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Microbiological Research 183 (2016) 8–18

Contents lists available at ScienceDirect

Microbiological Research

j ourna l h omepa ge: www.elsev ier .com/ locate /micres

ibberellins in Penicillium strains: Challenges for endophyte-plantost interactions under salinity stress

na Lúcia Leitãoa,∗, Francisco J. Enguitab

MEtRICs, Departamento de Ciências e Tecnologia da Biomassa, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica,829-516 Caparica, PortugalFaculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisboa 1649-028, Portugal

r t i c l e i n f o

rticle history:eceived 9 November 2015ccepted 14 November 2015vailable online 1 December 2015

a b s t r a c t

The genus Penicillium is one of the most versatile “mycofactories”, comprising some species able to pro-duce gibberellins, bioactive compounds that can modulate plant growth and development. Althoughplants have the ability to synthesize gibberellins, their levels are lower when plants are under salinitystress. It has been recognized that detrimental abiotic conditions, such as saline stress, have negative

eywords:enicilliumlantsalinityibberellins

effects on plants, being the availability of bioactive gibberellins a critical factor for their growth underthis conditions. This review summarizes the interplay existing between endophytic Penicillium strainsand plant host interactions, with focus on bioactive gibberellins production as a fungal response thatallows plants to overcome salinity stress.

© 2015 Elsevier GmbH. All rights reserved.

ymbiotic interactions

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82. Gibberellins as modulators of plant-endophyte interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.1. Fungal endophytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .92.2. Gibberellins as phytohormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.3. Molecular details of gibberellin action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3. Fungi as gibberellin producers: biosynthetic gene clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104. Genus Penicillium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115. Saline stress, gibberellins and Penicillium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

5.1. Salinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .135.2. Interactions between Penicillium and plants through gibberellins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145.3. Putative gibberellin biosynthetic genes in Penicillium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

6. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

. Introduction

Fungi are important microbial factories of bioactive extracel-ular metabolites or “extrolites”, namely secondary metabolites.

community or industrial sector (Brakhage, 2013; Correa et al., 2014;Kim et al., 2014; Leitão and Enguita, 2014; Quang et al., 2014).

The gibberellins, hormones synthesized by plants, are interest-ing extrolites also produced by some strains of fungi like Fusariumsacchari, Fusarium konzum, Fusarium subglutinans, Aspergillus fumi-

nzymes, immunosuppressive agents, antitumor agents, antibi-tics, vitamins, and pigments, are examples of the representativeanoply of products with increasing interest either for scientific

∗ Corresponding author. Fax: +351 212948543.E-mail address: aldl@fct.unl.pt (A.L. Leitão).

ttp://dx.doi.org/10.1016/j.micres.2015.11.004944-5013/© 2015 Elsevier GmbH. All rights reserved.

gatus, Penicillium janthinellum and Penicillium resedanum, amongothers (Troncoso et al., 2010; Khan et al., 2011b, 2015a,b). The gib-berellins are related by their chemical structure (resulting from

isoprene polymerization) and their biosynthesis pathway (originin hydroxymethyl-glutaryl coenzyme A), but only few of them arebioactive. One of these bioactive gibberellins is the gibberellic acid,often called GA3; curiously some fungal strains are able to produce

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igher quantities of GA3 when compared to plants (Hedden et al.,001).

The use of fungal endophytes and their extrolites can be anxcellent opportunity to minimize the negative effect of abioticactors, such as salinity, on crop yield. The term “plant-growth-romoting-fungi” was established to designate some rhizosphereungi able to promote a direct effect on plant growth upon root col-nization or by the treatment with their metabolites (Hossain et al.,014). Recent studies have revealed that Penicillium endophyteould supply gibberellins to plant host, which is particularly impor-ant when plant is under biotic or abiotic stress. In this review, weummarize recent discoveries on the endophytic Penicillium strainsnd plant host interactions, with emphasis on bioactive gibberellinss response to salt stress.

. Gibberellins as modulators of plant-endophytenteractions

.1. Fungal endophytes

Fungal endophytes refer to the fungi which invade or live insidehe tissues of plants without causing apparent harm to themChandra, 2012). They were described by the first time in 1904 inhe darnel, Lolium temulentum (Freeman, 1904), but they did noteceive much attention until the recent development of screeningechnologies that revealed their great potential as a main source ofxtrolites with promising agricultural and pharmaceutical appli-ations (Tan and Zou, 2001; Kusari et al., 2012). Therefore, theelationship between the endophyte and the plant is generally con-idered mutualistic because the endophyte significantly improvesost plant tolerance to abiotic stresses such as drought and water-eficit or biotic factors such as insects, vertebrate herbivores andematodes, along with increased resistance and promoting plantrowth, nutrients uptake, and water resource use; and in turn thelant provides the microorganism with nutrients, protection, andfficient dissemination (Schardl et al., 2004). However, the ideahat there are no neutral interactions but rather that endophyte-ost relationship is a balanced symbiotic continuum ranging fromutualism through commensalism to parasitism, is gaining follow-

rs (Aly et al., 2011). In fact, when inside the plant, fungi assume quiescent state until environmental conditions are favorable forheir growth. The fungi have the ability to colonize the plant mostlyy association with but in some cases can live inside the plantither penetrating inside the root cortex or in the aerial parts ofhe plant, due to their extracellular enzymatic system (Waqas et al.,012; Khan et al., 2013a). After colonization fungi grow well in thepoplastic washing fluid of the host (Chandra, 2012).

