Accepted Manuscript Title: Differential expression of SofDIR16 and SofCAD genes in smut resistant and susceptible sugarcane cultivars in response to Sporisorium scitamineum Authors: Elena S´anchez-Elordi, Roberto Contreras, Roberto de Armas, Mario C. Benito, Borja Alarc´on, Eliandre de Oliveira, Carlos del Mazo, Eva M. D´ıaz-Pe˜na, Roc´ıo Santiago, Carlos Vicente, Mar´ıa E. Legaz PII: DOI: Reference:
S0176-1617(18)30147-0 https://doi.org/10.1016/j.jplph.2018.04.016 JPLPH 52774
To appear in: Received date: Revised date: Accepted date:
25-10-2017 27-4-2018 30-4-2018
Please cite this article as: S´anchez-Elordi Elena, Contreras Roberto, de Armas Roberto, Benito Mario C, Alarc´on Borja, de Oliveira Eliandre, del Mazo Carlos, D´ıaz-Pe˜na Eva M, Santiago Roc´ıo, Vicente Carlos, Legaz Mar´ıa E.Differential expression of SofDIR16 and SofCAD genes in smut resistant and susceptible sugarcane cultivars in response to Sporisorium scitamineum.Journal of Plant Physiology https://doi.org/10.1016/j.jplph.2018.04.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Differential expression of SofDIR16 and SofCAD genes in s m u t r e s i s t a n t and
susceptible
sugarcane
cultivars
in response to Sporisorium
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scitamineum. Elena Sánchez-Elordia, Roberto Contrerasb, Roberto de Armasc, Mario C. Benitob, Borja Alarcóna, Eliandre de Oliveirad, Carlos del Mazoa, Eva M. Díaz-Peñaa, Rocío Santiagoe, Carlos Vicentea and María E. Legaza*
Intercellular Communication in Plant Symbiosis Team, Faculty of Biology, Complutense
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a
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University, 28040 Madrid, Spain
Department of Genetics, Faculty of Biology, Complutense University, 28040 Madrid, Spain
c
Department of Plant Biology, University of Havana, Cuba
d
Plataforma de Proteómica, Parc Cientific de Barcelona, Universitat de Barcelona, Barcelona,
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b
Department of Biochemistry and Department of Geographical Sciences, Universidade
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e
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Spain
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Federal de Pernambuco, Recife, Brazil
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Correspondence
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*Corresponding author, e-mal:
[email protected]
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ABSTRACT Proteomic profiling of the stalk of a smut resistant and a susceptible sugarcane cultivars revealed the
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presence of dirigent and dirigent-like proteins in abundance in the pool of high molecular mass (HMMG) and mid-molecular mass (MMMG) glycoproteins, produced as part of the defensive response to the fungal smut pathogen. Quantitative RT-PCR analysis showed that expression levels of SofDIR16 (sugarcane dirigent16) and SofCAD (sugarcane cinnamyl alcohol dehydrogenase) were higher in the smut resistant My 55-14 cultivar than in the sensitive B 42231 cultivar prior to
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infection. Inoculation with fungal sporidia or water decreased the level of SofCAD transcripts in My
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55-14, indicating that regulation of SofCAD expression does not take part of the specific response to
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smut infection. In contrast, SofDIR16 expression was almost nullified in My 55-14 after inoculation
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with fungal sporidia, but not after water injection. It is proposed that the decreased expression of dirigent proteins induces the formation of lignans, which are involved in the defense response of the
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smut resistant My 55-14 cultivar.
Key words: cinnamoyl alcohol dehydrogenase, dirigent protein, lignans, lignin, Sporisorium
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scitamineum, sugarcane.
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1.
Introduction Smut, caused by the fungus Sporisorium scitamineum, is an important disease of sugarcane,
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a major sugar and biomass producer in the tropics and subtropics, including Cuba. The disease causes losses as high as 40% of the total yield of the crop (Chinea and Rodríguez, 1994), affecting both plant growth and juice quality (Martinez et al., 2000). Sugarcane smut is characterized by a long whip-like sorus at the apex of an infected stalk, in which billions of
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teliospores are produced (Waller, 1970; Lemma et al., 2015). Resistance to smut has been first
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correlated with chemical modifications such as pre-formed flavonoids in buds (Lloyd and Naidoo,
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1983; Martinez et al., 1999) and surface wax component. Later studies reported the
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accumulation of free or conjugated polyamines in buds, first expanded leaves, roots and stalks (Legaz et al., 1998; Piñón et al., 1999) and the production of several glycoproteins in the stalk
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juice, affecting spore germination. Different fractions of these glycoproteins prevent the correct
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arrangement of microtubules and cause nuclear fragmentation defects, inhibit cell polarization by
teliospore
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impeding the correct functionality of the actomyosin complex, t h e r e b y agglutination
and
germination inhibition,
accompanied
by
causing enzymatic
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h y d r o l y s i s a n d disassembly of the teliospore cell wall and release of the protoplast (Legaz et al., 2005; Millanes et al., 2005; Sánchez- Elordi et al., 2016a).
