View source for Journal:Fertilization following pollination predominantly decreases phytocannabinoids accumulation and alters the accumulation of terpenoids in Cannabis inflorescences

{{DISPLAYTITLE:Journal:Fertilization following pollination predominantly decreases phytocannabinoids accumulation and alters the accumulation of terpenoids in ''Cannabis'' inflorescences}}
{{Infobox journal article
|name         = 
|image        = 
|alt          = <!-- Alternative text for images -->
|caption      = 
|title_full   = Fertilization following pollination predominantly decreases phytocannabinoids accumulation and alters the accumulation of terpenoids in ''Cannabis'' inflorescences
|journal      = ''Frontiers in Plant Science''
|authors      = Feder, Carni L.; Cohen, Oded; Shapira, Anna; Katzir, Itay; Peer, Reut; Guberman, Ohad; Procaccia, Shiri; Berman, Paula; Flaishman, Moshe; Meiri, David
|affiliations = Technion-Israel Institute of Technology, Agricultural Research Organization of Israel
|contact      = Email: dmeiri at technion dot ac dot il
|editors      = Taglialatela-Scafati, Orazio
|pub_year     = 2021
|vol_iss      = '''12'''
|at           = 753847
|doi          = [https://doi.org/10.3389/fpls.2021.753847 10.3389/fpls.2021.753847]
|issn         = 1664-462X
|license      = [http://creativecommons.org/licenses/by/4.0/ Creative Commons Attribution 4.0 International]
|website      = [https://www.frontiersin.org/articles/10.3389/fpls.2021.753847/full https://www.frontiersin.org/articles/10.3389/fpls.2021.753847/full]
|download     = [https://www.frontiersin.org/articles/10.3389/fpls.2021.753847/pdf https://www.frontiersin.org/articles/10.3389/fpls.2021.753847/pdf] (PDF)
}}

==Abstract==
Over the last few decades, a growing body of evidence has increasingly showed the therapeutic capabilities of ''[[Cannabis]]'' plants. These capabilities have been attributed to the specialized secondary metabolites stored in the glandular [[trichome]]s of female [[inflorescence]]s, mainly [[Cannabinoid|phytocannabinoids]] and [[terpenoid]]s. The accumulation of these metabolites in the flower is versatile and influenced by a largely unknown regulation system, attributed to genetic, developmental, and environmental factors. As ''Cannabis'' is a [[Dioecy|dioecious]] plant, one main factor is fertilization after successful pollination. Fertilized flowers are considerably less potent, likely due to changes in the contents of phytocannabinoids and terpenoids.

This study examined the effect of fertilization on metabolite composition by crossbreeding [[Tetrahydrocannabinol|Δ<sup>9</sup>-tetrahydrocannabinol]] (Δ<sup>9</sup>-THC)- or [[cannabidiol]] (CBD)-rich female plants with different male plants: THC-rich plants, CBD-rich plants, or the original female plant induced to develop male pollen sacs. We used advanced analytical methods to assess the phytocannabinoid and terpenoid content, including a newly developed semi-quantitative analysis for terpenoids without analytical standards. 

We found that fertilization significantly decreased phytocannabinoid content. For terpenoids, the subgroup of [[Monoterpene|monoterpenoids]] had similar trends to the phytocannabinoids, proposing both are commonly regulated in the plant. The [[Sesquiterpene|sesquiterpenoids]] remained unchanged in the THC-rich female plants and had a trend of decreasing in the CBD-rich female plants. Additionally, specific phytocannabinoids and terpenoids showed an uncommon increase in concentration following fertilization with particular male plants. 

Our results demonstrate that although the profile of phytocannabinoids and their relative ratios were kept, fertilization substantially decreased the concentration of nearly all phytocannabinoids in the plant regardless of the type of fertilizing male plant. Our findings may point to the functional roles of secondary metabolites in ''Cannabis''.

Keywords: ''Cannabis'', cannabinoids, terpenoids, secondary metabolites, chromatography/mass spectrometry, analytical methods, gas chromatography, high pressure liquid chromatography

==Introduction==
''[[Cannabis sativa]]'' L. (''[[Cannabis]]'') has been known as a medicinal plant since ancient times.<ref>{{Cite journal |last=Bonini |first=Sara Anna |last2=Premoli |first2=Marika |last3=Tambaro |first3=Simone |last4=Kumar |first4=Amit |last5=Maccarinelli |first5=Giuseppina |last6=Memo |first6=Maurizio |last7=Mastinu |first7=Andrea |date=2018-12 |title=Cannabis sativa: A comprehensive ethnopharmacological review of a medicinal plant with a long history |url=https://linkinghub.elsevier.com/retrieve/pii/S0378874118316611 |journal=Journal of Ethnopharmacology |language=en |volume=227 |pages=300–315 |doi=10.1016/j.jep.2018.09.004}}</ref> During the last two decades, many studies have added to the growing evidence for its therapeutic effects in a wide range of conditions such as neurodegenerative disorders<ref>{{Cite journal |last=Fernández-Ruiz |first=Javier |date=2019-05 |title=The biomedical challenge of neurodegenerative disorders: an opportunity for cannabinoid-based therapies to improve on the poor current therapeutic outcomes: Cannabinoids and neuroprotection |url=https://onlinelibrary.wiley.com/doi/10.1111/bph.14382 |journal=British Journal of Pharmacology |language=en |volume=176 |issue=10 |pages=1370–1383 |doi=10.1111/bph.14382 |pmc=PMC6487558 |pmid=29856067}}</ref><ref>{{Cite journal |last=Cassano |first=Tommaso |last2=Villani |first2=Rosanna |last3=Pace |first3=Lorenzo |last4=Carbone |first4=Antonio |last5=Bukke |first5=Vidyasagar Naik |last6=Orkisz |first6=Stanislaw |last7=Avolio |first7=Carlo |last8=Serviddio |first8=Gaetano |date=2020-03-06 |title=From Cannabis sativa to Cannabidiol: Promising Therapeutic Candidate for the Treatment of Neurodegenerative Diseases |url=https://www.frontiersin.org/article/10.3389/fphar.2020.00124/full |journal=Frontiers in Pharmacology |volume=11 |pages=124 |doi=10.3389/fphar.2020.00124 |issn=1663-9812 |pmc=PMC7069528 |pmid=32210795}}</ref>, pain<ref>{{Citation |last=Starowicz |first=Katarzyna |last2=Finn |first2=David P. |date=2017 |title=Cannabinoids and Pain: Sites and Mechanisms of Action |url=https://linkinghub.elsevier.com/retrieve/pii/S1054358917300443 |work=Advances in Pharmacology |language=en |publisher=Elsevier |volume=80 |pages=437–475 |doi=10.1016/bs.apha.2017.05.003 |isbn=978-0-12-811232-8 |access-date=2021-11-24}}</ref>, epilepsy,<ref>{{Cite journal |last=Franco |first=Valentina |last2=Bialer |first2=Meir |last3=Perucca |first3=Emilio |date=2021-03 |title=Cannabidiol in the treatment of epilepsy: Current evidence and perspectives for further research |url=https://linkinghub.elsevier.com/retrieve/pii/S0028390820305104 |journal=Neuropharmacology |language=en |volume=185 |pages=108442 |doi=10.1016/j.neuropharm.2020.108442}}</ref> multiple sclerosis<ref>{{Cite journal |last=Rice |first=Jessica |last2=Cameron |first2=Michelle |date=2018-08 |title=Cannabinoids for Treatment of MS Symptoms: State of the Evidence |url=http://link.springer.com/10.1007/s11910-018-0859-x |journal=Current Neurology and Neuroscience Reports |language=en |volume=18 |issue=8 |pages=50 |doi=10.1007/s11910-018-0859-x |issn=1528-4042}}</ref>, and more.<ref>{{Cite journal |last=Gonçalves |first=Joana |last2=Rosado |first2=Tiago |last3=Soares |first3=Sofia |last4=Simão |first4=Ana |last5=Caramelo |first5=Débora |last6=Luís |first6=Ângelo |last7=Fernández |first7=Nicolás |last8=Barroso |first8=Mário |last9=Gallardo |first9=Eugenia |last10=Duarte |first10=Ana |date=2019-02-23 |title=Cannabis and Its Secondary Metabolites: Their Use as Therapeutic Drugs, Toxicological Aspects, and Analytical Determination |url=http://www.mdpi.com/2305-6320/6/1/31 |journal=Medicines |language=en |volume=6 |issue=1 |pages=31 |doi=10.3390/medicines6010031 |issn=2305-6320 |pmc=PMC6473697 |pmid=30813390}}</ref> These therapeutic abilities have been attributed to the secondary metabolites biosynthesized in ''Cannabis''<ref>{{Cite journal |last=Andre |first=Christelle M. |last2=Hausman |first2=Jean-Francois |last3=Guerriero |first3=Gea |date=2016-02-04 |title=Cannabis sativa: The Plant of the Thousand and One Molecules |url=http://journal.frontiersin.org/Article/10.3389/fpls.2016.00019/abstract |journal=Frontiers in Plant Science |volume=7 |at=19 |doi=10.3389/fpls.2016.00019 |issn=1664-462X |pmc=PMC4740396 |pmid=26870049}}</ref>, with more than 500 different secondary metabolites having been identified.<ref>{{Cite journal |last=ElSohly |first=Mahmoud A. |last2=Slade |first2=Desmond |date=2005-12 |title=Chemical constituents of marijuana: The complex mixture of natural cannabinoids |url=https://linkinghub.elsevier.com/retrieve/pii/S002432050500891X |journal=Life Sciences |language=en |volume=78 |issue=5 |pages=539–548 |doi=10.1016/j.lfs.2005.09.011}}</ref><ref name=":0">{{Cite journal |last=Flores-Sanchez |first=Isvett Josefina |last2=Verpoorte |first2=Robert |date=2008-10 |title=Secondary metabolism in cannabis |url=http://link.springer.com/10.1007/s11101-008-9094-4 |journal=Phytochemistry Reviews |language=en |volume=7 |issue=3 |pages=615–639 |doi=10.1007/s11101-008-9094-4 |issn=1568-7767}}</ref> These metabolites belong to several groups of compounds, including [[Cannabinoid|phytocannabinoids]], [[terpenoid]]s, and [[flavonoid]]s. 

