July 31, 2015

Tutorial: Mass spectrometry in plant science, part 4 – measuring ethylene gas by GC-MS

In this post I would like to discuss a specific application of mass spectrometry to plant science, that of measuring the concentration of ethylene gas in head space samples. Ethylene gas is a plant hormone with numerous roles in plant development and defense. Climacteric fruits produce ethylene when they enter the ripening phase, and so for agronomists it can be very important to measure ethylene in order to monitor the progression of fruit development. Its chemical structure can be represented as CH2=CH2 and is the simplest alkene in nature. Because it is so volatile and present at such low concentrations, it is difficult to measure. A number of dedicated infrared and laser detectors can be found on the market. They may or may not offer the kind of stability and reproducibility that is required for precision analysis. For labs that do not have the funds to buy a dedicated, high end detector,  the default analytical technique to measure ethylene is a standard GC with a packed column connected to a FID (flame ionization detector). This technique has the virtue of being highly reproducible and sensitive enough for most applications. It takes advantage of the fact that ethylene produces an electrical signal when it burns at the detector, unlike the oxygen and nitrogen in the air which it is dissolved in. This is a tremendous advantage over mass spectrometry in this case because due to an unfortunate cosmic coincidence, both ethylene and nitrogen gas have a molecular mass of 28, meaning they are indistinguishable at unit resolution. This can make it nearly impossible to use mass spectrometry to pick out low levels of ethylene in a head space sample where the nitrogen is a million times as concentrated. And in practice it isn't so easy to separate the two on normal stationary column phases. An aluminum oxide column is probably the best phase currently available. However, nowadays, a GC system hooked up to a mass spectrometer is probably far more common that one connected to a FID, so many labs that might like to analyze ethylene production from plants have an analytical tool that is perhaps too sophisticated to get the job done, at least without a few tricks.  Here I offer a modest technical innovation that makes GC-MS nonetheless a viable technique that is comparable to a GC-FID for this task and represents another way that mass spectrometry can enrich our investigations in the plant sciences.

Conn et al. "Convergent evolution of strigolactone perception enabled host detection in parasitic plants"

Caitlin E. Conn1, Rohan Bythell-Douglas2, Drexel Neumann1, Satoko Yoshida3, Bryan Whittington4, 4, Ken Shirasu3, Charles S. Bond2, Kelly A. Dyer1, David C. Nelson1,*
James H. Westwood
1Department of Genetics, University of Georgia, Athens, GA 30602, USA
2School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia 6009, Australia
3RIKEN Center for Sustainable Resource Science, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
4Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061, USA
Science 31 July 2015: Vol. 349 no. 6247 pp. 540-543 DOI: 10.1126/science.aab1140

Here's an interesting story on the perception of strigolactones (plant branching hormones) and how the normal physiological process of sensing these hormones has been co-opted  by sneaky parasites. Conn et al. find that a modern day clade of an ancient paralog of the Arabidopsis strigolactone receptor D14 is overrepresented in the genomes of certain parasitic plants. While derived from the same ancient paralog KAI2, the KAI2d clade members are used by obligate parasites to locate host plants. KAI2d induces germination in Orobanchaceae members by sensing strigolactones in host plant root exudates so that these parasites only germinate when a suitable host is near. In other words, while D14 is used by plants like Arabidopsis to sense their own strigolactone production and respond accordingly by initiating branching, parasitic plants use a very similar receptor with a common evolutionary origin to sense the same hormone but in this case to locate a host to parasitize. In a strange case of convergent evolution, both modern day clades (D14 and KAI2d) function as strigolactone sensors but for very different ends.

