80 mV lower than those previously agreedon the basis of conventional redox titra-tion. Brinkert et al. [4] reasoned that thebicarbonate ion could explain the differ-ence – bubbling with inert gas woulddeplete the sample of carbon dioxideand therefore of bicarbonate, HCO3

�,from the carbonic anhydrase reaction:

CO2 þ H2O fi HCO3� þ Hþ

By contrast, the electrochemical cell ofShibamoto et al. [7] would not beexpected to remove the bicarbonate thatinteracts with the iron atom guiding elec-tron transfer from QA (Figure 1). Accord-ingly, Brinkert et al. carried out their redoxtitration on photosystem II reactioncentres, this time in the presence andabsence of 1 mM bicarbonate. Withoutbicarbonate the Em value was �60 mVin the Mn-containing preparation and+64 mV with Mn depleted. With bicarbon-ate present, both Em values dropped, aspredicted, to �124 mV with Mn and to�22 mV without [4]. Brinkert et al. alsodemonstrate that accumulation of QA

��

at high light intensity decreases bindingof bicarbonate to photosystem II [4].

The Safety ValveA clear inference is that return of the elec-tron from QA

�� to pheophytin can occurwhen bicarbonate is in place and whenQB is present as QBH2. This transfer isuphill, but the hill is less steep than the‘cliff’ presented when bicarbonate isabsent. Furthermore, this reduction ofpheophytin produces a high proportionof its triplet state, which convertsground-state oxygen to singlet oxygen,a toxic product. Depletion of CO2 will thusdo two things. First, a slow-down of theBenson–Calvin–Bassham cycle of CO2

assimilation will feed back, preventing oxi-dation of QBH2 and thus allowing QA

�� nooption for forward electron transport.Second, removal of the bicarbonate ionwill increase the Em of QA, favouring asafer back-reaction to chlorophyll or tothe donor side of photosystem II throughcytochrome b559 [8].

Origins of OxygenPhotosynthesis first evolved in a worlddevoid of free oxygen [9], and purple bac-terial photosynthesis today occurs onlyunder anoxic conditions. There the gluta-mate adjacent to the iron atom (Figure 1) isfixed in place – and harmful singlet oxygencannot be produced. When the first cya-nobacterium learned to use its quinone-containing reaction centre to make oxy-gen [10–12], QA

�� had urgently to find asafe back-reaction. Replacement of theglutamate by a removable bicarbonateion provided the solution.

In explaining the CO2-requirement of pho-tosystem II, Brinkert et al. also finally lay torest the ghost of the long-abandoned the-ory that primary photochemistry splitsCO2 to give O2 plus a C atom thatbecomes hydrated. The real primary eventis transmembrane electron transfer, andCO2 assimilation itself does not requirelight at all. Well, we knew that. Even so,the wrong model persisted in popular sci-ence writing, and it is satisfying to under-stand, now, that CO2 tunes redoxchemistry at the interface of our biospherewith energy from the sun.

AcknowledgmentsJ.F.A. holds an Emeritus Research Fellowship of the

Leverhulme Trust. J.N. acknowledges support from

the CREST program of the Japan Science and Tech-

nology Agency.

1Research Department of Genetics, Evolution, and

Environment, Darwin Building, University College London,

Gower Street, London WC1E 6BT, UK2School of Biological and Chemical Sciences, Queen Mary

University of London, Mile End Road, London E1 4NS, UK

*Correspondence: j.f.allen@ucl.ac.uk (J.F. Allen) and

j.nield@qmul.ac.uk (J. Nield).@Twitter: @ProfJohnAllen

http://dx.doi.org/10.1016/j.tplants.2016.11.011

References1. Umena, Y. et al. (2011) Crystal structure of oxygen-evolving

photosystem II at a resolution of 1.9 Ångström. Nature 473,55-U65

2. Kato, Y. et al. (2009) Spectroelectrochemical determinationof the redox potential of pheophytin a, the primary electronacceptor in photosystem II. Proc. Natl. Acad. Sci. U. S. A.106, 17365–17370