To date, the most characterized are phytocannabinoids, lipophilic compounds made of isoprene units (five-carbon building blocks)<ref name=":8" />, which are almost exclusive to the ''Cannabis'' plant.<ref name=":9">{{Cite journal |last=Gülck |first=Thies |last2=Møller |first2=Birger Lindberg |date=2020-10 |title=Phytocannabinoids: Origins and Biosynthesis |url=https://linkinghub.elsevier.com/retrieve/pii/S1360138520301874 |journal=Trends in Plant Science |language=en |volume=25 |issue=10 |pages=985–1004 |doi=10.1016/j.tplants.2020.05.005}}</ref> More than 140 different phytocannabinoids have been found to accumulate to various extents in glandular [[trichome]]s that are located in the aerial parts of the plant and mostly on the female flowers, which are arranged in a cluster on the stem of the [[inflorescence]].<ref name=":8">{{Cite journal |last=Hanuš |first=Lumír Ondřej |last2=Meyer |first2=Stefan Martin |last3=Muñoz |first3=Eduardo |last4=Taglialatela-Scafati |first4=Orazio |last5=Appendino |first5=Giovanni |date=2016 |title=Phytocannabinoids: a unified critical inventory |url=http://xlink.rsc.org/?DOI=C6NP00074F |journal=Natural Product Reports |language=en |volume=33 |issue=12 |pages=1357–1392 |doi=10.1039/C6NP00074F |issn=0265-0568}}</ref> Phytocannabinoids can be classified into several subclasses according to their chemical structure, including the [[Tetrahydrocannabinol|Δ<sup>9</sup>-tetrahydrocannabinol]] (THC) and [[cannabidiol]] (CBD) families, as well as [[cannabinol]] (CBN), [[cannabigerol]] (CBG), [[cannabichromene]] (CBC), and many others.<ref name=":0" /><ref name=":1">{{Cite journal |last=Berman |first=Paula |last2=Futoran |first2=Kate |last3=Lewitus |first3=Gil M. |last4=Mukha |first4=Dzmitry |last5=Benami |first5=Maya |last6=Shlomi |first6=Tomer |last7=Meiri |first7=David |date=2018-12 |title=A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis |url=http://www.nature.com/articles/s41598-018-32651-4 |journal=Scientific Reports |language=en |volume=8 |issue=1 |pages=14280 |doi=10.1038/s41598-018-32651-4 |issn=2045-2322 |pmc=PMC6155167 |pmid=30250104}}</ref> 

Terpenoids, found in many other plants, represent a second large group of metabolites. These metabolites are closely related to phytocannabinoids, sharing the same isoprenoid precursor and built up by branched isoprene units.<ref name=":2">{{Cite journal |last=Booth |first=Judith K. |last2=Yuen |first2=Macaire M.S. |last3=Jancsik |first3=Sharon |last4=Madilao |first4=Lufiani L. |last5=Page |first5=Jonathan E. |last6=Bohlmann |first6=Jörg |date=2020-09 |title=Terpene Synthases and Terpene Variation in Cannabis sativa |url=https://academic.oup.com/plphys/article/184/1/130-147/6117797 |journal=Plant Physiology |language=en |volume=184 |issue=1 |pages=130–147 |doi=10.1104/pp.20.00593 |issn=0032-0889 |pmc=PMC7479917 |pmid=32591428}}</ref> Terpenoids are responsible for the fragrance and taste of the plant and are suggested to also have defensive roles. They may also contribute to the therapeutic effects attributed to ''Cannabis''.<ref name=":4">{{Citation |last=Russo |first=Ethan B. |last2=Marcu |first2=Jahan |date=2017 |title=Cannabis Pharmacology: The Usual Suspects and a Few Promising Leads |url=https://linkinghub.elsevier.com/retrieve/pii/S1054358917300273 |work=Advances in Pharmacology |language=en |publisher=Elsevier |volume=80 |pages=67–134 |doi=10.1016/bs.apha.2017.03.004 |isbn=978-0-12-811232-8 |access-date=2021-11-24}}</ref> 

Another group of metabolites worth mentioning is flavonoids. Among this group, which is widespread in the plant kingdom, there are three specific prenylated flavonoids, termed Cannflavins A, B, and C, which are unique to ''Cannabis'' and show potent anti-inflammatory abilities.<ref>{{Cite journal |last=Radwan |first=Mohamed M. |last2=ElSohly |first2=Mahmoud A. |last3=Slade |first3=Desmond |last4=Ahmed |first4=Safwat A. |last5=Wilson |first5=Lisa |last6=El-Alfy |first6=Abir T. |last7=Khan |first7=Ikhlas A. |last8=Ross |first8=Samir A. |date=2008-10 |title=Non-cannabinoid constituents from a high potency Cannabis sativa variety |url=https://linkinghub.elsevier.com/retrieve/pii/S0031942208003518 |journal=Phytochemistry |language=en |volume=69 |issue=14 |pages=2627–2633 |doi=10.1016/j.phytochem.2008.07.010 |pmc=PMC4888767 |pmid=18774146}}</ref><ref>{{Cite journal |last=Rea |first=Kevin A |last2=Casaretto |first2=José A. |last3=Al-Abdul-Wahid |first3=M. Sameer |last4=Sukumaran |first4=Arjun |last5=Geddes-McAlister |first5=Jennifer |last6=Rothstein |first6=Steven J. |last7=Akhtar |first7=Tariq A. |date=2019-08 |title=Biosynthesis of cannflavins A and B from Cannabis sativa L |url=https://linkinghub.elsevier.com/retrieve/pii/S0031942218303819 |journal=Phytochemistry |language=en |volume=164 |pages=162–171 |doi=10.1016/j.phytochem.2019.05.009}}</ref><ref>{{Cite journal |last=Erridge |first=Simon |last2=Mangal |first2=Nagina |last3=Salazar |first3=Oliver |last4=Pacchetti |first4=Barbara |last5=Sodergren |first5=Mikael H. |date=2020-10 |title=Cannflavins – From plant to patient: A scoping review |url=https://linkinghub.elsevier.com/retrieve/pii/S0367326X2030294X |journal=Fitoterapia |language=en |volume=146 |pages=104712 |doi=10.1016/j.fitote.2020.104712}}</ref>

