Subscriber access provided by GRIFFITH UNIVERSITY
Article
RNA-Seq Analysis Reveals Transcriptional Changes in root of maize seedlings treated with two increasing concentrations of a new biostimulant Sara Trevisan, Alessandro Manoli, Laura Ravazzolo, Clizia Franceschi, and Silvia Quaggiotti J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03069 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 44
Journal of Agricultural and Food Chemistry
1
RNA-Seq Analysis Reveals Transcriptional Changes in root of maize seedlings treated with
2
two increasing concentrations of a new biostimulant
3
Sara Trevisan1* Alessandro Manoli1, Laura Ravazzolo1, Clizia Franceschi2 and Silvia Quaggiotti1
4 5
1
6
University of Padua, Agripolis, Viale dell'Università, 16, 35020 Legnaro (PD), Italy
7 8
2
9
Department of Agriculture, Food, Natural resources, Animals and Environment (DAFNAE),
ILSA S.p.A. Via Quinta Strada 28, 36071 Arzignano (Italy)
*Corresponding author:
[email protected] 10 11
Running title: Effect of APR® on maize root transcriptome.
12
ACS Paragon Plus Environment
1
Journal of Agricultural and Food Chemistry
Page 2 of 44
13 14
Abstract
15
Biostimulants are a wide range of natural or synthetic based products containing substances and/or
16
microorganisms that can stimulate plant processes to improve nutrient uptake, nutrient efficiency,
17
tolerance to abiotic stress, and crop quality (http://www.biostimulants.eu/). The use of biostimulants
18
is proposed as an advanced solution to face the demand for sustainable agriculture, by ensuring
19
optimal crop performances and better resilience to environment changes. The proposed approach
20
would like to predict and characterize the function of natural compounds as biostimulants.
21
In this research, plant growth assessments and transcriptomic approaches are combined to
22
investigate and understand the specific mode(s) of action of APR®, a new product provided by the
23
ILSA group (Arzignano, Vicenza). Maize seedlings (B73) were kept in a climatic chamber and
24
grown in a solid medium to test the effects of two different combinations of the protein hydrolysate
25
APR® (A1 and A1/2). Data on root growth evidenced a significant enhancement of the dry weight of
26
both roots and root/shoot ratio in response to APR®.
27
Transcriptomic profiles of lateral roots of maize seedlings treated with two increasing
28
concentrations of APR® were studied by mRNA-sequencing analysis (RNA-seq). Pairwise
29
comparisons of the RNA-seq data identified a total of 1006 differentially expressed genes between
30
treated and control plants. The two APR® concentrations were demonstrated to affect the expression
31
of genes involved in both common and specific pathways. Based on the putative function of the
32
isolated DEGs, APR® has been proposed to enhance plant response to adverse environmental
33
conditions.
34 35
Keywords
36
RNA-Seq, Zea mays L., biostimulant, gene expression, root.
37
ACS Paragon Plus Environment
2
Page 3 of 44
Journal of Agricultural and Food Chemistry
38 39
Introduction
40
Abiotic stresses including drought, temperature extremes, flooding, salinity, and toxic metals
41
strongly compomises crop productivity. Furthermore, the expected global climate changes will
42
enlarge the impact of stresses even further.1, 2 To cope with these concerns, conventional agriculture
43
has been increasing its dependence on chemical fertilizers and pesticides, with severe effects on
44
natural ecosystem and human health.3 To reorient conventional agriculture toward sustainability the
45
use survey of non-toxic, natural, active substances is requested together with the assessment of
46
their effectiveness in protection and stimulation of plants performances under suboptimal and even
47
harmful conditions .4-5-6 The global market of plant biostimulants has been projected to reach to
48
more than €800 million in 2018 with annual growth potential in 10% and more. 7-9 According to the
49
European Biostimulants Industry Council (EBIC), more than 6 million hectares were annually
50
treated with biostimulant products within European countries, which represent the largest market
51
for this sector.8 Biostimulants were initially used in order to promote plant growth in horticulture,
52
where the term “biostimulant” was apparently coined in 1990s.10 Nowadays, they are always more
53
frequently used also in conventional crop production, to both mitigate environmental impact and
54
enhance crop quality, nutrient efficiency and abiotic stress tolerance.11
55
In this work, the biostimulant activity of a collagen derived-protein thermal hydrolysate, namely
56
APR®, developed by ILSA S.p.A (Arzignano, Vicenza, Italy), on maize seedlings was investigated
57
through both growth and transcriptomic analyses. Protein hydrolysates (HPs) are obtained by
58
chemical, thermal and/or enzymatic protein hydrolysis from a variety of both vegetal and animal
59
residues.10,12 HPs has been demonstrated to stimulate nutrient uptake, to enhance yield of several
60
horticultural crops and to trigger carbon and nitrogen metabolisms by eliciting nitrogen uptake and
61
consequently crop performances.11,13-20 Several biostimulants have been suggested to have
62
phytohormone-like activities, by hypothesising that some HPs might both contain precursor of
63
phytohormonal biosynthesis or directly promote auxin- and gibberellin-like effects.19, 21-23 Chelating ACS Paragon Plus Environment
3
Journal of Agricultural and Food Chemistry
Page 4 of 44
64
functions with role in decreasing plant toxicity to heavy metals and antioxidant activity are also
65
described for some amino acids found as component of HPs.9,11 Finally, HPs application seems also
66
to stimulate plant tolerance to environmental stresses, including salinity, drought, alkalinity,
67
temperature and nutrient deficiency. 19,20,25-28 While the knowledge on the benefits of HPs on plants
68
is steadily improving, deeper investigations on molecular mechanisms that regulate beneficial
69
effects of HPs application on plant are needed. In this scenario, deciphering the dynamic profiles of
70
the transcriptome in response to APR® will be central to characterize the mode of action of the
71
newly proposed biostimulant.
