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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

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Journal of Agricultural and Food Chemistry

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RNA-Seq Analysis Reveals Transcriptional Changes in root of maize seedlings treated with

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two increasing concentrations of a new biostimulant

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Sara Trevisan1* Alessandro Manoli1, Laura Ravazzolo1, Clizia Franceschi2 and Silvia Quaggiotti1

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1

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University of Padua, Agripolis, Viale dell'Università, 16, 35020 Legnaro (PD), Italy

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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]

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Running title: Effect of APR® on maize root transcriptome.

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Abstract

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Biostimulants are a wide range of natural or synthetic based products containing substances and/or

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microorganisms that can stimulate plant processes to improve nutrient uptake, nutrient efficiency,

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tolerance to abiotic stress, and crop quality (http://www.biostimulants.eu/). The use of biostimulants

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is proposed as an advanced solution to face the demand for sustainable agriculture, by ensuring

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optimal crop performances and better resilience to environment changes. The proposed approach

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would like to predict and characterize the function of natural compounds as biostimulants.

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In this research, plant growth assessments and transcriptomic approaches are combined to

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investigate and understand the specific mode(s) of action of APR®, a new product provided by the

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ILSA group (Arzignano, Vicenza). Maize seedlings (B73) were kept in a climatic chamber and

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grown in a solid medium to test the effects of two different combinations of the protein hydrolysate

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APR® (A1 and A1/2). Data on root growth evidenced a significant enhancement of the dry weight of

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both roots and root/shoot ratio in response to APR®.

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Transcriptomic profiles of lateral roots of maize seedlings treated with two increasing

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concentrations of APR® were studied by mRNA-sequencing analysis (RNA-seq). Pairwise

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comparisons of the RNA-seq data identified a total of 1006 differentially expressed genes between

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treated and control plants. The two APR® concentrations were demonstrated to affect the expression

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of genes involved in both common and specific pathways. Based on the putative function of the

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isolated DEGs, APR® has been proposed to enhance plant response to adverse environmental

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conditions.

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Keywords

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RNA-Seq, Zea mays L., biostimulant, gene expression, root.

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Introduction

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Abiotic stresses including drought, temperature extremes, flooding, salinity, and toxic metals

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strongly compomises crop productivity. Furthermore, the expected global climate changes will

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enlarge the impact of stresses even further.1, 2 To cope with these concerns, conventional agriculture

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has been increasing its dependence on chemical fertilizers and pesticides, with severe effects on

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natural ecosystem and human health.3 To reorient conventional agriculture toward sustainability the

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use survey of non-toxic, natural, active substances is requested together with the assessment of

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their effectiveness in protection and stimulation of plants performances under suboptimal and even

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harmful conditions .4-5-6 The global market of plant biostimulants has been projected to reach to

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more than €800 million in 2018 with annual growth potential in 10% and more. 7-9 According to the

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European Biostimulants Industry Council (EBIC), more than 6 million hectares were annually

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treated with biostimulant products within European countries, which represent the largest market

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for this sector.8 Biostimulants were initially used in order to promote plant growth in horticulture,

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where the term “biostimulant” was apparently coined in 1990s.10 Nowadays, they are always more

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frequently used also in conventional crop production, to both mitigate environmental impact and

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enhance crop quality, nutrient efficiency and abiotic stress tolerance.11

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In this work, the biostimulant activity of a collagen derived-protein thermal hydrolysate, namely

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APR®, developed by ILSA S.p.A (Arzignano, Vicenza, Italy), on maize seedlings was investigated

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through both growth and transcriptomic analyses. Protein hydrolysates (HPs) are obtained by

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chemical, thermal and/or enzymatic protein hydrolysis from a variety of both vegetal and animal

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residues.10,12 HPs has been demonstrated to stimulate nutrient uptake, to enhance yield of several

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horticultural crops and to trigger carbon and nitrogen metabolisms by eliciting nitrogen uptake and

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consequently crop performances.11,13-20 Several biostimulants have been suggested to have

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phytohormone-like activities, by hypothesising that some HPs might both contain precursor of