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De Armas et al. (2007) have correlated the susceptibility or resistance to smut to changes in
free phenolic compound levels and phenylalanine ammonia lyase (PAL) and peroxidase activities. Accumulation and modification of phenolics as well as their polymerization to form lignin have been observed in plant tissues (Benhamou, 2004). The most common of these modifications consisted of the esterification of phenols to cell-wall polysaccharides (de 3
Ascensão and Dubery, 2000). Santiago et al. (2009) showed that the resistance of the My 55-14 cultivar to smut was based on the highest increase in the inoculated to uninoculated plant ratio of cell-wall-bound caffeic and syringic acids compared with the same, unchanged ratio, in the
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susceptible cv B 42231. Several findings suggested the induction of the phenylpropanoid pathway metabolism by S. scitamineum as an important aspect of sugarcane post-infection resistance mechanism (Sundar et al. 2012).The biosynthesis of phenylpropanoid results in a variety of products including lignin, flavonoids and hydroxycinnamic acids. Some of these compounds behave in plants
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as phytoalexins, antiherbivory compounds, antioxidants, ultraviolet protectants, pigments and
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aroma compounds (Dixon, 2001). Que et al. (2011) identified 23 differentially expressed
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proteins related mainly to chloroplast structural organization, photosynthesis, signal
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transduction, protein folding and resistance to smut in plants infected with S. scitamineum. genes
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Additionally, Schaker et al. (2016) reported a n i n c r e a s e in expression o f
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i n v o l v e d in hormone biosynthesis and signalization, production of siRNAs and epigenetic regulation, and Barnabas et al. (2016) identified 53 glycoproteins related to defense responses
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to smut infection in sugarcane, including several oxido-reductases, and in particular a cinnamoyl alcohol dehydrogenase.
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Structural resistance to pathogens is usually associated with lignification. The major enzymes
involved in the monolignol biosynthesis include c innamoyl alcohol dehydrogenase (CAD)
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that catalyzes the reduction of cinnamoyl aldehyde to cinnamoyl alcohol before polymerization into the lignin polymer (Bonawitz and Chapple, 2010). CAD is coded by a multigene family, of which one of them is primarily responsible for lignin biosynthesis. Amino acid sequence alignment of CADs from Saccharum and other genetically related species indicated a high similarity in conserved domain and binding residue characteristics to alcohol dehydrogenases 4
(Trabucco et al., 2013). Selman-Housein et al. (1999) reported that two copies of CAD genes were found in the sugarcane genome, one of them encoding a sinapyl alcohol dehydrogenase (SAD), which reduces sinapaldehyde to sinapyl alcohol. Santiago et al. (2012)
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showed that inoculation of sugarcane stalks with the smut pathogen elicited lignification and produced significant increases in CAD and SAD levels.
Dirigent (DIR) proteins represent a family of plant proteins, which are induced in response to biotic and abiotic stresses and play a role in directing the stereoselective biosynthesis of (+) or (-) pinoresinol from coniferyl alcohol monomers and the stereoselective
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biosynthesis of (+)-pinoresinol from coniferyl alcohol monomers (Davin et al., 1997) and for
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the stereo-selective coupling of monolignol radicals to produce lignan or lignin (Fig. 1).
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Lignins and lignans are produced by partition of the monolignols pool, p-coumaryl, coniferyl
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and sinapyl alcohol, towards specific biosynthetic pathways (Lewis and Yamamoto, 1990; Lewis and Davin, 1999; Lewis et al., 1999). Lignins are structural cell wall components,
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whereas lignans are ubiquitous molecules involved in plant defense (Davin and Lewis, 2000).
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It is proposed that monolignols occur as free molecules or glucosides, the storage form of
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monolignols, and that the storage pool might be used to produce lignans or “defense lignin”, a barrier mechanically reinforced to impede the penetration of the pathogen (Wang et al., 2013).
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There are only few reports DIR proteins from sugarcane. Damaj et al. (2010) isolated
SHDIR16 that is highly expressed in the sugarcane stalk and potentially involved in lignification
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and defense responses. Jin-Long et al. (2012) reported a novel DIR in sugarcane stalks, expressed in response to drought, salt and oxidative stresses, and Nobile et al. (2017) studied the transcriptional profiles of DIR genes in sugarcane mature internodes. The present work aims first at profiling the glycoproteins of the stalk juice of the smut resistant My 55-14 and susceptible B 42231 sugarcane cultivars for detection of DIR 5
proteins and, second, at correlating the changes in expression levels of SofCAD16 and SofDIR16 genes with the smut resistance response following inoculation with S. scitamineum in the two cultivars for possible involvement of the lignin/lignan biosynthesis pathway.
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2. Material and methods 2.1. Plant material
Two sugarcane cultivars, one s m u t susceptible (B 42231), and one resistant (My 55-14) (Ordosgoitti et al., 1983) were imported from Cuba and environmentally adapted in
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Madrid (Spain). Plants were cultured in a greenhouse at the Real Jardín Botánico Alfonso XIII
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of Complutense University under natural light and controlled irrigation. Thirty 12-month-old
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sugarcane plants per cultivar were used for each biological replicate of the experiment. Six plants
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were used as the control (non-inoculated plants), twelve plants for water-injection (six plants collected after 3 h injection and six after 6 h injection) and twelve plants for sporidia-inoculation
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(six plants collected after 3 h inoculation and six after 6 h inoculation). In total, ninety
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plants (for 3 biological replicates) were used per cultivar, as shown in Fig. 2.
