), in order to achieve pure and defined compounds data. The new compounds were finally identified as kaempferol 3-O-[4-O-α-l-(3-O-acetic)-rhamnopyranosyl-6-O-β-d-xylopyranosyl]-β-d-glucopyranoside, kaempferol 3-O-[4-O-α-l-(2-O-acetic)-rhamnopyranosyl-6-O-β-d-xylopyranosyl]-β-d-glucopyranoside and kaempferol 3-O-[4-O-α-l-(2,3-O-diacetic)-rhamnopyranosyl-6-O-β-d-xylopyranosyl]-β-d-glucopyranoside.


Introduction
The genus Ornithogalum, also known as the "Star of Beth-Lehem", belongs to the family Asparagaceae, which includes about 250-300 species [1]. These include wild and cultivated species that are widely distributed across Europe, Asia (reaching as far east as Afghanistan), Africa and Madagascar [2][3][4]. Most members of this genus are herbaceous perennials, spring-and summer-flowering bulb plants. In recent decades, the African varieties of the plant (along with some others) have been grown commercially and sold as cut flowers and potted plants in South Africa, the USA, the Netherlands and Israel. The plant's potential as a cut flower and garden plant is severely hampered by its susceptibility to bacterial soft rot caused by Pectobacterium carotovorum (Pc) species. Several attempts have been made to minimize soft-rot disease through the use of plant activators that induce systemic resistance and there have also been efforts to develop resistant clones through molecular and classical breeding [5][6][7]. Such strategies have involved the induction and accumulation of secondary metabolites, resulting in reduced bacterial pressure and multiplication, or direct interference with bacterial virulence [8]. In this context, external application of methyl jasmonate (MJ) has been shown to activate the jasmonic-acid signaling pathway, which plays a central role in the regulation of secondary-metabolite biosynthesis in tomato (Solanum lycopersicum) [9][10][11][12]. In O. dubium and Zantedeschia aethiopica (another ornamental monocot), defense elicitation with exogenous MJ has been shown to reduce disease symptoms and lead to increased accumulation of polyphenolic compounds following Pc infection [6,13].
Plant phenolics are considered to be the most abundant secondary metabolites isolated from plants. To date, over 8000 phenolic structures (with simple molecular structures or polymeric structures) have been discovered [14,15]. Most polyphenols appear in nature as glycosides with one or more glycosidic moieties. They are involved in essential processes, such as growth and reproduction, and many help protect plants from biotic and abiotic stress [14][15][16][17]. Flavonoids are known to play important roles in plant tissues, including providing protection against UV-B radiation, as antioxidants, as antifeedants and as phytoalexins [18][19][20][21][22]. Some flavonoids are synthesized in response to plant pathogens [15,16,21,23].
Here, two Ornithogalum species, O. dubium, which is highly susceptible to soft rot, and O. thyrosoides, which is relatively resistant, were crossed to yield interspecific F1 hybrids with different levels of resistance to the soft-rot pathogen. In correlation with their observed resistance to soft rot, these hybrids produced metabolites in response to infection with Pc, and those metabolites exhibited patterns of UV absorption that are typical of flavonoids. Three flavonoids were further purified from leaf extracts and mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy were used to identify them as three novel kaempferol O-tri-glycosides.

Results and Discussion
Interspecific breeding was carried out between two Ornithogalum accessions: O. dubium (#49_60) and O. thyrosoides (#36_1) [24]. Following embryo rescue, the cross yielded two F1 hybrids, designated #2_28 and #2_32, and micropropagation protocols were used to clone those hybrids ( Figure 1D) for further analysis [25]. Infiltration with Pc showed that both hybrids are less sensitive to soft-rot infection than the parent #49_60, with #2_28 being the more resistant ( Figure 1A). Following infiltration with Pc, leaves were extracted with aqueous methanol, as described previously [13], and the levels of phenolic compounds in the extracts were determined, revealing an inverse correlation between sensitivity to Pc and levels of phenolics (expressed as catechin equivalents, Figure 1B). All extracts were then separated and characterized using reversed phase high performance liquid chromatography (RP-LC) and photo diode array detection, and a unique profile of phytochemicals was observed for each plant line ( Figure 1C). Each of the plant lines had a typical color phenotype: The pollen donor was white, the female flower orange and the F1 hybrids were light orange ( Figure 1D). Pc infection was found to induce the production of several compounds as a part of the general plant response to the bacterium with more than two-fold increases in the compounds assigned Peak Numbers 1, 2 and 3. Hyphenated and complementary spectral analyses were used to further characterize these three molecules.

