HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses

14 Pages·2016·6.65 MB·English

Journal of Experimental Botany, Vol. 67, No. 14 pp. 4297–4310, 2016
doi:10.1093/jxb/erw212 Advance Access publication 23 May 2016
RESEARCH PAPER
HvPap-1 C1A protease actively participates in barley
proteolysis mediated by abiotic stresses
Blanca Velasco-Arroyo1,*, Mercedes Diaz-Mendoza1,*, Jacinto Gandullo1, Pablo Gonzalez-Melendi1,
M. Estrella Santamaria1, Jose D. Dominguez-Figueroa1, Goetz Hensel2, Manuel Martinez1,
Jochen Kumlehn2 and Isabel Diaz1,† D
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1 Centro de Biotecnologia y Genomica de Plantas, Universidad Politecnica de Madrid, Autovia M40 (km 38), Pozuelo de Alarcon, 28223 lo
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Madrid, Spain d
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2S ePelalannt dR, eGparotedruscletbiveen B, iGoleorgmy,a nLyeibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstr.3, 06466 Stadt d from
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* These authors contributed equally to this work ttps
† Correspondence: [email protected] ://a
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Received 9 May 2016; Accepted 10 May 2016 m
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Editor: Christine Foyer, Leeds University p.c
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Abstract /artic
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Protein breakdown and mobilization from old or stressed tissues to growing and sink organs are some of the meta- -a
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bolic features associated with abiotic/biotic stresses, essential for nutrient recycling. The massive degradation of pro- s
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teins implies numerous proteolytic events in which cysteine-proteases are the most abundant key players. Analysing c
the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and t/67
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grain yield and quality. In this study, dark and nitrogen starvation treatments were selected to induce stress in barley. 4
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Results show that C1A proteases participate in the proteolytic processes triggered in leaves by both abiotic treat- 2
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ments, which strongly induce the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Differences /2
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in biochemical parameters and C1A gene expression were found when comparing transgenic barley plants overex- 9
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pressing or silencing the HvPap-1 gene and wild-type dark-treated leaves. These findings associated with morpho- 78
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logical changes evidence a lifespan-delayed phenotype of HvPap-1 silenced lines. All these data elucidate on the b
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role of this protease family in response to abiotic stresses and the potential of their biotechnological manipulation to g
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control the timing of plant growth. s
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Key words: Abiotic stress, barley, C1A proteases, cysteine-proteases, darkness, proteolysis, stay-green phenotype. 24
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Introduction r 2
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Plants respond to different environmental stresses by repro- alternative strategies by inducing premature senescence and
gramming the expression of subsets of genes depending on early flowering. Plant proteolysis associated with these physi-
each stress-promoting feature. Once these molecular events ological processes is essential for plant survival, via promo-
are initiated, a complex physiological network is activated, tion of recycling of nutrients from stressed tissues to growing
triggering metabolic pathways that finally impact on the or sink tissues. The outcome involves a massive degrada-
plant physiology (Suzuki et al., 2014; Zmienko et al., 2015). tion of macromolecules, dismantling of cellular structures,
Generally, to overcome abiotic/biotic stresses, plants develop mainly chloroplasts, and the subsequent mobilization of
© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
4298 | Velasco-Arroyo et al.
mineral nutrients and nitrogen-containing molecules to sus- acid fragmentation are also up-regulated in senescent leaves
tain further growth and development (Hörtensteiner, 2007; (Buchanan-Wollaston, 1997; Yang and Ohlrogge, 2009; Avila-
Diaz-Mendoza et  al., 2014). Up-regulation of proteases, Ospina et al., 2014; Christiansen and Gregersen, 2014; Orsel
from plastidial and nuclear genomes, are needed for protein et al., 2014; Sakamoto and Takami, 2014).
breakdown throughout stress responses, implying a complex In cereals, abiotic stresses induce degradation of leaf nitro-
traffic of proteins, peptides and amino acids among subcel- gen-containing macromolecules (mainly proteins) to free
lular compartments (Roberts et al., 2012; Carrion et al., 2013; amino acids (glutamate, glutamine and asparagine, among
Diaz-Mendoza et al., 2014). In particular, cysteine proteases others) prior to being phloem-loaded and transported to
(CysProt) of the C1A papain family are the predominantly developing grains. This process provides most of the nitro-
up-regulated plant proteases (Roberts et al., 2012; Diaz and gen needed for grain filling. Particularly, chloroplast proteins
Martinez, 2013; Diaz-Mendoza et al., 2014). C1A CysProt including Rubisco (D-Ribulose-1,5-bisphosphate carboxy-
genes are strongly expressed in response to multiple stresses, lase/oxygenase), Calvin cycle enzymes and proteins involved
such as darkness, drought, nutrient starvation, extreme tem- in photosynthetic light reactions are the main targets for pro-
peratures, salt and pest and pathogen attack (Rabbani et al., teases and represent the first source of transportable nitrogen D
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2003; Parrott et al., 2010; Guo and Gan, 2012; Diaz-Mendoza (Masclaux-Daubresse et al., 2007, 2010; Feller et al., 2008). w
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et al., 2014; Kempema et al., 2015). However, changes in Variations in the stress type and timing may disturb the active lo
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protease gene expression do not necessarily lead to changes photosynthetic period leading to alterations in efficient nitro- de
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in protease activity, probably due to parallel alterations in gen and micronutrient mobilization and, therefore, affecting fro
the expression of genes encoding protease inhibitors (Diaz- crop quality and yield (Gregersen et al., 2013; Distelfeld et al., m
