Fungal Genetics and Biology 70 (2014) 86–93

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Fungal Genetics and Biology

journal homepage: www.elsevier .com/ locate/yfgbi

Intracellular pH responses in the industrially important fungusTrichoderma reesei

http://dx.doi.org/10.1016/j.fgb.2014.07.0041087-1845/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding authors at: VTT Technical Research Centre of Finland, Espoo,Finland (M. Valkonen); Laboratory of Biotechnology, National Institute of Chemis-try, 1000 Ljubljana, Slovenia (M. Bencina).

E-mail addresses: mari.valkonen@vtt.fi (M. Valkonen), mojca.bencina@ki.si(M. Bencina).

Mari Valkonen c,⇑, Merja Penttilä c, Mojca Bencina a,b,⇑a Laboratory of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Sloveniab Centre of Excellence EN-FIST, 1000 Ljubljana, Sloveniac VTT Technical Research Centre of Finland, Espoo, Finland

a r t i c l e i n f o

Article history:Received 8 May 2014Accepted 8 July 2014Available online 19 July 2014

Keywords:Genetically encoded pH sensor RaVCRatiometric indicatorpH homeostasisFilamentous fungus Trichoderma reeseiDual sequential scanning microscopy

a b s t r a c t

Preserving an optimal intracellular pH is critical for cell fitness and productivity. The pH homeostasis ofthe industrially important filamentous fungus Trichoderma reesei (Hypocrea jecorina) is largelyunexplored. We analyzed the impact of growth conditions on regulation of intracellular pH of the strainRut-C30 and the strain M106 derived from the Rut-C30 that accumulates L-galactonic acid—fromprovided galacturonic acid—as a consequence of L-galactonate dehydratase deletion. For live-cellmeasurements of intracellular pH, we used the genetically encoded ratiometric pH-sensitive fluorescentprotein RaVC. Glucose and lactose, used as carbon sources, had specific effects on intracellular pH ofT. reesei. The growth in lactose-containing medium extensively acidified cytosol, while intracellular pHof hyphae cultured in a medium with glucose remained at a higher level. The strain M106 maintainedhigher intracellular pH in the presence of D-galacturonic acid than its parental strain Rut-C30. Acidicexternal pH caused significant acidification of cytosol. Altogether, the pH homeostasis of T. reeseiRut-C30 strain is sensitive to extracellular pH and the degree of acidification depends on carbon source.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

The filamentous fungus Trichoderma reesei (anamorph ofHypocrea jecorina) is one of the most efficient industrial hemicellu-lase and cellulase producer (Schuster and Schmoll, 2010). It hassignificant importance as production host for industrial enzymesin general and for enzyme preparations used in hydrolysis oflignocellulosic raw materials in biorefinery applications. Theimpact of environmental factors and culture conditions on theproduction of hydrolytic enzymes by T. reesei has been investigatedand it has been seen that the carbon source, temperature, light andexternal pH (pHex) significantly affect fungal enzyme production(Adav et al., 2011; Aro et al., 2005; Jun et al., 2013; Kubiceket al., 2009; Pakula et al., 2005; Schmoll et al., 2010). Earlier reportshave shown that certain xylanases are produced at neutral pH,while acidic pH favors cellulase production (Bailey et al., 1993).Unlike some other filamentous fungi such as Aspergillus niger andA. nidulans, which tolerate broad extracellular pH ranges, T. reesei

favors more acidic pH, and its growth and enzyme production isimpaired at alkaline pH values of above 6.5 (Adav et al., 2011; Liet al., 2013).

Research on pH homeostasis of yeast Saccharomyces cerevisiaehas shown that cytosolic pH (pHi) is an important regulator of cellfitness (Aabo et al., 2011; Orij et al., 2012, 2011). A decrease of pHi

reduces cell vitality and consequently decreases production yieldsof S. cerevisiae. Low pHex (reviewed in Mattanovich et al. (2004)) orproduction of organic acids such as xylonic acid (Toivari et al.,2012) poses pH stress for the cells. Moreover, near neutral pHi

has been linked directly to more efficient production of lactic acid(Valli et al., 2006). There appear to be intrinsic differences in pHtolerance between different fungal species. The robustness of pHhomeostasis is a trademark of certain industrial strains, e.g. forthe citric acid producing strain A. niger. It has been shown thatthe pHi in the A. niger hyphae can be slightly above neutral evenwhen the pHex is as low as 1.8 (Bagar et al., 2009; Hesse et al.,2002).

