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Scheme illustrating the key experiment of J.-B. Boussingault of 1838, performed with pea (Pisum sativum) plants that were raised in sterile sand (A). O D sand without mineral salts; KP D sand with potassium and phosphate; KPS D sand with potassium, phosphate and nitrate. The root system of a pea plant (B) is characterized by numerous nodules that contain nitrogen-fixing bacteria (adapted from ref. 9).  

Scheme illustrating the key experiment of J.-B. Boussingault of 1838, performed with pea (Pisum sativum) plants that were raised in sterile sand (A). O D sand without mineral salts; KP D sand with potassium and phosphate; KPS D sand with potassium, phosphate and nitrate. The root system of a pea plant (B) is characterized by numerous nodules that contain nitrogen-fixing bacteria (adapted from ref. 9).  

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In 1963, a monograph by Thomas D. Luckey entitled Germfree Life and Gnotobiology was published, with a focus on animals treated with microbes and reference to the work of Louis Pasteur (1822–1895). Here, we review the history and current status of plant gnotobiology, which can be traced back to the experiments of Jean-Baptiste Boussingault (1801–18...

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... sciences can be traced back to the work of the French chemist and agricultural scientist Jean-Baptiste Boussingault . In 1838, he performed a set of experiments by transplanting legumes in sand that had been sterilized, but did not contain any nitrogen. Boussingault observed that the legumes continued to grow under germ (and N)-free conditions (Fig. 1A). Accordingly, he concluded that these plants fix atmospheric nitrogen via an unknown mecha- nism. Based on this finding, the French chemist discovered that, following the growth of legume crops such as peas (Pisum sativum) (Fig. 1B), an increase in soil nitrogen (N) occurrs. 8 Decades later, it was discovered that root modules that ...
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... contain any nitrogen. Boussingault observed that the legumes continued to grow under germ (and N)-free conditions (Fig. 1A). Accordingly, he concluded that these plants fix atmospheric nitrogen via an unknown mecha- nism. Based on this finding, the French chemist discovered that, following the growth of legume crops such as peas (Pisum sativum) (Fig. 1B), an increase in soil nitrogen (N) occurrs. 8 Decades later, it was discovered that root modules that contain N 2 -fixing bacteria (Rhizobium sp) are responsible for this posi- tive effect on the nutrition of crop plants. 8,9 As mentioned above, Julius Sachs, who referred to the work of Boussingault, was the first to consider bacteria ...
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... the shadow of this anthropocentric research that is signifi- cant for human health, plant gnotobiology developed. This branch of the botanical sciences can be traced back to the work of the French chemist and agricultural scientist Jean-Baptiste Boussingault . In 1838, he performed a set of experiments by transplanting legumes in sand that had been sterilized, but did not contain any nitrogen. Boussingault observed that the legumes continued to grow under germ (and N)-free conditions (Fig. 1A). Accordingly, he concluded that these plants fix atmospheric nitrogen via an unknown mecha- nism. Based on this finding, the French chemist discovered that, following the growth of legume crops such as peas (Pisum sativum) (Fig. 1B), an increase in soil nitrogen (N) occurrs. 8 Decades later, it was discovered that root modules that contain N 2 -fixing bacteria (Rhizobium sp) are responsible for this posi- tive effect on the nutrition of crop plants. 8,9 As mentioned above, Julius Sachs, who referred to the work of Boussingault, was the first to consider bacteria as plant- associated microbes. 2,10 Since the outer leaf surface of most land plants (embryophytes) is large, whereas the internal areas, such as the lumen of the vascular bundles and the intercellular spaces, are small, green, sessile organisms "wear their guts on the out- side." The aerial parts of green plants, notably the leaves, are col- onized by bacteria at densities of up to 10 million microbes per cm 2 (refs. 11, 12). Recent studies with germ-free (gnotobiotic) land plants (bryophytes, angiosperms etc.) have shown that many of these epiphytic prokaryotes, such as methylobacteria, secrete hormones (auxin, cytokinines), and hence are beneficial to their sessile host. Accordingly, they have been classified as growth-promoting phytosymbionts 7,13 Hence, not only animals, but also plants are superorganisms or chimeras composed of pro-and eukaryotic cells. Green plants benefit from their outer coat of "hardworking" microbial partners and are, like humans, characterized by a meta-genome. In addition to epiphytic microbes, endophytes may also be importance as symbionts of green plants. This topic is beyond the scope of the present ...