72
RNA-sequencing (RNA-seq) has emerged as a powerful and revolutionary tool to provide high
73
resolution analyses of plant transcriptional dynamics during various aspects of growth and
74
development through sequencing of their associated cDNA (complementary DNA) populations.
75
Transcriptome analyses of the effects of biostimulants on plants highlighted global changes in genes
76
transcription across multiple processes, pathways, and cell functions.
77
of mechanisms of action of biostimulants under stress or non-stress conditions is still lacking.
78
In the current study, the effects on growth of two different APR® concentrations were assessed in
79
both roots and shoots. Subsequently, RNA-Seq analysis has been applied on lateral roots, to dissect
80
the root transcriptomic profiles in response to the treatments. Gene ontology enrichment analysis of
81
RNA-seq data and KEGG pathways analysis helped in deciphering the complexity of network of
82
metabolic and signalling pathways involved in the regulation of root responses to APR®.
83
To the best of our knowledge this is the first reported RNA-seq based study to examine the effect of
84
a solid protein hydrolysate obtained by thermal hydrolysis on maize root transcriptome. These next-
85
generation sequencing data contribute to the available information on the molecular effects exerted
86
by biostimulants on plant root.
29-32
However, the knowledge
87 88
Materials and Methods
89
Preparation of hydrolysates from tanned bovine hides ACS Paragon Plus Environment
4
Page 5 of 44
Journal of Agricultural and Food Chemistry
90
APR® has been obtained by a process of thermobaric hydrolysis applied on trimmings and shavings
91
of bovine hides previously tanned with wet-blue technology. The hydrolysis process was made in
92
spherical rotating autoclaves by high-pressure steam and followed by a low temperature
93
dehydration systems.
94 95
Experimental design and plant growth analyses
96
The experiments were carried out on Zea mays L. (B73) seedlings. Seeds were germinated on moist
97
paper towels at 25ºC for 72 hours. Seedlings were transplanted into vases (16 cm diameter, 20 cm
98
height, volume max. 4 L), each contained 3.5 L of river sand. Five days before the transplanting
99
into the pots, two different amounts of APR® were added in one application, together with 50 ml of
100
distilled water, to the soil mixture: APR® high concentration (A1), APR® medium concentration
101
(A1/2) and a control grown medium (C) (Table 1). The amount of APR®, before being distributed in
102
the vases, was divided into three sub-units for each concentration and then gradually applied every
103
5 cm of sand. The amount of APR®, as well as, the method of its application was first determined in
104
preliminary studies (Ertani, personal communication). Plants were irrigated with 100ml of a
105
modified Hoagland solution every 2 days, according to the following composition (µM): Ca(NO3)2
106
(200), KNO3 (200), MgSO4 (200), KH2PO4 (40), FeNaEDTA (10), H3BO3 (4.6), MnCl2 (0.9),
107
ZnCl2 (0.09), CuCl2 (0.036), NaMoO4 (0.01). Plants were placed in a climatic chamber for 15 days;
108
a day/night cycle of 14 h/10 h at 25°C/18°C air temperature, 70/90% relative humidity, and 280
109
µmol m−2 s−1 photon flux density were utilized as standard conditions. A completely random design
110
was used to analyze data. The experiment was carried using three plastic trays each with six vases
111
for each treatment (one plant for each cell). For the plant growth measurements (root and shoot dry
112
weight; root/shoot ratio), six plants from each plot were chosen for treatment, in three independent
113
biological repetitions. For the determination of the dry weight, samples, divided into root and shoot
114
fractions respectively, were transferred in an oven and dried at 80°C for 72 h. When statistically
115
significant differences were found (ANOVA test), the subsequent test for comparison of treatment ACS Paragon Plus Environment
5
Journal of Agricultural and Food Chemistry
Page 6 of 44
116
means was performed using the Tukey test. Data were analyzed using the program Statistix v 8.0
117
(analytical software, Tallahassee, FL, US).