The fungal endophyte-plant host relationship seems to beightly dependent on genetic, physiological and environmentalontrol (Kogel et al., 2006). Despite of that, there is no doubt thatn the case of mutualistic interaction the presence of the endo-hyte helps to mitigate the effects of plant stresses, which requires

continual metabolic interaction between fungus and plant host.ndophytic fungi have a strong tolerance towards plantı́s metabo-ites due to their ability to transform and detoxify them with theoncomitant production of extrolites, some of them with greatharmaceutical potential as bioactive compounds (Kusari et al.,012; Khan et al., 2015b).

In some cases from endophyte-host relationship resultsetabolites that are produced simultaneously by the plant and the

ungus, like the phytohormones gibberellins (Takeda et al., 2015).

wo opposite theories tried to explain this curious phenomenon.ne supports the idea that endophyte evolved gibberellins biosyn-

hetic pathways independently from plants, based on the highonservation of gibberellins cluster organization in Phaeosphaeria

cal Research 183 (2016) 8–18 9

spp. and Sphaceloma manihoticola, two distantly related fungalspecies. The differences between plants and fungi at biochemi-cal and genetic levels strengthens that higher plants and fungihave evolved their biosynthetic pathways to gibberellins indepen-dently (MacMillan, 1997; Hedden et al., 2001; Yamaguchi, 2008;Bomke and Tudzynski, 2009). The other one point out that duringthe co-evolution of microorganisms and their host plants, endo-phytes undergo genetic modification, for instance by host genetransfer, that allow them to adapt successfully to the plant microen-vironments, which could be also corroborated by the lack of plantresponse against the presence of endophytes (Chapman and Ragan,1980; Germaine et al., 2004).

2.2. Gibberellins as phytohormones

Gibberellins were first identified as phytohormones in the 1930sbased on an over-growth rice seedling due to infections by Fusariumfujikuroi (teleomorph Gibberella fujikuroi) a pathogenic rice fun-gus (Ogas, 2000). These fungal secondary metabolites have beenreported to play a pivotal role in plant growth and developmentprocesses, such as regulation of gene expression in the cereal, seedgermination, stem elongation, flowering and fruit development. Inthe presence of gibberellins, plants are able to alter their physiol-ogy and biochemistry in rapid response to environmental changes(Olszewski et al., 2002). Gibberellins were merely isolated andidentified as plant hormone from extracts of higher plants in themid-50s by British scientists (Lang, 1956; Radley, 1956). The knowl-edge of gibberellic acid (GA3) structure from G. fujikuroi openedthe window for new studies that culminated with the discoverythat gibberellins were diterpenoid compounds (Birch et al., 1958;Cross et al., 1959). Further studies were done, most of them withthe mutant BI-41a (GA-deficient mutant of G. fujikuroi blockedat an early step of the pathway), and bring to light the biosyn-thetic pathway of gibberellic acid in the G. fujikuroi (Bearder et al.,1974; Bearder, 1983). During several years, gibberellin pathwaywas only reported in the F. fujikuroi. Detailed characterization atchemical, biochemical and genetic levels in F. fujikuroi has beenreported (Cerda-Olmedo et al., 1994; Tudzynski, 2005; Bomke andTudzynski, 2009). The GA3 biosynthesis, for example, involve twoearly cyclization reactions, from geranylgeranyl diphosphate toent-kaurene, followed by several oxidative reactions catalyzed bycytochrome P450 monooxygenases to render the final product,19–10 �-lactone (Keller and Hohn, 1997; Tudzynski and Holter,1998).

The gibberellins are small molecules of a large group of tetra-cyclic diterpenoid carboxylic acids, being defined by their chemicalstructure based on the ent-gibberellane carbon skeleton andassigned gibberellin “numbers” depending on chronological orderof their identification. Nowadays, there are 136 known gibberellinsproduced by fungi, plants and even bacteria. Nevertheless, only asmall number of them, such as GA1, GA3, GA4 and GA7 are promi-nent bioactive (Davies, 2004).

2.3. Molecular details of gibberellin action

As phytohormones, gibberellins regulate critical steps in theplant life cycle. Their physiological action is mainly exerted bycounteracting the inhibitory effect of DELLA proteins, a family ofnuclear negative regulators that restrict plant growth probably bytranscriptional reprogramming (Fig. 1) (Sun, 2011). DELLA proteinsare expressed under osmotic or temperature stress, repressing theplant growth. Exposure of Arabidopsis thaliana to salt stress trig-

gers a reduction in bioactive gibberellins, promotes DELLA (group oftranscriptional regulators) accumulation and consequently DELLA-mediated growth restriction (Achard et al., 2006). Although it isnot yet clear whether gibberellins response is dependent on a

10 A.L. Leitão, F.J. Enguita / Microbiological Research 183 (2016) 8–18

Fig. 1. Action mechanism of gibberellins. Gibberellins (GA) action is exerted by binding to the GID1 nuclear receptor, and subsequent recruitment of DELLA proteins. Thef uitin