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... the shadow of this anthropocentric research that is signifi- cant for human health, plant gnotobiology developed. This branch of the botanical sciences can be traced back to the work of the French chemist and agricultural scientist Jean-Baptiste Boussingault . In 1838, he performed a set of experiments by transplanting legumes in sand that had been sterilized, but did not contain any nitrogen. Boussingault observed that the legumes continued to grow under germ (and N)-free conditions (Fig. 1A). Accordingly, he concluded that these plants fix atmospheric nitrogen via an unknown mecha- nism. Based on this finding, the French chemist discovered that, following the growth of legume crops such as peas (Pisum sativum) (Fig. 1B), an increase in soil nitrogen (N) occurrs. 8 Decades later, it was discovered that root modules that contain N 2 -fixing bacteria (Rhizobium sp) are responsible for this posi- tive effect on the nutrition of crop plants. 8,9 As mentioned above, Julius Sachs, who referred to the work of Boussingault, was the first to consider bacteria as plant- associated microbes. 2,10 Since the outer leaf surface of most land plants (embryophytes) is large, whereas the internal areas, such as the lumen of the vascular bundles and the intercellular spaces, are small, green, sessile organisms "wear their guts on the out- side." The aerial parts of green plants, notably the leaves, are col- onized by bacteria at densities of up to 10 million microbes per cm 2 (refs. 11, 12). Recent studies with germ-free (gnotobiotic) land plants (bryophytes, angiosperms etc.) have shown that many of these epiphytic prokaryotes, such as methylobacteria, secrete hormones (auxin, cytokinines), and hence are beneficial to their sessile host. Accordingly, they have been classified as growth-promoting phytosymbionts 7,13 Hence, not only animals, but also plants are superorganisms or chimeras composed of pro-and eukaryotic cells. Green plants benefit from their outer coat of "hardworking" microbial partners and are, like humans, characterized by a meta-genome. In addition to epiphytic microbes, endophytes may also be importance as symbionts of green plants. This topic is beyond the scope of the present ...

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... Efforts are underway to improve the efficacy of PGPB that are available to farmers as alternatives to expensive and environment-damaging fertilizers. 1,2 Pantoea ananatis is a Gram-negative bacterium of the Enterobacteriacea family that occurs in plant tissues mostly as a phytopathogen. 3 Previous studies have established the taxonomy of this species, placing it in the class Gammaproteobacteria and family Enterobacteriaceae, under the diverse genus Pantoea, which contains approximately 20 different species with varying applications and properties. ...
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A bacterium growing on infected leaves of Hydrocotyle umbellata, commonly known as dollarweed, was isolated and identified as Pantoea ananatis. An ethyl acetate extract of tryptic soy broth (TSB) liquid culture filtrate of the bacterium was subjected to silica gel chromatography to isolate bioactive molecules. Indole was isolated as the major compound that gave a distinct, foul odor to the extract, together with phenethyl alcohol, phenol, tryptophol, N-acyl-homoserine lactone, 3-(methylthio)-1-propanol, cyclo(L-pro-L-tyr), and cyclo(dehydroAla-L-Leu). This is the first report of the isolation of cyclo(dehydroAla-L-Leu) from a Pantoea species. Even though tryptophol is an intermediate in the indoleacetic acid (IAA) pathway, we were unable to detect or isolate IAA. We investigated the effect of P. ananatis inoculum on the growth of plants. Treatment of Lemna paucicostata Hegelm plants with 4 × 10⁹ colony forming units of P. ananatis stimulated their growth by ca. five-fold after 13 days. After 13 days of treatment, some control plants were browning, but treated plants were greener and no plants were browning. The growth of both Cucumis sativus (cucumber) and Sorghum bicolor (sorghum) plants was increased by ca. 20 to 40%, depending on the growth parameter and species, when the rhizosphere was treated with the bacterium after germination at the same concentration. Plant growth promotion by Pantoea ananatis could be due to the provision of the IAA precursor indole.