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phytohormonal biosynthesis or directly promote auxin- and gibberellin-like effects.19, 21-23 Chelating ACS Paragon Plus Environment

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functions with role in decreasing plant toxicity to heavy metals and antioxidant activity are also

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described for some amino acids found as component of HPs.9,11 Finally, HPs application seems also

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to stimulate plant tolerance to environmental stresses, including salinity, drought, alkalinity,

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temperature and nutrient deficiency. 19,20,25-28 While the knowledge on the benefits of HPs on plants

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is steadily improving, deeper investigations on molecular mechanisms that regulate beneficial

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effects of HPs application on plant are needed. In this scenario, deciphering the dynamic profiles of

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the transcriptome in response to APR® will be central to characterize the mode of action of the

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newly proposed biostimulant.

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RNA-sequencing (RNA-seq) has emerged as a powerful and revolutionary tool to provide high

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resolution analyses of plant transcriptional dynamics during various aspects of growth and

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development through sequencing of their associated cDNA (complementary DNA) populations.

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Transcriptome analyses of the effects of biostimulants on plants highlighted global changes in genes

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transcription across multiple processes, pathways, and cell functions.

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of mechanisms of action of biostimulants under stress or non-stress conditions is still lacking.

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In the current study, the effects on growth of two different APR® concentrations were assessed in

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both roots and shoots. Subsequently, RNA-Seq analysis has been applied on lateral roots, to dissect

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the root transcriptomic profiles in response to the treatments. Gene ontology enrichment analysis of

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RNA-seq data and KEGG pathways analysis helped in deciphering the complexity of network of

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metabolic and signalling pathways involved in the regulation of root responses to APR®.

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To the best of our knowledge this is the first reported RNA-seq based study to examine the effect of

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a solid protein hydrolysate obtained by thermal hydrolysis on maize root transcriptome. These next-

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generation sequencing data contribute to the available information on the molecular effects exerted

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by biostimulants on plant root.

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However, the knowledge

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Materials and Methods

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Preparation of hydrolysates from tanned bovine hides ACS Paragon Plus Environment

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APR® has been obtained by a process of thermobaric hydrolysis applied on trimmings and shavings

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of bovine hides previously tanned with wet-blue technology. The hydrolysis process was made in

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spherical rotating autoclaves by high-pressure steam and followed by a low temperature

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dehydration systems.

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Experimental design and plant growth analyses

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The experiments were carried out on Zea mays L. (B73) seedlings. Seeds were germinated on moist

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paper towels at 25ºC for 72 hours. Seedlings were transplanted into vases (16 cm diameter, 20 cm

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height, volume max. 4 L), each contained 3.5 L of river sand. Five days before the transplanting

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into the pots, two different amounts of APR® were added in one application, together with 50 ml of

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distilled water, to the soil mixture: APR® high concentration (A1), APR® medium concentration

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(A1/2) and a control grown medium (C) (Table 1). The amount of APR®, before being distributed in

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the vases, was divided into three sub-units for each concentration and then gradually applied every

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5 cm of sand. The amount of APR®, as well as, the method of its application was first determined in

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preliminary studies (Ertani, personal communication). Plants were irrigated with 100ml of a

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modified Hoagland solution every 2 days, according to the following composition (µM): Ca(NO3)2

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(200), KNO3 (200), MgSO4 (200), KH2PO4 (40), FeNaEDTA (10), H3BO3 (4.6), MnCl2 (0.9),

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ZnCl2 (0.09), CuCl2 (0.036), NaMoO4 (0.01). Plants were placed in a climatic chamber for 15 days;

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a day/night cycle of 14 h/10 h at 25°C/18°C air temperature, 70/90% relative humidity, and 280

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µmol m−2 s−1 photon flux density were utilized as standard conditions. A completely random design

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was used to analyze data. The experiment was carried using three plastic trays each with six vases

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for each treatment (one plant for each cell). For the plant growth measurements (root and shoot dry

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weight; root/shoot ratio), six plants from each plot were chosen for treatment, in three independent

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biological repetitions. For the determination of the dry weight, samples, divided into root and shoot

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fractions respectively, were transferred in an oven and dried at 80°C for 72 h. When statistically

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significant differences were found (ANOVA test), the subsequent test for comparison of treatment ACS Paragon Plus Environment

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means was performed using the Tukey test. Data were analyzed using the program Statistix v 8.0

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(analytical software, Tallahassee, FL, US).