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Mendoza et al., 2014; Kidric et al., 2014). 2014). Small-grain cereals such as barley, wheat and rice, ttp
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The role of C1A proteases in response to abiotic stresses mobilize up to 90% of the nitrogen from vegetative tissues to ://a
is clearly shown in Arabidopsis plants constitutively express- the grains (Gregersen et al., 2008). Understanding these pro- c
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ing CysProt genes from wheat (TaCP) and sweet potato teolytic pathways, in particular chloroplastic protein degrada- e
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(SPCP2). Overexpressing plants showed enhanced tolerance tion, may extend the photosynthetic period, as occurs in ‘stay ic
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to drought and/or salt stress and higher enzymatic activity green’ phenotypes, and modify the total nitrogen content of u
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than the wild-type (WT) plants (Chen et al., 2010; Zang et al., the grain. Thus, the manipulation of key factors involved in .co
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2010). In contrast, the sweet potato SPCP3 CysProt ectopi- protein remobilization such as C1A proteases, seems a prom- /jx
cally expressed in Arabidopsis plants caused altered pheno- ising strategy to improve the outcome of senescence. b/a
typic traits and increased sensitivity to drought stress (Chen Barley is an excellent model for studying proteolysis rtic
et al., 2013). Furthermore, suppression of the CaCP protease induced by stress within monocots, given the numerous le-a
in pepper plants retarded salt- and osmotic-induced leaf research analyses focused on this species and the comprehen- bs
stress responses (Xiao et al., 2014), and expression of an anti- sive knowledge of the whole C1A CysProt family (Martinez trac
sense construct of the cathepsin H-like protease gene BoCP5 et al., 2009; Diaz-Mendoza et al., 2014). Besides, barley has t/6
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delayed floret senescence in broccoli (Eason et al., 2005). been recently considered a translational model for adaptation /14
Altogether, these data suggest that different CysProt may play to climate change (Pérez-López et al., 2012; Dawson et al., /42
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opposite roles in the same or similar physiological processes. 2015). 7
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In this context, the interplay between CysProt and their spe- In this work, we demonstrate the contribution of barley 19
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cific inhibitors, cystatins, is very relevant and remains subject C1A proteases in the proteolytic processes induced in leaves 7
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to a complex regulatory crosstalk dependent on the specific by the abiotic treatments of darkness and nitrogen starva- b
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treatment inducing plant stress. In the last decade, transgenic tion. We have generated barley transgenic plants overexpress- g
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expression of phytocystatins has been used to improve plant ing and silencing the stress-induced HvPap-1 cathepsin F-like e
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behaviour under biotic and abiotic stresses although little has protease to characterize the stress-response features triggered t o
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been published about the pathways involved (Martinez et al., by the biotechnological modification of proteolytic path- 24
2009; 2012). Recently, transgenic soybean and Arabidopsis ways. A senescence-delayed phenotype observed in HvPap-1 N
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plants overexpressing a rice cystatin displayed enhanced silenced lines during natural and induced senescence demon- e
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drought tolerance by altering strigolactone pathways (Quain strates the great potential of this genetically engineered plant. b
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et al., 2014). r 2
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Most abiotic stresses are closely related to plant senes- 18
Materials and methods
cence and activate the down-regulation of genes involved in
the photosynthetic process in parallel to the up-regulation of Plant material and growth conditions
genes responsible for chlorophyll breakdown in chloroplasts
Grains of barley (Hordeum vulgare L. cv. ‘Golden Promise’) were
(Gregersen et al., 2008; Krupinska et al., 2012). Chloroplasts germinated in trays with vermiculite (to induce severe stress condi-
regulate the onset of plant senescence by generating reac- tions) or soil (to induce moderate stress conditions), watered daily
tive oxygen species (ROS) and modifying the oxidative state and incubated at 22 ºC under 16 h light/8 h dark photoperiod for 7
d in Sanyo MLR-350-H chambers. Seven-day-old plants were incu-
of electron transporters (Keech et al., 2007; Queval and
bated in continuous darkness at 22 ºC or with 16 h light/8 h dark
Foyer, 2012, Baxter et al., 2014). In addition, genes associ-
photoperiod (control plants). In parallel, 7-d-old plants were grown
ated with mitochondrial electron transport, β-oxydation of in pots filled with vermiculite and watered daily with complete
fatty acids, glutamine and asparagine synthesis and nucleic Hoagland nutrient solution (Hoagland, 1920) for control plants,
Barley HvPap-1 protease participates in response to abiotic stresses | 4299
or with Hoagland nutrient solution without any nitrogen source to (Oñate-Sanchez and Carbajosa, 2008) and digestion with DNase.
induce severe senescence, at 22 ºC and 16 h/8 h light/dark photoper- cDNAs were synthesized from 2 µg of RNA using High Reverse
iod. Whole plant leaves were harvested after 3 and 7 d of treatment Transcription kit (Applied Biosystems) following the manufacturer’s
(darkness, nitrogen starvation and control), frozen in liquid nitrogen instructions. Quantitative real-time PCR (qPCR) analyses were per-
and stored at −80 ºC until further analysis. formed by triplicate samples by means of a CFX96 real-time system
Barley transgenic lines overexpressing or silencing the barley (BioRad) using the SYBR Green detection system. Quantification
HvPap-1 gene (OE Pap1 and KD Pap1, respectively) were gener- was standardized to barley cyclophilin (HvCycl) mRNA levels fol-
ated in collaboration with the IPK- Gatersleben, Plant Reproductive lowing Diaz-Mendoza et al. (2014). The primers used are shown in
Biology Group and molecularly analysed as described by Diaz- Supplementary Table S1 at JXB online.
Mendoza et al. (2016). Transgenic plants were grown in soil under
the same temperature and photoperiod conditions described above.