Ongoing research is focused on matching specific metabolites found in the plant and their therapeutic capabilities. To this end, specialized analytical methods have been developed in order to obtain precise knowledge on all the components of the plant and the effects they are responsible for. Currently, more than 90 phytocannabinoids and 100 terpenoids are routinely identified and quantified to obtain an overall chemical profile of each [[Cheomotype|chemovar]] used for a medicinal purpose.<ref name=":1" /><ref name=":6">{{Cite journal |last=Shapira |first=Anna |last2=Berman |first2=Paula |last3=Futoran |first3=Kate |last4=Guberman |first4=Ohad |last5=Meiri |first5=David |date=2019-09-03 |title=Tandem Mass Spectrometric Quantification of 93 Terpenoids in Cannabis Using Static Headspace Injections |url=https://pubs.acs.org/doi/10.1021/acs.analchem.9b02844 |journal=Analytical Chemistry |language=en |volume=91 |issue=17 |pages=11425–11432 |doi=10.1021/acs.analchem.9b02844 |issn=0003-2700}}</ref> In parallel to the search for specific biological activities of the secondary metabolites, broad ongoing research is focused upon the elucidation of ''in planta'' metabolites’ biosynthesis, transport, and accumulation pathways. 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Lacey |date=2020-01 |title=Cannabis glandular trichomes alter morphology and metabolite content during flower maturation |url=https://onlinelibrary.wiley.com/doi/10.1111/tpj.14516 |journal=The Plant Journal |language=en |volume=101 |issue=1 |pages=37–56 |doi=10.1111/tpj.14516 |issn=0960-7412}}</ref><ref>{{Cite journal |last=McGarvey |first=Peter |last2=Huang |first2=Jiahao |last3=McCoy |first3=Matthew |last4=Orvis |first4=Joshua |last5=Katsir |first5=Yael |last6=Lotringer |first6=Nitzan |last7=Nesher |first7=Iris |last8=Kavarana |first8=Malcolm |last9=Sun |first9=Mingyang |last10=Peet |first10=Richard |last11=Meiri |first11=David |date=2020-12 |title=De novo assembly and annotation of transcriptomes from two cultivars of Cannabis sativa with different cannabinoid profiles |url=https://linkinghub.elsevier.com/retrieve/pii/S0378111920306958 |journal=Gene |language=en |volume=762 |pages=145026 |doi=10.1016/j.gene.2020.145026}}</ref>, and have been integrated into a genomic database for ''Cannabis'' (CannabisGDB).<ref>{{Cite journal |last=Cai |first=Sen |last2=Zhang |first2=Zhiyuan |last3=Huang |first3=Suyun |last4=Bai |first4=Xu |last5=Huang |first5=Ziying |last6=Zhang |first6=Yiping Jason |last7=Huang |first7=Likun |last8=Tang |first8=Weiqi |last9=Haughn |first9=George |last10=You |first10=Shijun |last11=Liu |first11=Yuanyuan |date=2021-05 |title=CannabisGDB: a comprehensive genomic database for Cannabis Sativa L |url=https://onlinelibrary.wiley.com/doi/10.1111/pbi.13548 |journal=Plant Biotechnology Journal |language=en |volume=19 |issue=5 |pages=857–859 |doi=10.1111/pbi.13548 |issn=1467-7644 |pmc=PMC8131054 |pmid=33462958}}</ref> Biosynthetic pathways are being unraveled, and recently more than 30 ''Cannabis''-specific terpenoid synthases have been characterized.<ref name=":2" /><ref name=":3" /><ref>{{Cite journal |last=Booth |first=Judith K. |last2=Page |first2=Jonathan E. |last3=Bohlmann |first3=Jörg |date=2017-03-29 |editor-last=Hamberger |editor-first=Björn |title=Terpene synthases from Cannabis sativa |url=https://dx.plos.org/10.1371/journal.pone.0173911 |journal=PLOS ONE |language=en |volume=12 |issue=3 |pages=e0173911 |doi=10.1371/journal.pone.0173911 |issn=1932-6203 |pmc=PMC5371325 |pmid=28355238}}</ref><ref>{{Cite journal |last=Allen |first=Keith D. |last2=McKernan |first2=Kevin |last3=Pauli |first3=Christopher |last4=Roe |first4=Jim |last5=Torres |first5=Anthony |last6=Gaudino |first6=Reggie |date=2019-09-12 |editor-last=Hamberger |editor-first=Björn |title=Genomic characterization of the complete terpene synthase gene family from Cannabis sativa |url=https://dx.plos.org/10.1371/journal.pone.0222363 |journal=PLOS ONE |language=en |volume=14 |issue=9 |pages=e0222363 |doi=10.1371/journal.pone.0222363 |issn=1932-6203 |pmc=PMC6742361 |pmid=31513654}}</ref><ref>{{Cite journal |last=Zager |first=Jordan J. |last2=Lange |first2=Iris |last3=Srividya |first3=Narayanan |last4=Smith |first4=Anthony |last5=Lange |first5=B. Markus |date=2019-08 |title=Gene Networks Underlying Cannabinoid and Terpenoid Accumulation in Cannabis |url=https://academic.oup.com/plphys/article/180/4/1877-1897/6117720 |journal=Plant Physiology |language=en |volume=180 |issue=4 |pages=1877–1897 |doi=10.1104/pp.18.01506 |issn=0032-0889 |pmc=PMC6670104 |pmid=31138625}}</ref> In addition, the environmental and developmental factors that affect metabolite accumulation are also studied, such as light levels<ref>{{Cite journal |last=Hawley |first=Dave |last2=Graham |first2=Thomas |last3=Stasiak |first3=Michael |last4=Dixon |first4=Mike |date=2018-11 |title=Improving Cannabis Bud Quality and Yield with Subcanopy Lighting |url=https://journals.ashs.org/view/journals/hortsci/53/11/article-p1593.xml |journal=HortScience |volume=53 |issue=11 |pages=1593–1599 |doi=10.21273/HORTSCI13173-18 |issn=0018-5345}}</ref><ref>{{Cite journal |last=Magagnini |first=Gianmaria |last2=Grassi |first2=Gianpaolo |last3=Kotiranta |first3=Stiina |date=2018-06-12 |title=The Effect of Light Spectrum on the Morphology and Cannabinoid Content of Cannabis sativa L. |url=https://www.karger.com/Article/FullText/489030 |journal=Medical Cannabis and Cannabinoids |language=en |volume=1 |issue=1 |pages=19–27 |doi=10.1159/000489030 |issn=2504-3889 |pmc=PMC8489345 |pmid=34676318}}</ref><ref>{{Cite journal |last=Namdar |first=Dvory |last2=Charuvi |first2=Dana |last3=Ajjampura |first3=Vinayka |last4=Mazuz |first4=Moran |last5=Ion |first5=Aurel |last6=Kamara |first6=Itzhak |last7=Koltai |first7=Hinanit |date=2019-06 |title=LED lighting affects the composition and biological activity of Cannabis sativa secondary metabolites |url=https://linkinghub.elsevier.com/retrieve/pii/S0926669019301086 |journal=Industrial Crops and Products |language=en |volume=132 |pages=177–185 |doi=10.1016/j.indcrop.2019.02.016}}</ref>, soil types, and harvest time.<ref name=":5">{{Cite journal |last=Meier, C.; Mediavilla, V. |year=1998 |title=Factors influencing the yield and the quality of hemp (Cannabis sativa L.) essential oil |url=http://www.internationalhempassociation.org/jiha/jiha5107.html |journal=Journal of the International Hemp Association |volume=5 |issue=1 |pages=16–20}}</ref><ref>{{Cite journal |last=Bernstein |first=Nirit |last2=Gorelick |first2=Jonathan |last3=Zerahia |first3=Roei |last4=Koch |first4=Sraya |date=2019-06-17 |title=Impact of N, P, K, and Humic Acid Supplementation on the Chemical Profile of Medical Cannabis (Cannabis sativa L) |url=https://www.frontiersin.org/article/10.3389/fpls.2019.00736/full |journal=Frontiers in Plant Science |volume=10 |pages=736 |doi=10.3389/fpls.2019.00736 |issn=1664-462X |pmc=PMC6589925 |pmid=31263470}}</ref><ref>{{Cite journal |last=Chandra |first=Suman |last2=Lata |first2=Hemant |last3=ElSohly |first3=Mahmoud A. |date=2020-06-26 |title=Propagation of Cannabis for Clinical Research: An Approach Towards a Modern Herbal Medicinal Products Development |url=https://www.frontiersin.org/article/10.3389/fpls.2020.00958/full |journal=Frontiers in Plant Science |volume=11 |pages=958 |doi=10.3389/fpls.2020.00958 |issn=1664-462X |pmc=PMC7333344 |pmid=32676092}}</ref> The increasing information on the impact of these different factors on metabolite accumulation has the prospect of developing specific chemovars harboring a pre-planned group of metabolites.<ref name=":7">{{Cite journal |last=Romero |first=P. |last2=Peris |first2=A. |last3=Vergara |first3=K. |last4=Matus |first4=J.T. |date=2020-09 |title=Comprehending and improving cannabis specialized metabolism in the systems biology era |url=https://linkinghub.elsevier.com/retrieve/pii/S0168945220301771 |journal=Plant Science |language=en |volume=298 |pages=110571 |doi=10.1016/j.plantsci.2020.110571}}</ref>