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ormation of the ternary complex, GA-GID1-DELLA, facilitates the action of the ubiq6S proteasome. Gibberellins can be synthesized directly by plants or by endophytver the plant growing signals. Under abiotic stress conditions, several gibberellin-

ascade of interactions involving gibberellins and other hormoneignalling pathways, the regulation of expression or activity ofranscriptions factors involved in gibberellin metabolism genesould represent one mechanism of stress tolerance in plants. Athe same time, the abiotic form of stress is able to induce a seriesf enzymes which are involved in the inactivation of gibberellins,amely gibberellin-oxidases (Rieu et al., 2008). Active forms of gib-erellins (named GA1, GA3, GA4 and GA7) act via GID1 proteins, aamily of specific nuclear receptors that play an important role inegulating different developmental processes in plants (Ueguchi-anaka et al., 2007; Voegele et al., 2011). Gibberellin binding toID1 receptor induces a conformational change in the protein thatakes it prone to interact with the N-terminal domain of DELLA

epressors. Gibberellins of fungal origin share the same functionalharacteristics of the plant gibberellins, since they are identical inheir chemical structure (Khan et al., 2013b). After the interactionith gibberellins, the GID1-DELLA complex is subsequently ubiq-itinated by the ubiquitin-transferase SCF, and thus targeted forrotein degradation mediated by the 26S proteasome. In conse-

uence, the gibberellin action results in reduced levels of the DELLAepressor and a stimulation of plant growth (Fig. 1).

transferase SCF, which acts over DELLA proteins and induces their degradation viaroorganisms, namely fungi, counteracting the inhibitory effects of DELLA proteinsating enzymes are produced, such as gibberellin-oxidases (GA2ox).

The detailed molecular mechanism of the gibberellin-GID1-DELLA interaction has been characterized by X-ray crystallographystudies (Fig. 2) (Murase et al., 2008; Shimada et al., 2008). Com-plexes containing gibberellins GA3 and GA4, the GID1 receptor andthe N-terminal DELLA domain have been resolved at high resolu-tion, showing an intimate interaction between the receptor andthe gibberellin molecule, established in a deep protein pocket andbased mainly on hydrophobic interactions (Fig. 2) (Murase et al.,2008). However, the lack of the structural information on the apo-receptor prevented to understand the dynamics of its interactionwith the gibberellin ligand and the subsequent binding to the N-terminal DELLA domain.

3. Fungi as gibberellin producers: biosynthetic geneclusters

Several species of fungi belonging to the geni Fusarium,Aspergillus and Penicillium have been currently characterized as

gibberellin producers (Tudzynski, 2005). The canonical pathwayfor gibberellin biosynthesis in fungi was originally described inF. fujikuroi and their molecular details and involved enzymesreviewed elsewhere (Tudzynski, 2005; Bomke and Tudzynski,

A.L. Leitão, F.J. Enguita / Microbiological Research 183 (2016) 8–18 11

Fig. 2. Structural determinants for the gibberellin action as determined by X-ray crystallography. (A) ribbon representation of the GA3-GID1-DELLA (PDB code: 2ZSH),showing the GID1 structure with the binding pocket where gibberellin GA3 is located, and also the N-terminal domain of DELLA. The graph was prepared by using theCCP4MG software (McNicholas et al., 2011). (B) planar diagram of molecular interactions (Ligplot diagram) occurring in the substrate binding pocket of GID1 receptori etweed residua

2sdlctw(abt(

tlc4GgBdbPagpctab2tpioees

nvolved in the recognition of gibberellin GA3. Atoms involved in hydrogen bonds botted lines. Hydrophobic interactions are depicted only by the number and type of

nd Swindells, 2011).

009). Gibberellins, like other diterpenoid compounds, areynthesized starting from geranyldiphosphate (GDP), farnesyliphosphate (FDP) and geranylgeranyl diphosphate (GGDP). This

ast compound is a precursor for gibberellins and also for somearotenoids and ubiquinones. In fungi and plants, GGDP is cyclizedo produce ent-kaurene, the first gibberellin-specific precursor,hich will suffer sequential oxidations to generate GA12-aldehyde

Fig. 2). Fungal gibberellin biosynthetic pathway will convert GA12-ldehyde into GA14-aldehyde by an oxidation reaction catalyzedy a cytochrome P450 protein. Further oxidation and desatura-ion reactions will produce the gibberellins GA1, GA3, GA4 and GA7Tudzynski, 2005; Bomke and Tudzynski, 2009).

Despite the biochemical characterization of gibberellin biosyn-hetic pathway in fungi, the genetic background is comparativelyess known. In F. fujikuroi the gibberellin biosynthetic cluster isomprised by seven clustered genes encoding four cytochrome P-50 oxidoreductases (P450-1, P450-2, P450-3 and P450-4), twoGDP synthases (Ent-kaur-16-ene synthase, CPS/KS, and geranyl-eranyl diphosphate synthase, GGS2), and a GA4 desaturase (DES).esides species belonging to the Fusarium genus, there are only twoocumented cases of the genetic characterization of a gibberelliniosynthetic gene cluster in Sphaceloma (Bomke et al., 2008) andhaeosphaeria (Kawaide et al., 1997, 2000). The evolutionary mech-nisms by which these fungi acquired the gibberellin biosyntheticene clusters are not yet clear. An increasing number of evidencesointed out that the presence of homologous biosynthetic genelusters in distantly related fungi can probably result from horizon-al gene transfer (Slot and Rokas, 2011). Interestingly, recent datalso indicated the presence of defective or truncated gibberelliniosynthetic clusters in some Fusarium species (Wiemann et al.,013). These incomplete clusters are probably related with adap-ive phenomena in these fungal species, which exerted selectionressure for specific gene deletion (Malonek et al., 2005). In Fusar-

um mangiferae, Fusarium circinatum and some strains of Fusarium

xysporum, the gibberelling biosynthetic cluster is present but itsxpression prevented by a non-functional promoter (Wiemannt al., 2013). Silent biosynthetic clusters like those observed inome Fusarium species are very common among fungi, and can

n the receptor and the ligand are depicted for each amino acid and represented bye involved. The panel was designed and edited by the Ligplot+ software (Laskowski

open new possibilities of genetic manipulation for the production ofsecondary metabolites like gibberellins (Leitão and Enguita, 2014).