From the article:
Obligate parasitic plants in the Orobanchaceae germinate after sensing plant hormones, strigolactones, exuded from host roots. In Arabidopsis thaliana, the α/β-hydrolase D14 acts as a strigolactone receptor that controls shoot branching, whereas its ancestral paralog, KAI2, mediates karrikin-specific germination responses. We observed that KAI2, but not D14, is present at higher copy numbers in parasitic species than in nonparasitic relatives. KAI2 paralogs in parasites are distributed into three phylogenetic clades. The fastest-evolving clade, KAI2d, contains the majority of KAI2 paralogs. Homology models predict that the ligand-binding pockets of KAI2d resemble D14. KAI2d transgenes confer strigolactone-specific germination responses to Arabidopsis thaliana. Thus, the KAI2 paralogs D14 and KAI2d underwent convergent evolution of strigolactone recognition, respectively enabling developmental responses to strigolactones in angiosperms and host detection in parasites.

July 17, 2015

Stapelia asterias in bloom: this flower stinks

I have this beautiful succulent on my balcony, Stapelia asterias, which is currently blooming. I always appreciated the tiger-like pattern to the petals, but as a member of the carrion flower group, its odor is apparently supposed to resemble rotting meat to attract carrion flies, its pollination vector. As a bonus, it also smells like poop. I learned this one day when I cut some freshly opened flowers and brought them inside to admire. It is better to admire them through a closed window as it turns out. Would a carrion flower by any other name smell as putrid?

July 8, 2015

Magnard et al. "Biosynthesis of monoterpene scent compounds in roses"

Jean-Louis Magnard1, Aymeric Roccia1,2, Jean-Claude Caissard1, Philippe Vergne2, Pulu Sun1, Romain Hecquet1, Annick Dubois2, Laurence Hibrand-Saint Oyant3, Frédéric Jullien1, Florence Nicolè1, Olivier Raymond2, Stéphanie Huguet4, Raymonde Baltenweck5, Sophie Meyer5, Patricia Claudel5, Julien Jeauffre3, Michel Rohmer6, Fabrice Foucher3, Philippe Hugueney5,*, Mohammed Bendahmane2,*, Sylvie Baudino1,*

1Laboratoire BVpam, EA3061, Université de Lyon/Saint-Etienne, 23 Rue du Dr Michelon, F-42000, Saint-Etienne, France 
2Laboratoire Reproduction et Développement des Plantes UMR Institut National de la Recherche Agronomique (INRA)–CNRS, Université Lyon 1-ENSL, Ecole Normale Supérieure de Lyon, 46 Allée d’Italie, 69364 Lyon Cedex 07, France. 
3INRA, Institut de Recherche en Horticulture et Semences (INRA, AGROCAMPUS-OUEST, Université d’Angers), SFR 4207 QUASAV, BP 60057, 49071 Beaucouzé Cedex, France
4Génomiques Fonctionnelles d’Arabidopsis, Unité de Recherche en Génomique Végétale, UMR INRA 1165–Université d’Evry Val d’Essonne–ERL CNRS 8196, Evry, France
5INRA, Université de Strasbourg, UMR 1131 Santé de la Vigne et Qualité du Vin, 28 Rue de Herrlisheim, F-68000 Colmar, France
 6Université de Strasbourg–CNRS, UMR 7177, Institut Le Bel, 4 Rue Blaise Pascal, 67070 Strasbourg Cedex, France

This outstanding report by Magnard, et al addresses the origin of monoterpenoid scent compounds in rose petals. Roses breed for sale as cut flowers are arguably the world's most important ornamental plants, and descriptions of their scents has inspired poets and artists for centuries or longer. However, the aggressive breeding which has improved the color and longevity of cut flowers has also occasionally resulted in hybrids which lack the same aromatic bouquets of more classic cultivars such as Papa Mailland. Magnard and co-workers exploited these induced genetic differences by comparing high and low scent varieties to identify differentially expressed genes that correlated with the accumulation of monoterpene alcohols typical of high quality rose scents. Geraniol, for instance, is an important monoterpene alcohol in rose oil, and geraniol synthase has been identified in basil, cinnamon, and many other plant species.  Ordinarily, we would just look at rose EST databases and look for terpene synthases, which are now some of the best studied catalysts in  the field of natural products with well known signature motifs at the amino acid level.That should lead us to a likely geraniol synthase candidate in rose.