This study examined the effect of an additional factor, the fertilization of ''Cannabis'' flowers following pollination of the pistil. Fertilization of flowers is a key step in the plant life cycle. Successful pollination activates a series of events followed by fertilization and embryogenesis. This includes the development of an ovary on one hand, together with senescence and abscission of floral organs, degradation of macromolecules, and recycling of different nutrients on the other hand.<ref>{{Cite journal |last=O'Neill |first=Sharman D. |date=1997-06 |title=POLLINATION REGULATION OF FLOWER DEVELOPMENT |url=https://www.annualreviews.org/doi/10.1146/annurev.arplant.48.1.547 |journal=Annual Review of Plant Physiology and Plant Molecular Biology |language=en |volume=48 |issue=1 |pages=547–574 |doi=10.1146/annurev.arplant.48.1.547 |issn=1040-2519}}</ref><ref>{{Cite journal |last=Tripathi |first=Siddharth Kaushal |last2=Tuteja |first2=Narendra |date=2007-11 |title=Integrated Signaling in Flower Senescence: An Overview |url=http://www.tandfonline.com/doi/abs/10.4161/psb.2.6.4991 |journal=Plant Signaling & Behavior |language=en |volume=2 |issue=6 |pages=437–445 |doi=10.4161/psb.2.6.4991 |issn=1559-2324 |pmc=PMC2634333 |pmid=19517004}}</ref><ref>{{Cite journal |last=Borghi |first=Monica |last2=Fernie |first2=Alisdair R. |date=2020-08 |title=Outstanding questions in flower metabolism |url=https://onlinelibrary.wiley.com/doi/10.1111/tpj.14814 |journal=The Plant Journal |language=en |volume=103 |issue=4 |pages=1275–1288 |doi=10.1111/tpj.14814 |issn=0960-7412}}</ref> ''Cannabis'' is a [[Dioecy|dioecious]] plant, harboring either female or male reproductive organs. It is also a wind-pollinated plant, in which the pollination of flowers is not dependent on specific animal pollinators. Phytocannabinoids are most abundant in the female flower [[inflorescence]]s.<ref name=":0" /> Fertilized flowers, harboring seeds, are considerably less potent. Hence the term “''sinsemilla'',” Spanish for “without seed,” that defines plants associated with high psychoactive effects.<ref>{{Cite journal |last=Potter |first=David J. |last2=Hammond |first2=Kathy |last3=Tuffnell |first3=Shaun |last4=Walker |first4=Christopher |last5=Di Forti |first5=Marta |date=2018-04 |title=Potency of Δ 9 -tetrahydrocannabinol and other cannabinoids in cannabis in England in 2016: Implications for public health and pharmacology |url=https://onlinelibrary.wiley.com/doi/10.1002/dta.2368 |journal=Drug Testing and Analysis |language=en |volume=10 |issue=4 |pages=628–635 |doi=10.1002/dta.2368}}</ref> In addition, it is a common work practice by ''Cannabis'' growers to eliminate male plants growing in a field to maintain the unfertilized inflorescences and maximize the phytocannabinoid concentrations. Therefore, it is likely that the content of secondary metabolites such as phytocannabinoids and terpenoids changes following the pollination and fertilization of ''Cannabis'' inflorescences. However, although mentioned in a few studies<ref name=":4" /><ref name=":5" /><ref name=":11">{{Cite web |last=Potter, D. |date=March 2009 |title=The Propagation, Characterisation and Optimisation of Cannabis sativa L. as a Phytopharmaceutical |url=https://extractionmagazine.com/wp-content/uploads/2018/06/THE-PROPAGATION-CHARACTERISATION-AND-OPTIMISATION-OF-CANNABIS-SATIVA-L-AS-A-PHYTOPHARMACEUTICAL.pdf |format=PDF |publisher=King’s College London}}</ref>, this phenomenon was not studied in depth. In the last few years, an increasing number of ''Cannabis'' growers are moving from using cuttings from female “mother plants” to seeds. Even though the seeds are usually feminized, around 5–10% will be males, and thus the question about the effect of pollination on the phytocannabinoids and terpenoids expression becomes critical.

In order to gain more insight into the ''Cannabis'' metabolite regulation pathway, this work studied the effect of flower fertilization on the plant’s secondary metabolite accumulation. We used indoor growing methods together with analytical procedures in order to investigate the effect of fertilization on metabolite composition and concentration in ''Cannabis'' inflorescences, and specify which metabolites are affected and to what extent.

==Materials and methods==
===Chemicals and reagents===
[[Liquid chromatography–mass spectrometry]] (LC/MS)-grade acetonitrile (catalog number 1.00029), methanol (1.06035), and water (1.15333); and [[gas chromatography]] (GC) headspace-grade dimethyl sulfoxide (DMSO) (1.01900) were purchased from Mercury Scientific and Industrial Products Ltd. (Rosh Haayin, Israel). Ethanol, (catalog number 052541), acetic acid (010778) and n-Hexane (091484) were obtained from BioLab Ltd. (Jerusalem, Israel). Phytocannabinoid analytical standards (>98%) CBG, THC, CBD, CBC, CBN, [[cannabigerolic acid]] (CBGA), [[tetrahydrocannabinolic acid]] (THCA), [[cannabidiolic acid]] (CBDA), cannabinolic acid (CBNA), [[cannabichromenic acid]] (CBCA), [[delta-8-Tetrahydrocannabinol|Δ<sup>8</sup>-tetrahydrocannabinol]] (Δ<sup>8</sup>-THC), [[tetrahydrocannabivarin]] (THCV), [[cannabidivarin]] (CBDV), cannabidivarinic acid (CBDVA), and [[cannabicyclol]] (CBL) were purchased from Sigma-Aldrich (Rehovot, Israel); [[cannabichromevarin]] (CBCV) was purchased from Cayman Chemical (Ann Arbor, MI, United States). Terpenoid analytical standards (>95% unless stated otherwise) were purchased from Sigma-Aldrich (Rehovot, Israel); [[valencene]] (>80% pure), α- and β-curcumene (>90% pure), [[Phellandrene|α-phellandrene]], and [[sabinene]] were purchased from Extrasynthese (Genay, France); a mixture of [[Alkane#Linear alkanes|''n''-alkanes]] was purchased from Sigma R 769 (40 mg/mL, C8-C20, Saint Louis, MO, United States) for semi-quantitative analysis.