4. Genus Penicillium

Penicillium belongs to the phylum Ascomycota, however itstaxonomic characterization is still a matter of discussion andthe difficulties in identifying most Penicillium species requiresmultidisciplinary approaches. Clarification of species concepts inthe genus Penicillium was supported mainly by morphologicalcharacteristics. Raper and Thom, for example, based Penicillium tax-onomy classification on the combination of macroscopical (suchas colony texture and color) with micromorphological features(Raper and Thom, 1949). In Raper and Thom classification, Penicilliathat produce monoverticillate conidiophores were included in theMonoverticillata group and this group was divided into nine series(genus subdivision). Later in Pitt’s classification modifications onseries were performed and sections were introduced in subgenusbased on the presence of a swelling at the stipe apex (Pitt, 1979).Despite of direct identification of pure Penicillium species beingpossible by image analysis (Dorge et al., 2000); conidial color, pro-duction of ascomata and ascospores, shape and ornamentation ofconidia and growth rates on solid media remain relevant parame-ters for species identification (Houbraken et al., 2012). Houbrakenet al. (2011) based on a multigene approach redefined the genusPenicillium using single name nomenclature and including bothasexual and sexual reproducing species. They proposed a sectionalclassification and subdivided Penicillium into two subgenera and 25sections (Houbraken et al., 2011).

The genus Penicillium has received much attention due to havethe best known producer of the antibiotic penicillin, P. chryso-genum. Later, mycotoxins became focused upon as they appearedin the processing or ripening of human foods, being a major riskfor human health due to their cytotoxicity (Keller and Hohn, 1997).

However, Penicillium species are also well known as potential toolin the environment field, since they have the ability to degrade, orto remove a wide variety of compounds and heavy metals (Leitão,2009). Furthermore, recent studies demonstrated that the bio-

12 A.L. Leitão, F.J. Enguita / Microbiological Research 183 (2016) 8–18

Fig. 3. Gibberellin biosynthetic pathway in Fusarium fujikuroi and putative gibberellin biosynthetic genes in several Penicillium species. A, gibberellin biosynthetic pathwayas characterized in F. fujikuroi including the involved enzymes; GGDP, geraryl-geranyl diphosphate; EK, ent-kaneurin; EKA, ent-kaneuroic acid; GA14-ald, GA-14 aldehyde.B, sequence alignment of P450-1 protein from F. fujikuroi with its putative orthologs in several Penicillium species. Conserved residues along the sequence are depicted in redboxes.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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ogical activity of Penicillium strains can reduce the genotoxicitynduced by several toxic compounds (Pereira et al., 2014; Romero-guilar et al., 2014).

The success of Penicillium strains is mainly due to their occur-ence in various food and feedstuffs (Santini et al., 2014), indoornvironments such as air, dust and damp building materials (Changt al., 1995; Scott et al., 2004; McMullin et al., 2014; Visagie et al.,014), as well as in the marine (Gong et al., 2014; Kim et al.,014; Liao et al., 2014; Quang et al., 2014; Guo et al., 2015; Parkt al., 2015) and soil (Leitão et al., 2007; Aly et al., 2011; Gongt al., 2014; Moore-Kucera et al., 2014; Tansakul et al., 2014)nvironments. Moreover, the catabolic capacity of these microor-anisms due to the relative unspecificity of their enzymes togetherith their limited growth requirements, diversity of secondaryetabolites production and high ability to form extended mycelial

etworks allow them to survive in an inhospitable environment.t has been suggested that the products originated from fungal

etabolic machinery supply them with a chemical arsenal thatncreases its fitness under challenging ecological conditions. Inact, these characteristics are shared by several fungal speciesnd could be a serious advantage in terms of natural selection.n the other hand, symbiotic interactions with other organismso-occuring in the same habitat have a significant impact in thecosystem. For instance, it is known that Penicillium species aremportant phosphate-solubilizing microorganisms; this capacityllowed Penicillium oxalicum I1 to promote maize growth whenungus was inoculated in the plant (Gong et al., 2014).

Some Penicillium species are considered to be plant pathogensue to their capacity to potentially produce mycotoxins that arehen consumed by humans and animals. For instance, Penicil-ium expansum, Penicillium italicum and Penicillium digitatum, majorostharvest pathogens of pome and citrus, produce the polyke-ide lactone, namely patulin (Li et al., 2015); however patulin isot required by P. expansum to successfully infect apples (Ballestert al., 2015; Li et al., 2015). Interesting, cell-free filtrate of Penicil-ium GP15-1 increased systemic resistance against cucumber leafnfection by the anthracnose pathogen Colletotrichum orbiculareHossain et al., 2014). Other example is the penicisteroid A isolatedrom the culture extracts of the Penicillium chrysogenum QEN-4S strain that colonizes an unidentified marine red algal specieselonging to the genus Laurencia. This polyoxygenated steroidhowed moderate antifungal activity against Alternaria brassicaend potent activity against Aspergillus niger (Gao et al., 2011).ecently, a study conducted with a P. janthinellum strain showedhat its inoculation in tolerant Solanum lycopersicum reduced cel-ular superoxide anions in aluminum stress (Khan et al., 2015c).dditionally, the effect of fungal strain in the tomato plant was com-ared to exogenous gibberellic acid and a similar bio-prospectiveotential was described. Based on these results, the application ofiochemically active endophyte was proposed to increase metalhytoextraction and ensure crop physiological homeostasis.