... Similarly, Bacillus megaterium with Azotobacter chroococcum are found to promote cucumber growth by producing cytokinins. Cytokinins are responsible for cytokinesis, vascular cambium sensitivity, root apical dominance, and vascular differentiation [27,28]. Under stressed conditions, plants often produce ethylene that can inhibit specific processes that cause premature senescence, such as root elongation or nitrogen fixation in legumes. ...
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Plant probiotic bacteria are a versatile group of bacteria isolated from different environmental sources to improve plant productivity and immunity. The potential of plant probiotic-based formulations is successfully seen as growth enhancement in economically important plants. For instance, endophytic Bacillus species acted as plant growth-promoting bacteria, influenced crops such as cowpea and lady's finger, and increased phytochemicals in crops such as high antioxidant content in tomato fruits. The present review aims to summarize the studies of Bacillus species retaining probiotic properties and compare them with the conventional fertilizers on the market. Plant probiotics aim to take over the world since it is the time to rejuvenate and restore the soil and achieve sustainable development goals for the future. Comprehensive coverage of all the Bacillus species used to maintain plant health, promote plant growth, and fight against pathogens is crucial for establishing sustainable agriculture to face global change. Additionally, it will give the latest insight into this multifunctional agent with a detailed biocontrol mechanism and explore the antagonistic effects of Bacillus species in different crops.
... To gain insights into the role of specific plant endophytes that exist as a part of the microbiome, experimental validations have been carried out in gnotobiotic systems. Such systems offer the advantage of meticulous investigation on the relationships between plant microbiota phenotypes and genotypes, and the impact of stress factors (M€ uller et al., 2016;Kutschera and Khanna, 2016). Initially, a gnotobiotic system was developed to decipher the intricacies of tomato rhizosphere colonization by Pseudomonas spp. ...
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Under natural conditions plants are not individual entities; they are associated with diverse microbiota to form the plant holobiont. The concept of plant holobiont is being actively explored to address the issues related to plant's health. Endophytes are a class of plant-associated microbes, which reside within the internal tissues of plants. They have been ubiquitously reported in all plants investigated so far. The plant-endophyte interactions may exhibit different modes of symbiotic association, ranging from beneficial (mutualism), neutral (commensal), to even pathogenic. Although we have a fair idea of the factors affecting plant-microbe interactions, the intricacies involved in fine-tuning their association are just beginning to unfold. Some of the pertinent questions surrounding the plant-endophyte symbiosis include: how are endophytes different from other beneficial microbes like rhizo-bia, mycorrhizae, and rhizobacteria? What mechanisms ensure that endophytes gain an unsurpassed entry and colonization into plants without eliciting a strong defense reaction? Why do different strains of the same microbial species enter into diverse modes of symbi-otic association with plants? What factors cause the switch in the lifestyle of endophytes? In the present review, these questions have been addressed in the light of recent data and finally, concluded with gaps in endophyte research, which could be deliberated in future endeavors.
... These novel apparatuses enabled plant scientists of his time to perform quantitative experiments at an unprecedented accuracy, and provided novel insights into the physiology of green organisms, from aquatic plants via mosses to crop species (beans etc.). These studies were recently re-evaluated in a modern context (Kutschera and Khanna 2016). ...
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One century ago, the German chemist and botanist Wilhelm Pfeffer (1845–1920) died, shortly after finishing his last lecture at the University of Leipzig. Pfeffer was, together with Julius Sachs (1832–1897), the founder of modern plant physiology. In contrast to Sachs, Pfeffer’s work was exclusively based on the principles of physics and chemistry, so that with his publications, notably the ca. 1.600 pages-long Handbuch der Pflanzenphysiologie (2. ed., Vol. I/II; 1897/1904), experimental plant research was founded. Here we summarize Pfeffer’s life and work with special emphasis on his experiments on osmosis, plant growth in light vs. darkness, gravitropism, cell physiology, photosynthesis and leaf movements. We document that Pfeffer was the first to construct/establish constant temperature rooms (growth chambers) for seed plants. Moreover, he pioneered in outlining the carbon-cycle in the biosphere, and described the effect of carbon dioxide (CO2)-enhancement on assimilation and plant productivity. Wilhelm Pfeffer pointed out that, at ca. 0.03 vol% CO2 (in 1900), photosynthesis is sub-optimal. Accordingly, due to human activities, anthropogenic CO2 released into the atmosphere promotes plant growth and crop yield. We have reproduced Pfeffer’s classical experiments on the role of CO2 with respect to plant development, and document that exhaled air of a human (ca. 4 vol% CO2) strongly promotes growth. We conclude that Pfeffer not only acted as a key figure in the establishment of experimental plant physiology. He was also the discoverer of the phenomenon of CO2-mediated global greening and promotion of crop productivity, today known as the “CO2-fertilization-effect”. These topics are discussed with reference to climate change and the most recent findings in this area of applied plant research.
