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PlantaAn International Journal of PlantBiology ISSN 0032-0935 PlantaDOI 10.1007/s00425-015-2299-z

Early transcriptional changes in Betavulgaris in response to low temperature

Vita Maria Cristiana Moliterni, RobertaParis, Chiara Onofri, Luigi Orrù, LuigiCattivelli, Daniela Pacifico, CarlaAvanzato, et al.

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ORIGINAL ARTICLE

Early transcriptional changes in Beta vulgaris in responseto low temperature

Vita Maria Cristiana Moliterni1 • Roberta Paris2 • Chiara Onofri2 •

Luigi Orru1 • Luigi Cattivelli1 • Daniela Pacifico2 • Carla Avanzato3 •

Alberto Ferrarini3 • Massimo Delledonne3 • Giuseppe Mandolino2

Received: 17 December 2014 / Accepted: 7 April 2015

� Springer-Verlag Berlin Heidelberg 2015

Abstract

Main conclusion Major metabolic pathways and genes

affected by low-temperature treatment were identified

and a thorough picture of the early transcriptional

changes in sugar beet plantlets upon cold stress was

given.

Sugar beet (Beta vulgaris L.) is an important source of

sugar and bioethanol production in temperate areas

worldwide. In these areas, plantlet survival and sucrose

yield of mature plants can be seriously limited by low

temperatures, especially when plantlets are exposed to

freezing temperatures (below 0 �C) at the early develop-

mental stages. This frequently occurs when the crop is

sown in early spring or even in autumn (autumn sowing) to

escape drought at maturity and pathogen outbreaks. The

knowledge of molecular responses induced in plantlets

early upon exposure to low temperature is necessary to

understand mechanisms that allow the plant to survive and

to identify reactions that can influence other late-appearing

traits. In this work, a wide study of sugar beet transcrip-

tome modulation after a short exposure to a cold stress,

mimicking what is experienced in vivo by young plantlets

when temperature drops in the early spring nights, was

carried out by high-throughput sequencing of leaves and

root RNAs (RNA-Seq). A significant picture of the earliest

events of temperature sensing was achieved for the first

time for sugar beet: the retrieval of a great amount of

transcription factors and the intensity of modulation of a

large number of genes involved in several metabolic

pathways suggest a fast and deep rearrangement of sugar

beet plantlets metabolism as early response to cold stress,

with both similarities and specificities between the two

organs.

Keywords Cold stress � RNA-Seq � Stress-inducedtranscriptome � Sugar beet � Transcription factors

Abbreviations

ABA Abscisic acid

AF Antifreeze protein

AP2/ERF Apetala2/ethylene responsive

BR Brassinosteroid

CBF/

DREB

C-repeat binding factor/dehydration

responsive element- binding factor

COR Cold responsive

DE Differentially expressed

FPKM Fragments per kilobase of exon per million

fragments mapped

FunCat Functional Catalogue

GO Gene ontology

PR Pathogenesis-related

ROS Reactive oxygen species

TF Transcription factor

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00425-015-2299-z) contains supplementarymaterial, which is available to authorized users.

& Giuseppe Mandolino

giuseppe.mandolino@entecra.it

1 Consiglio per la ricerca e la sperimentazione in agricoltura e

l’analisi dell’economia agraria, Centro di ricerca per la

genomica vegetale, via San Protaso 302,

29017 Fiorenzuola d’Arda, Italy

2 Consiglio per la ricerca e la sperimentazione in agricoltura e

l’analisi dell’economia agraria, Centro di ricerca per le

Colture Industriali, via di Corticella 133, 40128 Bologna,

Italy

3 Dipartimento di Biotecnologie, Universita degli Studi di

Verona, Ca Vignal 1, Strada Le Grazie 15, 37134 Verona,

Italy

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DOI 10.1007/s00425-015-2299-z

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Introduction

Sugar beet (Beta vulgaris L.) is an important source for

sugar production in the temperate areas of the world, and is

the main one in Europe; in fact, 20 % of the world’s supply

of sugar derives from sugar beet (Agribusiness Handbook

2009). Moreover, the production of sugar is not only de-

voted to the food industry, but also to the production of

ethanol. Even though sugar beet is still considered a high-

income crop, it requires careful agronomical practices and

preservation from biotic and abiotic stress, to maintain its

profitability and ability to produce high-quality taproots.

Though its cultivated area has decreased by over 30 %

in Europe since 2000, the sugar beet root yield of modern

varieties has increased (FAOSTAT data), indicating that

these are more evolved and able to cope with different

environments and growth conditions. These varieties are

the result of several breeding strategies like the introgres-

sion of resistance traits, also from wild sources (Panella

and Lewellen 2007; Biancardi et al. 2012), based on the

increased knowledge of the genetic traits for high sugar

production and accumulation in the roots.