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2.2. Teliospore germination and plant inoculation Teliospores of S. scitamineum collected from an infected field of cv. B 154 42231 cv. in
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Cuba were rinsed three times in sterile distilled water containing 50 µM streptomycin sulphate and incubated as described previously (Santiago et al., 2009). Single sporidial colonies were
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isolated and re-incubated. Serial dilutions of the resultant cultures were made in potato dextrose broth (PDB) and grown on potato dextrose agar (PDA) and incubated as before. Single sporidial colonies were then isolated and incubated on PDA plates. In order to determine the mating type of each isolate, random mating experiments were performed. Mating reaction was evidenced by the appearance of aerial mycelium and isolates were arbitrarily 6
designed as either plus or minus. As a control, thirty six stalks per cultivar (twelve for each biological replicate) were injected with 50 µL of distilled water using a Hamilton syringe. In the same way, thirty six stalks were inoculated with 50 µL of a suspension containing 2·106 sporidia
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mL-1 of a 1:1 mixture (plus and minus) of isolated mating cell types (Santiago et al., 2009). Injection with distilled water or inoculation with smut sporidia was carried out into the stalk 3 cm above the first leaf with a visible dewlap, in the apical portion of the stalk through the leaf sheath (Santiago et al., 2012). Groups of six plants for each replicate were collected at 3 h or 6 h post-injection/inoculation. Eighteen plants were not injected/inoculated and used as a
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control, with six for each biological replicate.
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2.3. Sample collection
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Stalk segments of 3 cm (1.5 cm above and below the point of injection/inoculation) were sampled from the stalks at 3 h or 6 h post-injection/inoculation. The third developed leaves were
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sampled at 6 h post-injection/inoculation, selecting three sections of 10 cm: a basal section (10
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cm from the basis), a central section (from 25 to 35 cm from the basis) and an apical section (from 50 to 60 cm from the basis). Stalk segments from meristematic and leaf tissues from the
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six plants of each replicate were immediately ground in liquid nitrogen to a fine powder for RNA
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isolation. Samples were collected at 3 h and 6 h post-inoculation based on previous work demonstrating that the increase in lignin content occurred after 2 h of sporidial inoculation
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(Santiago et al., 2012). Different pools of samples were analyzed by RT-PCR. The first pool consisted of meristematic tissue from six stalks of non-inoculated plants. The second and third pools consisted of meristematic tissue from six stalks of plants collected after 3 h and 6 h of water injection. The fourth and fifth pools consisted of meristematic tissue from six stalks from plants collected after 3 h and 6 h of sporidial inoculation. Since the sugarcane smut fungus infects the meristem portion and spreads systemically inside the culm, pools of leaf sections from 7
plants collected at 6 h post-injection/inoculation were analyzed the same way (Fig.2). The response has also been analyzed in foliar tissue since previous work of the group showed that the transport of fungal elicitors to the leaves triggered changes in phenol content and in enzymatic
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activities of PAL and POX (de Armas et al., 2007). The complete experiment (from injection/inoculation to genetic analysis) was repeated three times to obtain three independent biological replicates.
2.4. Proteomic analysis of the stalk proteins fractions, HMMG and MMMG stalk protein fractions
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For purification of the stalk protein fractions, including HMMG and MMMG, six uninoculated
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stalks from both B 42231 and My 55-14 cultivars were cut and mechanically crushed using a
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manual stuffing box. The crude juice from each cv. was mixed in a pool and centrifuged at 5000
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g for 15 min at 2 ºC. Supernatants were filtered through a filter paper (Vicente et al., 1991) and chromatographed through two Sephadex G10 and G50 columns (Sigma-Aldrich® Chemical
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Co, St. Louis, MO) to obtain High Molecular Mass Glycoproteins (HMMG) and Mid
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Molecular Mass Glycoproteins (MMMG), as described previously (Millanes et al., 2005).The
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eluted fractions, HMMG and MMMG, were monitored for carbohydrates according to Dubois et al. (1956) and for proteins according to Lowry et al. (1951). The eluted MMMG and
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HMMG fractions for both My 55-14 and B 42231 cultivars. were analyzed by SDS-PAGE electrophoresis, according to standard procedures (Laemmli, 1970), with gels stained with
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Coomassie blue (Sigma-Aldrich® Chemical Co, St. Louis, MO) to detect all the present proteins, whether or not they are glycoproteins. Proteomic analysis was performed following the protocol described by Frias et al. (2010). HMMG and MMMG fractions were precipitated overnight using acetone at -20 °C. The pellet was dried in a vacuum centrifuge. One-dimensional electrophoresis was performed under denaturing conditions on 12.5% (MMMG) or 8% SDS8
PAGE (HMMG). Proteins in gel were digested with trypsin. Excised gel spots were washed sequentially with ammonium bicarbonate buffer and acetonitrile (J.T. Baker, Deventer, The Netherlands). Proteins were reduced and alkylated, respectively, by treatment with 10 mM DTT
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solution for 30 min, followed by treatment with a 100 mM solution of iodine acetamide. After sequential washings with buffer and acetronitrile, proteins were digested overnight at 37 °C with 0.27 nM of trypsin. Tryptic peptides were extracted from the gel matrix with 10% (v/v) formic acid and acetonitrile, and the extracts were pooled and dried in a vacuum centrifuge. All chemicals were of analytical grade (Sigma-Aldrich® Chemical Co.).