3. Müh, F. et al. (2012) Light-induced quinone reduction inphotosystem II. Biochim. Biophys. Acta. 1817, 44–65

4. Brinkert, K. et al. (2016) Bicarbonate-induced redox tuningin photosystem II for regulation and protection. Proc. Natl.Acad. Sci. U. S. A. 113, 12144–12149

5. Johnson, G.N. et al. (1995) A change in the midpointpotential of the quinone QA in photosystem II associatedwith photoactivation of oxygen evolution. Biochim. Bio-phys. Acta. 1229, 202–207

6. Deisenhofer, J. et al. (1995) Crystallographic refinement at2.3 Å resolution and refined model of the photosyntheticreaction centre from Rhodopseudomonas viridis. J. Mol.Biol. 246, 429–457

7. Shibamoto, T. et al. (2010) Species-dependence of theredox potential of the primary quinone electron acceptorQA in photosystem II verified by spectroelectrochemistry.FEBS Lett. 584, 1526–1530

8. Nishimura, T. et al. (2016) The N-terminal sequence of theextrinsic PsbP protein modulates the redox potential of Cytb559 in photosystem II. Sci. Rep. 6, 21490

9. Knoll, A.H. et al. (2016) Life: the first two billion years. Philos.Trans. R. Soc. B Biol. Sci. 371, 20150493

10. Allen, J.F. (2005) A redox switch hypothesis for the origin oftwo light reactions in photosynthesis. FEBS Lett. 579, 963–968

11. Cardona, T. et al. (2012) Charge separation in photosystemII: a comparative and evolutionary overview. Biochim. Bio-phys. Acta. 1817, 26–43

12. Fischer, W.W. et al. (2016) Evolution of oxygenic photosyn-thesis. Annu. Rev. Earth Planet. Sci. 44, 647–683

ForumThe First DarwinianPhylogenetic Tree ofPlantsUwe Hoßfeld,1,2

Elizabeth Watts,1 andGeorgy S. Levit1,2,*

In 1866, the German zoologist ErnstHaeckel (1834–1919) published thefirst Darwinian trees of life in thehistory of biology in his book Gen-eral Morphology of Organisms. Wetake a specific look at the first phy-logenetic trees for the plant king-dom that Haeckel created as part ofthis two-volume work.

Evolutionary Foundations andNew VisualizationsErnst Haeckel (Figure 1) was a strong pro-ponent of integrating Darwin's ideas intoexisting research traditions in comparativeanatomy and morphology. Haeckel wasalso an efficient propagandist whose ideas

Trends in Plant Science, February 2017, Vol. 22, No. 2 99

Figure 1. Ernst Haeckel in His Laboratory in theBuitenzorg Botanical Gardens on the Island oJava, 1901 (courtesy: archive U.H.).

100 Trends in Plant Science, February 2017, Vol. 22, N

f

became very widespread. Following thepublication of Darwin's seminal Origin ofSpecies (1859) it became important tosee existing research fields in the light ofDarwin's ideas, and they were applied earlyon to investigating the connection betweenontogeny and phylogeny [1]. Haeckel usedcomparative anatomy and embryology as ameans of substantiating Darwin's theory ofcommon descent. Like Johann FriedrichMeckel the Younger (1781–1833) and hisschool of anatomy and pathology, Haeckelalso placed great theoretical emphasis onthe parallelism between the stages of indi-vidual development of the embryo to anadult and the serial development of lowerforms of animals to higher forms of animalsthat became apparent through studies incomparative anatomy and systematics. Infact, Haeckel used the German termEntwicklung (development) to describe bothtypes of development, thus highlighting thesimilarity between individual development(embryology) and phylogenetic ‘develop-ment’ over time. In addition to these twoparallels, he identified a third, which wasbased on systematics. In the threefold par-allelism of the phyletic (paleontological),biontic (individual) and systematic