. Saline stress, gibberellins and Penicillium

.1. Salinity

Salinity is the word that describes soils that enclose high concen-rations of water-soluble salts, mainly NaCl, which causing seriousgricultural yield losses. It is estimated that 20 % of the world’s cul-ivated fields and approximately half of the arable soil are affectedy salinity (Sairam and Tyagi, 2004). If we consider that in 2050

he population will increase 2.3 billion, representing an increase of0% of food crop production demands a new approach for threat-ning food security worldwide is essential (FAO, 2009). Salinitys hostile to most forms of life because it is responsible for an

cal Research 183 (2016) 8–18 13

imbalance of cellular ion homeostasis, which requires a quicklyosmotic adjustment via morphological flexibility and biosynthesisof secondary metabolites such as compatible solutes, which accu-mulation in plants at the millimolar range play an important rolein plant tolerance to salt stress (Chen and Murata, 2011; Nounjanet al., 2012). Despite that, the ability of Penicillium strains to toleratehigh concentrations of NaCl is known. In fact, the genera Penicilliumis representative of the pan-global stable mycobiota in hypersalineenvironment (Butinar et al., 2011).

In plants high salinity inhibits the growth of root and shootsystems by limiting the availability of water and micronutrientscausing cellular damage and modulating several processes. Besidesthe great effort canalized to the compatible solutes (for examplebetaine (Gao et al., 2004), glycinebetaine (Chen and Murata, 2011),trehalose (Nounjan et al., 2012) and proline (Strizhov et al., 1997;Nounjan et al., 2012)), phytohormone (abscisic acid (ABA), jas-monate (JA), brassinosteroid (BR) and gibberellic acid (GA) (Genget al., 2013; Ismail et al., 2014; Julkowska and Testerink, 2015))and enzymes (as ascorbate peroxidase, glutathione peroxidase,catalase, polyphenol oxidase (Sofo et al., 2015)) biosynthesis, thesalinity has additional negative effects on the cellular energy sup-ply, photosynthesis and redox homeostasis, since plants mustassimilate Na+ and Cl− (Zhu et al., 2010; Jacoby et al., 2011;Muller et al., 2014). When plants are under salinity conditions, thedecrease in photosynthesis can be mainly attributed to lower CO2availability through stomatal closure, being the control of respira-tion rates depend on substrate supply and biochemical regulation.It is suggested that the variability in respiratory responses may varysignificantly between species (Jacoby et al., 2011).

The influx of sodium ions by root epidermal and cortical cellsthrough nonselective cation channels (NSCCS) induces depolar-ization of the plasma membrane, reducing potassium ions (K+)channels uptake through inward-rectifying (Shabala and Cuin,2008). To prevent additional influx of sodium ions two deacti-vation mechanisms may be involved: by NSCC channels throughcAMP/cGMP-dependent signals or by high affinity potassiumtransporter (HKAT) channel. As a consequence of osmotic stress,activation of mechanosensitive calcium channels results in an addi-tional influx of protons and calcium ions (Ismail et al., 2014). Thecytosolic concentration of calcium (Ca2+) increase inducing reactiveoxygen species (ROS) production through NADPH oxidase stimula-tion and activating Ca2+ calmodulim-dependent kinases. The Ca2+

calmodulim-dependent kinases stimulates the plasma-membraneH+-ATPases activity among others enzymes, restoring membranevoltage and inhibiting depolarization-activated NSCCS (Klobus andJanicka-Russak, 2004; Shabala et al., 2006; Ismail et al., 2014;Julkowska and Testerink, 2015). On the other hand, Ca2+ and ROSmodulates the release of abscisic acid (ABA), a phytohormone thatregulates several plant biological processes such as growth, biosyn-thesis of compatible solutes, control of stomatal closure, amongothers (Ismail et al., 2014).

In a recent and very interesting review, it was proposed thatdepending on the timing of the events triggered by the sodium ionan adaptive/acclimation responses or the sodium accumulation inthe cytoplasm might occur. The adaptive responses could involvemechanisms of sequestration into vacuole and extrusion of sodiumas well as the constraint of jasmonate (JA) signalling. Meanwhile, adelay in the activation and, consequently, also in the deactivation,of “salinity signalling” through the generation and dissipation oftriggered calcium-dependent signal relative to a signal transmittedby ROS will originate the activation of JA signaling and thus leadingto cell death (Ismail et al., 2014).

It is out of the scope of this review to describe the plant cellu-lar mechanisms and molecular responses to high salinity, but forthose readers who are interested in this aspect we recommend thereview published elsewhere (Hasegawa et al., 2000; Sairam and

14 A.L. Leitão, F.J. Enguita / Microbiological Research 183 (2016) 8–18

Table 1Gibberellins production by Penicillium strains and effect on plant growth under salinity stress.