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For mass spectrometry analysis, purified and reconstituted tryptic peptides were injected for
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chromatographic separation in a reverse-phase capillary C18 column (75 µm of internal diameter
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and 15 cm length, PepMap column, LC Packings, Thermo Fisher Scientific Inc, Barcelona, mass
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Spain). The eluted peptides were subsequently analyzed on a nano-ESI-QTOF
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spectrometer (Q TOF Global, Micromass-Waters, Parc Tecnològic del Vallès, Barcelona, Spain),
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according to the method described by Frias et al. (2010). Data were generated in a PKL file format and submitted for database searching using the MASCOT server. Only tandem
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mass (MS/MS) spectra of +2 and +3 charged ions were searched against the NCBI nonredundant
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protein sequence database (Pruitt et al., 2005).
SofDIR16 and SofCAD expression analysis using quantitative reverse transcriptase-PCR
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2.6.
(qRT-PCR) For RNA isolation, ground tissue (300-400 mg) from each group (stalks, and basal, medial and apical leaf sections) was homogenized in 1.0 mL TRIzol reagent (InvitrogenTM, Life Technologies, Inc., Thermo Fisher Sci., Spain) and incubated for 10 min at room temperature. The supernatant was extracted with chloroform (0.2 mL) by incubation for 3 min at room 9
temperature and centrifuged for 15 min at 12000 g. RNA was purified from the aqueous phase of TRIzol-chloroform extract using the PureLink® RNA Mini Kit (Ambion, Thermo Fisher Sci., Spain), according to the manufacturer’s protocol.RNA was quantified by measuring ultraviolet
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absorbance at 260 nm/280 nm (NanoDrop® ND2421000) and stored at -80 ºC (at a final concentration of 200 ng µL-1).
The cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Thermo Fisher, Spain). qPCR samples (10 µL) contained 2.5 µL of synthesized cDNA, 0.6 µL of a mixture of forward and reverse primers at 10 µM each, 1.9 µL of
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autoclaved water and 5 µL of Fast SYBR Green Master Mix (Applied Biosystems, Thermo
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Fisher, Spain). qPCR reactions were performed in duplicate, using a 7900HT Fast Real-Time
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PCR System (Applied Biosystems) with the following cyclic conditions: one cycle at 50 ºC for 2
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min, one cycle at 95 ºC for 10 min, and 40 two-step cycles at 95 ºC for 15 sec and 60 ºC for 1
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min. qRT-PCR data analysis was performed at the Genomic Unit of the Complutense
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University. SofDIR16 and SofCAD expression levels in meristematic and leaf tissues were normalized to the levels in meristem from non-inoculated My 55-14 plants.
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Normalization and standard deviation calculations of the samples were made according to the Relative Standard Curve Method for Quantification (Applied Biosystems), using the comparative
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CT Method (∆∆CT Method).
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Primer sequences (Sigma-Aldrich® Chem. Co, St. Louis, USA) are as follows:
SofDIR16,
5´-GGCCAATCTGAGAAGCGTCCT-3´
(forward)
and
5´-
CACCACCGCCTCGTTAAG-3´ (reverse); SofCAD, 5´-GCTGCGTCGTCGAACCGTGAG-3´ (forward), and 5´-CGGCAAGAACGCGACAAG-3´ (reverse); and 18S ribosomal RNA reference,
5´-TCAACGAGGAATGCCTAGTAAGC-3´
(forward)
and
5´-
ACAAAGGGCAGGGACGTAGTC -3´ (reverse). 10
A completely conserved region among ribosomal 18S gene sequences from rye, wheat, barley and rice was selected as an internal constitutively expressed control that would be useful not only for this experiment but for future expression studies on such species.
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2.7. Statistical analysis
Statistical analysis was performed using the multiple ANOVA test followed by post hoc analysis with Tukey’s honest significant differences test. Differences were considered to be
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significant at p < 0.05.
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3. Results and Discussion
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Transcriptome analysis of sugarcane has permitted the identification of 1,460 genes associated
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with plant response to pathogens, including those associated with hypersensitive response (HR) and systemic acquired resistance (SAR), as well as resistance (R) and pathogenesis related (PR)
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genes (Wanderley-Nogueira et al., 2012). Among the 16 classes of PR genes studied, the PR-9
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class (peroxidases class) was the most abundant. This class contributes to plant disease resistance through deposition of lignin, conferring resistance against a broad spectrum of pathogens, as well
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as the accumulation of reactive oxygen species (ROS) (Van Loon and Van-Strien, 1999), 1,3-
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glucanases related hydrolysis of cell wall glucans (Ebel and Cosio, 1994), and thaumatin-like factors that permeate fungal membranes (Vigers et al., 1991).