The pHi measurements with pH-sensitive fluorescent dyes(reviewed in Han and Burgess (2010)) often suffer from problemsassociated with dye loading and dye sequestration within organ-elles. The penetration of dyes into cells varies significantly withdifferent dyes and cell types. Fluorescence of pH-sensitive fluores-cent proteins (reviewed by Bencina (2013)), on the other hand, is

M. Valkonen et al. / Fungal Genetics and Biology 70 (2014) 86–93 87

stable, species-independent, and rarely toxic to cells. A geneticallyencoded pH sensor can be targeted to subcellular compartmentsand monitored non-invasively in living cells using fluorescencemicroscopy, fluorescence spectroscopy, or flow cytometry (Bagaret al., 2009; Maresová et al., 2010; Miesenböck et al., 1998; Orijet al., 2009; Ullah et al., 2012; Valkonen et al., 2013). pH indicatorsbased on pHluorin (Miesenböck et al., 1998) are ratiometric byexcitation; the measurement relies on changes in the excitationspectrum upon pH. Therefore, intracellular sensor concentrationsand problems encountered by photobleaching do not affect themeasurement (O’Connor and Silver, 2007). pHluorin has been usedextensively for the analysis of pHi in yeasts (Maresová et al., 2010;Orij et al., 2009; Ullah et al., 2013; Valkonen et al., 2013). Bagaret al. (2009) were the first that used the genetically encoded ratio-metric pH sensor in filamentous fungi. They developed and charac-terized an improved version of the pHluorin, called RaVC, for theanalysis of pHi in A. niger and measured pH within individual livinghyphae using dual excitation confocal laser scanning microscopy.

The current work aims to obtain insight on the pH homeostasisin living hyphae of T. reesei. For improving enzyme production, thepH homeostasis mechanisms should be taken into considerationsince many protein synthesis processes are susceptible to pH. InA. niger, a mathematical model has been built in order to predictthe behavior of pH homeostasis in citric acid producing conditions(García and Torres, 2011). To our knowledge, the regulation ofintracellular pH of T. reesei has not been studied before. Weassessed the pHi of the industrial T. reesei Rut-C30 strain andM106 strain that originates from Rut-C30. Due to deletion ofL-galactonate dehydrogenase (Dlgd1) (Kuorelahti et al., 2006)the M106 strain accumulates L-galactonic acid, converted fromD-galacturonic acid added to the medium (Kuivanen et al., 2012).We also evaluated the impact of lactose and glucose as carbonsources on pHi. In addition, our results showed that there wereno differences in the pHi within different regions of hyphae, i.e.pHi gradients were absent, in the living hyphae of T. reesei. Thegrowth conditions, external pH and carbon sources notably influ-ence the pH homeostasis of T. reesei.

2. Experimental section

2.1. Strains and vector construction

A RaVC gene (Bagar et al., 2009) was cloned into an expressionvector pMV119 using yeast recombinational cloning (Oldenburget al., 1997). Initially, the RaVC gene was amplified by polymerasechain reaction (PCR) from a pMOJ009 vector with 30 bp overlap-ping ends to an A. nidulans gpdA, glyceraldehyde-3-phosphatase,promoter and a trpC, tryptophan synthase terminator. The forwardand reverse primers were: 50-GACTAACAGC TACCCCGCTT GAGCA-GACAT CATGGTGAGC AAGGGCGAGG AGCTGTTCAC CGGGGTG and50-CAGTAACGTT AAGTGGATCC CCGCGGACTA GTTTATTTGT ATA-GTTCATC CATGCCATGT GTAATCC, respectively. An A. nidulans ace-tamidase (amdS) gene (within the same transforming cassettes asthe RaVC gene) was used as a selection marker for T. reeseitransformation. The RaVC expression construct was targeted to aspecific locus using �1.5 kbp sequences flanking the hfb2 gene.For transformation into the T. reesei, pMV119 plasmid DNA wasdigested with PmeI and a fragment containing the RaVC expressioncassette, the amdS marker and the hfb2 flanking sequences, wasused for transformation. The RaVC was transformed into theT. reesei strains M106, with deleted L-galactonate dehydratase(Dlgd1) (Kuorelahti et al., 2006) and the strain, RutC-30(Montenecourt and Eveleigh, 1979). The transformation was per-formed according to Penttilä et al. (1987). The transformants,where the hfb2 gene was replaced by the RaVC expression cassette,

were screened by PCR. Transformants with similar fluorescencelevels and morphology were selected for further experiments.Based on determination of dry weight and media acidification after48 h, differences between the parental Rut-C30 and M106 strains,and the RaVC transformants were not significant.

2.2. Media and growth conditions

To analyze the effect of carbon sources on the pHi, the RutC-30and M106 strain were cultured in a minimal medium (5 g l�1

(NH4)2SO4, 15 g l�1 KH2PO4, 0.6 g l�1 MgSO4, 0.6 g l�1 CaCl2, 5 mg l�1

FeSO4 � 7H2O, 1.6 mg l�1 MnSO4 � H2O, 1.4 mg l�1 ZnSO4 � 7H2O,3.7 mg l�1 CoCl2) (pH 5) (Penttilä et al., 1987) with 20 g l�1 glucose,or 20 g l�1 lactose as a carbon source. For testing the impact ofL-galactonate accumulation on pHi, the fungi were grown in mini-mal medium with 10 g l�1

D-xylose (pH 5) for 24 h, after which10 g l�1

D-galacturonate (sodium salt, Sigma) was added.To determine the influence of extracellular pH (pHex) on pHi, the

strains were grown in the Verduyn minimal medium (Verduynet al., 1992) with 20 g l�1 glucose and buffered to pH 4–7 withK-biphtalate or K-phosphate (K-hydrogen phthalate/HCl for pH 4;K-hydrogen phthalate/NaOH for pH 5; K-dihydrogen phosphate/NaOH for pH 6, 7 and 8). All cultivations were performed in 24-wellplates at 30 �C, with shaking, for 24–48 h. Sorbic acid was used fora weak acid stress. The M106 strain was grown in the minimalmedium with 20 g l�1 glucose and treated with 5 or 10 mM sorbicacid. For exposure to the weak acid stress, samples were analyzedbefore treatment and for 10 min following treatment.

