Physiology of Methylotrophs Living in the Phyllosphere

microorganisms Review Physiology of Methylotrophs Living in the Phyllosphere Hiroya Yurimoto , Kosuke Shiraishi and Yasuyoshi Sakai * Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan; [email protected] (H.Y.); [email protected] (K.S.) * Correspondence: [email protected] Abstract: Methanol is abundant in the phyllosphere, the surface of the above-ground parts of plants, and its concentration oscillates diurnally. The phyllosphere is one of the major habitats for a group of microorganisms, the so-called methylotrophs, that utilize one-carbon (C1) compounds, such as methanol and methane, as their sole source of carbon and energy. Among phyllospheric microorganisms, methanol-utilizing methylotrophic bacteria, known as pink-pigmented facultative methylotrophs (PPFMs), are the dominant colonizers of the phyllosphere, and some of them have recently been shown to have the ability to promote plant growth and increase crop yield. In addition to PPFMs, methanol-utilizing yeasts can proliferate and survive in the phyllosphere by using unique molecular and cellular mechanisms to adapt to the stressful phyllosphere environment. This review describes our current understanding of the physiology of methylotrophic bacteria and yeasts living in the phyllosphere where they are exposed to diurnal cycles of environmental conditions. Keywords: methanol; methylotrophs; phyllosphere; diurnal adaptation; plant growth promotion Citation: Yurimoto, H.; Shiraishi, K.; Sakai, Y. Physiology of Methylotrophs 1. Introduction Living in the Phyllosphere. In nature, methanol is ubiquitous. Its main origin is considered to be the methyl ester Microorganisms 2021, 9, 809. groups of pectin, one of the major components of the plant cell wall [1,2]. Methanol is https://doi.org/10.3390/ produced through the hydrolysis of pectin methyl esters by pectin methylesterase, and microorganisms9040809 released from the plant stomata. Once released, methanol can be utilized by microorganisms living in the phyllosphere, defined as the aerial parts of plants, or emitted into the Academic Editors: Françoise Bringel, atmosphere as a volatile organic compound whose global emission is estimated to be 100 Tg Emilie Muller and Stéphane Vuilleumier per year [1,3,4]. The atmospheric concentration of methanol has been reported to fluctuate depending on the opening and closing of stomata [5]; however, the amount of methanol in Received: 18 March 2021 the phyllosphere had not been quantified. Recently, we revealed that the concentration of Accepted: 7 April 2021 Published: 12 April 2021 methanol available for microorganisms on the surface of plant leaves also oscillates during the daily light–dark cycle. Results showed that the methanol concentration in the phyllo- Publisher’s Note: MDPI stays neutral sphere was higher in the dark period and lower in the light period, which was opposite with regard to jurisdictional claims in to atmospheric methanol (Figure1)[ 6,7], suggesting that phyllospheric microorganisms published maps and institutional affil- utilize the methanol hydrolyzed from the plant pectin in a direct manner, rather than using iations. methanol present in the air. A wide variety of microorganisms colonize the phyllosphere and the area of soil surrounding plant roots (rhizosphere). Interactions between plants and microbes have been considered to affect not only the growth and proliferation of both organisms, but also the ecosystem and global environment. The total area of the global phyllosphere Copyright: © 2021 by the authors. 9 2 Licensee MDPI, Basel, Switzerland. is estimated to be 10 km , twice as large as the surface of the earth, and such space 26 27 This article is an open access article could be colonized by bacterial populations of 10 –10 cells, as well as lower numbers distributed under the terms and of archaea and fungi [8]. While plant–rhizobia and plant–mycorrhizae interactions in the conditions of the Creative Commons rhizosphere have been thoroughly investigated, studies of plant–microbe interactions in Attribution (CC BY) license (https:// the phyllosphere have been limited to those involving plant pathogens. As such, positive creativecommons.org/licenses/by/ and neutral interactions between phyllospheric microbes and their host plants have been 4.0/). closely researched only in the last decade [9,10]. Microorganisms 2021, 9, 809. https://doi.org/10.3390/microorganisms9040809 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, x FOR PEER REVIEW2 of 12 Microorganisms 2021, 9, 809 2 of 11 FigureFigure 1. 