118
For the molecular analysis, lateral roots were harvested and subsequently transferred into liquid
119
nitrogen and conserved at -80°C. Roots from one plant were pooled and represent a single sample
120
out of the three biological replicates (each of six plants). Plants used for sampling roots were
121
excluded from subsequent biomass sampling.
122 123
RNA isolation and sequencing library preparation
124
Three biological replicates were used for all RNA-Seq experiments from each treatment. Pooled
125
lateral root tissues were ground in liquid nitrogen, and total RNA was isolated with the TRIzol
126
reagent (Invitrogen, San Giuliano Milanese, Italy). RNA quality was assessed via agarose gel
127
electrophoresis and a Bioanalyzer (Agilent RNA 6000 Nano Chip; Agilent Technologies, Santa
128
Clara, CA, USA). For all samples, a RIN (RNA integrity number) ≥8.0 was detected.
129
cDNA libraries for Illumina sequencing were constructed according to the instructions of the
130
manufacturer (TruSeq RNA Sample Preparation; Illumina, San Diego CA, USA).
131 132
Processing and mapping of sequencing reads
133
Base calling was performed using the Illumina Pipeline and sequences were trimmed with ERNE.33
134
Quality trimming removed low quality and ambiguous nucleotides of sequence ends and adapter
135
contamination. TopHat was used to map and annotate the sequences on the B73 reference genome
136
(RefGen_v2;
137
Cufflinks software was used for the analysis of differentially expressed genes (DEGs).34-35 A single
138
unified assembly from each individual Cufflinks assembly was created thanks to Cuffmerge .
139
Transcripts with a false discovery rate (FDR) of ≤0.05 were taken as highly significant DEGs.
ftp://ftp.gramene.org/pub/gramene/maizesequence.org/release-5b/assembly/;
and
140 141
Gene Ontology (GO) and gene enrichment analysis ACS Paragon Plus Environment
6
Page 7 of 44
Journal of Agricultural and Food Chemistry
142
The PLAZA web tool (http://bioinformatics.psb.ugent.be/plaza/) was used to identify GO terms
143
within the different subset of genes.
144
Data
145
(http://bioinformatics.psb.ugent.be/plaza/) for the molecular function, biological processes and
146
cellular component with default settings.36-37
147
The pathways regulated in the A1, A1/2 and control groups were identified using the Kyoto
148
Encyclopedia
149
(http://www.genome.jp/kegg/). The pathways that showed the most differentially expressed genes
150
were identified using KEGG mapper (http://www.genome.jp/kegg/tool/map_pathway2.html).
for
gene
of
ontology
Genes
enrichment
and
Genomes
were
(KEGG)
computed
database
within
for
PLAZA
pathway
2.5
annotation
151 152
Hierarchical clustering
153
For each treatment, transcript relative abundance was estimated using the percentage of normalized
154
counts in each fraction relative to the total number of normalized counts. The mean of the relative
155
abundance per fraction was then calculated and their Euclidean distances computed with the hclust
156
function from the stats R package (R Development Core Team, 2012). Clustering was computed
157
with Ward's minimum variance method and a heat map generated with the gplots R package (2012)
158
(http://CRAN.R-project.org/package=gplots).
159 160
RESULTS
161
APR® triggers morphological responses in roots
162
Biostimulants are assumed to enhance the growth of plants. In this experiment, the biomass
163
production of the maize plants was assessed after 14 days of APR® treatments. The effects of two
164
increasing APR® doses (A1/2 and A1) on root and shoot dried weight were then evaluated.
165
For each condition, three biological replicates (RI, RII and RIII) were analyzed. Replicates were
166
first observed separately (Supporting Information S1). Replicates RII and RIII showed a statistically
167
significant enhancement of root biomass in the APR®-supplied plants, but the differences among ACS Paragon Plus Environment
7
Journal of Agricultural and Food Chemistry
Page 8 of 44
168
the treatments in replicate RI were not significant. No statistically significant differences were
169
obtained from the leaf-biomass analysis, while Root/Shoot (R/S) ratios, defined as the ratio of
170
belowground dry biomass (root) to the aboveground dry biomass (stem and leaves), followed the
171
previously observed root biomass behaviour .