===Experimental design===
The effect of fertilization was tested on two ''Cannabis sativa'' L. female plants. Female strains 333 THC-rich (15% THCA, 0.07% CBDA) and 423 CBD-rich (0.33% THCA, 9% CBDA) were subjected to fertilization, and two male plants strains—319 THC-rich (progeny of high THC landrace Highland Thai, Seedsman seeds) and 405 CBD-rich (progeny of Cherry CBD)—were used as pollen donors. In addition, the female plants were subjected to a sex conversion treatment<ref>{{Cite journal |last=Mohan Ram |first=H. Y. |last2=Sett |first2=R. |date=1982-12 |title=Induction of fertile male flowers in genetically female Cannabis sativa plants by silver nitrate and silver thiosulphate anionic complex |url=http://link.springer.com/10.1007/BF00275107 |journal=Theoretical and Applied Genetics |language=en |volume=62 |issue=4 |pages=369–375 |doi=10.1007/BF00275107 |issn=0040-5752}}</ref><ref>{{Cite journal |last=Small |first=Ernest |last2=Naraine |first2=Steve G. U. |date=2016-02 |title=Expansion of female sex organs in response to prolonged virginity in Cannabis sativa (marijuana) |url=http://link.springer.com/10.1007/s10722-015-0253-3 |journal=Genetic Resources and Crop Evolution |language=en |volume=63 |issue=2 |pages=339–348 |doi=10.1007/s10722-015-0253-3 |issn=0925-9864}}</ref>, and these induced males were also used as pollen donors to fertilize the two female plants. In order to achieve pollen sacs, 45-days old rooted cutting, 30 cm size female plants were sprayed daily until completely moist with ethylene inhibitor (Sodium Thiosulfate 0.5%) for five days prior to transferring to short day conditions. The female plants that were sex changed are referred to as males or induced-males. The female ''Cannabis'' plants were grown, three plants for each treatment, under an 18/6 light/dark regime (800 μmol), 23–27°C for 30 days before being transferred to flowering chambers with a 12/12 light/dark regime (500 μmol), 23–27°C for up to either 42 (six weeks) or 56 (eight weeks) days before some inflorescences were removed for chemical analysis. Female plants were grown in small flowering chambers (1 m<sup>2</sup>) in the presence of a single pollen donor. All plants were grown in 1 L pots on a mixture of pit/coconut 70%/30% soil, respectively.
l
The inflorescences of the treated plants, averaging 3–4 apical inflorescences per plant, were harvested and dried for 24–48 hours at 40°C until they reached a moisture content of 12% weight for weight (w/w). The inflorescences were ground to a fine powder using an electric grinder, then 98–103 mg were weighed and extracted with 1 mL ethanol. Samples were sonicated in an ultrasonic bath for 30 minutes, agitated in an orbital shaker at 25°C for 20 minutes, centrifuged at 20,000 x g for 5 minutes, then the samples were dissolved and diluted x20 in ethanol and filtered through a 0.22 μm Polytetrafluoroethylene syringe filter (Lumitron Ltd., Petah Tikva, Israel) prior to analysis.

===Phytocannabinoid identification and quantification===
Phytocannabinoid analyses for high concentrations of THC and CBD were performed using a Thermo Scientific UltiMate 3000 [[High-performance liquid chromatography#Pump pressure|ultra-high-performance liquid chromatography]] coupled with an [[Ultraviolet–visible spectroscopy|ultraviolet-visible]] [[Chromatography detector|diode array detector]] (UHPLC/UV) system. All other phytocannabinoids were identified and quantified by a similar UHPLC instrument coupled with a Q Exactive Focus Hybrid [[Quadrupole]]-[[Orbitrap]] MS (Thermo Scientific, Bremen, Germany), as previously described by Berman ''et al.''<ref name=":1" /> and Milay ''et al.''<ref>{{Cite journal |last=Milay |first=Looz |last2=Berman |first2=Paula |last3=Shapira |first3=Anna |last4=Guberman |first4=Ohad |last5=Meiri |first5=David |date=2020-10-15 |title=Metabolic Profiling of Cannabis Secondary Metabolites for Evaluation of Optimal Postharvest Storage Conditions |url=https://www.frontiersin.org/article/10.3389/fpls.2020.583605/full |journal=Frontiers in Plant Science |volume=11 |pages=583605 |doi=10.3389/fpls.2020.583605 |issn=1664-462X |pmc=PMC7593247 |pmid=33178249}}</ref> In short, chromatographic separation was achieved using a HALO C18 Fused-Core column (2.7 μm, 150 × 2.1 mm), with a HALO guard column (2.7 μm, 5 × 2.1 mm), and a ternary A/B/C multistep gradient (solvent A: water with 0.1% acetic acid; solvent B: acetonitrile with 0.1% acetic acid,; and solvent C: methanol). Identification and absolute quantification of phytocannabinoids were performed by external calibrations, as previously described by Berman ''et al.''<ref name=":1" /> Sixteen analytical standards (CBDVA, CBDA, CBCA, CBNA, CBGA, THCA, CBDV, CBD, CBC, CBN, CBG, THC, Δ8-THC, CBL, THCV, CBCV) were used for direct quantification and semi-quantification of additional phytocannabinoids. All extracted samples were injected and analyzed by an [[electrospray ionization]] (ESI)-LC/MS analysis, diluted at ratios of 1:9, 1:99, and 1:999 v/v ''Cannabis'' extract to ethanol.

===Terpenoids identification and quantification===
Profiling of terpenoids was performed using a modification of the [[Headspace gas chromatography for dissolved gas measurement|static headspace]] [[Gas chromatography–mass spectrometry#GC-tandem MS|gas chromatography–tandem mass spectrometry]] (SHS-GC/MS/MS) method by full evaporation technique.<ref name=":6" /> SHS-injections were performed by PAL RTC robotic tool (CTC Analytics, Swaziland) with 30 minute incubation time, temperature of 140°C, and 1,000 μL injection volume of the gas phase. Gas chromatographic separation was achieved in 74 minutes using a TRACE 1310 GC (Thermo Fisher Scientific, Bremen, Germany) equipped with a 30 m × 0.25 mm × 0.25 μm capillary DB-35MS UI column (Agilent Technologies, United States). MS/MS compound detection was performed by a TSQ 8000 Evo triple quadrupole mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). For terpenoids analyses, 10 mg of each ground ''Cannabis'' sample was weighed in duplicates in a 20 mL HS amber vial with 1.2 g of glycerol and sealed by a magnetic cap. Solutions for the construction of the calibration curves were prepared in hexane, and then 10 μL for each calibration level was added to amber vials with 1.2 g of glycerol in the same manner as the samples.

Some of the terpenoids were calculated semi-quantitatively based on the calibration curves of terpenoids with commercially available analytical standards with similar MS spectral characteristics and retention times. Identification of these terpenoids was performed by spectral searching against the NIST library (version 2.2) and relative Kovats retention indices using a mixture of ''n''-alkanes run under the same chromatographic conditions (for full details, see Supplementary material, Tables 2 and 3).

===Statistical analysis===
Statistical analyses were conducted using GraphPad Prism software version 8.2.1 (GraphPad Inc.). Differences between samples in phytocannabinoid and terpenoid concentrations were analyzed using two-way ANOVA followed by Dunnett’s multiple comparison test. ''P''-values were corrected for multiple testing using the Tukey ''post hoc'' test. A value of at least ''p'' ≤ 0.05 was considered significant for all tests (*''p'' ≤ 0.05, **''p'' ≤ 0.01, ***''p'' ≤ 0.001, ****''p'' ≤ 0.0001). Outliers were defined as data points greater than two standard deviations from the mean (9.6 for THCA and 9.5 for CBDA).

==Results==
===Phytocannabinoids quantity predominantly decreases after fertilization===
Mature inflorescences (six or eight weeks post-flower-induction) from female ''Cannabis'' plants of two distinct chemovars (Figures 1A and B), THC-rich (Type I) and CBD-rich (Type III), were subjected to fertilization by three different male ''Cannabis'' plant types: THC-rich (Figures 1C and D), CBD-rich (Figures 1E and F), or the original female plant induced to develop male pollen sacs by application of ethylene inhibitor (Figure 1). Induced-male plants (Figures 1G and H) were genetically identical to the female plants, and they had a distinct change in the sex of the flowers after treatment and a larger number of inflorescences compared to males (Figure 1I). Specific fertilization was achieved by incubation of the individual plants. (Figure 1J)