Penicillium strain Gibberellin (GA) Plant growth effect under salinity stress References

P. citrinum KACC43900 GA1 1.95 ng/ml Can promote Ixeris repens growth (shootlength, plant length)

Khan et al. (2008)

GA3 3.83 ng/mlGA4 6.03 ng/mlGA5 0.365 ng/mlGA7 2.35 ng/mlGA9 0.65 ng/mlGA12 0.11 ng/mlGA15 0.72 ng/mlGA19 0.67 ng/mlGA20 0.30 ng/mlGA24 1.40 ng/ml

P. funiculosum LHL06 GA1 1.53 ng/ml Can promote soybean growth (shoot length,shoot fresh/dry biomass, chlorophyll content,photosynthesis rate, leaf area)

Khan et al. (2011a)

GA4 9.34 ng/mlGA8 1.21 ng/mlGA9 37.87 ng/ml

P. minioluteum LHL09 GA4 12.84 ng/ml Can promote soybean growth (shoot length,shoot fresh/dry biomass, chlorophyll content,leaf area)

Khan et al. (2011b)

GA7 48.912 ng/ml

Penicillium sp. SJ-2-2 GA1 1.185 ng/ml Can promote cucumber growth (shoot length,plant height, chlorophyll content, leaf area)

You et al. (2012)

GA3 1.255 ng/mlGA4 3.497 ng/mlGA7 1.357 ng/mlGA9 0.530 ng/mlGA12 0.335 ng/mlGA19 0.011 ng/mlGA20 0.033 ng/mlGA24 0.838 ng/mlGA34 0.049 ng/ml

Penicillium sp. LWL3 GA1 5.33 ng/ml Can promote sitiens growth (shoot length,shoot fresh biomass, photosynthesis rate)

Waqas et al. (2012)

GA3 3.42 ng/ml

P. janthinellum LK5 GA3 1.2 ng/ml Khan et al. (2013a,b)GA4 10.19 ng/mlGA7 0.71 ng/mlGA12 13.98 ng/ml

P. resedanum LK6 GA1 7.1 ng/ml Can promote pepper growth (shoot length,shoot dry weight, photosynthesis rate)

Khan et al. (2015a,b,c)

GA3 13.9 ng/mlGA4 19.159 ng/mlGA7 1.12 ng/ml

TDyscrcrspnp

5g

e

GA9 2.2 ng/mlGA12 1.93 ng/mlGA20 1.68 ng/ml

yagi, 2004; Ismail et al., 2014; Julkowska and Testerink, 2015).espite of plant salinity stress research advances in the recentears, an understanding of the temporal dynamic nature of tran-criptional events is still lacking. Gibberellins are an example oflassical growth promoting hormone; however, salt stress inducedepression of the gibberellin signaling pathways resulting in lowerell cycle (West et al., 2004). GA biosynthesis and signaling haveecently been shown to be necessary during the late phases of thealt response to promote recovery (Geng et al., 2013). Therefore, GAresence is also a critical factor under salt stress, justifying an alter-ative approach to prevent its absence. Can symbiotic interactionlant-fungus supply gibberellins?

.2. Interactions between Penicillium and plants through

ibberellins

Although various Penicillium species have been reported asndophytics (Spurr and Welty, 1975; Collado et al., 1999; Larran

et al., 2001; Cao et al., 2002; Peterson et al., 2005), earlier than 2008very little was known about these symptomless microorganismsliving inside host plant and gibberellins production under salin-ity stress. A strain of Penicillium citrinum isolated from dune plantIxeris repenes was described for the first time as a possible advan-tage for plants at saline environment (Khan et al., 2009). P. citrinumKACC43900 promoted I. repenes growth by the production of bioac-tive gibberellins in the rhizosphere (Khan et al., 2009). Later, severalstudies with different Penicillium strains have been reported usinga low gibberellins biosynthesis mutant rice cultivar, Waito-C forplant growth-promoting verification. Waito-C is a GA-deficient ricemutant, which lacks GA 3�-hydroxylase, and consequently is hin-dering GA1 synthesis from GA20 (Ahmad et al., 2010). In all of thesestudies were confirmed that fungal strains supplied plant growthpromotion to Waito-C. In the case of Penicillium funiculosum LHL06

and Penicillium minioluteum LHL09, isolated from Glycine max. L.,stimulated Waito-C growth by secretion of bioactive gibberellins.In the culture filtrates of LHL06 and LHL09 strains GA1 and GA4 and

A.L. Leitão, F.J. Enguita / Microbiological Research 183 (2016) 8–18 15

Table 2Putative gibberellin biosynthetic enzymes identified by homology with the proteins from F. fujikuroi.

Species Putative gibberellin biosynthetic enzyme MycocosmProtein ID

Hit lenght % Identity E-value

Penicillium bilaiae ATCC 20851 v1.0 Ent-kaurene oxidase (P450-4) Penbi1|369049 163 34.90% 6.72E-65CPS-KS Ent-kaur-16-ene synthase (CPS/KS) Penbi1|369053 243 40.30% 8.86E-107Cytochrome P450 monooxygenase (P450-3) Penbi1|375870 110 35.37% 3.89E-51GA14-synthase (P450-1) Penbi1|375870 150 41.10% 3.95E-74GA20 oxidase (P450-2) Penbi1|375870 150 42.25% 8.78E-78Geranylgeranyl diphosphate synthase (ggs2) Penbi1|416244 90 48.13% 4.96E-53GA4 desaturase (des) Penbi1|481848 36 39.56% 5.69E-09