For imaging the spatial distribution of RaVC and analysis of pHi

gradient, the T. reesei was grown on the minimal medium with20 g l�1 lactose solidified with 1.5% (w/v) agar. 72-h-old culturesof T. reesei were used for experiments. Agar bearing a leading edgeof the colony was cut out and carefully placed, hypha side down,on a glass coverslip. The plasma membrane and vacuoles werestained with SynaptoRed C2 dye (25 lM) for 15–30 min (Fischer-Parton et al., 2000).

2.3. Confocal laser scanning microscopy

A Leica TCS SP5 laser scanning microscope mounted on a LeicaDMI 6000 CS inverted microscope (Leica Microsystems, Germany)equipped with an HCX PL APO 63� (NA 1.4) oil immersion objec-tive was used for imaging. For pH indicator RaVC recording, amethod described by Bagar et al. (2009) was adopted. For sequen-tial excitation, a 50 mW 405-nm diode laser and a 476-nm line of a25 mW argon laser were used. Laser powers of 3% and 10% wereused for the diode laser and the argon laser, respectively. Succes-sive images excited at 405 and 476 nm were captured. The fluores-cence emission was detected at 500–550 nm.

2.4. pH measurements

The pHi was measured using the pH-sensitive fluorescent pro-tein RaVC. Fluorescence intensities, after excitation at 405 and476 nm, were recorded and converted to pH values using a customwritten software program incorporating Eqs. (1) and (2) (Bagaret al., 2009).

Ri ¼ ½Fð405 nmÞi � Fð405 nmÞbackground�=½Fð476 nmÞi� Fð476 nmÞbackground� ð1Þ

where F(405 nm)i and F(476 nm)i are the fluorescence intensities ofthe region of interest. F(405 nm)background and F(405 nm)background

denote the average background fluorescence intensities.

pH ¼ pKa � log10 Ri � RmaxpH

� �.Rmin

pH � Ri

� �h ið2Þ

88 M. Valkonen et al. / Fungal Genetics and Biology 70 (2014) 86–93

where Ri is the emission ratio at a given pH, and RpHmin and RpH

max arelimits for the ratio at extreme acidic (pH 5.2) and alkaline (pH 8.5)pH, respectively. These limits were individually determined for eachset of experiments described in next section. The RpH

min and RpHmax

were obtained by incubating mycelia in buffer solutions with thepH values of 5.2 and 8.5 with freshly prepared 50 lM nigericinfor approx. 30 min.

2.5. In situ calibration

An in situ calibration curve, as described by Bagar et al. (2009)with some modifications, was used to calculate pKa. Briefly, myce-lia were washed and resuspended in buffer solutions with a pHrange of 5.2–8.5. The ionophore nigericin (50 lM) was added andthe cells were incubated at least 30 min to equilibrate pHi withpHex. The calibration buffer contained 50 mM MES, 50 mM HEPES,50 mM KCl, 50 mM NaCl, 200 mM ammonium acetate, 10 mMNaN3, 10 mM 2-deoxyglucose, and 50 lM nigericin. The pH wasadjusted with NaOH or HCl (described by Brett et al. (2005)). Aftermycelia had been treated for 30 min with nigericin, fluorescenceintensities after excitation at the 405 and 476 nm were recordedand ratios were calculated using Eq. (1). The mean ratio was con-verted to pH using Eq. (2).

2.6. Image processing and statistical analysis

A representative pseudocolored image of T. reesei hypha—calculated from sequentially acquired raw images—was selectedfrom a set of at least five pseudocolored images at given conditions.Different colors were assigned to defined pH values in accordancewith the in situ calibration curve calculated with Eqs. (1) and (2)(blue, alkaline; red, acidic). Each point of ratio/pH on a graphwas calculated from mean fluorescence intensities of region ofinterest from a pair of two images collected at the 405- and 476-nm excitations. All microscopic data are shown as the mean witha standard deviation calculated from at least ten images per exper-iment. Each set of experiments was independently repeated threetimes. Graphs were prepared with GraphPad Prism 5.0 softwarepackage (http://www.graphpad.com/) that was also used for statis-tical analysis. Student’s unpaired two tailed t-test (assumed Gauss-ian distribution of values) was used for statistical comparisonbetween groups and p values of <0.05 (⁄), <0.01 (⁄⁄), <0.001 (⁄⁄⁄)were considered statistically significant; p > 0.05 was consideredas not significant (ns). Raw confocal images without processingwere analyzed with the ImageJ (http://rsbweb.nih.gov/ij/).