[[File:Fig1 Feder FrontPlantSci2021 12.jpg|1000px]]
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{| border="0" cellpadding="5" cellspacing="0" width="1000px"
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  | style="background-color:white; padding-left:10px; padding-right:10px;" |<blockquote>'''Fig. 1''' Study design. Differences in inflorescences between THC-rich and CBD-rich plants without ('''A''', '''B''') and after fertilization with either THC-rich male (strain 319) ('''C''', '''D''') or CBD-rich male (strain 405) ('''E''', '''F''') and their respective induced-male plants ('''G''', '''H'''). ('''I''') Representative image of a male donor plant (strain 405). To capture images, the plants were placed on the same white background and photographed individually. ('''J''') Experimental design. Female ''Cannabis'' plants of two distinct types, THC- or CBD-rich chemovars, were subjected to fertilization by three different male ''Cannabis'' plants: THC- or CBD-rich plants, and an induced-male plant achieved by application of ethylene inhibitor. Female and male plants were incubated together for six to eight weeks. The profile of their secondary metabolites was analyzed by UHPLC/UV and ESI-LC/MS for phytocannabinoids and SHS-GC/MS/MS for terpenoids.</blockquote>
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Fertilization resulted in a predominantly significant decrease of overall total phytocannabinoids concentration in inflorescences for both the THC-rich and CBD-rich females, by all three types of males (Figure 2A). The concentration of the phytocannabinoids was analyzed by UHPLC/UV and ESI-LC/MS. (The full list of the 95 phytocannabinoids quantified, as named by Berman ''et al.''<ref name=":1" />, is displayed in Supplementary material, Table 1.) A sharper decrease was detected in the THC-rich chemovar female, exhibiting an average 75% decrease, while CBD-rich females showed a 60% decrease in phytocannabinoid contents after fertilization. Next, we investigated changes in quantities of individual phytocannabinoids (Figures 2B–E). For the THC-female, fertilization caused a reduction in the abundant phytocannabinoids, whose concentrations in the plant were above 0.02%, except for the phytocannabinoid CBCA, which had an increase of about 50% when the plant was fertilized with an induced male (Figure 2B). Additional phytocannabinoids, whose concentrations in the plant were 0.001–0.2%, were also mostly reduced upon fertilization. The concentrations of CBC, cannabichromevarinic acid (CBCVA), and 373-15c were increased when fertilized by the induced male (Figure 2C). When THCA was excluded as an outlier, as its concentration is 15-fold higher, the less abundant phytocannabinoids 331-18b, CBG, CBDA, and 331-18d were significantly reduced upon fertilization. Similarly, for the CBD-female, fertilization caused a reduction in both the abundant (Figure 2D) and additional phytocannabinoids (when CBDA is excluded as an outlier) (Figure 2E), except for the concentrations of THCA and THC that increased after fertilization with the induced male.


[[File:Fig2 Feder FrontPlantSci2021 12.jpg|1000px]]
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  | style="background-color:white; padding-left:10px; padding-right:10px;" |<blockquote>'''Fig. 2''' Phytocannabinoids quantity predominantly decreases after fertilization with all types of males. ('''A''') Total phytocannabinoid concentrations and ('''B'''–'''E''') Individual phytocannabinoid concentrations after fertilization relative to unfertilized control. Abundant phytocannabinoid concentrations were considered > 0.2% ('''B''', '''D''') and additional phytocannabinoid concentrations were 0.001–0.2% ('''C''', '''E''') in the unfertilized plants. Data are presented as mean ± SEM (''n'' = 4–6, %w/w) and statistically analyzed by two-way ANOVA followed by Dunnett’s multiple comparison test (*''p'' ≤ 0.05; **''p'' ≤ 0.01; ***''p'' ≤ 0.001; ****''p'' ≤ 0.0001). Significance in '''C''' and '''E''' was calculated after excluding THCA and CBDA, respectively, from the data.</blockquote>
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===Terpenoids quantity decreases after fertilization in the cannabidiol-rich female plant but varies in the THC-rich female plant===
In addition to assessing the phytocannabinoid contents, we quantified over 100 terpenoid compounds. The THC- and CBD-rich female plants differed in their profile of terpenoids before fertilization (Supplementary material, Figure 1). About half of the quantified terpenoids had pure analytical standards available and were analyzed as previously described.<ref name=":6" /> However, out of a total of 113 terpenoids detected using (SHS-GC/MS/MS), 63 terpenoids in either the THC-rich or the CBD-rich plants did not have commercially available standards (for a full list, see Supplementary material, Table 4). Some of these terpenoids demonstrated significant changes after fertilization, therefore, we assessed them with a newly developed semi-quantitative analysis (Figure 3). In this manner, we quantified terpenoids such as [[Guaiene|δ-guaiene]] and [[Bisabolene|trans-α-bisabolene]] (denoted as 81 and 93, respectively). The semi-quantitative analysis is based on the calibration curves of terpenoids with commercially available analytical standards, relying primarily on similar MS spectral characteristics and also on retention times (Supplementary material, Figure 2).


[[File:Fig3 Feder FrontPlantSci2021 12.jpg|1200px]]
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  | style="background-color:white; padding-left:10px; padding-right:10px;" |<blockquote>'''Fig. 3''' Terpenoid profiles of ''Cannabis'' strains before and after fertilization. Overlay of chromatograms of the unfertilized and fertilized samples of ('''A''') THC-rich and ('''B''') CBD-rich female plants were performed by the same scales [retention time (RT); relative abundance of the signal intensity; weight of the samples (10 mg)], showing monoterpenoids on the left and sesquiterpenoids on the right. <sup>*</sup> Terpenoids that were semi-quantified.</blockquote>
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The total amount of terpenoids in the inflorescences was found to be chemovar-specific (Figure 4A). The high-CBD female plants exhibited two to threefold higher concentrations of terpenoids, both in the unfertilized and all three types of fertilized plants, compared to the THC-rich female plants. Upon fertilization, there were no significant changes in terpenoid accumulation in the THC-rich female. In the CBD-rich female plants, there was no significant change when fertilized with a THC-rich male plant, but fertilization with a CBD-rich male or an induced male resulted in a significant reduction in total terpenoids. The profile of terpenoids in plants is highly variable<ref name=":2" />, and being mostly volatile compounds, they are also more susceptible to changes due to sample preparation procedure, e.g., the freshness of samples.<ref>{{Cite journal |last=Livingston |first=Samuel J. |last2=Quilichini |first2=Teagen D. |last3=Booth |first3=Judith K. |last4=Wong |first4=Darren C. J. |last5=Rensing |first5=Kim H. |last6=Laflamme‐Yonkman |first6=Jessica |last7=Castellarin |first7=Simone D. |last8=Bohlmann |first8=Joerg |last9=Page |first9=Jonathan E. |last10=Samuels |first10=A. Lacey |date=2020-01 |title=Cannabis glandular trichomes alter morphology and metabolite content during flower maturation |url=https://onlinelibrary.wiley.com/doi/10.1111/tpj.14516 |journal=The Plant Journal |language=en |volume=101 |issue=1 |pages=37–56 |doi=10.1111/tpj.14516 |issn=0960-7412}}</ref> We detected an overall fertilization-dependent decrease in total terpenoid accumulation only in the CBD-rich plant, while the THC-rich plant showed a mixed trend of changes, either reduction or no significant change.


[[File:Fig4 Feder FrontPlantSci2021 12.jpg|1200px]]
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  | style="background-color:white; padding-left:10px; padding-right:10px;" |<blockquote>'''Fig. 4''' Terpenoid quantity varies after fertilization. Terpenoid concentrations as quantified by SHS-GC/MS/MS of ('''A''') total identified terpenoids, ('''B''') total monoterpenoids and ('''C''') total sesquiterpenoids. Data are reported as mean ± SEM of terpenoid concentrations (n = 3–4, ppm). Statistically significant differences between treatments and control (unfertilized) were calculated by two-way ANOVA followed by Dunnett’s multiple comparison test (*''p'' ≤ 0.05; **''p'' ≤ 0.01; ****''p'' ≤ 0.0001).</blockquote>
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Out of 113 terpenoids detected, 31 were monoterpenoids, built up by two isoprene units (10 carbons), and the rest were sesquiterpenoids, built up by three isoprene units (15 carbons).<ref name=":6" /> To further evaluate the influence of fertilization on terpenoid accumulation after fertilization, we analyzed these two distinct subgroups. Monoterpenoid concentrations were significantly reduced for the THC-rich female by 60–80% upon fertilization with all three types of male plants; for the CBD-rich female, there was a significant 50% reduction except for when fertilized by the induced-male, which left the concentrations unchanged (Figure 4B). The concentration of sesquiterpenoids was unchanged for the THC-rich female, but there was a trend of reduced concentrations in the CBD-rich fertilized female, which was statistically significant when fertilized with the CBD-rich or the induced male (Figure 4C).