... Based on his earlier work on blue lightinduced promotion of virulence in pathogenic bacteria, 10 he now focussed his attention on root nodules in Pisum sativum and related plants. 36 The senior scientist provided data indicating that crop yield in legumes, such as fava bean (Vicia faba) and garden pea (P. sativum), is enhanced when the soil microbes (rhizobia) were irradiated with blue light before inoculation of the seeds that are prepared for planting in moist substrate. ...
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The American biologist Winslow Russel Briggs (1928–2019) was a global leader in plant physiology, genetics and photobiology. In this contribution, we try to share our knowledge of the remarkable career of this outstanding scientist. After earning his PhD at Harvard (Cambridge, Massachusetts), he started his independent research program at Stanford University (California). Among many major contributions was his elegant experiment that conclusively demonstrated the role of auxin transport in the phototropic bending response of grass coleoptiles. During subsequent years as Professor of biology at Harvard University, Briggs focused on phytochrome and photomorphogenesis. In 1973, he re-located to Stanford to become Director of the Department of Plant Biology, Carnegie Institution for Science, and faculty member in the Biology Department at Stanford University. After his retirement (1993), he continued his research on “light and plant development” as an emeritus at Carnegie until the day of his death on February 11, 2019. Through his long research career, Briggs stayed at the cutting edge by re-inventing himself from a plant physiologist, to biochemist, geneticist, and molecular biologist. He made numerous discoveries, including the LOV-domain photoreceptor phototropin. Winslow Briggs, who was also a naturalist and gifted pianist, inspired and promoted the work of generations of young scientists – as mentor, colleague and friend.
... Despite the fact that Haeckel (1866) introduced the Kingdom Protista and characterized the few bacteria known at that time as Monera, bacteriology was in its infancy. It should be noted that Haeckel (1866) argued that all living beings alive today descended in some way from bacteria-like microbes (Kutschera 2016). Today, Haeckel's hypothesis is well established, i.e., life started about 3500 million years ago with the emergence of simple microbes, and subsequently, aquatic microorganisms were the sole inhabitants of the planet about 80% of the time that living beings have existed on Earth (Kutschera 2017a, b;Martin 2017;Spang et al. 2017). ...
... Gnotobiology, i.e., the study of developmental patterns of animals in the absence or presence of a defined mixture of microbes (i.e., germ-free or axenic vs. non-sterile controls), became an established discipline in the 1950s (Luckey 1963, Kutschera andKhanna 2016). At that time, due to a systems biology approach, it became possible to raise germ-free mice (Mus musculus), and other mammals, in the laboratory. ...
... The microbiome of typical land plants is large, but is essentially restricted to the outer surfaces of the root system (in the soil) and the above-ground phytosphere (shoot, i.e., stem, leaves, flowers). In contrast to the human microbiome, which also comprises numerous microbes in the blood stream (Kowarsky et al. 2017), the plant microbiome is external and not yet as well characterized (Kutschera 2007, 2015b, Vorholt 2012, Kutschera and Khanna 2016. However, as Vandenkoornhuyse et al. (2015) have pointed out, the microbiota of plants, which essentially consists of bacteria and fungi, interacts with their green host organism in a variety of ways (nutrient uptake, resistance to pathogenic organisms, etc.; see also Faure et al. 2018). ...