Moderate stress mediated by darkness was induced in 7-d-old trans- Immunoblot analysis
genic and control plants grown in soil as described above. Whole Protein extracts were prepared from frozen leaf samples as indi-
plant leaves were harvested after 7, 14 and 21 d of treatment (dark- cated above. After separation on SDS-polyacrylamide gels (12%,
ness/control), frozen into liquid nitrogen and stored at −80 ºC until w/v) according to Laemmli (1970), proteins were electro-transferred
further analysis. onto nitrocellulose membrane (GE Healthcare) and blocked in PBS
(phosphate buffered saline) antisera buffer containing 5% (w/v) pow- Do
dered skimmed milk, for 3 h. Immunoblotting was performed with w
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Photosynthetic pigment measurements anti-peptide polyclonal antibodies specifically selected against each lo
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Chlorophyll a and b, total chorophyll and carotenoids (xanthophylls protease. Supplementary Table S2 shows the peptide sequences used de
aunndd ecra raobtieontiecs )s twreesrsee qs uaanndt icfioendt irno lW coTn adnitdio tnras.n 1sg0e0n micg l ionfe sle ianvceusb wateerde tboit gs ebty t hPei naendtaib Aodniteisb. oAdlyl pSreorvteicaesse. aTnhteib poodliyecsl ownearel apnrotidbuocdeyd ainga rianbs-t d fro
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ground in a mortar with liquid nitrogen and suspended in 15 ml of the Large Subunit of Rubisco (anti-LSR) was supplied by Agrisera. h
80% (v/v) acetone in photo-protected tubes. After centrifugation at Optimal dilutions of primary antibodies were adapted to each pro- ttp
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13 000 g for 2 min, the absorbance of 1 ml of the supernatant was tease. Peroxidase-conjugated anti-rabbit IgG (Sigma) diluted at 1:10 ://a
measured at 470, 663 and 646 nm, for carotenoids, chlorophyll a 000 (v/v) was used as a secondary antibody for detection with ECL c
a
and chlorophyll b, respectively, using a UV-vis spectrophotometer Plus (GE Healthcare). d
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(UltroSpecTM 3300pro, Amersham Bioscience). Pigment content m
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was calculated using the extinction coefficients and equations deter- .o
mined by Lichtenthaler (1987). Specimen processing for microscopy up
Thin strips of leaves cut from control and senescence-induced barley .co
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Protein quantification and protease activities plants were fixed in a freshly prepared solution of 4% (w/v) formal- /jx
dehyde in PBS first at room temperature under vacuum until the b
/a
Tbyo tgarl isnodluinbgl ep plarontte itnisss uweesr ei ne xltirqaucitde dn fitrroomge tnr ebateefodr aen tdh ec oandtdroitli olena voefs sapnedc idmeheynds rsaatnekd, itnh aen s eorvieersn oifg hint carte 4a sºiCn.g S caomnpcelenst rwaetiroen wsa osfh emde itnh aPnBoSl rticle
500 µl of extraction buffer (150 mM NaCl, 50 mM sodium phos- in water as follows: methanol 30%, 50% and 70% (v/v) for 30 min -a
b
phate, pH 6 and 2 mM EDTA). After centrifugation at 16 300 g for each and methanol 100% for 90 min (with three changes) at 4 ºC. s
10 min at 4 ºC, the supernatant was used for protein quantification Specimens were progressively infiltrated in LR White resin (Agar trac
according to the method of Bradford (1976), with bovine serum Scientific) in a series of mixtures of methanol:LR White (v/v) with t/6
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albumin as standard. increasing concentrations of the resin (2:1; 1:1; 1:2) for 1 h each at /1
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Protease activities were assayed by measuring the hydrolysis of 4 ºC, then left in pure resin with 0.5% (v/v) benzoin- methyl- ether /4
substrates containing the AMC (7-amino-4-methyl coumarin) as a catalyst, overnight at 4 ºC. Polymerization in capsules was per- 29
fluorophore carried out in microtiter plate format. The stand- formed under UV light at −20 ºC for 2 d and at 22 ºC for 1 d. 1–2 μm 7/2
ard assay volume was 100 µl, using 5 µg of barley protein extract thin sections were cut from the polymerized blocks in a Leica EM 19
and the corresponding substrate added to a final concentration of UC6 ultramicrotome. 77
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0.25 mM. Cathepsin B-like and L/F-like activities were assayed using 0
b
Z-RR-AMC (N-carbobenzoxy-Arg-Arg-AMC) and Z-FR-AMC y
(N-carbobenzoxy-Phe-Arg-AMC) substrates, respectively. For these Structural analysis and immunofluorescence detection of gu
CysProts a buffer containing 0.1 M of sodium phosphate pH 6.5, HvPap-1, HvPap-16 and HvPap-19 es
and 10 mM cysteine, 10 mM EDTA and 0.1% (v/v) Brij 35 was used. Thin sections of 1–2 μm were carefully collected on water drops t on
Legumain-like activity was determined using the substrate Z-AAN- on 10-well Teflon-printed slides (Fisher Scientific Inc.), let dry and 2
4
AMC (N-carbobenzoxyloxy-Ala-Ala-Asn-7-AMC) at 100 µM con- stored at room temperature until further use. To assess any possi- N
centration, in 50 mM HEPES buffer (pH 7.5) containing 2.5 mM ble structural rearrangements at the subcellular level in the stressed ov
e
dithiothreitol (DTT). Trypsin-like activity was analysed using Z-R- samples vs the controls, the sections were stained with 0.05% (w/v) m
b
AMC (N-carbobenzoxy-Arg-AMC) and elastase-like activity using toluidine blue O (TBO, Panreac), rinsed in distilled water, mounted e
MeOSAAPV-AMC (MeOSuc-Ala-Ala-Pro-Val-AMC) in buffer and observed on a Zeiss Axiophot microscope under bright field. r 2
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Tris-HCl 0.1 M, pH 7.5. The reaction was incubated at 30 ºC for Photographs were taken with a Leica DFC300 FX CCD cam- 18
1 h and emitted fluorescence was measured with a 365 nm excita- era using the Leica Application Suite 2.8.1 build 1554 acquisition
tion wavelength filter and a 465 nm emission wavelength filter. All software.