172
The subsequently statistical analyses were performed considering all the three replicates (RI, RII
173
and RIII) per treatment (A1 and A1/2) respect to the control plants (C), to avoid any misguided
174
interpretation of data. Plants supplemented with APR® produced roots with a statistically significant
175
increased biomass (Fig. 1). The effect was dose-dependent, with a significantly greater root biomass
176
(20%) for the half-supplied plants (A1/2). A modest but significant effect (12%) on root biomass was
177
recorded for the plant grown in presence of the complete dose (A1). Shoot biomass was unchanged
178
in the three growth conditions.
179
APR® treatments resulted in significantly higher mean R/S ratios, with no significant difference
180
between the two set of plants (A1 and A1/2).
181
Data indicate that APR® preferentially enhanced root rather than shoot growth.
182 183
RNA sequencing and mapping of maize lateral root transcriptome
184
To obtain a global view of the transcriptome response of maize to APR®, high-throughput RNA
185
sequencing using Illumina sequencing technology was performed on whole RNAs (RNA-Seq)
186
extracted from mature root tissues (lateral roots).
187
RNA sequencing of the 9 samples (two plants-set grown in presence of different APR® doses,
188
namely A1 and A1/2, one control plants-set and three biological replicates) yielded a total of 422.6
189
million reads, translating to a mean of 47 million reads per sample (Table 2). On average, the RNA-
190
Seq experiments yielded between 21 and 62 million reads per sample. Among all reads, 83 to 90%
191
mapped to unique positions in the maize reference genome. Gene expression levels were quantified
192
and reported as fragments per kilobase of transcript per million mapped reads (FPKM), estimated
193
using Cufflinks32. ACS Paragon Plus Environment
8
Page 9 of 44
Journal of Agricultural and Food Chemistry
194
The expression of 29,332 transcripts (FPKM > 1.0) was detected in the mature root (control)
195
whereas APR® treated plants had the highest number of expressed genes with 29,441 (A1) and
196
29,553 (A1/2) expressed genes.
197 198
Global Transcriptome Changes after Plant Treatment with APR® and
199
Hierarchical Cluster Analysis
200 201
To reveal the transcriptome changes influenced by APR®, differentially expressed genes (DEGs)
202
were identified by pair-wise comparison of samples collected (A1/2 vs C; A1 vs C; A1 vs A1/2).
203
Cuffdiff analysis revealed 1006 DEGs, among which only 2.8% (29 DEGs) were shared by all
204
comparison groups (Fig. 2).
205
When controlling false discovery rate (FDR) at 5%, 290 and 643 genes were differentially
206
expressed in response to the half-dose of APR® and whole concentration, respectively (Fig. 2).
207
Complete lists of differently expressed genes treated with both the doses are shown in
208
Supplementary Table S1.
209
Considering the genes differentially regulated by the highest APR® concentration respect to the
210
control, 643 DEGs were almost equally divided in up (329 DEGs) and down (314 DEGs) -regulated
211
(Fig. 2 and Fig. 3).
212
The number of DEGs isolated in the comparison between the highest concentration versus the
213
control was markedly higher than the number of differentially expressed genes detected by the half-
214
dose experiment (290 DEGs), suggesting a greater impact of the higher APR® dose on root
215
transcriptome. It is noteworthy that the half dose resulted in the up-regulation of the 80.7% of the
216
DEGs identified (Fig. 3).
217
Both the applications (A1 and A1/2) affect gene expression regulating the transcription of a
218
consistent part of them (48% and 54% respectively) to a small degree (fold change > 0.5 or < 2).
ACS Paragon Plus Environment
9
Journal of Agricultural and Food Chemistry
Page 10 of 44
219
A stronger effect (fold change ≥2 or ≤ 0.5) is evident on the remaining transcripts (52% and 46%
220
for A1 and A1/2, respectively) (Fig. 4 and Fig. 5). Further examination of the results showed that 129
221
DEGs were co-regulated by the two applications, with most of them (96%) showing the same (i.e.,
222
increased or decreased relative expression) in both the thesis. Only four of them
223
(GRMZM2G037454, GRMZM2G026930, GRMZM5G833699, GRMZM2G063287) were found to
224
have a conflicting pattern of expression.
225
A total of 514 genes was differentially exclusively regulated by A1 concentration, while 161 DEGs
226
were unique to A1/2 concentration (Fig. 2).
227
The comparison between the two APR® treatments (A1 vs A1/2) identified 492 transcripts with
228
significant different expression values, 207 of them showing a conserved expression pattern (94
229
DEGs increased and 113 DEGs decreased relative expression respect to control sample), thus
230
suggesting a dose-effect for the studied product. A discrepancy in expression profiles was, instead,
231
recorded for 87 DEGs (A1>C, A1/2C, A1 10 or FC