3. Results

3.1. RaVC displays pH sensitivity in hyphal samples

To image spatial distribution and temporal changes of pHi

within hyphae of T. reesei we used the genetically encoded ratio-metric pH sensor RaVC, which was expressed in cytosol of the T.reesei Rut-C30 and M106 strains. In 48-h old hyphae of Rut-C30(Fig. S1) and M106 (Fig. 2) RaVC fluorescence was primarilydetected in the cytosol. Images of growing hyphae were capturedafter excitation at 405- and 476-nm using dual excitation confocalmicroscopy (Fig. S1A) and ratio images were calculated (Fig. S1B).An in situ calibration of RaVC was performed to correlate the calcu-lated ratio of fluorescence intensities with pH (Fig. 1). Hyphae werepermeabilized with nigericin in calibration buffers with pH valuesranging from 5.2 to 8.5 for 30 min. This incubation period was suf-ficient to equalize pHi with pHex. Ratios of emission intensitiesfrom regions of interest were calculated based on Eq. (1) and plot-ted against pHex (Fig. 1A). RpH

min and RpHmax were calculated from the

emission intensities at pH 5.2 and 8.5 for each set of experiments.The in situ determined pKa for RaVC was 7.1 ± 0.1, and it was calcu-lated from the logarithmic plot of the obtained standard/calibra-tion curve (Fig. 1C). The pKa of RaVC is similar to that ofpHluorin, a parent protein from which RaVC is derived. The RaVCprotein has already been tested in the filamentous fungus A. nigerand the codon optimization of RaVC and its pH sensitivity weredescribed previously (Bagar and Bencina, 2012; Bagar et al., 2009).

The pKa, RpHmin, and RpH

max were used to calculate the pHi withinhyphae using Eq. (2). Results are presented as pseudocoloredimages (Fig. 1B and S1B). The range of pH values that can be reli-ably measured is limited by the properties of RaVC. At pH valueslower than 5.5, the RaVC values exhibited a plateau (Fig. 1A) andits fluorescence deteriorated at pH values above 8.5 as determinedpreviously for RaVC (Bagar et al., 2009) and pHluorin (Mahon,2011; Miesenböck et al., 1998; Orij et al., 2009). The optimal linearresponse to pH falls between 5.5 and 8.0. Therefore, calculated pHvalues that are below pH 5.5 or above 8.5 are not shown on plots,and are shown as black on pseudocolored images. The RaVC is aratiometric probe; therefore the calculated pH values are indepen-dent on concentration of the expressed probe. This makes theprobe extremely valuable for in situ or in vivo analysis (Bencina,2013), where the expression of probe varies over time, e.g. at earlytime points of cultivation, and where the exact concentration ofprobe could not be determined. The fluorescence intensities ofprobe were high enough at 48-h of cultivation and later; earlierthe fluorescence was less intense. According to the results of in situcalibration the RaVC fluorescence, in a combination with dual exci-tation confocal microscopy, can be used to study pHi in livinghyphae of T. reesei.

3.2. pHi gradients are absent in growing hyphae

We analyzed whether transverse and longitudinal pHi gradientsare present within regions of hyphae growing by hyphal tip exten-sion on plates on solid agar medium. The ratio images of 72-h oldM106 growing hyphae were captured over time period of 4 min,and pHi values were calculated (Fig. 2, Movie S1). Although minorfluctuations in the pHi were observed, no clear pHi gradient wasdetected along the lengths and cross-sections of apical (Fig. 2,Movie S1) and subapical regions (data not shown) of the T. reeseihyphae. Similar results were recorded for the Rut-C30 strain. Ourresults support the conclusion that pHi gradient cannot be consid-ered as a general feature of polarized cells.

We also observed that under the described growth conditionson solid media, some of the RaVC protein was in addition to itscytosolic localization also sequestered in tubular organelles closeto the tip of the hyphae (Fig. 2) and spherical organelles in the sub-apical parts of the hyphae, which are most likely vacuoles, as themembrane surrounding these organelles can be stained with themembrane-selective dye SynaptoRed C2 (data not shown). Thesetubular and spherical organelles were more acidic than the sur-rounding cytoplasm. Their pH was below 5.5. The exact value couldnot be determined, since it was outside the linear detection rangeof RaVC.