assays were carried out in triplicate. Blanks were used to account for For immunofluorescence the sections on the 10-well slides were
spontaneous breakdown of substrates and results were expressed as hydrated with PBS for 5 min and unspecific binding sites were
nmol of hydrolysed substrate per mg of protein per min (nmol mg−1 blocked by 10 min incubation with 5% (w/v) BSA (bovine serum
min−1). The system was calibrated with known amounts of AMC in albumin) in PBS. They were subsequently incubated with 20 µl
a standard reaction mixture. drops per well of a rabbit-raised antibody to the CysProt (HvPap-1,
HvPap-16, HvPap-19) applied 1:50 (v/v) in PBS, for 1 h at room tem-
perature in a humid chamber. After two washes of 15 min in PBS,
Quantitative real-time PCR analysis
an Alexa Fluor 488 anti-rabbit antibody (Molecular Probes) was
Total RNA was extracted from frozen barley leaves by the phe- applied in a 1:25 solution in 2.5% (w/v) BSA in PBS, for 45 min at
nol/chloroform method, followed by precipitation with 8 M LiCl room temperature in a humid chamber and darkness. Subsequent to
4300 | Velasco-Arroyo et al.
another two washes of PBS for 15 min each, the slides were mounted C1A proteases and proteolytic patterns are modified in
in a 50:50 (v/v) solution of glycerol/PBS. barley leaves under induction of severe senescence
The activity of two main protease groups, cysteine- and
Confocal imaging of HvPap-1, HvPap-19 and HvPap-16
serine-proteases, was analysed using specific substrates for
Serial sections were collected on a Leica SP8 confocal microscope
cathepsin L/F- and B-like and legumain (CysProt) or trypsin
using the laser excitation lines of 488 nm (to detect the proteases) and
and elastase (serine-proteases), respectively. Members of
633 nm (to detect the red autofluorescence from the chlorophyll). All
series were captured under the same conditions (pinhole size, gain, both protease groups, in particular trypsin, cathepsin L/F-
offset, magnification). The management of the series was performed and B-like, participated in the degradation of leaf proteins.
with either the LAS-AF-Lite 3.1.0_8587 or Fiji software. To com- Only cathepsin L/F- and B-like activities were significantly
posite the corresponding figure in Adobe Photoshop CS3, the maxi-
increased in senescent leaves (up to 2.5- and 3.4-fold under 7
mum projections of the green and red channels were overlaid for each
d of darkness and nitrogen starvation treatment, respectively)
treatment shown. Only the automatic levels were adjusted.
compared to the non-treated controls (Fig. 2).
The expression profile of the 41 C1A CysProt barley
Starch quantification D
genes and the 13 genes encoding cystatins in 7 d of darkness- o
Thirty grams of fresh leaves from darkness-treated transgenic and w
induced leaf senescence was previously assessed by qPCR by n
WT barley lines were used for total starch quantification with lo
STA20 Kit (Sigma) following the manufacturer’s recommendations. our group (Diaz-Mendoza et al., 2014). Based on these data, ade
Dgluilcuotisoen sst awnedrea rcda rcruierdve o. uMt eaass unreecmesesnartsy wtoe rfiet pinetrofo lrimneeadr istyix otfi mthees tehprseine cFa-tlhikeep s(Hinv LP-alpik-1e (gHenveP)a, po-n4e, -c6a tahnedp s-i1n6 H ge-lnikees) (, HonvPe acpa-t1h2- d from
for each sample. After calculations, starch content was expressed as gene) and one cathepsin B-like (HvPap-19 gene) were selected h
grams of transformed starch per 100 grams of initial fresh weight. ttp
to study their expression under stresses induced by continu- s
ous darkness and by the absence of nitrogen at 3 and 7 d. The ://a
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Data analysis results, expressed as mRNA levels normalized to the consti- ad
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Statistical differences among treatments and/or lines were analysed tutively active barley cyclophilin gene, revealed that the cath- m
by one-way ANOVA followed by Tukey’s (HSD) multiple compari- epsin F-, B- and H-like genes were highly induced in stressed ic.o
son test performed using the soft R Project (v.3.1.2) package. leaves vs controls. This induction was dependent on the treat- up.c
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ment with some differences on the expression time course. m
Results Small alterations on the expression of HvPap-4, HvPap-6 /jxb
Structural and physiological changes in leaves under and HvPap-16 cathepsin L-like were also detected after dark /artic
treatment and nitrogen deprivation (Fig. 3A, B). le
severe stress Immunoblot assays using antibodies against peptides of -ab
s
For comparison purposes, firstly two stress treatments were the HvPap-1, HvPap-16 and HvPap-19 CysProt were per- tra
c
used to analyse barley leaf responses. Seven-day-old plants formed after checking peptide specificity to avoid cross-reac- t/6
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were grown in vermiculite under continuous darkness or in tivity (Diaz-Mendoza et al., 2016). Results from immunoblots /1
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hydroponic cultures without any nitrogen source, for 3 and 7 indicated a link between transcript and protein accumula- /4
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d. Chlorophyll, carotenoids and protein contents were ana- tion patterns in most of the samples (Fig. 3). As previously 97
lysed as standard parameters of abiotic stress. A significant reported by Cambra et al. (2012), the protein profile of /21
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decrease in chlorophyll level was observed in darkness- and HvPap-1 showed two bands of different size corresponding 77
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nitrogen-depleted leaves, at both time points. This effect was to the immature protein (40 kDa), the same size of the inac- 0
b
particularly striking in plants grown under dark conditions in tive recombinant protein expressed in Escherichia coli, and y g
comparison to controls (Fig. 1A, B). Similarly, a significant the mature processed form (26 kDa). Both bands increased ue
s
reduction in the chlorophyll/carotenoid ratio was observed their signal in senescing leaf samples at 7 d of treatment. t o
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after 7 d of treatment, which was particularly conspicuous in A similar induction pattern was observed for the active form 2
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leaves grown under nitrogen starvation. The level of total sol- of the HvPap-19 cathepsin B-like protein while no differences N
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uble proteins was also reduced in the treated plants vs controls were detected for HvPap-16. The two treatments (darkness v
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(Fig. 1A, B). An early yellowing phenotype found in darkness- and nitrogen starvation) also altered Rubisco levels in barley mb
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and nitrogen-starved leaves was mainly observed in the leaf leaves. A clear depletion of the Rubisco enzyme was found r 2
apex (see Supplementary Fig. S1), paralleled by strong subcel- by a specific antibody against its large subunit (LS), more 01
8
lular rearrangements in the mesophyll, as seen on toluidine strikingly in leaves grown under 7 d of continuous darkness
blue O (TBO)-stained sections (Fig. 1C). After 7 d in dark- (Fig. 3C).