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The founders of modern biology (Jean Lamarck, Charles Darwin, August Weismann etc.) were organismic life scientists who attempted to understand the morphology and evolution of living beings as a whole (i.e., the phenotype). However, with the emergence of the study of animal and plant physiology in the nineteenth century, this “holistic view” of the living world changed and was ultimately replaced by a reductionistic perspective. Here, I summarize the history of systems biology, i.e., the modern approach to understand living beings as integrative organisms, from genotype to phenotype. It is documented that the physiologists Claude Bernard and Julius Sachs, who studied humans and plants, respectively, were early pioneers of this discipline, which was formally founded 50 years ago. In 1968, two influential monographs, authored by Ludwig von Bertalanffy and Mihajlo D. Mesarović, were published, wherein a “systems theory of biology” was outlined. Definitions of systems biology are presented with reference to metabolic or cell signaling networks, analyzed via genomics, proteomics, and other methods, combined with computer simulations/mathematical modeling. Then, key insights of this discipline with respect to epiphytic microbes (Methylobacterium sp.) and simple bacteria (Mycoplasma sp.) are described. The principles of homeostasis, molecular systems energetics, gnotobiology, and holobionts (i.e., complexities of host–microbiota interactions) are outlined, and the significance of systems biology for evolutionary theories is addressed. Based on the microbe—Homo sapiens—symbiosis, it is concluded that human biology and health should be interpreted in light of a view of the biomedical sciences that is based on the holobiont concept.
... 6 His detailed account of the external and internal structure of an economically relevant invertebrate ( Figure 2B) documents that Sachs was not only a botanist but also a zoologist, with a broad perspective on organismic interactions. 7 Throughout his career as a professional scientist in Prague, Tharandt, Poppelsdorf/Bonn, Freiburg and Würzburg, Sachs published four comprehensive textbooks: the Handbuch der Experimental-Physiologie der Pflanzen, 1865, (Handbook of Experimental Plant Physiology), 8 the Lehrbuch der Botanik, 1868, (Textbook of Botany, 4th edition in 1874), 1 the Geschichte der Botanik, 1875, (History of Botany), 9 and, his magnum opus, the Vorlesungen über Pflanzen-Physiologie, 1882, (2nd ed. 1887) (Lectures on Plant Physiology) 10 . ...
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One hundred and fifty years ago, Julius von Sachs’ (1832–1897) monumental Lehrbuch der Botanik (Textbook of Botany) was published, which signified the origin of physiological botany and its integration with evolutionary biology. Sachs regarded the physiology of photoautotrophic organisms as a sub-discipline of botany, and introduced a Darwinian perspective into the emerging plant sciences. Here, we summarize Sachs’ achievements and his description of sexuality with respect to the cellular basis of plant and animal biparental reproduction. We reproduce and analyze a forgotten paper (Gutachten) of Sachs dealing with Die Akademische Frau (The Academic Woman), published during the year of his death on the question concerning gender equality in humans. Finally, we summarize his endorsement of woman’s rights to pursue academic studies in the natural sciences at the University level, and conclude that Sachs was a humanist as well as a great scientist.
... Since, for instance, sugarcane plants harbor in their intercellular spaces large populations of endophytic bacteria (Beijerinckia, Herbaspirillum, etc.), and, in addition to the PGPRs, the PPFMs or methylobacteria (genus Methylobacterium) likewise live attached to these green organisms (from the flowers via the leaves/stem down to the root tips), it is fair to interpret land plants as superorganisms. The well-known soil-borne mycorrhizas (fungi associated with the root system) should also be mentioned in this context, since Mereschkowsky (1905Mereschkowsky ( , 1910Mereschkowsky ( , 1920 discussed these organisms in some detail and published a scheme illustrating their possible evolutionary development (Kutschera 2007;Kutschera and Khanna 2016). 9 Conclusions: Symbiogenesis as the "Big Bang" ...