3.3. Lactose as carbon source triggers cytosolic acidification

Lactose containing media are often used with T. reesei in indus-trial scale production of hydrolytic enzymes. On the other hand,expression of hydrolases is repressed by the presence of glucose(Mach-Aigner et al., 2010; Parajó et al., 1998; Schaffner andToledo, 1991; Warzywoda et al., 1983). We analyzed the pH withinhyphae of the Rut-C30 and M106 strains cultured in liquid non-buffered minimal media with glucose, or lactose as carbon sources.Additionally, pH of the culture supernatants was measured. The

Fig. 1. The in situ calibration of the RaVC sensor determined by sequential dualexcitation confocal microscopy. (A) A plot of the fluorescence ratio versus pH. 48-h-old T. reesei M106 cells expressing RaVC were washed with the pH-adjusted buffers,resuspended, and treated with freshly prepared nigericin to equalize pHex with pHi.The ratio (Ri) between the emission intensities (collected between 500 and 550 nm)at 405- and 476-nm excitation for each region of interest was calculated using Eq.(1). Each calculated Ri value is presented as point. (B) Pseudocolored images ofpermeabilized T. reesei M106 hyphae, calculated from images taken at 405- and476-nm excitation. (blue, alkalinic; red, acidic) (C) A logarithmic plot of thefluorescence intensities ratio R0 calculated by [(Ri � RpH

min)/(RpHmax � Ri)] versus pH.

(For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

Fig. 3. Carbon source effects on pHi. (A) The M106 and Rut-C30 strains were grownfor 48 h in liquid minimal medium containing glucose and lactose as carbonsources. The measured pHex of 48-h-old cultivations is depicted below the graph.The pHi for the M106 and Rut-C30 strains in lactose medium was not calculatedsince the pHi was below 5.5. (B) The emission intensities ratios for the M106 andthe Rut-C30 strain grown in medium with lactose or glucose. The M106 and theRut-C30 were grown in a minimal liquid medium, containing lactose or glucose, for48 h before analysis. Each point on a graph presents an intracellular pH (pHi) (A)calculated from a ratio (Ri) (B) from average fluorescence intensities of selectedregions collected at 405- and 476-nm excitations. Line at (B) indicates the lowerrange of linearity determined from the calibration curve (see Fig. 1).

M. Valkonen et al. / Fungal Genetics and Biology 70 (2014) 86–93 89

initial media pH was set to 5. After 48-h of cultivation, the pHof supernatants was between 2.4 and 3.7 depending on the car-bon source (Fig. 3). The time point for analysis of pHi – 48-h of

Fig. 2. The pHi analysis of the growing hyphal tip. (A) Images of RaVC fluorescence after eof the fluorescence intensities of RaVC after a pixel-by-pixel calculation using Eqs. (1) andwas grown on agar plates with a minimal medium with 20 g l�1 lactose. Scale bar = 20 l

cultivation – was selected based on fungal growth and expressionof pH probe. In addition, at that time point approximately 50% car-bon was still detected in media.

The pHi of the M106 strain grown in the glucose containingmedium was 6.2 (±0.3), which was significantly higher than thepHi of the parental strain Rut-C30, 5.7 (±0.3) (Fig. 3A). On the otherhand, the pHi of both strains grown in the medium with lactosewas below 5.5. This is below the linearity of RaVC; therefore, onlymean ratio is depicted (Fig. 3B). The Ri of Rut-C30 was 0.59 and was

xcitation at 405- and 476-nm at defined time points. (B) Pseudocolored ratio images(2). A growing hyphal tip from a margin of a 48-h-old M106 colony is shown. M106m.

90 M. Valkonen et al. / Fungal Genetics and Biology 70 (2014) 86–93

significantly higher than the Ri of M106 (being 0.47). Takentogether, the results show that the carbon source clearly affectedthe pHi of T. reesei.

3.4. D-Galacturonic acid has an impact on intracellular pH of Rut-C30

The engineered M106 strain converts D-galacturonic acid pres-ent in the medium to L-galactonate with D-xylose as a carbonsource. The strain accumulates L-galactonic acid into the culturemedium, but also intracellular accumulation can be observed.The parental strain Rut-C30, in which L-galactonate dehydrataseis present, on the other hand, converts D-galacturonic acid viagalactonic acid further to pyruvate in the cellular metabolism,and intracellular L-galactonate was not detected (Kuivanen et al.,2012).

An effect of D-galacturonic acid on pHi in T. reesei was examined.According to Kuivanen et al. (2012) the optimal xylose concentra-tion for L-galactonic acid synthesis is between 6 and 11 g l�1; andL-galactonate accumulation in the M106 strain is highest 24 h afterthe addition of D-galacturonic acid. Therefore the M106 and theRut-C30 strains were grown in non-buffered D-xylose (10 g l�1)containing medium for 24 h. After that, D-galacturonic acid wasadded to the cultivations and the hyphae were imaged at 6 and24 h after D-galacturonic acid addition. The pHi in hyphae of theM106 strain dropped only slightly, from 6.6 (±0.1) in 6-h sampleto 6.4 (±0.2) (p < 0.01) in 24-h sample (Fig. 4A). Interestingly, thepHi of the Rut-C30 decreased from 6.7 (±0.1) to 6.0 (±0.2) duringthe same time period (Fig. 4B). As a comparison, the pHi of myceliacultured in the D-xylose minimal medium (without D-galacturonicacid addition) for 48 h was 6.4 (±0.2) and 6.3 (±0.1) (p > 0.05; notsignificant) for the strains M106 and Rut-C30, respectively. Alto-gether, the D-galacturonic acid and consequent accumulation ofL-galactonate had no apparent effect on pH homeostasis of theM106 strain. On the other hand, the D-galacturonic acid triggeredacidification of Rut-C30 hyphae. The pH of the culture media (pHex)was also determined. 24 h after the addition of D-galacturonic acidthe pHex was 3.9 for both strains (Fig. 4).