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Transcriptome and proteome profile analyses of sugarcane under smut stress have provided
insight into differentially expressed genes associated with the molecular mechanism of interaction between the sugarcane and pathogen (Barnabas et al., 2016; Schaker et al., 2016; Wu et al., 2013, You-Xiong et al., 2011). Glycoproteins, containing a heterofructan as glycidic moiety, are also produced by sugarcane in response to smut infection. They represent two different pools of glycoproteins the 11
high molecular-mass glycoproteins (HMMG) and mid molecular-mass (MMMG) glycoproteins (Legaz et al., 2005). These glycoproteins affect polarization of the cytoplasm during smut spore germination (Millanes et al., 2005, 2008) and impair germ tube protrusion and germination of the
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spores (Sánchez-Elordi et al., 2016a and b). The inhibition of smut teliospore germination by HMMG and MMMG could be specifically related to actin polymerization and a functional actomyosin complex (Sánchez-Elordi, 2016b).
Other proteins such as enzymes involved in lignin biosynthesis pathway (namely CAD) have been implicated in the resistance of the sugarcane to the smut pathogen (Schaker et al., 2016;
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You- Xiong et al., 2011).
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3.1. MMMG and HMMG fractions of stalk juice of smut resistant and susceptible cultivars share
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DIR proteins
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Previous analysis of both HMMG and MMMG by capillary electrophoresis revealed that the
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MMMG fraction contains two cationic and four anionic components, whereas only one cationic
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and four anionic proteins are separated from the HMMG fraction (Legaz et al., 1998). In our study, we performed proteomic analysis of both HMMG and MMMG fractions from the stalk
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juice of My 55-14 and B 42231 cultivars using only plants that are non-inoculated with smut to
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detect the difference in protein composition between the smut resistant and sensitive cultivars. The main components of HMMG are shown in Fig. 3A, two from the susceptible cv. (bands 1 and
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2) and four (bands 3-6) from the resistant cv. At least three prominent components (bands 1, 2 and 3) were visible by Coomassie blue staining in MMMG from B 42231 cv. and six (bands 4-9) from My 55-14 cv. (Fig. 3B). Each band was excised from the gel and digested with trypsin. Peptides were separated by liquid chromatography and subsequently analyzed on a nano-ESI-QTOF mass spectrometer (Micromass-Waters). Data were submitted for database search in the MASCOT server and searched against the NCBI non-redundant protein sequence database (Frias 12
et al., 2010; Pruitt et al., 2005). Proteins putatively identified from HMMG and MMMG derived from the stalk juice of both cultivars are shown in Tables 1 and 2, respectively. Bands from HMMG of both cultivars are
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shown in Fig. 3A. Band 1 from B 42231 and band 3 from My 55-14 corresponded to DIR or DIRlike proteins from Saccharum hybrid cv. CP72-1210. Proteins from B 42231 MMMG (bands 1-3 in Fig. 3B) and My 55-14 MMMG (bands 4-6 in Fig. 3B) were identified as DIR, putative DIR protein or DIR-like proteins with a molecular mass of about 20 kDa from Saccharum hybrid cv. CP72-1210. DIR proteins direct the outcome of the coupling of monolignol coniferyl alcohol into
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(+) or (-) pinoresinol, the first intermediate in the enantiocomplementary pathways of lignin
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biosynthesis. In the absence of DIR proteins, pinoresinol is a relatively minor product in the lignin
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biosynthetic pathway (Davin et al., 1997). A transcriptome and genome-wide survey of DIR
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domain-containing proteins in sugarcane permitted their classification into 64 groups according to
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phylogenetic and sequence alignment analyses (Nobile et al., 2017).
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Other minor proteins were identified from the proteomic analysis of HMMG and MMMG of the stalk juice of My 55-14 and B42231 cultivars. These proteins play roles including symbiosis,
hypersensitive
responses,
and
signal
transduction
and
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microsporogenesis,
differentiation. Heat shock 70 protein seems to prevent accumulation of heat denatured protein
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aggregates or facilitate protein reactivation following stress (Waters et al., 1996). Bands 2 and 4 from HMMG of both cultivars shared about 11% similarity with a porin. Putative proteins of
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bands 5 and 6 from My 55-14 HMMG were propyl carboxypeptidase-like protein, putative serine peptidase, heat shock 70 protein or hypothetical protein SORBIDRAFT_03g035680, all of them from the Poaceae family. Bands 7-9 from My 55-14 MMMG did not appear in the susceptible cv. (Fig. 3 B). Band 7 was not identified, and band 8 was slightly expressed and shared 3% similarity with a cysteine protease 1 from Zea mays. Proteins of band 9 corresponded to a methionine 13
synthase 2 enzyme from Hordeum vulgare subsp. vulgare (Table 2 ).
3.2. SofCAD expression pattern in the smut resistant and susceptible cultivars in response to
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sporidial inoculation is similar to that of water injection and is non-specific Expression levels of SofCAD (involved in lignin biosynthesis) were analyzed in both smut resistant and susceptible sugarcane cultivars in meristematic and leaf tissues following sporidial inoculation and water injection. In meristematic tissues, SofCAD expression in the resistant cv. (My 55-14) were three times higher than in the susceptible cv. (B 42231) control plants (Fig. 4).