ness, the chloroplasts looked smaller and round-shaped losing To analyse the subcellular localization of these C1A
their typical peripheral location within the cell and forming CysProt in stressed leaves, immunofluorescence was per-
aggregates. Under nitrogen starvation, small chloroplasts kept formed with the same specific antibodies used for immunoblot
their peripheral position although assumed a rather spheri- experiments. The maximum projection of confocal Z series is
cal shape with spaces among them (Fig. 1C). All these phe- shown in Fig. 4. Green fluorescence signal from the CysProt
notypic, cell structure and physiological parameters confirm HvPap-1, HvPap-19 and HvPap-16 was mainly localized to
strong alterations associated with severe senescence occurring the epidermis in control leaves (Fig. 4A–C). After 7 d without
under darkness and nitrogen starvation treatments. any nitrogen source the labelling of HvPap-1 and HvPap-19
Barley HvPap-1 protease participates in response to abiotic stresses | 4301
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Fig. 1. Biochemical and cell structural changes in barley leaves after severe stress treatments. (A) Chlorophyll content, chlorophyll/carotenoids ratio and 29
total protein content of barley leaves grown in vermiculite under continuous darkness or with 16 h/8 h photoperiod for 3 and 7 d. Data are means ±SE 7/2
of six measurements. (B) Chlorophyll content, chlorophyll/carotenoids ratio and total protein levels of barley leaves grown in Hoagland nutrient solution 19
with or without nitrogen source for 3 and 7 d. Data are means ±SE of six measurements. Significant differences between control and treated plants are 77
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indicated with capital letters (3 d) and small letters (7 d) (P<0.05, HSD). (C) Structural cell changes of barley leaves grown under continuous darkness, 0
nitrogen starvation or control conditions for 7 d. Leaves were stained with 0.05% (w/v) toluidine blue O and observed on a Zeiss Axiophot microscope by
under bright field. (This figure is available in colour at JXB online.) gu
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was observed in a high number of small vesicles within mes- induced by the described stress treatments and because it is t o
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ophyll cells (Fig. 4D, E). As seen on TBO-stained samples a member of the barley cathepsin F-like family, which has 24
(Fig. 1C), the chloroplasts looked more spherical and smaller been thus far poorly studied in plants. For that, transgenic No
v
than those from the controls. Besides, their distribution was plants overexpressing (OE Pap1 lines) or silencing (KD Pap1 e
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different, with large spaces between them at the periphery of lines) the HvPap-1 gene previously generated were analysed be
the cell. A more striking phenotype was observed after 7 d in (Diaz-Mendoza et al., 2016). The phenotypes of the selected r 2
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darkness. HvPap-1 and HvPap-19 CysProt localized to larger transgenic lines revealed differences throughout the plant 18
patches and the chloroplasts clustered together (Fig. 4G, H). growth cycle in comparison to WT plants since the first stages
The red autofluorescence from the chlorophyll was weaker in of development (see Supplementary Fig. S2). OE Pap1 lines
the senescence-induced specimens than in the controls. The presented early yellowish symptoms in the apex of the old-
localization pattern of HvPap-16 was restricted to epidermal est leaves at 3 weeks of growth while evidences of stress were
layers in both treatments (Fig. 4F, I), as in the controls. not observed either in KD Pap1 lines or in the WT plants at
this time point (Supplementary Fig. S2). After 5 weeks, OE
Pap1 lines showed lower numbers of green leaves than the
Transgenic barley lines overexpressing or silencing the
silencing or WT ones and this feature became more promi-
HvPap-1 CysProt
nent in the following week. At 9 weeks, when the spikes had
To investigate the in vivo behaviour of C1A CysProt, the appeared, a clear delay in natural senescence in the KD Pap1
HvPap-1 gene was selected based on its high expression levels lines with respect to the overexpressing and non-transformed
4302 | Velasco-Arroyo et al.