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In 1905, Constantin S. Mereschkowsky (1855–1921) proposed that the green organelles (chloroplasts) of algae and land plants evolved from ancient, once free-living cyanobacteria. This endosymbiotic hypothesis was based on numerous lines of evidence. In a 1910 paper, Mereschkowsky argued that the time has come to introduce a new theory on the origin of living beings; since Darwin’s era, so many new findings have accumulated that now an alternative, anti-selectionist theory of evolution has to be established. Based on the principle of symbiosis (i.e., the union of two different organisms whereby both partners mutually benefit), Mereschkowsky coined the term “symbiogenesis theory,” which is based on an analogy between the feeding process of amoebae and cellular events that may have occurred in the ancient oceans. Mereschkowsky’s symbiogenesis hypothesis explains the origin of chloroplasts from archaic cyanobacteria, with respect to plant evolution. In 1927, the Russian cytologist Ivan E. Wallin (1883–1969) proposed that the mitochondria of eukaryotic cells are descendants of ancient, once free-living bacteria. Here, I outline the origin and current status of the Mereschkowsky–Wallin concept of symbiogenesis (primary and secondary endosymbiosis) and explain why it is compatible with the Darwin–Wallace principle of natural selection, which is described in detail. Nevertheless, largely due to the work of Lynn Margulis (1938–2011), symbiogenesis is still considered today as an Anti-Darwinian research program. I will summarize evidence indicating that symbiogenesis, natural selection, and the dynamic Earth (plate tectonics) represent key processes that caused major macro-evolutionary transitions during the 3500-million-year-long history of life on Earth.
... Hence, in our investigation, we first explored the effects of soil extracts (i.e., naturally occurring mixtures of microbes) on the development of the root system. We used autoclaved (sterile) media as controls, according to the principles of plant gnotobiology (Requena et al. 1997;Kutschera 2002;Kutschera and Khanna 2016). Then, we raised Arabidopsis seedlings in the absence/presence of p h y t o h o r m o n e -s e c r e t i n g m e t h y l o b a c t e r i a (Methylobacterium sp.) and documented their effects on root development. ...
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In numerous experimental studies, seedlings of the model dicot Arabidopsis thaliana have been raised on sterile mineral salt agar. However, under natural conditions, no plant has ever grown in an environment without bacteria. Here, we document that germ-free (gnotobiotic) seedlings, raised on mineral salt agar without sucrose, develop very short root hairs. In the presence of a soil extract that contains naturally occurring microbes, root hair elongation is promoted; this effect can be mimicked by the addition of methylobacteria to germ-free seedlings. Using five different bacterial species (Methylobacterium mesophilicum, Methylobacterium extorquens, Methylobacterium oryzae, Methylobacterium podarium, and Methylobacterium radiotolerans), we show that, over 9 days of seedling development in a light-dark cycle, root development (hair elongation, length of the primary root, branching patterns) is regulated by these epiphytic microbes that occur in the rhizosphere of field-grown plants. In a sterile liquid culture test system, auxin (IAA) inhibited root growth with little effect on hair elongation and significantly stimulated hypocotyl enlargement. Cytokinins (trans-zeatin, kinetin) and ethylene (application of the precursor ACC) likewise exerted an inhibitory effect on root growth but, in contrast to IAA, drastically stimulated root hair elongation. Methylobacteria are phytosymbionts that produce/secrete cytokinins. We conclude that, under real-world conditions (soil), the provision of these phytohormones by methylobacteria (and other epiphytic microbes) regulates root development during seedling establishment.
... 27 Finally, we want to stress that root development is, under natural conditions, also regulated by epiphytic microbes that inhabit the rhizosphere. 4,28,29 The interaction between the micronutrient B and bacteria that live in (or are attached to) the root system is unknown. Hence, more integrative "plant-nutrientsmicrobe"-studies are required to resolve the puzzle of root development during the evolution of land plants. ...
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Experimental work has shown that Boron (i.e., Boric acid, B) is an essential and multifunctional microelement for vascular plant development. In addition to its other functions, which include xylem development and lignin biosynthesis, we now know that B is involved in phytohormone-signaling and influences the mechanical properties of intercellular pectins. From these data, we conclude that B played an important role during the evolutionary development of lignified tissues, and that it may have been involved in the evolution of vascular plant roots, as hypothesized by D. H. Lewis in 1980. Herein, we review the data pertaining to Lewis' hypothesis, present experimental results on the role of B in root (vs. rhizoid) formation in sunflower vs. a liverwort, and describe the appearance of roots in the fossil record. Open questions are addressed, notably the lack of our knowledge concerning soil microbes and their interactive roles with the micronutrient Boron during root formation.