Penicillium fellutanum ATCC 48694 v1.0 Ent-kaurene oxidase (P450-4) Penfe1|374742 103 34.33% 9.80E-47CPS-KS Ent-kaur-16-ene synthase (CPS/KS) Penfe1|374742 113 35.42% 1.72E-54Cytochrome P450 monooxygenase (P450-3) Penfe1|403578 171 45.72% 3.95E-81GA14-synthase (P450-1) Penfe1|403578 82 41.84% 1.35E-50GA20 oxidase (P450-2) Penfe1|417921 47 43.12% 1.76E-12Geranylgeranyl diphosphate synthase (ggs2) Penfe1|424093 216 42.27% 7.29E-72GA4 desaturase (des) Penfe1|424093 172 42.57% 1.12E-90

Penicillium glabrum DAOM 239074 v1.0 Ent-kaurene oxidase (P450-4) Pengl1|107178 112 37.46% 6.11E-52CPS-KS Ent-kaur-16-ene synthase (CPS/KS) Pengl1|107178 144 42.99% 3.76E-76Cytochrome P450 monooxygenase (P450-3) Pengl1|345507 96 47.76% 3.36E-60GA14-synthase (P450-1) Pengl1|374226 64 38.55% 7.71E-16GA20 oxidase (P450-2) Pengl1|400029 126 40.38% 8.45E-60Geranylgeranyl diphosphate synthase (ggs2) Pengl1|401725 142 36.79% 3.39E-62GA4 desaturase (des) Pengl1|436008 177 39.51% 6.39E-71

Penicillium janthinellum ATCC 10455 v1.0 Ent-kaurene oxidase (P450-4) Penja1|284697 35 34.65% 1.46E-07CPS-KS Ent-kaur-16-ene synthase (CPS/KS) Penja1|427667 154 45.16% 1.99E-82Cytochrome P450 monooxygenase (P450-3) Penja1|427667 149 43.44% 4.65E-88GA14-synthase (P450-1) Penja1|434515 115 44.92% 4.97E-54GA20 oxidase (P450-2) Penja1|445613 203 45.62% 9.72E-84Geranylgeranyl diphosphate synthase (ggs2) Penja1|447197 145 37.56% 6.09E-60GA4 desaturase (des) Penja1|459586 87 48.07% 2.11E-46

Penicillium raistrickii ATCC 10490 v1.0 Ent-kaurene oxidase (P450-4) Penra1|287118 191 43.51% 8.32E-115CPS-KS Ent-kaur-16-ene synthase (CPS/KS) Penra1|287118 171 40.05% 2.71E-110Cytochrome P450 monooxygenase (P450-3) Penra1|348500 99 37.79% 2.87E-48GA14-synthase (P450-1) Penra1|352910 139 34.32% 1.17E-57

hase (

Gt(pg2PlpWirbtLalwlaps(h2

fsf

GA20 oxidase (P450-2)

Geranylgeranyl diphosphate syntGA4 desaturase (des)

A4 and GA7 were detected, respectively, showing the capacity ofhese strains to produce bioactive gibberellins under salinity stressAhmad et al., 2010; Khan et al., 2011c). In another report an endo-hytic fungus, Penicillium sp. LWL3, was isolated from roots of fieldrown cucumber plants and secreted GA1 and GA3 (Waqas et al.,012). Bioactive gibberellins, GA3, GA4 and GA7, were isolated from. janthinellum LK5, an endophytic fungus inhabiting the roots of S.ycopersicum Mill (tomato plant) from fields located near Kyung-ook National University. P. janthinellum LK5 improves growth ofaito-C, as well as of ABA-deficient tomate under salinity, reduc-

ng sodium ion toxicity and incrementing calcium contents in itsoot as compared to control (Khan et al., 2013b). Recently, it haseen reported that endophytes could have effects comparable tohose of exogenous gibberellins. When the endophytic P. resedanumK6, isolated from Capsicum annuum L., and exogenous gibberelliccid treatments were applied on pepper plants significantly ame-iorated the negative effect of salt stress. A higher benefit effect

as observed by application of combined LK6 strain plus gibberel-ic acid treatment. Moreover, it also showed that LK6 strain had thebility to increase biomass, shoot length, chlorophyll content andhotosynthesis rate compared with the uninfected control underalinity stress, such as occurred in other Penicillium strains (Table 1)Khan et al., 2015b). However, the process by which these phyto-ormones are secreted into plant tissues is not known (Khan et al.,015c).

Comparative study on gibberellins production of Fusariumujikuroi and Penicillium sp., curiously revealed that Penicilliumtrains capacity is generally similar or higher than wild type F.ujikuroi. Early, it has been reported that bioactive GA production

Penra1|355675 83 41.50% 1.06E-26ggs2) Penra1|363110 112 47.66% 3.51E-58

Penra1|376286 194 35.93% 1.57E-60

capacity of a P. citrinum strain was much higher than F. fujikuroi(Khan et al., 2008). The Penicillium sp. SJ-2-2, a halophyte of healthyroots collected from a salt marsh of Suncheon Bay in South Korea,synthesized as much GA1 and GA3 than F. fujikuroi, and synthe-sized much more of GA4 and GA7 (You et al., 2012). Similarly, theP. resedanum LK6 was also reported to produce significantly higheramounts of GA1 and GA4 than the F. fujifuroi, and GA3 content wasat lower level (Khan et al., 2015a).