Fig. 4. The pH homeostasis response on D-galacturonic acid differs between M106and Rut-C30 strain. (A and B) The pHi values with representative ratio images(below) from growing hyphae expressing RaVC. (A) The M106 and (B) Rut-C30strains were grown in xylose-containing liquid minimal medium (pH 5) for 24 h,and D-galacturonic acid was added. The pHi was analyzed 0, 6 and 24 h afterD-galacturonic acid addition. As a control, a strain with no addition of D-galacturonicacid was used. The pHex for analyzed cultures is shown below the graph. Each pointon a graph presents an intracellular pH value (pHi) calculated from a ratio (Ri) fromaverage fluorescence intensities of selected regions collected at 405- and 476-nmexcitations. Line indicates pH value 6.5; ns stands for not significant p > 0.05.

3.5. Prolonged exposure to low pHex triggers a pHi decrease

In order to investigate the relationship between pHex and pHi,we grew T. reesei Rut-C30 and M106 strains in buffered Verduynmedia with pHex ranging from 4.0 to 7.0. The Verduyn mediawas used in studies of pHi regulation in yeast (Orij et al., 2009).The medium maintained the pHex at set value better than the med-ium typically used for cultivation of T. reseei. In this experimentalset up with glucose as a carbon source, T. reesei grew slow at thelowest pHex, with some mycelia detected only after 3 days of culti-vation. The slow growth was not a consequence of expressed RaVCprobe, since slow growth at the lowest pH was observed also forthe Rut-C30 parent strain. The pH of the growth media and pHi

of hyphae were measured 48 h after inoculation (Fig. 5). Differ-ences in the pHi for the cultures grown at different pHex wereobserved. The pH homeostasis of the M106 strain seemed moresensitive to pHex than that of Rut-C30, the M106 pHi decreasedwith decreasing pHex. In M106 grown at pHex 5, the calculatedpHi had decreased to 6.0 (±0.2) (Fig. 5A), while the pHi of Rut-C30 was 6.3 (±0.2) (Fig. 5B). The pHi for the cultures grown atthe lowest analyzed medium pH, pHex 4, was �5.4 for both theRut-C30 and M106 strains. It should be noted that the pHi withinmany hyphae was outside the range of a linear RaVC response topH in this condition (Fig. 5). These results show that when exposedto acidic pHex, T. reesei was inefficient in maintaining the pHi nearto neutral after 48-h of cultivation.

3.6. Sorbic acid causes intracellular acidification of T. reesei hyphae

Sorbic acid is a preservative that prevents the growth of micro-organisms in food or beverages (Ullah et al., 2012). It has beenshown that in A. niger sorbic acid decreases pHi and delays sporegermination and growth (Plumridge et al., 2004). We analyzedthe effects of sorbic acid on the pHi of the T. reesei strain M106 bytreating hyphae grown 48 h in non-buffered minimal medium onglucose with 10 mM sorbic acid. The pHi was measured beforethe addition of sorbic acid and then monitored for 10 min. The sor-bic acid treatment resulted in a clear cytoplasmic acidification frompH 6.4 (±0.2) to 5.6 (±0.2) (Fig. 5C). The acidification of cytosol afterthe addition of sorbic acid was recorded also for the Rut-C30 strain(data not shown). The pHi did not recover even after 30 min (datanot shown). The results are in accordance with previously pub-lished data on A. niger using the same method (Bagar et al., 2009).

4. Discussion

4.1. The pH sensor RaVC provides insights into the spatial dynamics ofpHi

The genetically encoded pH-sensitive fluorescent proteins haveproven immensely valuable for studies of pHi of yeast (Maresováet al., 2010; Orij et al., 2009; Ullah et al., 2013), filamentous fungi(Bagar and Bencina, 2012; Bagar et al., 2009), and higher eukary-otes (Bencina, 2013; Miesenböck et al., 1998). Therefore, we usedthe RaVC protein for live-cell imaging of pHi within the hyphaeof the filamentous fungus T. reesei. RaVC is genetically encoded,which avoids cell manipulations to introduce the pH indicator intoa cell that is needed when fluorescent dyes are used. Like pHluorinand pHluorin2, RaVC has bimodal absorbance/fluorescence spec-tral characteristics (Bagar et al., 2009; Mahon, 2011; Miesenböcket al., 1998), which enables the quantification of pHi with high spa-tial and temporal resolution within the pH range from 5.5 to 8.0(Fig. 1).