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Fig. 2. Proteolytic patterns of barley leaf after stress treatments. Proteolytic activities of barley leaves grown in vermiculite under continuous darkness m
or with 16 h/8 h photoperiod, or grown in Hoagland nutrient solution with or without nitrogen source for 3 and 7 d. Specific substrates to be degraded /jxb
by cathepsin L/F-like (Cat L/F) and B-like (Cat B), legumain (Leg), trypsin (Tryp) and elastase (Elast) were used. Data are means±standard error of six /a
measurements. Significant differences between control and treated plants are indicated with different letters (P<0.05, HSD). rtic
le
-a
b
plants was observed. This delayed-senescence phenotype of The phenotype of transgenic and control lines after 14 d s
HvPap-1 silenced lines was remarkable at 10–11 weeks, when of continuous darkness presented slower growth and were trac
KD Pap1 1175 and 1130 lines still maintained green leaves shorter than the same genotypes grown under dark/light pho- t/6
7
whereas OE Pap1 lines and WT plants turned yellow and toperiod (Fig. 6). However, the most interesting observation /14
completely dried out (Fig. 5). was the absence of brownish symptoms in the oldest leaf of /42
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whole darkened KD Pap1 lines. In contrast, severely stressed 7
/2
Transgenic barley HvPap-1 lines show alterations phenotypes were detected in the oldest leaf apex of OE 19
7
Pap1 lines as well as in the WT plants after 14 d of darkness 7
associated with stress mediated by darkness 8
0
(Fig. 6). The expression patterns of some CysProt in dark- b
y
The implication of the studied CysProt in response to treated and control leaves from selected overexpressing and g
u
darkness was analysed by comparing transgenic and non- silencing lines were investigated by qPCR assays. As shown es
transgenic lines. To mimic natural conditions, experimen- in Supplementary Fig. S4, Hv-Pap1 transcripts increased in t o
n
tal plant growth parameters were slightly modified with plants grown under darkness independently of the transgene 24
respect to the severe treatment previously used. Seven- insertion, although messenger levels were lower in knock- N
o
v
day-old plants were grown in soil and then subjected to down lines than in overexpressing or WT lines. The mRNA e
m
continuous darkness for 3, 7, 14 and 21 d. As expected, profile for other genes encoding CysProt was also analysed b
e
leaves from dark-treated plants were shorter than those in these transgenic plants. The expression of the HvPap-19 r 2
0
grown under photoperiod conditions. After 21 d of dark- gene was up-regulated in dark-treated OE Pap1 and KD Pap1 18
ness, the leaves were much smaller than in the controls lines. HvPap-6 and HvPap-12 genes presented similar expres-
and presented severe damage (see Supplementary Fig. S3). sion patterns under dark and control conditions in most over-
These phenotypic observations and the determination of expressing and silenced HvPap-1 lines. The HvPap-16 gene
standard parameters associated with stress (chlorophyll, was strongly repressed in response to darkness in all trans-
carotenoids and proteins) led us to select the 14 d time- genic lines and WT (see Supplementary Fig. S4).
point for further molecular, biochemical and physiologi- HvPap-1, HvPap-19, HvPap-6 and HvPap-16 proteases
cal studies. According to Keech et al. (2007) and Zmienko were detected by immunoblot in protein extracts from con-
et al. (2015), long-term treatment of continuous darkness trol and dark-treated leaves of transformed and non-trans-
(2 weeks) is suitable to study protease impact on chloro- formed plants, using specific antibodies. The HvPap-1 protein
plast degradation. increased not only in the overexpressing OE Pap1 lines in
Barley HvPap-1 protease participates in response to abiotic stresses | 4303
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Fig. 3. Transcripts and protein levels of C1A CysProt (cathepsin F-, H-, B- and L-like) in barley leaves after severe stress treatments. (A) Expression of t o
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CysProt genes (HvPap-1, -4, -6, -12, -16 and -19) in leaves grown in vermiculite under continuous darkness or with 16 h/8 h photoperiod, for 3 and 7 2
4
d. (B) Expression of CysProt genes (HvPap-1, -4, -6, -12, -16 and -19) in leaves grown in Hoagland nutrient solution with or without nitrogen source, N
for 3 and 7 d. Data were determined by qPCR and expressed as relative mRNA levels of C1A CysProt genes normalized to barley cyclophilin mRNA ov
e
content. (C) Protein accumulation pattern of CysProt in barley leaves after severe stress treatments using immunoblot assays. Recombinant CysProt m
proteins purified from E. coli cultures (rC) were used as control size. Molecular bands corresponding to inactive and active forms of CysProt are indicated be
by arrows. Rubisco protein pattern was analysed using a specific antibody against its Large Subunit (LS Rubisco). r 2
0
1
8
comparison with the WT, but also after the darkness treat- being more pronounced in the overexpressing HvPap-1 lines
ment. In contrast, HvPap-1 diminished in all KD Pap1 lines (Fig. 7).
(Fig. 7). A slight increase of HvPap-6 and HvPap-19 proteins
was also observed in leaves grown under darkness. No altera-
Physiological changes are associated with stress
tions in the HvPap-16 protein levels were detected in OE Pap1
mediated by darkness in HvPap-1 transgenic
lines compared to WT plants. Nevertheless, a clear increase
barley lines
in the inactive form of the HvPap-16 protease (upper band)
was detected in light-grown KD lines, whereas this inactive The total amount of soluble proteins was quantified in
form disappeared when plants were subjected to darkness. darkness-treated and non-treated transgenic lines as well
Additionally, Rubisco was slightly reduced in stressed leaves, as in non-transgenic controls. OE Pap1 lines did not show
4304 | Velasco-Arroyo et al.