o. 2

developments he recognized one of thegreatest and most important phenomenain living nature [2]. The explanation of this‘threefold genealogical parallel’ he called‘The fundamental law of organic develop-ment, or in short form the “biogenetic law”’.Haeckel wrote about the reciprocal causalrelationships in his Generelle Morphologieder Organismen (General Morphology ofOrganisms): ‘41. Ontogenesis is the shortand quick recapitulation of phylogenesis,controlled through the physiological func-tions of inheritance (reproduction) andadaptation (nutrition). 42. The organic indi-vidual. . .recapitulates through its fast andshort individual development the mostimportant of changes in form, which theancestors have gone through during theslow and long paleontological developmentfollowing the rules of inheritance and adap-tation’ [2,3].

At the same time Haeckel realized theproblems associated with this subject[4,5]. The ‘complete and faithful recapitu-lation’ becomes ‘effaced and shortened’because ‘ontogenesis always choosesthe most direct path’. In addition, the reca-pitulation becomes ‘counterfeited andchanged through secondary adaptations’and is therefore more easily identifiedwhen the ‘bion’ (individual) and its ances-tors developed in similar conditions ofexistence [2].

Convinced of this parallelism and the the-ory of common descent, Haeckel setabout illustrating the ‘true’ course of evo-lutionary descent with modification (devel-opment). His first attempt was undertakenin illustrating the development of plantsover time using a rooted phylogenetic tree(Figure 2).

General Morphology of Organisms(1866)In 1866, Haeckel published his two-vol-ume book General Morphology of Organ-isms, and with it he created the firstDarwinian phylogenetic trees (i.e., tree oflife and tree of plants) to illustrate the noveltheory of evolution. The subtitle of the book

was The General Principles of the OrganicForm-Science, Founded Mechanicallythrough the Theory of Descent asReformed by Charles Darwin (AllgemeineGrundzüge der organischen Formen-Wis-senschaft, mechanisch begründet durchdie von Charles Darwin reformierteDescendenz-Theorie). The first volumeThe General Anatomy of Organisms (Allge-meine Anatomie der Organismen) wasdedicated to Haeckel's teacher, the anat-omist Carl Gegenbaur (1826–1903). Thesecond volume entitled General Develop-mental History (Allgemeine Entwicklungs-geschichte) was dedicated to the‘founders of the theory of descent’ – Dar-win, Goethe, and Lamarck. This book isthe key to Haeckel's later work, its goalbeing to apply Darwin's theory to biology ingeneral, and to morphology in particular. Inhis book Haeckel goes through both theanimal and the plant kingdoms, concen-trating on morphology and phylogeny. Hecoined new terms for new avenues ofresearch, such as ecology, phylogeny,ontogeny, and phylum, concepts whichare still used today. Haeckel also pre-sented his first ideas on the relationshipbetween ontogeny and phylogeny, and heintroduced a systematic organization of theexisting groups of organisms based ongenealogy, rather than on the old typologi-cal ideas which were the prominent modelat the time [6–8].

Another important aspect of the first vol-ume of the book is Haeckel's attempt toestablish a promorphology – a generaltheory of basic forms. The second volumecan be seen as a first attempt to establishevolutionary morphology and evolutionaryembryology as new fields of research. Inthe second volume, Haeckel also formu-lated his ideas for a biological anthropol-ogy based on Darwin's theory of evolution[9,10].

The First Trees of LifeVisualization has always played a specialrole in biology because visual images werearguments themselves. Haeckel paid spe-cial attention to visualization throughout

Figure 2. Tree of Life of the Plant Kingdom; from General Morphology of Organisms, 1866(courtesy: archive U.H.). Haeckel illustrated the 19th century understanding of phylogenetic relationshipswithin the plant kingdom. In this rooted phylogenetic tree fungi and lichen are placed at the base of the tree of life,illustrating their ancient existence, while more recent plant groups such as gymnosperms and angiospermsappear adjacent to ferns in the crown of the tree.