===Individual terpenoid concentrations are differentially affected by fertilization===
Next, we set out to examine the accumulation of individual terpenoids in the plants (Figure 5) and found chemovar-specific differences. For the THC-rich female, the most abundant terpenoid was [[Caryophyllene|β-caryophyllene]], and its concentration was reduced upon fertilization (Figures 5A,B). For the CBD-rich female, the most abundant terpenoid was [[Bisabolol|α-bisabolol]], its concentration was above detection limit both before and after fertilization (Figures 5C,D). Moreover, we noticed that the terpenoid profile changed during the length of the flowering time, between six to eight weeks after fertilization. This was in contrast to the phytocannabinoids profile, which was more consistent between these two time-points (data not shown). For example, for the CBD-rich female, the sesquiterpenoid [[Caryophyllene|caryophyllene oxide]] had a very low concentration in the six-week flowering plant but became highly abundant in the eight-week plant (Figures 5C,D). Hence, in addition to chemovar-specific differences, differential accumulation was observed between six- and eight-week growth in the same chemovar.


[[File:Fig5 Feder FrontPlantSci2021 12.jpg|1200px]]
{{clear}}
{| 
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{| border="0" cellpadding="5" cellspacing="0" width="1200px"
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  | style="background-color:white; padding-left:10px; padding-right:10px;" |<blockquote>'''Fig. 5''' Individual terpenoid concentrations in THC- and CBD-rich female plants at six or eight weeks. Abundant terpenoids in the unfertilized female flowers and their concentrations at six weeks ('''A''', '''C''') and eight weeks ('''B''', '''D'''). Data are reported as mean ± SEM of terpenoid concentration (''n'' = 2–3, except THC-female fertilized by induced male (THC) at six weeks). Statistically significant differences between treatments and control (unfertilized) were calculated by two-way ANOVA, followed by Dunnett’s multiple comparison test (*''p'' ≤ 0.05; **''p'' ≤ 0.01; ***''p'' ≤ 0.001; ****''p'' ≤ 0.0001). Values presented without SEM exceeded the maximal detection limit (maximum limits of detection for terpenoids appear in Supplementary material, Table 5).</blockquote>
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As seen in Figure 5, numerous terpenoids significantly decreased following fertilization. However, several specific terpenoids showed an interesting increase in concentration after fertilization. For example, in the THC-rich female, members of the eudesmol family of sesquiterpenoids (α-, β-, and γ-eudesmol) were mostly undetected in the unfertilized plant, but their concentrations were significantly increased upon fertilization by the THC male plant only, both at six and eight weeks after fertilization (Figure 6A). Interestingly, the levels of these terpenoids were either reduced or unchanged in the CBD-rich female due to fertilization processes. In contrast, in the CBD-rich female, the monoterpenoid [[Linalool|linalool]] significantly increased upon fertilization by the induced male, but was reduced or unchanged following all other fertilization processes in both plant chemovars (Figure 6B).


[[File:Fig6 Feder FrontPlantSci2021 12.jpg|1200px]]
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{| border="0" cellpadding="5" cellspacing="0" width="1200px"
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  | style="background-color:white; padding-left:10px; padding-right:10px;" |<blockquote>'''Fig. 6''' Specific terpenoids are increased following fertilization. (''A'') α-, β-, and γ-eudesmol and ('''B''') linalool concentrations in THC-rich and CBD-rich females six and eight weeks after fertilization (''n'' = 2–3, except THC-female fertilized by induced male [THC] at six weeks). Statistically significant differences between treatments and control (unfertilized) were calculated by two-way ANOVA followed by Dunnett’s multiple comparison test (*''p'' ≤ 0.05; ****''p'' ≤ 0.0001).</blockquote>
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==Discussion==
The present study was designed to examine the influence of flower fertilization on the accumulation of ''Cannabis'' secondary metabolites. The primary outcome is the significant overall decrease in phytocannabinoid metabolites upon fertilization. This decrease was evident in almost all phytocannabinoids measured, regardless if those were the abundant ones or the relatively low accumulating components (Figure 2). Though the altogether amount of phytocannabinoids is drastically reduced, the ratio between the different compounds is kept and their profile in the plant remains principally unchanged.

Terpenoid concentrations mostly decreased but varied. While monoterpenoids had a similar decrease as portrayed by the phytocannabinoids, sesquiterpenoids exhibited a more diverse pattern, some of which increased and some decreased upon fertilization (Figure 3). However, examining specific metabolites can point to several phytocannabinoids or terpenoids that have an individual trend, suggesting a more complex regulatory network (Figures 4 and 5).

First, these results confirm that when the objective is to maintain high levels of phytocannabinoids, fertilization must be avoided. Apart from a physical separation between female and male flowers or vegetative reproduction, this goal could be achieved using advanced genetic manipulations that target female fertilization pathways.<ref>{{Cite journal |last=Huang |first=Jian |last2=Smith |first2=Ashley R. |last3=Zhang |first3=Tianyu |last4=Zhao |first4=Dazhong |date=2016-02-01 |title=Creating Completely Both Male and Female Sterile Plants by Specifically Ablating Microspore and Megaspore Mother Cells |url=http://journal.frontiersin.org/article/10.3389/fpls.2016.00030 |journal=Frontiers in Plant Science |volume=7 |doi=10.3389/fpls.2016.00030 |issn=1664-462X |pmc=PMC4740954 |pmid=26870055}}</ref><ref>{{Cite journal |last=Jung |first=Yu Jin |last2=Kim |first2=Dong Hyun |last3=Lee |first3=Hyo Ju |last4=Nam |first4=Ki Hong |last5=Bae |first5=Sangsu |last6=Nou |first6=Ill Sup |last7=Cho |first7=Yong-Gu |last8=Kim |first8=Myong Kwon |last9=Kang |first9=Kwon Kyoo |date=2020-09-11 |title=Knockout of SlMS10 Gene (Solyc02g079810) Encoding bHLH Transcription Factor Using CRISPR/Cas9 System Confers Male Sterility Phenotype in Tomato |url=https://www.mdpi.com/2223-7747/9/9/1189 |journal=Plants |language=en |volume=9 |issue=9 |pages=1189 |doi=10.3390/plants9091189 |issn=2223-7747 |pmc=PMC7570381 |pmid=32933074}}</ref><ref>{{Cite journal |last=Zhu |first=Haocheng |last2=Li |first2=Chao |last3=Gao |first3=Caixia |date=2020-11 |title=Applications of CRISPR–Cas in agriculture and plant biotechnology |url=https://www.nature.com/articles/s41580-020-00288-9 |journal=Nature Reviews Molecular Cell Biology |language=en |volume=21 |issue=11 |pages=661–677 |doi=10.1038/s41580-020-00288-9 |issn=1471-0072}}</ref>

Second, this study revealed the resemblance between monoterpenoids and phytocannabinoids accumulation patterns. Both secondary metabolite species are decreased upon fertilization, while sesquiterpenoids are differently influenced. Possible explanations for this similarity are common intracellular regulation pathways or shared morphological structures. From a cellular perspective, monoterpenoids and phytocannabinoids share the common biosynthetic precursor geranyl diphosphate (GPP) and are both biosynthesized in the plastid.<ref name=":2" /><ref name=":7" /> In contrast, sesquiterpenoids are synthesized in the cytosol from a different precursor (farnesyl pyrophosphate or FPP). This suggests that phytocannabinoids and monoterpenoids may share a common regulation mechanism, involving an enzymatic step upstream to GPP, such as GPP synthase (illustrated in Figure 7).