The RaVC protein expressed in T. reesei hyphae distributeswithin the cell similarly to what was observed in A. niger (Bagar

Fig. 5. Effect of pHex on the pHi. (A) The M106 and (B) Rut-C30 strains were grown in a buffered liquid medium (pH 4, 5, 6, and 7) for 48 h. The pHi values calculated fromfluorescence intensities ratios, and the representative pseudocolored ratio images (below) from growing hyphae expressing RaVC, are depicted. The pHex that the cultivationsreached during the experiment is indicated below the graphs. (C) The effect of sorbic acid on pHi of the M106 strain. The pHi of hyphae grown in minimal liquid medium withglucose (20 g l�1) for 48-h before and 10 min after treatment with the sorbic acid (10 mM). Each point on a graph presents a intracellular pH value (pHi) calculated from aratio (Ri) from average fluorescence intensities of selected regions collected at 405- and 476-nm excitations. Lines indicate pH values 6.0 and 6.5; ns stands for not significantp > 0.05.

M. Valkonen et al. / Fungal Genetics and Biology 70 (2014) 86–93 91

et al., 2009). As in A. niger, it is sequestered within tubular andspherical organelles. These organelles are more acidic than the sur-rounding cytoplasm, and they are in T. reesei more acidic than inA. niger (pH 6.2–6.5) (Bagar et al., 2009; Hesse et al., 2002).

The hallmark of filamentous fungi is polarized hyphal growth.Seiler and Plamann (Seiler and Plamann, 2003) identified many pro-teins that contribute to the polarized growth of Neurospora crassaand divided them in four groups regulating the (i) cytoskeleton,(ii) cell wall and membrane biosynthesis, (iii) signal transductionpathways, and (iv) secretory pathway. Existence of ion gradientsin polarized growing cells has been proposed as a mechanism forestablishing and maintaining cell polarity. For filamentous growthof Candida albicans, a long range gradient of phosphoinositide bis-phosphate has to be generated and maintained by the tip localizedPI(4)P-5-kinase (Vernay et al., 2012). Whether the polarized tip-growth requires a proton gradient has not been confirmed. Forexample, Robson et al. (1996) showed an alkaline pHi gradient atthe extending hyphal tip of N. crassa using a ratiometric pH sensi-tive dye. On the other hand, experiments carried out both withA. niger (Bagar et al., 2009) and N. crassa (Parton et al., 1997) failedto show any pHi gradients longitudinal or across the apical regionsof actively growing hyphae, which is in agreement with our results(Fig. 2, Movie S1). These results support the idea that a cytoplasmicpH gradient is not a general feature of cells growing by tip extensionand argue against a central regulatory role of cytoplasmic pH gradi-ents in supporting polarized tip growth.

4.2. External growth conditions affect the degree of intracellularacidification

As far as we know there is only one previous analysis of pHi

after growth on different carbon sources in filamentous fungi.Hesse et al. (2002) have shown that A. niger can maintain the pHi

values within a narrow range in the presence of various carbonsources. In yeast, the intracellular acidification is directly associ-ated with glucose starvation (Karagiannis and Young, 2001; Orijet al., 2009; Valkonen et al., 2013). Peters et al. (2013) identifiedpHi as a specific cellular signal involved in glucose sensing thatmanages the kinetics of proteosome storage granules’ formationand dissociation. In our experimental set up, there was stillapproximately 50% of the carbon source left in the cultures after48-h of growth, so the intracellular acidification that we observedin this study is not triggered by starvation. We studied pH homeo-stasis of the Rut-C30 and M106 strains grown in minimal medium

containing glucose or lactose. Lactose is a known inducer forproduction of hydrolytic enzymes in T. reesei. Glucose on the otherhand, represses expression of these enzyme genes. The Rut-C30strain is mutated in the glucose repressor gene cre1, which reducesglucose repression, but the strain is not fully derepressed (Mach-Aigner et al., 2010; Parajó et al., 1998; Schaffner and Toledo,1991; Warzywoda et al., 1983). Consequently, cellulase expressionlevels remain lower on glucose than on inducing media. The anal-ysis of cytosolic pH using the RaVC sensor revealed that the carbonsource in the cultivation medium affects the degree of intracellularacidification of T. reesei (Fig. 3). The pHi of mycelia grown in a med-ium with lactose was below the calculated value of 5.5, showing anunexpectedly strong effect on intracellular acidification.

The M106 strain was derived from the parent strain Rut-C30 bydeletion of L-galactonate dehydrogenase (Dlgd1) (Kuorelahti et al.,2006). The M106 strain, converts D-galacturonic acid – the mainmonomer of pectin and an attractive substrate for bioconversions– to L-galactonic acid, which is excreted to the culture mediumand also accumulates inside the cells (Kuivanen et al., 2012). Withrespect to the pHi, the reference strain Rut-C30 was more sensitiveto the presence of the D-galacturonic acid in the medium than theM106 strain (Fig. 4). L-Galactonic acid, due to high pKa (12.6 takenfrom ECMDB data base id no.: ECMDB00565), is undissociated anddiffuses from the cell with no apparent impact on the pHi of M106as shown in this study. The Rut-C30 strain, on the other hand, con-verts D-galacturonic acid to pyruvate and L-glyceraldehyde, whichboth are readily metabolized.