July 7, 2015

Fund my research and win the world cup

I have been seeking a faculty position for the last few years to expand my research activities, hopefully before my current contract runs out. I'm nearly out of time, and it's a little scary. So far, it looks pretty grim. If I start to really think about the statistics behind getting a tenured faculty position, it is depressing. It is a little like becoming a professional athlete in one of the big U.S. sports (football, baseball, and basketball; or soccer in the rest of the world). Starting out in age group play is a little like being an undergraduate, and moving into graduate school and then the first post-doctoral position is perhaps comparable to moving up into more competitive leagues in professional sports. Getting tenure would be like playing in the NFL or NBA. Winning the world cup would, to carry this absurd analogy further, be like winning a Nobel prize. Fortunately, I have a way to virtually guarantee a world cup victory, and all I ask is a mere faculty position.

Changes to the blog after first year: title change

I have only kept up a pace of about one post per two weeks during the first year of trying out blogging, which indicates the limited amount of free time I have to dedicate to this side interest if nothing else. This blog was originally entitled "Plant biology review", but Botany is essentially the biology of plants. Since there is a heavy emphasis on chemistry covered here, I have decided to change the title to "Chemical Botany" to reflect this fact and shorten it a bit. The URL will remain the same however, so any one reading this need not update their bookmarks.

Echinopsis pachanoi (San Pedro cactus) flowering on my balcony

Like most succulent cactus, San Pedro doesn't flower very often but produces large, showy flowers with a spectacular aromatic bouquet when it does. Unfortunately, the flowers are ephemeral and seldom last more than a day. I've had this specimen for about five years and this is the first flowering I've seen from it. Here is a brief sequence of photos taken every few weeks over the 7-8 week course of its development. Two buds were gnawed off by insects or birds (or bats?) and fell off early on, so only one made it to full flowering. It's a long time to wait for only a single day of this brilliant trumpet shaped flower, but the fragrance really is incredible. If any more bloom this summer, I will be sure to take the SPME fiber home from the lab with me for some volatile collection. I expect fruity esters typical of lily are well represented in its bouquet as well as abundant benzenoids. And, of course, a few terpenoids have to be there as well.
Note the dense, hairy bracts around the base of the flower during the early stages of development.

Click below to see how the story ends...

May 26, 2015

Tutorial: Mass spectrometry in plant science, part 3 – Calculating % atom labeling by mass spectrometry in isotopic labeling experiments

Labeling with stable isotopes is the best method for calculating the flux of metabolites through biological systems. 13C is the isotope of choice for most small organic metabolites, and labeling with 13CO2 under physiological conditions gives us the most realistic picture of how the plant's metabolism works in its natural environment. This approach is known as whole plant kinetics . In order to calculate the degree of label incorporation into the pool of metabolites following a labeling experiment, it is first necessary to extract those metabolites from labeled plant tissue and separate them by gas or liquid chromatography. We can then collect the spectra one by one as they elute from the column and enter the mass spectrometer. The resulting mass spectra tell us the pool sizes of the different isotopologs of a given metabolite (i.e. related chemical species which differ only in that they bear one or more 13C isotopes compared to the all 12C reference molecule). Depending on how the molecule fragments under a typical ionization technique like electron impact, it may be impossible to say where in the molecule these 13C isotopes are located by mass spectrometry; we can only say how many isotopic labels are present. We can however determine the relative abundances of labeled species that have 1, 2, 3, or more isotopes compared to a reference compound (usually unlabeled). But this spectral data still doesn’t tell us what we really would like to know following a labeling experiment: what percentage of the total carbon atoms in a metabolite pool are represented by 13C instead of 12C?