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Only the leaf basal section of B 42231 showed an expression higher than that found for My 55-
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14 (Fig. 5B). It is interesting to note that a gradient of expression of SofCAD was established
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between the different leaf sections of the resistant cv., from the highest expression at the leaf
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basal portion (the oldest) to the lowest at the leaf apical portion (the youngest) of control plants
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(Fig. 5A). A rapid decrease in transcript levels (40% change) of SofCAD in meristematic tissue
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of the resistant cv. was noted at 3 h of inoculation with compatible sporidia. This decrease was even higher (~ 70%) at 6 h post-infection. Water injection produced a similar response, that is, a
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reduction in expression levels, i.e. 1.5-fold and 7.7-fold in the resistant cv. at 3 h and 6 h post-
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injection, respectively. The response produced by the susceptible cv. was also a reduction of 1.6fold at 3 h and 1.8-fold at 6 h post-injection (Fig. 4). Sporidial inoculation of My 55-14 leaves, in
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meristematic tissues, caused a decrease in SofCAD expression levels (4.5-, 9.7- and 1.6-fold) in basal, central and apical leaf sections, respectively (Fig 5A). This decrease also occurred after water injection. Water injection and inoculation with smut sporidia produced a repression of the SofCAD gene only in the basal section (67% and 23% reduction, respectively) of B 42231 leaves, whereas an increase (70%) of this expression was observed after sporidial-inoculation in leaf central sections. However, no variation was detected in leaf apical sections (Fig. 5B). 14
The monolignols are differentially employed for both lignin and lignan biosynthesis. Previous work demonstrated a multistep model of lignin biosynthesis after inoculation of My 5514 and B 42231 sugarcane cultivars with smut sporidia (Santiago et al., 2012). It was shown that
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lignin accumulated about 29% in My 55-14 cv. and only 13% in B 42231 cv. after smut infection. Moreover, lignin accumulation was correlated to an increased activity of sinapyl alcohol dehydrogenase (SAD) in the resistant cv. It was proposed that resistance is due to an increase in SAD and not CAD activity, at least for 48 h post-inoculation. In our study, the SofCAD expression level was 3-fold higher in the smut resistant My 55-14 cv. than in the
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susceptible B 42231 cv. meristematic tissues (Figs. 4-5). Furthermore, previous results indicated
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that following sporidial inoculation of the stalk, at the level of insertion of the third leaf sheath,
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the increase in CAD and SAD activities was already evident in meristems at 3 h to become
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maximal after 6 h post-inoculation (Santiago et al., 2012). In our study, inoculation with both smut sporidia and water injection produced a decrease in SofCAD expression levels in My 55-14
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and B 42231, which could indicate that CAD repression appears non-specific and is a
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consequence of wounding. This corroborates previous results described by Santiago et al. (2012),
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who verified that CAD activity does not seem specifically relevant in lignin accumulation in My
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55-14 after smut inoculation.
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3.3. SofDIR16 is differentially expressed in the smut resistant and susceptible cultivars in response to sporidial inoculation and water injection Since DIR proteins were detected in both smut resistant and susceptible cultivars, expression of SofDIR16 was analyzed in meristematic and leaf tissues of sugarcane following sporidial inoculation and water injection. In contrast to SofCAD expression, SofDIR16 was expressed in the meristem and leaf sections in a constitutive way and higher in the smut resistant cultivar than 15
in the susceptible one in control, untreated plants (Fig. 6). It was also noted that SofDIR16 expression was highly up-regulated (~ 6.6-fold higher than in control tissues) in the susceptible B 42231 meristematic tissues at 3 h after sporidial inoculation, whereas it was down-regulated (~
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100- fold lower than in control tissues) in My 55-14 meristems at 3 h post-inoculation (Fig. 6). This pattern was similar for the 6 h inoculation point. At this time point, a reduction of about 40% in SofDIR16 expression was observed in the resistant cv. and an increase of 50% in the susceptible cv. (Fig. 6).
Water injection produced different effects on SofDIR16 expression levels depending on the
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inoculation time, i.e. 1.39- and 13.6-fold reduction at 3 h and an increase of 290- and 780-fold at
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6 h in the resistant My 55-14 and susceptible B 42231 cultivars, respectively (Fig. 6). It could be
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hypothesized that mechanical (but non pathogenic) wounding lead to a local rise in SofDIR16
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expression at 6 h post-injection. Water and wounding stresses were reported to act in a
D
synergistic way on lignin production, since wounding caused water stress in Daucus carota
TE
(Becerra-Moreno et al., 2015). This fact would explain why SofDIR16 expression in sugarcane increased after water injection at the point of application (meristem), whereas it decreased in
EP
remote areas (Fig. 7A and B). Moreover, an increased SofDIR16 expression was also observed in the basal leaf sections, whereas its expression was reduced in both central and apical sections of
CC
the smut resistant My 55-14 cv. This is most likely due to the signal reaching adjacent areas as a
A
result of a faster signal transmission in the resistant cultivar. On the other hand, the increased content of ABA and other inhibitors in the mature leaf zone (basal zone) might be associated with a loss of sensitivity to the pathogen (Bonga, 1982). This would result in a higher production of lignans in the smut susceptible B 42231 cv. than in the My 55-14 cv., which would require higher SofDIR16 activity (Fig. 5B). However, it appears that wound induced resistance (waterinjected control) is higher in My55-14 while resistance to the pathogen would be innate in this 16
resistant cv. In leaves, SofDIR16 expression levels decreased from the basal to the apical section in control plants in the resistant cv. (Fig. 7A). Smut sporidial inoculation severely reduced the level
SC RI PT
of SofDIR16 expression (85%, 99% and 90% in basal, central and apical leaf sections, respectively) in My 55-14 cv. (Fig. 7A). Water injection caused a 2.4-fold increase in SofDIR16 expression only in the basal section of leaves whereas it reduced SofDIR16 expression in both central and apical leaf sections (Fig. 7A). Leaves from the susceptible cv. showed lower SofDIR16 levels (30-fold) than those from the resistant cv. in control plants, with a similar
U
expression gradient, i.e. down-regulation of expression from basal to apical leaf sections.