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For the molecular analysis, lateral roots were harvested and subsequently transferred into liquid

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nitrogen and conserved at -80°C. Roots from one plant were pooled and represent a single sample

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out of the three biological replicates (each of six plants). Plants used for sampling roots were

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excluded from subsequent biomass sampling.

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RNA isolation and sequencing library preparation

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Three biological replicates were used for all RNA-Seq experiments from each treatment. Pooled

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lateral root tissues were ground in liquid nitrogen, and total RNA was isolated with the TRIzol

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reagent (Invitrogen, San Giuliano Milanese, Italy). RNA quality was assessed via agarose gel

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electrophoresis and a Bioanalyzer (Agilent RNA 6000 Nano Chip; Agilent Technologies, Santa

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Clara, CA, USA). For all samples, a RIN (RNA integrity number) ≥8.0 was detected.

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cDNA libraries for Illumina sequencing were constructed according to the instructions of the

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manufacturer (TruSeq RNA Sample Preparation; Illumina, San Diego CA, USA).

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Processing and mapping of sequencing reads

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Base calling was performed using the Illumina Pipeline and sequences were trimmed with ERNE.33

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Quality trimming removed low quality and ambiguous nucleotides of sequence ends and adapter

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contamination. TopHat was used to map and annotate the sequences on the B73 reference genome

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(RefGen_v2;

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Cufflinks software was used for the analysis of differentially expressed genes (DEGs).34-35 A single

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unified assembly from each individual Cufflinks assembly was created thanks to Cuffmerge .

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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

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Gene Ontology (GO) and gene enrichment analysis ACS Paragon Plus Environment

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The PLAZA web tool (http://bioinformatics.psb.ugent.be/plaza/) was used to identify GO terms

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within the different subset of genes.

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Data

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(http://bioinformatics.psb.ugent.be/plaza/) for the molecular function, biological processes and

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cellular component with default settings.36-37

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The pathways regulated in the A1, A1/2 and control groups were identified using the Kyoto

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Encyclopedia

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(http://www.genome.jp/kegg/). The pathways that showed the most differentially expressed genes

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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

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Hierarchical clustering

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For each treatment, transcript relative abundance was estimated using the percentage of normalized

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counts in each fraction relative to the total number of normalized counts. The mean of the relative

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abundance per fraction was then calculated and their Euclidean distances computed with the hclust

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function from the stats R package (R Development Core Team, 2012). Clustering was computed

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with Ward's minimum variance method and a heat map generated with the gplots R package (2012)

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(http://CRAN.R-project.org/package=gplots).

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RESULTS

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APR® triggers morphological responses in roots

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Biostimulants are assumed to enhance the growth of plants. In this experiment, the biomass

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production of the maize plants was assessed after 14 days of APR® treatments. The effects of two

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increasing APR® doses (A1/2 and A1) on root and shoot dried weight were then evaluated.

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For each condition, three biological replicates (RI, RII and RIII) were analyzed. Replicates were

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first observed separately (Supporting Information S1). Replicates RII and RIII showed a statistically

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significant enhancement of root biomass in the APR®-supplied plants, but the differences among ACS Paragon Plus Environment

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the treatments in replicate RI were not significant. No statistically significant differences were

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obtained from the leaf-biomass analysis, while Root/Shoot (R/S) ratios, defined as the ratio of

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belowground dry biomass (root) to the aboveground dry biomass (stem and leaves), followed the

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previously observed root biomass behaviour .

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The subsequently statistical analyses were performed considering all the three replicates (RI, RII

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and RIII) per treatment (A1 and A1/2) respect to the control plants (C), to avoid any misguided

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interpretation of data. Plants supplemented with APR® produced roots with a statistically significant

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increased biomass (Fig. 1). The effect was dose-dependent, with a significantly greater root biomass

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(20%) for the half-supplied plants (A1/2). A modest but significant effect (12%) on root biomass was

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recorded for the plant grown in presence of the complete dose (A1). Shoot biomass was unchanged

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in the three growth conditions.