May 8, 2015

Pandanus candelabrum as an indicator species for diamond rich kimberlite pipes in West African jungles

There is a report this month in the journal Economic Geology on a little known tropical plant species in the Pandanaceae family. In West Africa, it apparently grows in kimberlite rich soils. Since kimberlite is a mineral which is typically rich in diamonds, this is a significant discovery in economic botany. Unfortunately, the article, a single author contribution by Stephen Haggerty, is behind a pay wall, and I have not obtained a copy yet. There is a nice summary in Science by Eric Hand that explains some of the geology behind diamond formation, deep below the surface. I did not know, for instance, that diamonds are formed hundreds of kilometers below the surface and reach the top by being pushed through pipes of kimberlite in explosive events, sometimes at speeds greater than the speed of sound.

May 1, 2015

A U.S. News and World Report article glorifies Goodall's embrace of anti-GMO nonsense

Anna Medaris Miller, writing for U. S. News and World report, gives what I hope was unintended free advertising to a dismal new book on anti-GMO quackery by Steven M. Druker ("Altered Genes, Twisted Truth"-scared yet?) and endorsed by Jane Goodall, a once great scientist currently destroying her legacy with woo. Using what is now a formulaic, emotional, fact-free, "think of the children" marketing approach to peddle absolute non-sense to a public confused over a complex scientific topic, Druker displays an intellectual honesty comparable to those who sell books on secret cures for cancer your doctor doesn't want you to know about.

April 17, 2015

Strand et al. "Activation of cyclic electron flow by hydrogen peroxide in vivo"

Deserah D. Strand, Aaron K. Livingston, Mio Satoh-Cruz, John E. Froehlich, Veronica G. Maurino, and David M. Kramer

From the article:

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H2O2. We thus tested the hypothesis that CEF can be activated by H2O2 in vivo. CEF was strongly increased by H2O2 both by infiltration or in situ production by chloroplast-localized glycolate oxidase, implying that H2O2 can activate CEF either directly by redox modulation of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H2O2, suggesting activation of previously expressed CEF-related machinery. H2O2-dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H2O2. We propose that H2O2 is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants. 

April 9, 2015

González-Cabanelas et al. "The diversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate from the 2-C-methyl-D-erythritol 4-phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana"

Diego González-Cabanelas, Louwrance P. Wright, Christian Paetz, Nawaporn Onkokesung, Jonathan Gershenzon, Manuel Rodríguez-Concepción and Michael A. Phillips*
The Plant Journal, Volume 82, Issue 1, pages 122–137, April 2015 DOI:10.1111/tpj.12798

Today I would like to highlight a publication of my own, as self-serving as that might be. This is the product of a Master's project carried out by my former student Diego González-Cabanelas, who has since gone to a PhD program at the Max Planck Institute of  Chemical Ecology (Jena, Germany). In this report, we describe a metabolic "shunt" funneling carbon flux away from the main isoprenoid precursor pathway in the chloroplast when flux is elevated. That's pretty important information if you are trying to metabolically engineer this pathway and don't understand why so much of the expected flux seems to be disappearing when we use the standard 'upregulation of structural genes' approach. An unusual cyclic diphosphate intermediate in the pathway has been recently alleged to have signaling properties, activating defense gene expression in the nucleus. Naturally, this would require this metabolite to physically leave the confines of the plastid and interact with other factors in the cytosol, nucleus, or elsewhere to control gene expression. It is also quite strange that a metabolic intermediate in a pathway would be selected to play such a different parallel role in defense signaling, so the actual story may be much more complex. Due to our limited knowledge of how isoprenoid biosynthesis is regulated at the molecular level, we are still making early discoveries into how this biosynthetic network is controlled. I like to think this is one of them.