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Fauigto. fl4u. oOrevsecrelanyc oe f( rmeda)x iimn uomld bparorljeeyc tlieoanvse os fa cftoenr fo7c da lo sf edraiersk ndeusrsin,g N t hseta irmvamtiuonno oflru coorenstrcoel ntcreea ltomceanliztsa.t iCony soPf rCoty sHPvrPoat p(g-1re (eAn,) Da,n dG )t,h He vdPeatpec-1ti9o n(B o,f Ec,h Hlo)r oapnhdy ll strac
HvPap-16 (C, F, I). Epidermal cells, ep; mesophyll cells, ms. Bar, 50 μm. t/6
7
/1
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most KD Pap1 lines presented increased levels of protein in /4
2
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dark-treated and non-treated leaves in comparison with their 7
/2
corresponding WT (Supplementary Fig. S5B). Additionally, 1
9
7
the proteolytic activity pattern (cathepsin L/F- and B-like 7
8
CysProt) of OE, KD and WT plants grown in the darkness or 0 b
y
under control conditions was determined using specific sub- g
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strates. No significant differences on the cathepsin L/F-like e
s
activity were detected between transgenic and WT lines when t o
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plants were grown under light conditions. Under darkness, 2
4
the cathepsin L/F-like activity mostly decreased under dark- N
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ness into KD lines (Supplementary Fig. S6A). Similar data e
m
resulted from the measurements of cathepsin B-like activity b
e
(Supplementary Fig. S6B). r 2
0
Photosynthetic pigments were also determined in whole 1
8
aerial biomass of the plants after 14 d of darkness vs con-
trol conditions. Differences in the chlorophyll a levels were
Fig. 5. Natural senescence phenotypes of 10-week-old HvPap-1 nearly undetectable among OE, KD and WT when these
overexpressing (OE Pap1: 919 and 937 lines), silencing (KD Pap1: 1130 plants were grown under photoperiod conditions. A decrease
and 1175 lines) and WT barley plants. of chlorophyll a was observed in all dark-treated leaves com-
pared to the non-treated ones, particularly in the WT (see
significant differences in protein content as compared to non- Supplementary Fig. S7A). Similar results were found for the
transgenic lines when grown under photoperiod but a slight quantification of carotenoids in most lines (Supplementary
protein reduction was appreciated in some transgenic lines Fig. S7C). In contrast, the amount of chlorophyll b was dras-
grown in darkness (see Supplementary Fig. S5A). By contrast, tically reduced in treated and non-treated HvPap1 amiRNA
Barley HvPap-1 protease participates in response to abiotic stresses | 4305
accumulation was strongly reduced in 14 d dark-treated
leaves but no remarkable differences were detected between
transgenic and WT samples.
Discussion
Protein breakdown and mobilization are some of the major
metabolic features associated with abiotic stresses, essential
for nutrient recycling. Rubisco, the most abundant protein in
plants, is likely the major target for proteases when proteo-
lytic processes are activated (Theonen et al., 2007; van der
Hoorn, 2008; Krupinska et al., 2012; Martinez et al., 2012).
The identification of these proteases is crucial to under-
D
standing the physiological mechanisms behind the process o
w
in order to bioengineer plants with altered timing of senes- n
lo
cence, which is closely related to grain quality and yield. The ad
e
barley-C1A CysProt system is a promising model to analyse d
the role of proteases in plants subjected to abiotic treatments. fro
m
C1A CysProt are strongly up-regulated in response to mul- h
tiple stresses, and barley is a model crop whose genome has ttps
been sequenced and in which transgenic technology is well ://a
c
a
established. Besides, the whole family of C1A CysProt and d
e
their inhibitors (cystatins) has been identified in this species m
ic
(Martinez et al., 2009; Diaz-Mendoza et al., 2014). .o
u
Slow growth rates and yellowish leaf apices were the first p.c
o
phenotypes displayed by barley plants under severe dark- m
ness and nitrogen starvation treatments (see Supplementary /jx
b
Fwiags. oSb1s)e.r vReedd puactriaolnle li nto cthhleo rdoispmhyalnl talinndg ocfa rtohtee nceolild s tcrouncttuenret /article
(Fig. 1), probably due to microtubule rearrangements (Keech -ab
s
et al., 2010). These parameters are considered reliable indica- tra
c
tors of stress and senescence. In particular, chlorophyll abun- t/6
dance is a useful indicator of the chloroplast status because 7/1
4
it tends to remain constant in photosynthetically active leaves /4
2
(Sorin et al., 2015). Major subcellular rearrangements in mes- 9
7
Fig. 6. Phenotypes of WT, HvPap-1 overexpressing (OE Pap1: 919 and ophyll cells undergoing severe stresses involved changes in /2
1
937 lines) and silencing (KD Pap1: 1130 and 1175 lines) barley plants grown 9
chloroplast size and distribution within the cell. Chloroplasts 7
iwn hsooleil upnladnetrs dgarrokwnne susn odre lrig chotn/dtianruko puhso dtoarpkenreiosds foorr 1164 h d/8.  (hA )p Phhoetonpoetyripoed .o (fB ) lost their typical lenticular size and became more spherical 780 b
Detail of the oldest leaf apex grown under continuous darkness. and smaller. Under nitrogen starvation they still occupied the y g
cell periphery with spaces among them. After the darkness u
e
s
leaves in comparison with the WT and presented an unde- treatment they formed aggregates (Fig. 1C). These results t o
fined pattern in OE Pap1 lines (Supplementary Fig. S7B). are in accordance with previous publications, and demon- n 2
4
Additionally, the total chlorophyll content of the oldest strate that chlorophyll degradation is a common early event N
o
leaf was observed after 14 d of stress treatment, by detecting in stressed leaves and leaf senescence, closely related to plas- v
e
its autofluorescence under the confocal microscope. Since the tid disassembly (Krupinska et al., 2012; Carrion et al., 2014; m
b
e
apex of the leaf is older than the medium/basal part (seg- Hollmann et al., 2014). Keech et al. (2007) reported that r 2
ments 1 and 2, respectively, in Fig. 8), the highest chlorophyll metabolism in whole darkened Arabidopsis plants, leaves 01
8
fluorescence was generally found in the non-stressed seg- entered a ‘stand-by mode’ with low mitochondrial activity to
ment 2 (Fig. 8). A lower fluorescence emission was detected preserve active photosynthetic machinery, while in individual
in OE Pap1 lines than in WT. In contrast, autofluorescence darkened leaves the high mitochondrial activity provided
levels were increased in the KD Pap1 lines. Similar patterns, energy and carbon skeletons for a rapid degradation of cellu-
although less remarkable, were found in leaf tissues grown lar components. In this study, stress parameters measured in
under darkness, which correlated with alterations in the tissue whole darkened barley plants or in nitrogen-depleted plants
structures observed under bright field (Fig. 8). In addition, indicate that senescence was actually induced, since chloro-
Table 1 shows that the total amount of starch in OE Pap1 and phyll was degraded and chloroplasts were altered.