N
Inoculation with compatible smut sporidia increased two times SofDIR16 expression in the
A
basal section of B 42231 whereas no significant differences were observed in its central or apical
M
leaf sections compared to those of control tissues (Fig. 7B). In this case, water injection produced
D
a severe decrease of SofDIR16 levels (95%, 88% and 52% in basal, central and apical leaf
TE
sections, respectively). No increase was detected after water injection in any section of B 42231 leaves.
EP
Inoculation of with smut sporidia caused a different effect on SofDIR16 expression than
CC
water injection in both My 55-14 and B 42231 cultivars. Surprisingly, the pathogen prevented the increase in SofDIR16 expression, which was caused by mechanical wounding. A differential
A
response of plants to abiotic mechanical wounding or pathogen attack has been described (Rajendran et al., 2014). Moreover, it was observed that plants react differently to stresses occurring individually or combined, and the reaction to a complex situation involving different kinds of stresses cannot be easily predicted (Atkinson and Urwin, 2012; Rejeb et al., 2014). In the same way, our results evidenced that sporidial inoculation stimulated a different response in sugarcane when compared with the mechanical wounding stress. 17
3.4 A suggested role for SofDIR16 in lignin accumulation An important finding of this study is that SofDIR16 expression was almost nullified in the smut
SC RI PT
resistant My 55-14 cv. after sporidial inoculation, a phenomenon not observed with water injection. This is consistent with a specific defense mechanism in the resistant cv. triggered by the presence of the pathogen. Santiago et al. (2012) found that lignin accumulated in the My 5514 cv. after infection principally by means of an increased SAD activity. Once lignin production was ensured by SAD, a dramatic decrease in SofDIR16 expression could involve a redirection of
U
the plant metabolism to elaborate an adequate defense response (Smith and Stitt, 2007) to block
N
pathogen development, reinforcing as such the lignin accumulation strategy. This pattern of
A
response, not observed in the smut susceptible B 42231 cv., was triggered in the meristems of the
M
resistant My 55-14 cv. (Fig. 6) and transmitted to adjacent areas. We hypothesize that the smut
D
resistant cv. was able to detect the pathogen whereas the susceptible c v . could not distinguish
TE
clearly between the pathogen attack and water injection. In relation to this metabolic redirection, Ma and Liu (2015) found that TaDIR13, a dirigent
EP
protein from wheat, promoted lignan (and not lignin) biosynthesis and enhanced pathogen
CC
resistance. The decrease in S o f DIR16 expression in the smut resistant cv. could be directed to the reinforcement of a lignin barrier in response to the wound itself caused by injecting water or
A
pathogen (Fig. 7A). This may be related to the production of ROS after wounding caused by water injection in a manner similar to that described for wheat in response to injury, in which superoxide anion accumulated and increased exocellular peroxidase activity (Minibayeva et al., 2009). It could be possible that, in the absence of SofDIR16, the monolignol pool is rich with other metabolites (i.e. lignans), which possess antimicrobial activity as well as antioxidant and 18
cytotoxic capacities according to Rahman and Gray (2002). Thus, we consider that the resistance to smut infection could be related to lignan accumulation in the My 55-14 cv. as a result of a decrease in SofDIR16 expression. This hypothesis is supported by our preliminary results
SC RI PT
showing the inhibition of smut spore growth by a mixture of sugarcane lignans extracted from My 55-14 plants (data not shown). Recent studies on sugarcane proteins associated with defense mechanisms did not describe the involvement of DIR proteins (Souza et al., 2017; Su et al., 2016). The lack of previous evidence on the function of DIR proteins in the defense of sugarcane against the smut pathogen reflects the novelty and importance of the present work that describes
N
U
the possible involvement of SofDIR16 in lignan accumulation.
A
Acknowledgements
M
The authors would like to duly acknowledge the help of Ministerio de Economía y
D
Competitividad (Spain) for the financial support of the project BFU2009-11983. 487
TE
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FIGURE CAPTIONS Fig. 1. Schematic diagram showing the phenoxy radical coupling products from cynnamoil
A
CC
EP
TE
D
M
A
N
U
SC RI PT
alcohol and the role of dirigent proteins in the production of lignin or lignin precursors.
27
Fig. 2. Experimental design for tissue sampling and pooling after pathogen inoculation. Thirty 12-month-old sugarcane plants per cultivar were used for each replicate of the experiment. Six plants were used as the control (non-inoculated plants), twelve plants were used for water-
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injection (six plants collected after 3 h of inoculation and six plants collcted after 6 h of inoculation) and twelve plants were used for sporidia inoculation (six plants collected after 3 h
A
CC
EP
TE
D
M
A
N
U
of inoculation and six after 6 h of inoculation). Three biological replicates were used.