March 6, 2015

Tutorial: Mass spectrometry in plant science, part 2 – using isotopic labels to study plant metabolism

In this second entry on the use of mass spec in plant research, I want to introduce isotopic labeling and its power to inform us about plant metabolic processes quantitatively. By feeding an isotopically labeled substrate into the metabolic stream of an organism and observing it later in some downstream metabolic intermediate, we can gain insights into the rates at which these transformations take place inside the cell. Experimentally, we observe these isotopic labels as shifts to heavier m/z values in the mass spectrum of molecules that incorporate them. As isotopically labeled atoms introduced in our labeling experiment migrate through the different metabolites in a biochemical pathway, we see specific mass peaks rise and fall in a spectrum over time that are indicative of the turnover of that pool of metabolites. This is a complex subject that has been covered in detail with precisely the mathematical rigor you might expect (see for example publications by Wolfgang Wiechert on metabolic flux analysis). In this short monograph, I can only hope present how well mass spectral analysis lends itself to quantifying plant metabolism.

March 5, 2015

Zhang et al. "Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids"

Jiang Zhang, Sher Afzal Khan, Claudia Hasse, Stephanie Ruf, David G. Heckel, Ralph Bock*

Science 27 February 2015:Vol. 347 no. 6225 pp. 991-994 DOI:10.1126/science.1261680

Crop protection in large scale agriculture is a necessity given the many unintended consequences of traditional breeding. One of these consequences was the elimination of many natural plant defenses against insects, which consisted partly of the accumulation of bitter and poisonous secondary metabolites which made them unappealing for insects to eat. In our aeons long quest to make a better tasting tomato, our breeding experiments naturally resulted in food that tasted better to us by eliminating those same bitter, poisonous metabolites. And insects could not thank us more for our efforts, as they find our domesticated plants even tastier than their wild type cousins. A considerable amount of scientific energy has therefore been invested in devising strategies that protect crops intended to fill our pantries from their natural enemies: the rest of nature.

February 17, 2015

New edition of Taiz and Zeiger's classic text now available "Plant Physiology and Development" (Sinauer Assoc. Publishing)

When I took Plant Physiology as an undergraduate twenty years ago, we used Taiz and Zeiger's 1991 first edition text of the same name. The chapter on terpenoids was written by Jonathan Gershenzon, who at the time was a lab director for Prof. Rodney Croteau at the Institute for Biological Chemistry at Washington State University. Reading that chapter changed my life, as I became interested in this family of natural products as a result and later completed my PhD in Rod's lab. Later as  apost-doc I went to work for Jonathan, who by then had become a Director at the Max Planck Institute for Chemical Ecology in Jena, Germany. It was a profoundly rewarding experience. In fact, I am still studying plant terpenoid biosynthesis as a struggling independent researcher in the harsh landscape of Spain in the age of austerity. It is a profoundly difficult experience. All of those experiences, both good and challenging, are indirectly the result of reading that influential chapter as an undergraduate and feeling a fascination for a subject that no other really compared to at that time.

February 13, 2015

Keith Kloor: "Agricultural researchers rattled by demands for documents"

Until now I have resisted any temptation to address political issues in the modest endeavor that is this blog. Instead, I have focused exclusively on recent reports in the plant literature that might have importance to plant science professionals or students of plant biology. I also include research seminars from eminent scientists that visit our institute. In addition, I have a number of educational posts either up or in the works, which I hope to complete between awaiting manuscripts that want finishing. Today, however, I would like to mention a political issue with some relevance to plant science since it impacts the way we conduct research and reveals an important chasm between the public understanding of what we do and what we do. I refer to a recent news item by journalist Keith Kloor published this week in Science.