After 48 h of cultivation, the pHex of the cultures on differentcarbon sources decreased from 5 to around 3. The decrease of pHof culture medium during cultivation has been observed also inother studies (Bailey and Viikari, 1993; Bailey et al., 2002). Weexamined whether acidic pHex has an effect on pH homeostasisand consequently on the pHi of T. reesei. The pHi is a tightly regu-lated physiological parameter in all cellular systems. For instance,A. niger can withstand substantial shifts in pH without loss of via-bility during citric acid production. Besides, A. niger can grow atpHex values ranging from 2.5 to 8.5, while pHi remains unaffected(Bagar et al., 2009). The pH homeostasis of the studied T. reeseistrains is not as robust as that of A. niger. At pHex above 4, thepHi of Rut-C30 is maintained reasonably well but clear acidificat-ion of pHi was observed in the medium with initial pH 4 comparedto pH 7 (Fig. 5). Different pHi responses on low pHex was deter-mined for yeast (Orij et al., 2009; Valli et al., 2005; Valkonenet al., 2013). Thus, it is likely that the intracellular acidification is

92 M. Valkonen et al. / Fungal Genetics and Biology 70 (2014) 86–93

at least partially linked to a decrease in pHex, and the type ofcarbon source only delineates a degree of pHi decrease. In thisrespect, lactose would be the least favored carbon source sincethe pHi drops below 5.5. A lactose-associated cytosolic acidificationcould be caused by different speed and metabolic routes for carbonutilization (reviewed by Seiboth et al. (2007b), Ivanova et al. (2013)and Seiboth et al. (2007a, 2005)), which might contribute to theenergy pool to maintain an optimal pHi. The slower growth ofT. reesei on lactose compared to glucose containing media indicatesa slow utilization and conversion of lactose (Fekete et al., 2008),which might explain the observed behavior of pH homeostasis inthe presence of lactose.

The cytosolic acidification of T. reesei could be associated withinflux of protons during the uptake of nutrients as has been shownfor yeasts (Guimarães et al., 2008). In addition, the excretion oforganic acids during growth acidifies growth medium (Bailey andViikari, 1993; Bailey et al., 2002; Olejníková et al., 2011). As wehave shown here, low pHex at least partially shapes pHi of T. reesei.For A. niger, on the other hand, the pHi remains unchanged also atacidic pHex (Bagar et al., 2009). For yeast S. cerevisiae, carbon star-vation initiates significant decrease in pHi (Orij et al., 2009;Valkonen et al., 2013). It seems that the pHi regulation of A. niger,yeasts and T. reesei differs; nevertheless a direct comparison is dif-ficult since growth conditions differ between organisms. The acid-ification is normally counteracted by H+-ATPases (reviewed forA. niger (Bencina et al., 2009)) draining protons against a protongradient into the organelles, or into the medium. A transcriptomicanalysis of Trichoderma viride revealed that acidification of mediumtriggers the repression of P-type H+-ATPases (Trushina et al., 2013).It has been shown that during cultivation and at the acidification ofmedia, H+-ATPase activity decreases (Simkovic et al., 2007;Trushina et al., 2013) probably leading to acidification of the cyto-sol. Direct correlation between H+-ATPases activities and cytosolicacidification remains to be established.

Taken together, the external growth parameters—carbon sourceand pHex—affect the pHi of the hydrolase-hyperproducing strains,Rut-C30 and M106. The differences observed for T. reesei whencompared to A. niger and yeast S. cerevisiae may at least partly bedue to the fact that the RutC-30 strain is a heavily mutagenisedstrain. We could not exclude that mutations in the genome of thisindustrially important strain affect the pHi regulation. The effect ofmutations on pHi could be elucidated with detailed analysis pHhomeostasis of T. reesei wild type strains. The importance in sus-taining optimal pHi has been demonstrated with yeast in connec-tion with lactic acid production; higher pHi was associated withbetter productivity (Valli et al., 2006) and faster growth (Orijet al., 2012). For T. reesei, it has been shown that fed-batch andpH-controlled fermentations give better cellulase yields (Baileyand Tähtiharju, 2003) than batch fermentations where the nutrientdepletion and the medium acidification occur. So far, however,data on pHi in filamentous fungi in different culture conditions islimited and the maintenance of pHi might be important to ensureoptimal cellular activity during fermentations. It would be valuableto study the pHi of T. reesei (and other fungi) industrial strains inproduction conditions. Therefore, further analysis will be necessaryto evaluate a link between intracellular pH and productivity of theTrichoderma.

Acknowledgments

This research was supported by the Slovenian Research Agencyand the EN-FIST Centre of Excellence, the Academy of Finlandunder the Finnish Centre of Excellence in White Biotechnology—Green Chemistry program (Grant 118573). The authors wish tothank Dr. Marilyn Wiebe, Dr. Juha-Pekka Pitkänen, and MScYvonne Nygård for their help in the design of the experiments.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.fgb.2014.07.004.

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