1.Colonization Colonization of methanol-utilizing of methanol-utilizing methylotrophs inmethylotro the phyllospherephs where in the methanol phyllosphe concentrationsre where oscillate methanol concentrationsdiurnally. Methanol-utilizing oscillate bacteria diurnally. (one-carbon Methanol-utiliz (C1) bacteria, pink-pigmenteding bacteria facultative (one-carbon methylotrophs (C1) (PPFMs)) bacteria, and pink- pigmentedyeasts (C1 yeasts) facultative colonize the surfacemethylotrophs of plant leaves (PPFMs)) and acquire nutrients and ye producedasts (C1 by yeasts) plants. Concentrations colonize ofthe methanol surface of plant in the phyllosphere oscillate diurnally, with lower concentrations in the light period (L) and higher concentrations in the leavesdark period and (D). acquire After the nutrients leaf printing onproduced the agar medium by plants. containing Concentrations methanol as the sole of carbon methanol source, pink-pigmented in the phyllosphere oscillatecolonies were diurnally, observed (leftwith panel lower photo). concentrationsCandida boidinii cells in expressing the light the period fluorescent (L) protein and higher Venus proliferate concentrations on in theArabidopsis dark period thaliana leaves (D). (right After panel the photo). leaf printing on the agar medium containing methanol as the sole carbon source, pink-pigmentedAmong phyllosphericcolonies were microorganisms, observed (left methanol-utilizing panel photo). bacteria, Candida known boidinii as pink- cells expressing the fluorescentpigmented protein facultative Venus methylotrophs proliferate (PPFMs), on Arabidopsis are the dominant thaliana colonizers leaves of(right plant panel leaf photo). surfaces (Figure1)[ 11,12]. Methylotrophs are a diverse group of microorganisms that utilize reduced one-carbon (C1) compounds, such as methanol and methane, as their sole sources of carbon and energy. C1-utilizing microorganisms include bacteria, archaea, and A wide varietyfungi. of microorganisms Most methylotrophic fungi colonize are yeasts. the PPFMs phyllosphere are members ofand the genusthe areaMethylobac- of soil surrounding plant rootsterium (rhizosphere)., although some have Interactio recently beenns reclassifiedbetween into plantsMethylorubrum and microbes[13], and some have of been these are known to have the ability to promote plant growth [14,15]. Along with PPFMs considered to affect(C1 not bacteria), only somethe methylotrophicgrowth and yeasts proliferation (C1 yeasts), which of both belong organisms, to the genera Candidabut also the ecosystem and globaland Komagataellaenvironment., also colonize The total the phyllosphere area of (Figurethe global1)[ 6]. These phyllosphere yeasts can grow is vigor- estimated ously on methanol-containing media and have been used as hosts for heterologous protein 9 2 to be 10 km , twiceproduction as large using as the strong surface and regulatable of the methanol-inducedearth, and such gene spacepromoters could [7,16 be–19 ].colonizedBe- by bacterial populationscause of theirof 10 intracellular26–1027 cells, dynamics, as well these yeastsas lower have also numbers been used of asmodel archaea organisms and fungi to investigate the molecular and cellular mechanisms of the development and degradation [8]. While plant–rhizobiaperoxisomes, and which plant–mycorrhizae are essential organelles for interactions methanol metabolism. in the rhizosphere have been thoroughly investigated, studies of plant–microbe interactions in the phyllosphere have been limited to those involving plant pathogens. As such, positive and neutral interactions between phyllospheric microbes and their host plants have been closely researched only in the last decade [9,10]. Among phyllospheric microorganisms, methanol-utilizing bacteria, known as pink- pigmented facultative methylotrophs (PPFMs), are the dominant colonizers of plant leaf surfaces (Figure 1) [11,12]. Methylotrophs are a diverse group of microorganisms that utilize reduced one-carbon (C1) compounds, such as methanol and methane, as their sole sources of carbon and energy. C1-utilizing microorganisms include bacteria, archaea, and fungi. Most methylotrophic fungi are yeasts. PPFMs are members of the genus Methylo- bacterium, although some have recently been reclassified into Methylorubrum [13], and some of these are known to have the ability to promote plant growth [14,15]. Along with PPFMs (C1 bacteria), some methylotrophic yeasts (C1 yeasts), which belong to the genera Candida and Komagataella, also colonize the phyllosphere (Figure 1) [6]. These yeasts can grow vigorously on methanol-containing media and have been used as hosts for heterologous protein production using strong and regulatable methanol-induced gene promot- ers [7,16–19]. Because of their intracellular dynamics, these yeasts have also been used as Microorganisms 2021, 9, 809 3 of 11

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