Zhou et al. "Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis "

Xiangjun Zhou, Ralf Welsch, Yong Yang, Daniel Álvarez, Matthias Riediger, Hui Yuan, Tara Fish, Jiping Liu, Theodore W. Thannhauser, and Li Li

Phytoene synthase (PSY) has long been considered the rate limiting step in the formation of carotenoids, C40 isoprenoid compounds which act as auxiliary pigments in photosynthesis, quenching highly excited chlorophyll and preventing photooxidative damage to chloroplasts. In the human diet, carotenoids are considered both essential nutrients (beta-carotene serves as provitamin A) as well as generally beneficial antioxidant neutraceuticals due to their electron donating (and free radical scavenging) properties. Increasing the accumulation of carotenoids in crops has been a biotechnological goal for some time, and it is clear that PSY modulation will be an important part of this process. But the control of flux though the long and fairly complex carotenoid biosynthetic pathways is tightly regulated, resisting obvious attempts at increasing carotenoid accumulation through simple overexpression. Here Zhou et al. provide an important advance in understanding the posttranscriptional regulation of PSY in plants, which is mediated by the OR (Orange) protein and a small group of partially redundant, related proteins (OR-like) that regulate PSY activity through protein-protein interactions using a combination of yeast 2-hybrid assays, bimolecular fluorescence complementation, and mutant analysis.

Early Edition: Xiangjun Zhou, doi: 10.1073/pnas.1420831112
The original article can be found here

From the article:

February 11, 2015

Boutanaev et al. "Investigation of terpene diversification across multiple sequenced plant genomes"

Alexander M. Boutanaev, Tessa Moses, Jiachen Zi, David R. Nelson, Sam T. Mugford, Reuben J. Peters, and Anne Osbourn

This actually came out the end of last year, but since terpene synthases have been a theme lately, this one is well worth including. Boutanaev et al. used comparative genomics to identify associations between two groups of enzymes that are important for making terpenes: terpene synthases (TS), which generate olefinic hydrocarbons or alcohols from prenylated diphosphate precursors of varying chain lengths, and cytochrome P450s (CYPs), which introduce various oxygen functionalities into the carbon skeleton through regiospecific oxidation reactions once the diphosphate has been removed.  By comparing TS/CYP gene pairs from many sequenced plant genomes, they determined that the evolution of terpenoid biosynthetic pathways, which requires TS and CYP activities among others, has occurred differently in monocot and dicot lineages. In monocots, individual TY and CYP genes were recombined in dynamic genome rearrangements whereas new terpenoid diversity was acquired in dicots by gene duplication and subfunctionalization of TS/CYP pairs. Their approach also facilitated the identification of functional pairing of TS and CYP genes in previously unknown gene clusters.

vol. 112 no. 1 Alexander M. Boutanaev,  E81–E88, doi: 10.1073/pnas.1419547112
The original paper can be seen here 

From the article


Plants produce an array of specialized metabolites, including chemicals that are important as medicines, flavors, fragrances, pigments and insecticides. The vast majority of this metabolic diversity is untapped. Here we take a systematic approach toward dissecting genetic components of plant specialized metabolism. Focusing on the terpenes, the largest class of plant natural products, we investigate the basis of terpene diversity through analysis of multiple sequenced plant genomes. The primary drivers of terpene diversification are terpenoid synthase (TS) “signature” enzymes (which generate scaffold diversity), and cytochromes P450 (CYPs), which modify and further diversify these scaffolds, so paving the way for further downstream modifications. Our systematic search of sequenced plant genomes for all TS and CYP genes reveals that distinct TS/CYP gene pairs are found together far more commonly than would be expected by chance, and that certain TS/CYP pairings predominate, providing signals for key events that are likely to have shaped terpene diversity. We recover TS/CYP gene pairs for previously characterized terpene metabolic gene clusters and demonstrate new functional pairing of TSs and CYPs within previously uncharacterized clusters. Unexpectedly, we find evidence for different mechanisms of pathway assembly in eudicots and monocots; in the former, microsyntenic blocks of TS/CYP gene pairs duplicate and provide templates for the evolution of new pathways, whereas in the latter, new pathways arise by mixing and matching of individual TS and CYP genes through dynamic genome rearrangements. This is, to our knowledge, the first documented observation of the unique pattern of TS and CYP assembly in eudicots and monocots.