It has been reported that plants react to mycorrhization by GAs-secreting Penicillium strains, altering the ABA, JA and salicylic acidlevels, as well as the accumulation of isoflavones and the enzymesactivities involved in the removal of ROS, catalases and peroxidases.Additionally, endophyte treatment improve plant nutrition balanceas a consequence of higher nitrogen and phosphorus solubilizationand K+, Mg2+ and Ca2+ levels, which in turn might limit/inhibit theuptake of Na+. Despite of antagonistic behavior described in the lit-erature in what concern to JA, ABA and enzymes, which needs tobe clarified at “omics” levels, endophytic association has not onlyre-programmed the plant for higher growth but also significantlyameliorated the effect of salt induced stress (Khan et al., 2011a;Khan et al., 2011b; Waqas et al., 2012; Khan et al., 2013a). The mech-anism by which endophyte treatment augments host response tosalinity stress is still not clearly understood (Khan et al., 2015a).

5.3. Putative gibberellin biosynthetic genes in Penicillium

In F. fujikuroi genome the genes involved in the main steps ofgibberellin biosynthesis are clustered together (Linnemannstonset al., 1999). The gibberellin cluster in this microorganism is com-

1 biologi

poylg(aegtga2tda1dia

6

rtfamulwpsdhbescodttimmrpohphcorecptpttba

6 A.L. Leitão, F.J. Enguita / Micro

osed by seven genes: four genes encoding cytochrome P450xidoreductases (named from 1 to 4) involved in different hydrox-lation steps of the gibberellin nucleus, two GGDP synthase genesocated in tandem (Ent-kaur-16-ene synthase, CPS/KS, and geranyl-eranyl diphosphate synthase, ggs2), and a GA4 desaturase genedes), encoding the enzyme which converts GA4 to GA7. Avail-ble genomic data from JGI Mycocosm genomic resource (Grigorievt al., 2014) allowed us to localize putative gibberellin biosyntheticenes in 5 out of the 14 available complete genomes belonging tohe Penicillium genus (Table 2). Interestingly, one of the analyzedenomes belongs to P. janthinellum which has been previously char-cterized as a gibberellin producer (Khan et al., 2013; Khan and Lee,013). The sequence homology of the putative gibberellin biosyn-hetic enzymes in different Penicillium species showed a higheregree of homology in the group of the cytochrome P450 enzymes,s the ent-kaurene oxidase (P450-4) and the GA14-synthase (P450-) (Fig. 3). Also as depicted in Table 2, putative CPS/KS proteins fromifferent Penicillium species showed a high homology with the orig-

nal enzyme from F. fujikuroi, whereas the desaturase enzymes (des)re comparatively less conserved.

. Conclusions and future perspectives

There is increasing interest in the discovery of naturally occur-ing chemical molecules for stimulating plant growth in ordero increase agricultural crops yield. Endophytic fungi are widelyound in almost all kinds of plants, and their species compositionnd number seems to be affected by ages of plants and environ-ental among other factors. Fungi like Penicillium species can be

sed as a readily renewable and inexhaustible source of extro-ite compounds that can improve plants under negative biotic as

ell as abiotic conditions. It is now commonly accepted that thishenomenon is observed when endophytic fungi-plants are underaline stress. In this environmental condition gibberellins are pro-uced by Penicillium strains. Structures GA1, GA3, GA4 and GA7ave been identified with function as growth hormones producedy Penicillium strains as salinity stress resistance response. Inter-sting, the rhizobacterium Pseudomonas putida H-2-3 is also able toecrete gibberellin when soybean is under saline and drought stressonditions, improving the plant growth (Kang et al., 2014). On thether hand, it has been shown that plants reduce gibberellins pro-uction in the response to abiotic stress, reducing growth in orderhat plant can focus its energy and carbon resources on resistinghe stress (Colebrook et al., 2014). If we considered that all liv-ng organism have as priority growth and survival, the capacity of

icroorganisms to produce metabolites that are plant secondaryetabolites could be a way that microorganisms found to embar-

ass the host plants?. Endophytic Penicillium strains draw fromlant the water, food and physical protection against biotic and abi-tic adverse conditions, which allow them to live within the plantopping by favorable conditions to completely colonize the host-lant. Meanwhile, endophytic symbiosis resulted in significantlyigher assimilation of nutrient like phosphorus, sulfur, magnesium,alcium and potassium as compared to control plants; besides sec-ndary metabolites that endophyte may produce. It has been alsoeported that endophytic fungi biomass could constitute an inter-sting nitrogen source for plant. Furthermore, several Penicilliumultures revealed the presence of indole acetic acid, other importanthytohormone (Khan et al., 2011b; Waqas et al., 2012). Addi-ionally, it was described that Penicillium endophytes can helpedlant to re-program its responses to saline stress by regulating

he endogenous phytohormones and enzymes to minimize cellularoxicity (Khan et al., 2013a; Khan et al., 2015c). Such interactionsetween native endophytic fungi and plant host could be a reli-ble methodology to the plant salt stress, since chemical solutions

cal Research 183 (2016) 8–18

could be harmful to organisms in the soil, reducing the biodiver-sity of ecosystems. The major concern is to predict environmentalchanges, endophyte-host interactions, as well as plant-microflorasystem development. Nevertheless, we believe that application ofbioactive gibberellins Penicillium strains producers is a promiseenvironmental friendly strategy of improving plant growth andameliorating damage cause by salt stress in cultivation crops.

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