Tr

his career and was renowned for his artis-tic abilities. For example, his drawings ofthe natural world are world-famous. Fur-thermore, Haeckel invested a great deal inthe visualization of the very concept ofevolution as well.

In General Morphology he published atotal of eight phylogenetic trees anddivided all living organisms into three king-doms – animals, plants, and protists.Haeckel claimed that evolution and devel-opment affected everything from inorganicmatter (monads in the roots of a tree) toman. His intention was to revolutionizebiology on the basis of Darwin's theoryof descent with modification (evolution).However, in contrast to Darwin himself,Haeckel's objective was not only to visu-alize the principle of divergence but also tosuggest phylogenetic trees illustrating thereal phylogenetic relationships betweendifferent organismic groups. In his laterworks (History of Creation, 1868, andEvolution of Man, 1874) he continued todevelop his initial ideas, but he alwaysemphasized that his trees are only ‘heuris-tic hypotheses’ and should not be seen asa dogma. Haeckel's phylogenetic treesserved as a template for many phyloge-netic trees to follow. In later years Haeckelalso used his diagrams for a different pur-pose, namely to show the degree of evo-lutionary progress among the races ofmankind [9]. Over the past few decadesresearch on the reconstruction of the treeof life based on genetic findings has beenremarkably active, and knowledge on thesubject is expanding exponentially [11,12].

Concluding RemarksWhile General Morphology did not becomethe immediate success that Haeckel hadhoped for, his Darwin lectures turned outto be a greater success, attracting morethan 200 students in the winter semesterof 1867/68 (a third of the total studentbody at Jena University at the time). Hislectures were taken down in shorthandand were later published as NatürlicheSchöpfungsgeschichte in 1868 (trans-lated in 1876 as The History of Creation).

ends in Plant Science, February 2017, Vol. 22, No. 2 101

Here he also made some revisions basedon the criticism he had received from CarlGegenbaur and Thomas H. Huxley. Thisbook was written in an accessible styleand, together with the Anthropogenieoder Entwicklungsgeschichte des Men-schen of 1874 (English translation:Anthropogeny or Evolution of Man),became a great success. It was trans-lated into many languages and soldextremely well, and made an importantcontribution to the popularization ofthe theory of evolution in Europe andbeyond [6].

Haeckel's attempt to visualize evolutionand his initial phylogenetic trees gave riseto all current phylogenetic reconstructions.

AcknowledgmentsThe work of G.L. is supported by the Russian Foun-

dation for the Humanities, project 16-03-00555.

1Arbeitsgruppe Biologiedidaktik, Friedrich-Schiller-

Universität Jena, 07743 Jena, Germany2ITMO University, 191187 St. Petersburg, Russia

*Correspondence: gslevit@corp.ifmo.ru (G.S. Levit).

http://dx.doi.org/10.1016/j.tplants.2016.12.002

References1. Rensing, S.A. (2016) (Why) does evolution favour embryo-

genesis? Trends Plant Sci. 21, 562–573

2. Haeckel, E. (1866) Generelle Morphologie der Organismen.I: Allgemeine Anatomie der Organismen. II: AllgemeineEntwicklungsgeschichte der Organismen. Teildruck unterdem Titel: Prinzipien der generellen Morphologie der Organ-ismen, Verlag von Georg Reimer

3. Hoßfeld, U. et al. (2016) Haeckel reloaded: 150 Jahre ‘Bio-genetisches Grundgesetz’. Biol. Unserer Zeit 46, 190–195

4. Ulrich, W. (1968) Ernst Haeckel: ‘Generelle Morphologie’,1866 (Fortsetzung und Schluß). Zool. Beitr. 14, 213–311

5. Uschmann, G. (1966) 100 Jahre ‘Generelle Morphologie’.Biol. Rundsch. 5, 241–252

6. Hoßfeld, U. and Olsson, L. (2003) The road from Haeckel.The Jena tradition in evolutionary morphology and the originof ‘evo-devo’. Biol. Philos. 18, 285–307

7. Richards, R.J. (2008) The Tragic Sense of Life: ErnstHaeckel and the Struggle over Evolutionary Thought, Uni-versity of Chicago Press