NMR Analyses of Compound 1
Additional cleanup of the compound was performed using solid-phase extraction (SPE) through a single-use sep-pak™ C-18, prior to NMR analyses. Indeed, the NMR spectra supported the identification of Compound 1 as kaempferol-O-tri-glycoside (1, Figure 3      The connectivity of the kaempferol, the three sugar moieties, and the acetate unit was deduced using HMBC correlations (Figure 4). Glc in this molecule was linked to the hydroxyl at C-3 of kaempferol with a 3 J C-H correlation between the H Glc -1 and C-3 of the aglycone. It was also deduced from an HMBC cross-peak, that the anomeric protons of Xyl and Rha are correlated with C Glc -6 and C Glc -4, respectively. Accounting for all the spectroscopic data described above, the structure of 1 was confirmed as  The connectivity of the kaempferol, the three sugar moieties, and the acetate unit was deduced using HMBC correlations (Figure 4). Glc in this molecule was linked to the hydroxyl at C-3 of kaempferol with a 3 JC-H correlation between the HGlc-1 and C-3 of the aglycone. It was also deduced from an HMBC cross-peak, that the anomeric protons of Xyl and Rha are correlated with CGlc-6 and CGlc-4, respectively. Accounting for all the spectroscopic data described above, the structure of 1 was

Spectral Analysis of Compound 2
Comparison of the mass spectra ( (Tables 1 and 2, Figure 3).
The 1 H-NMR spectra of Compounds 1 and 2 were almost identical except for signals arising from the rhamnose acetate moiety. In the NMR analysis, it was concluded that Compound 2 is an isomer of Compound 1 in which the acetate group is attached to position 2 rather than position 3 of the   Figure 3).
The 1 H-NMR spectra of Compounds 1 and 2 were almost identical except for signals arising from the rhamnose acetate moiety. In the NMR analysis, it was concluded that Compound 2 is an isomer of Compound 1 in which the acetate group is attached to position 2 rather than position 3 of the rhamnose moiety. To conclude, compound 2 was identified as kaempferol

Spectral Analysis of Compound 3
The spectral data acquired for Compound 3 resembled the spectral data for Compounds 1 and

General
All solutions were prepared in DDW (double distilled water), unless indicated otherwise. All materials were purchased, from Sigma-Aldrich, unless otherwise indicated. HPLC grade methanol, ethanol, acetonitrile, ethyl acetate, hexane were purchased from Baker (Phillipsburg, NG, USA). All tissue culture materials were purchased from Duchefa (Haarlem, The Netherlands).
RP-LC-MS analyses were performed using Accela High-Speed LC system coupled with linear trap quadrupole (LTQ) Orbitrap Discovery hybrid mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with electrospray ionization source. The mass spectrometer was operated in both negative and positive ionization modes, and ion source was set as follows: Spray voltage 3 kV, capillary temperature 250 • C, ion-transfer optics parameters were optimized using automatic tune option, sheath gas rate (arb) 35, and auxiliary gas rate (arb) 15. Mass spectra were acquired in the m/z 150-2000 Da range. The LC-MS 3 analysis was performed in data depending acquisition mode. Data were analyzed using Xcalibur software (Thermo Fisher Scientific Inc., Waltham, MA, USA, version 1.4 SR1).

Plant Material, Establishment of Cell Cultures, Plants and Bacterial Infection
Two species of Ornithogalum, O. dubium (#49_60) and O. thyrsoides (#95/36/1) were crossed to produce F1 interspecific hybrids, of which two lines were selected designated as #2_28 and #2_32 and cloned for further analyses [24]. Cell cultures and infection methods were executed according to previous works [6,13]. Briefly, 20 micropropagated plantlets of each of the F1 hybrids were inoculated

Conclusions
The induction of the synthesis of flavonoids in Ornithogalum hybrids following infection with the soft-rot pathogen Pc revealed three novel kaempferol O-tri-glycosides. The levels of these compounds correlated with increased resistance to Pc infection in the parent line O. thyrosoides (#36_1), and in the F1 hybrids #2_28 and #2_32. The results suggest that interspecific breeding may be a practical approach to fight bacterial soft rot in ornamental flower bulbs.
Author Contributions: A.G. performed the physiological experiments, K.S. performed the isolation of the compounds. H.E.G. did NMR and data analysis, Z.K. analyzed the MS results; I.Y., K.S., and Z.K. designed the experiments and contributed to the writing of the study.

Funding:
We thank the chief Scientist for Agriculture grant number 256-0996 for financial support.