[[File:Fig7 Feder FrontPlantSci2021 12.jpg|1200px]]
{{clear}}
{| 
 | style="vertical-align:top;" |
{| border="0" cellpadding="5" cellspacing="0" width="1200px"
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  | style="background-color:white; padding-left:10px; padding-right:10px;" |<blockquote>'''Fig. 7''' Phytocannabinoids and terpenoids biosynthesis pathways. Fertilization affects the MEP pathway in an enzymatic step upstream to GPP synthase. MEP, Methylerythritol phosphate; MVA, Mevalonate; G3P, Glyceraldehyde 3-phosphate; DMAPP, Dimethylallyl diphosphate; IPP, Isopentenyl diphosphate; GPP, Geranyl diphosphate; FPP, Farnesyl diphosphate; GPPs, GPP synthase; FPPS, FPP synthase; TPSs, Terpene synthases.</blockquote>
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Alternatively, from a morphological perspective, previous studies have shown that although phytocannabinoids, monoterpenoids, and sesquiterpenoids are all biosynthesized and accumulated in the glandular trichomes, their distribution differentiates during trichome development and between trichome types. A recent study by Booth ''et al.''<ref name=":2" /> showed an increase in the ratio of monoterpenoids relative to sesquiterpenoids when flowers are maturing. Another study by Livingston ''et al.''<ref name=":3" /> showed that monoterpenoids are accumulated in both pre-stalked and stalked trichomes, while sesquiterpenoids are abundant in sessile trichomes. Phytocannabinoids are accumulated in both types of trichomes, but the stalked type composed 80–90% of the total trichomes in the mature flower. A common accumulation pattern of monoterpenoids and phytocannabinoids during flower development was also previously demonstrated.<ref>{{Cite journal |last=Aizpurua-Olaizola |first=Oier |last2=Soydaner |first2=Umut |last3=Öztürk |first3=Ekin |last4=Schibano |first4=Daniele |last5=Simsir |first5=Yilmaz |last6=Navarro |first6=Patricia |last7=Etxebarria |first7=Nestor |last8=Usobiaga |first8=Aresatz |date=2016-02-26 |title=Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes |url=https://pubs.acs.org/doi/10.1021/acs.jnatprod.5b00949 |journal=Journal of Natural Products |language=en |volume=79 |issue=2 |pages=324–331 |doi=10.1021/acs.jnatprod.5b00949 |issn=0163-3864}}</ref> Parallel accumulation and decrease of phytocannabinoids and monoterpenoids in contrast to sesquiterpenoids may suggest that trichome types are differently affected by fertilization, and hence the diversity in metabolite accumulation.

An additional major finding depicted in this study is the somewhat dependent outcome of the fertilization process on the pollen donor plant. Both THC- or CBD-rich male plants, whether naturally occurring or female-induced, had a different impact on the metabolite concentration in the female after fertilization. For instance, fertilization by the induced-male led to an increase of specific phytocannabinoids (Figure 2): THC and THCA in the CBD female, and CBC, CBCA, CBCVA, and 373-15C in the THC female. The exact mechanism by which these phytocannabinoids are increased is not yet clear. It may be the result of altered regulation of synthesis enzymes, for example the upregulation of THCA synthase or CBCA synthase.<ref name=":10" /> A previous study found over 10,000 genes are differentially expressed upon masculinization of female plants<ref>{{Cite journal |last=Adal |first=Ayelign M. |last2=Doshi |first2=Ketan |last3=Holbrook |first3=Larry |last4=Mahmoud |first4=Soheil S. |date=2021-01 |title=Comparative RNA-Seq analysis reveals genes associated with masculinization in female Cannabis sativa |url=http://link.springer.com/10.1007/s00425-020-03522-y |journal=Planta |language=en |volume=253 |issue=1 |pages=17 |doi=10.1007/s00425-020-03522-y |issn=0032-0935 |pmc=PMC7779414 |pmid=33392743}}</ref>, but it is not clear how these genes are related to phytocannabinoid expression in the fertilized female plant. A donor-dependent effect was also detected in the specific increase in the eudesmol family components, which were highly increased in the THC-rich female upon fertilization by the THC-rich male plant (Figure 6A) and a parallel specific increase in linalool in the CBD-rich female fertilized by the induced male (Figure 6B). However, regardless of the type of male plant used for fertilization, the overall profile of the phytocannabinoids in the fertilized female plant remained unaltered, i.e., no new phytocannabinoids that were not expressed in the unfertilized plant were discovered, and the relative ratio between the different phytocannabinoids was mostly kept. Interestingly, though the density of phytocannabinoids and terpenoids in males is minor (data not shown) compared to the female flowers, with high potency female plants showing 10 to 20 times more THC than corresponding males<ref>{{Cite book |last=Clarke, R.; Merlin, M. |year=2016 |title=Cannabis: Evolution and Ethnobotany |url=https://www.ucpress.edu/book/9780520292482/cannabis |edition=First |publisher=University of California Press |pages=456 |isbn=9780520292482}}</ref>, male plants also possess a distinct profile of these compounds.

==Conclusion==
Here, we used highly advanced analytical methods to thoroughly assess the composition of 95 phytocannabinoids and 113 terpenoids in the inflorescences of female plants fertilized by different males, including the female plant itself induced to develop male pollen sacs. We found that fertilization significantly decreased phytocannabinoids content, while terpenoids were differentially affected. To further elucidate the effect of fertilization on the secondary metabolite accumulation, future studies that follow the gene expression of enzymes upstream to GPP after fertilization may allow exposing master regulators of the biochemical pathways. In addition, better characterization of the morphological changes following fertilization may shed light on how different trichome types are affected by fertilization. Finally, the variance in metabolites observed by fertilization with different male plants may suggest that the pollen itself or the developing embryo influence the female sporophyte.

Altogether, one must remember that these specialized secondary metabolites have an important role ''in planta'', increasing the plant fitness to the environment.<ref>{{Cite journal |last=Huchelmann |first=Alexandre |last2=Boutry |first2=Marc |last3=Hachez |first3=Charles |date=2017-09 |title=Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest |url=https://academic.oup.com/plphys/article/175/1/6-22/6117034 |journal=Plant Physiology |language=en |volume=175 |issue=1 |pages=6–22 |doi=10.1104/pp.17.00727 |issn=0032-0889 |pmc=PMC5580781 |pmid=28724619}}</ref> The substantial decrease in phytocannabinoids and terpenoids after fertilization may point to their functional roles in the plant. The actual functions of phytocannabinoids and terpenoids in ''Cannabis'' were only sparsely studied, mainly suggesting roles in defense against biotic or abiotic factors<ref name=":11" />, protection from UV radiation<ref>{{Cite journal |last=Eichhorn Bilodeau |first=Samuel |last2=Wu |first2=Bo-Sen |last3=Rufyikiri |first3=Anne-Sophie |last4=MacPherson |first4=Sarah |last5=Lefsrud |first5=Mark |date=2019-03-29 |title=An Update on Plant Photobiology and Implications for Cannabis Production |url=https://www.frontiersin.org/article/10.3389/fpls.2019.00296/full |journal=Frontiers in Plant Science |volume=10 |pages=296 |doi=10.3389/fpls.2019.00296 |issn=1664-462X |pmc=PMC6455078 |pmid=31001288}}</ref>, prevention of desiccation<ref name=":9" />, or induction of cell death in leaves.<ref>{{Cite journal |last=Morimoto |first=Satoshi |last2=Tanaka |first2=Yumi |last3=Sasaki |first3=Kaori |last4=Tanaka |first4=Hiroyuki |last5=Fukamizu |first5=Tomohide |last6=Shoyama |first6=Yoshinari |last7=Shoyama |first7=Yukihiro |last8=Taura |first8=Futoshi |date=2007-07 |title=Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells |url=https://linkinghub.elsevier.com/retrieve/pii/S0021925819780661 |journal=Journal of Biological Chemistry |language=en |volume=282 |issue=28 |pages=20739–20751 |doi=10.1074/jbc.M700133200}}</ref> The observed dynamics of the accumulation of these metabolites during flower development and fertilization may point to their different roles along the plant’s life cycle.

==Supplementary material==
The supplementary material for this article can be found online at [https://www.frontiersin.org/articles/10.3389/fpls.2021.753847/full#supplementary-material frontiersin.org].

==Acknowledgements==
We thank Dr. Ari Feder for helpful discussions and insights.

===Author contributions===
PB, MF, and DM: conception and design. OC, IK, and RP: plant maintenance, manipulations, fertilization, and harvest. CL: acquisition and ESI-LC/MS analysis and interpretation of data. AS: acquisition of GC data and development of semi-quantitative GC methodology. OG: extraction and sample preparation of Cannabis samples and UHPLC/UV analysis. CL, AS, and SP: figure preparation. CL, SP, PB, MF, and DM: writing, review, and revision of the manuscript. MF and DM: study supervision. All authors contributed to the article and approved the submitted version.

===Data availability statement===
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

===Conflict of interest===
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

==References==
{{Reflist|colwidth=30em}}

==Notes==
This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added. The original article lists references in alphabetical order; however, this version lists them in order of appearance, by design.

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