8. Rieppel, O. (2016) Phylogenetic Systematics: Haeckel toHennig, Taylor and Francis

9. Hoßfeld, U. (2016) Geschichte der Biologischen Anthropo-logie in Deutschland, Franz Steiner Verlag

10. Kutschera, U. (2016) Haeckel's 1866 tree of life and theorigin of eukaryotes. Nat. Microbiol. 1, 16114

11. Cracraft, J. and Donoghue, M.J., eds (2004) Assemblingthe Tree of Life, Oxford University Press

12. Dayrat, B. (2003) The roots of phylogeny: how did Haeckelbuild his trees? Syst. Biol. 52, 515–527

102 Trends in Plant Science, February 2017, Vol. 22, No. 2

ForumModification of DNACheckpoints toConfer AluminumToleranceThomas Eekhout,1,2

Paul Larsen,3,* andLieven De Veylder1,2,*

Although aluminum (Al) toxicityrepresents a global agriculturalproblem, the biochemical targetsfor Al remain elusive. Recentlyidentified Arabidopsis mutantswith increased Al tolerance provideevidence of DNA as one of the maintargets of Al. This insight couldlead the way for novel strategiesto generate Al-tolerant cropplants.

Al Toxicity Represents aWorldwide Agricultural ProblemIdentified already 100 years ago as animportant agricultural problem [1], alumi-num (Al) toxicity represents an importantlimitation to worldwide crop productionthat is only rivaled by salinity stress. Altoxicity is pH-dependent and manifestsitself in acid soils (pH <5.5) that are pre-dominantly found in developing countriesin South America, Central Africa, andSouthwest Asia, as well as in easternNorth America, Australia, and throughoutEurope [2]. Industrial pollution and modernfarming practices serve to increase therange and impact of Al toxicity becauseboth contribute to soil acidification.

Al is the third most abundant (�7%) ele-ment in the Earth's crust and the mostabundant metal, distinguishing Al toxicityfrom heavy metal stresses that generallyarise from soil contamination. Under acidicconditions, the mineral form of Al dissolvesto release the soluble Al3+ species, whichis highly toxic to plants. The most evident

symptom and important consequence ofAl toxicity is root growth inhibition, whichoccurs even at micromolar concentra-tions. In conjunction with the root growthphenotypes, Al toxicity causes a reductionin nutrient uptake that eventually results innutritional deficiency in shoots and leaves,and leads to an overall reduction in shootbiomass and crop yield. Because of itsthreat to food security, it is imperative tounderstand the molecular reasons under-lying Al toxicity because this knowledgemay lead to new mechanisms to engineerAl-tolerant crop plants.

Will the Real Target of Al RevealItself?The development of Al tolerance mecha-nisms requires a thorough knowledge ofthe biochemical targets of Al3+. BecauseAl3+ is expected to interact promiscuouslywith anionic sites, it is still debatablewhether primary lesions of Al toxicity exist,and if so whether they are located withinthe apoplast or symplast. One primaryexternal target that has been put forwardis the cell wall, where Al3+ is suggested toincrease cell-wall rigidity by crosslinkingpectin. Intracellular Al3+ has been sug-gested to cause oxidative stress, thuspromoting lipid peroxidation. Other sug-gested targets include the cytoskeleton,ion transporters, and mitochondria [3](Figure 1).

Theoretically, the identification of mutantswith increased growth in the presence of Alshould help to pinpoint the primary target(s) of Al3+ because such mutants may rep-resent alterations in biochemical targets ofAl3+ or mechanisms required for detectingor coping with this damage. However, thisstrategy has fallen short because the mostcommon way to increase growth in thepresence of Al is to increase the ability toexclude Al from the root tip through therelease of high levels of di- and tricarboxylicacids such as malate and citrate. Theseorganic acids form stable complexes withionic Al, effectively excluding Al3+ fromplant tissue through chelation. Such resis-tance mechanisms often involve the