28
Fig. 3. Proteomic analysis of HMMG and MMMG glycoproteins of sugarcane stalk juice of a smut resistant and a susceptible cultivars. In A, Coomassie blue-stained SDS-PAGE (12.5% gel) of HMMG from sugarcane stalk juice. Lane 1, molecular mass standard; lane 2, proteins
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from susceptible B 42231 cultivar; lane 3, proteins from My 55-14 resistant cultivar. In B, Coomassie blue-stained SDS-PAGE (8% gel) of MMMG from sugarcane stalk juice. Lane 1, molecular mass standard; lane 2, proteins from B 42231 cultivar; lane 3, proteins from My 55-
A
CC
EP
TE
D
M
A
N
U
14 cultivar.
29
Fig. 4. SofCAD expression analysis in the sugarcane stalk meristem of a smut resistant and a susceptible cultivars at 3 h and 6 h post-inoculation with the smut sporidia, as determined by qRT-PCR. Data was normalized to the constitutive SofCAD level before injection or
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inoculation. Dashed boxes, control plants; white boxes, water injected; and black boxes, inoculation with compatible sporidia. All data points are means ± SE (n=3). Vertical bars represent the standard error of the means. Different letters indicate significant differences (p
42963 10% EPS 57 Saccharum hybrid 62 > 20199 5% cultivar Q117 57
Saccharum officinarum 21782
Saccharum hybrid 59 > 20269 11% cultivar CP72-1210 45
Variovorax paradoxus 221 > 42963 11% EPS 57 Variovorax paradoxus 181 > 40603 9% S110 57 198 > Zea mays 56763 8% 44 Oryza sativa Japonica 191 > 56450 7% Group 44 133 > Sorghum bicolor 57702 4% 44 Oryza sativa Japonica 113 > 55400 5% Group 44 Hordeum vulgare 93 > 55378 3% subsp. vulgare 44 Oryza sativa Japonica 46 > 56771 1% Group 44 48 > Spinacia oleracea 72558 1% 45
U N
A
M
β-amylase
gi|169777
D
Predicted protein Putative serine peptidase Heat shock 70 protein
A
CC
EP
TE
Band Cultivar Protein
34
Table 2. Protein identification in MMMG fraction from database searching in the MASCOT server. Protein description
Source
MM
Score Cober
1
B42231 MMMG gi|262285778
Dirigent-like protein
Saccharum hybrid cultivar CP72-1210
20269
115 > 11% 57
1
B42231 MMMG
Sugarcane Stem Lambda ZIPLOX Library (MCS) Saccharum hybrid cultivar Q117 cDNA clone MCSA211A08 5~ similar to dirigent, mRNA sequence.
Saccharum hybrid cultivar
18997
64 ≥ 64
2
B42231 MMMG gi|262285778
Dirigent-like protein
Saccharum hybrid cultivar CP72-1210
20269
84 > 11% 57
2
B42231 MMMG
3
B42231 MMMG gi|42454402
Putative dirigent protein
3
B42231 MMMG gi|1568639
Cu/Zn superoxide dismutase
3
B42231 MMMG
4
My55-14 MMMG gi|262285778
Dirigent-like protein
4
My55-14 MMMG gi|37700483
Dirigent
4
My55-14 MMMG
5
My55-14 MMMG gi|262285778
5
My55-14 MMMG gi|37700483
5
My55-14 MMMG
6
My55-14 MMMG gi|262285778
6
My55-14 MMMG
7
My55-14 MMMG
8
My55-14 MMMG gi|226496089
9
My55-14 MMMG gi|68655500
BQ535820
EP
CA279360
U
N
Dirigent-like protein Dirigent
Saccharum officinarum cDNA 5~, mRNA sequence.
Saccharum officinarum Saccharum officinarum
Triticum aestivum Saccharum officinarum
Saccharum hybrid cultivar CP72-1210 Saccharum hybrid cultivar Q117 Saccharum officinarum Saccharum hybrid cultivar CP72-1210 Saccharum hybrid cultivar Q117 Saccharum officinarum
84 > 64 99 > 20509 57 71 > 20424 57 115 > 21782 65 21782
20269 20199 21782 20269
6%
10% 13% 13% 10%
97 > 11% 45 54 > 5% 45 94 > 10% 64 102 > 11% 45
49 > 5% 45 102 > 21782 10% 64 20199
Dirigent-like protein
Saccharum hybrid cultivar CP72-1210
20269
61 > 11% 45
Saccharum hybrid cultivar SP80-3280 cDNA clone SCBFLB2093B08 5~, mRNA sequence.
Saccharum hybrid cultivar
27422
158 > 18% 64
Cysteine protease 1
Zea mays
51927
Methionine synthase 2 enzyme
Hordeum vulgare subsp. vulgare
84717
Unidentified 76 > 45 89 > 45
3% 2%
A
CC
Saccharum officinarum cDNA 5~, mRNA sequence.
TE
BQ535820
Saccharum officinarum cDNA 5~, mRNA sequence.
A
BQ535820
Saccharum officinarum cDNA 5~, mRNA sequence.
M
BQ535820
D
CF577328
SC RI PT
Protein access code/or EST
Band Cultivar Protein
35