KD Pap1 transgenic leaves grown under photoperiod was Another important parameter associated with abi-
approximately half that of WT leaves. As expected, starch otic stresses is the reduction of total protein content.
4306 | Velasco-Arroyo et al.
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Fig. 7. Protein patterns of C1A CysProt in transgenic and WT barley lines grown under darkness (D) or 16 h/8 h photoperiod (L) for 14 d and assayed a
d
by immunoblot. Proteins were extracted from leaves of WT, HvPap-1 overexpressing (OE Pap1: 919, 920, 932, 937 lines) and silencing (KD Pap1: 1128, e
d
1130, 1175, 1178 lines). Rubisco protein content was assayed using a specific antibody against its Large Subunit (LS Rubisco). fro
m
The significant decrease in protein levels in dark-treated of this CysProt in response to darkness can be inferred from http
a(Fnidg . n1itBro) gseung-gsetastrevde de iltehaevre sth ien inchoimbiptaiornis oonf tpor ottheein csoynnttrhoels- boof tihts feoxrpwraersdsi oannd i nr eovveerrseex gpernesestiinc ga popr rsoialecnhceisn. gT bhaer laelyte pralatinotns s://ac
a
sis or/and the activation of protease activities associated to could disturb stress progress, and thereby nutrient mobili- de
m
nutrient recycling. In this way, darkness and nitrogen star- zation. Transgenic overexpressing HvPap-1 barley lines did ic
vation clearly induced cathepsin L/F- and B-like activities not only exhibit high levels of mRNA and protein in control .ou
p
in leaves (Fig. 2). These data were supported by the up- leaves but also in response to darkness-induced treatment, .c
o
regulation of genes encoding barley C1A CysProt, particu- while opposite effects were observed in knockdown lines m
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larly HvPap-1, HvPap-12 and HvPap-19, and the detection (Fig. 7, Supplementary Fig. S6). The accumulation of the b
/a
of these proteases by immunoblot assays under the assayed HvPap-1 protease did not result in an increase in the proteo- rtic
experimental conditions (Fig. 3). The implication of differ- lytic activity. This might be due to the lack of a specific sub- le
-a
ent classes of CysProt cathepsin F, B-, L- and H-like, sug- strate to be exclusively degraded by cathepsin F-like enzymes, b
s
gested a functional redundancy of these proteases in protein since the one used in these proteolytic assays simultaneously tra
c
turnover upon treatments. So far, transcriptomic and prot- targeted cathepsin F- and L-like activities, and to compensat- t/6
7
eomic data from different authors have consistently assigned ing effects among protease activities. In senescing leaves of /1
4
a major role to members of all C1A CysProt groups during knockdown lines a clear decrease on protease activities, both /4
2
9
abiotic stresses induced in several plants species (Gregersen cathepsin L/F- and B-like classes (see Supplementary Fig. S6) 7
/2
et al., 2008; Martinez et al., 2012; Diaz-Mendoza et al., 2014; also suggests that accumulation of other proteases (Fig. 7) 1
9
Hollmann et al., 2014). Additionally, the subcellular locali- and/or alternatively protease inhibitors is altered. 77
8
zation of C1A CysProt revealed a dynamic trafficking of From a physiological point of view, the reduction of total 0 b
proteins to be degraded from the chloroplasts to the central protein paralleled to that of Rubisco was observed in most y g
u
vacuole. Senescence-associated vacuoles (SAVs) and the cen- of the darkness-treated OE Pap-1 lines vs control. These e
s
tral lytic vacuole, both containing chloroplastic proteins and results, together with the reduced autofluorescence emission t o
n
peptides, are enriched in CysProt activities during leaf senes- from the chlorophyll (Fig. 8) and the small amount of starch 2
4
cence (Otegui et al., 2005; Ishida et al., 2008; Carrion et al., detected in the overexpressing leaves (Table 1), indicate that N
o
2013; 2014). The immunofluorescence signal of HvPap-1 leaf senescence was accelerated. In contrast, the high pro- ve
m
and HvPap-19 was detected in small vesicles, probably SAVs, tein content and retarded loss of chlorophyll in the amiRNA b
e
within parenchyma cells of leaves undergoing senescence leaves, mostly detected in the apex of the oldest leaf, indicated r 2
0
(Fig. 4), which highly supports their involvement in the deg- a delay in the senescence process. The carbohydrate content 1
8
radation of chloroplastic proteins. In contrast, HvPap-1 and of barley leaves, mainly represented by low concentrations of
HvPap-19 were less abundant and mainly localized in epider- sucrose, starch, fructans and hexoses (Sicher et al., 1984), is
mal cells of control leaves. completely remobilized in response to darkness-induced treat-
HvPap-1 is a cathepsin F-like CysProt previously charac- ment and indicates that the photosynthetic partitioning was
terized to have an important role in grain filling and germina- similar in transgenic and WT leaves. As shown in Fig. 5 and
tion. It actively participates in the hydrolysis and mobilization Supplementary Fig. S4, a clear delayed-senescence phenotype
of storage proteins, mainly hordeins, controlling the grain of HvPap-1 amiRNA lines was observed both in barley plants
amino acid composition (Cambra et al., 2012; Diaz-Mendoza grown either under light/dark photoperiod, corresponding to
et al., 2016). In addition, the HvPap-1 gene is up-regulated in the natural lifespan, or under continuous darkness for 2 weeks
response to severe abiotic treatments. The functional relevance (Fig. 6). These phenotypes are presumably due to chloroplast