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APR® treatments resulted in significantly higher mean R/S ratios, with no significant difference

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between the two set of plants (A1 and A1/2).

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Data indicate that APR® preferentially enhanced root rather than shoot growth.

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RNA sequencing and mapping of maize lateral root transcriptome

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To obtain a global view of the transcriptome response of maize to APR®, high-throughput RNA

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sequencing using Illumina sequencing technology was performed on whole RNAs (RNA-Seq)

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extracted from mature root tissues (lateral roots).

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RNA sequencing of the 9 samples (two plants-set grown in presence of different APR® doses,

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namely A1 and A1/2, one control plants-set and three biological replicates) yielded a total of 422.6

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million reads, translating to a mean of 47 million reads per sample (Table 2). On average, the RNA-

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Seq experiments yielded between 21 and 62 million reads per sample. Among all reads, 83 to 90%

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mapped to unique positions in the maize reference genome. Gene expression levels were quantified

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and reported as fragments per kilobase of transcript per million mapped reads (FPKM), estimated

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using Cufflinks32. ACS Paragon Plus Environment

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The expression of 29,332 transcripts (FPKM > 1.0) was detected in the mature root (control)

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whereas APR® treated plants had the highest number of expressed genes with 29,441 (A1) and

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29,553 (A1/2) expressed genes.

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Global Transcriptome Changes after Plant Treatment with APR® and

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Hierarchical Cluster Analysis

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To reveal the transcriptome changes influenced by APR®, differentially expressed genes (DEGs)

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were identified by pair-wise comparison of samples collected (A1/2 vs C; A1 vs C; A1 vs A1/2).

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Cuffdiff analysis revealed 1006 DEGs, among which only 2.8% (29 DEGs) were shared by all

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comparison groups (Fig. 2).

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When controlling false discovery rate (FDR) at 5%, 290 and 643 genes were differentially

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expressed in response to the half-dose of APR® and whole concentration, respectively (Fig. 2).

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Complete lists of differently expressed genes treated with both the doses are shown in

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Supplementary Table S1.

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Considering the genes differentially regulated by the highest APR® concentration respect to the

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control, 643 DEGs were almost equally divided in up (329 DEGs) and down (314 DEGs) -regulated

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(Fig. 2 and Fig. 3).

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The number of DEGs isolated in the comparison between the highest concentration versus the

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control was markedly higher than the number of differentially expressed genes detected by the half-

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dose experiment (290 DEGs), suggesting a greater impact of the higher APR® dose on root

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transcriptome. It is noteworthy that the half dose resulted in the up-regulation of the 80.7% of the

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DEGs identified (Fig. 3).

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Both the applications (A1 and A1/2) affect gene expression regulating the transcription of a

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consistent part of them (48% and 54% respectively) to a small degree (fold change > 0.5 or < 2).


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A stronger effect (fold change ≥2 or ≤ 0.5) is evident on the remaining transcripts (52% and 46%

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for A1 and A1/2, respectively) (Fig. 4 and Fig. 5). Further examination of the results showed that 129

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DEGs were co-regulated by the two applications, with most of them (96%) showing the same (i.e.,

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increased or decreased relative expression) in both the thesis. Only four of them

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(GRMZM2G037454, GRMZM2G026930, GRMZM5G833699, GRMZM2G063287) were found to

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have a conflicting pattern of expression.

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A total of 514 genes was differentially exclusively regulated by A1 concentration, while 161 DEGs

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were unique to A1/2 concentration (Fig. 2).

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The comparison between the two APR® treatments (A1 vs A1/2) identified 492 transcripts with

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significant different expression values, 207 of them showing a conserved expression pattern (94

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DEGs increased and 113 DEGs decreased relative expression respect to control sample), thus

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suggesting a dose-effect for the studied product. A discrepancy in expression profiles was, instead,

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recorded for 87 DEGs (A1>C, A1/2C, A1 10 or FC

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