The main targets of sugar beet genetic improvement

have been the biotic (Cercospora and Rhizomania; De

Biaggi 2005; Skaracis and Biancardi 2005) and abiotic

(e.g. bolting and osmotic stress; Vastarelli et al. 2013) re-

sponses to stress. Breeding has also focused on the factors

that influence root quality and shape, given that presence in

the taproot of molassigenic elements (potassium, sodium,

alpha-amino nitrogen) are in turn increased by the occur-

rence of stress during the growth period.

Low-temperature stress is a significant cause of crop

quality and production losses in European and North Amer-

ican agriculture. The survival of young sugar beet plantlets

and the subsequent sucrose yield of mature plants are often

seriously limited by low temperatures, especially when

plantlets are exposed to freezing temperatures (below0 �C) atearly developmental stages. This can occur when the crop is

sown early (e.g. February in temperate countries like Italy),

with the aim of improving root production and escaping

drought periods at maturity or when the practice of ‘‘autumn

sowing’’ is adopted (like in Southern Europe) to anticipate

root harvest and escape drought and Cercospora outbreak.

The sensitivity of sugar beet plantlets to low tem-

peratures in open field especially depends on the growth

stage. A virtually total lethality has been reported for

plantlets at the cotyledon stage at a temperature of around

-2 �C, but at the three to four true-leaf stage, complete

destruction of the stand is reached only at -10 �C (Ste-

vanato 2005). Moreover, variability in Beta germplasm for

cold sensitivity and adaptability has also been reported

(Kirchhoff et al. 2012).

Therefore, it is important to achieve knowledge about

the mechanisms that are triggered in the plant by low

temperatures, as these can lead to a molecular and bio-

chemical cascade of reactions that can influence several

later-appearing traits, such as the increased tendency to

bolt, or the accumulation of molassigenic products in roots

(Hoffman 2010).

There are few genetic studies concerning cold response of

sugar beet; Wood (1952) reported a genetic variability for

this trait and a correlation of resistance to the root polari-

metric index that was not, however, later confirmed. The

same author also reported a correlation (0.59\ r\ 0.77)

between cold tolerance and Cercospora resistance.

Sugar beet genomics and post-genomics have recently

received a strong impulse from wide-spectrum metabolic,

proteomic and transcriptional analyses, which focus par-

ticularly on genes and proteins expressed in roots or during

germination and developmental processes (Bellin et al.

2002; Catusse et al. 2008) or upon exposure to multi-stress

conditions (Pestsova et al. 2008).

A reference genome sequence of sugar beet ‘RefBeet’

has recently been presented (Dohm et al. 2013), with a total

of 27,421 predicted protein-coding genes, of which 17,151

were functionally annotated by sequence homology. Be-

sides this achievement that will have a huge impact on

future functional studies, recently a reference transcriptome

assembly has also been published for sugar beet adult

plants under gibberellin treatment and vernalization (Mu-

tasa-Gottgens et al. 2012) and for elucidating the pathway

of accumulation of sucrose in taproots (Turesson et al.

2014).

It is well known that cold stress triggers a cascade of

molecular mechanisms leading to major physiological

consequences in the adult plant (Reeves et al. 2007; Abou-

Elwafa et al. 2012). Molecular studies took advantage of

the similarity between the flower induction mechanisms of

Arabidopsis thaliana and Beta vulgaris, but a detailed

study of the genes differentially expressed specifically

during the exposure to cold shock—as experienced by

young plants during temperature drops occurring in the late

winter/early spring nights—has never been carried out. The

modulation of gene expression occurring during the re-

sponse of young sugar beet plants to low temperature has

been described by Pestsova et al. (2008), but a high-

throughput analysis of the pathways involved in this re-

sponse is still due.

In this study, our aim was to identify major metabolic

pathways affected by low-temperature treatment in sugar

beet and the genes modulated by the early cold response in

the different organs. The approach followed was the global

analysis of sugar beet leaves and roots transcriptome in the

spring, diploid sugar beet cultivar Bianca, by de novo

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assembly and analysis of RNA-Seq data obtained by Illu-

mina technology.

Materials and methods

Plant material

Commercial seeds of the spring cultivar Bianca (KWS Saat

AG, Germany) were used for the experiments. Seedlings of

diploid Beta vulgaris ssp. vulgaris were germinated in

9 cm Petri dishes on wet paper, at 24 �C in the dark. After

1 week, seedlings (germination stage G, Fig. 1a) were

transferred to rock-wool pads on hydroponic trays (60 pads

per tray) and hydroponically grown in 10-l tanks of

Hoagland’s nutrient solution (Hoagland and Arnon 1950)

placed in growth chambers (Binder, Tuttlingen, Germany)

at 23 �C/17 �C air temperature (day/night), 60 % air rela-

tive humidity and a 14:10 photoperiod (150 lmol m-2 s-1

light intensity) for 4 weeks. Plantlets size and growth rate

were extremely uniform and over 90 % of them reached

the four-leaf stage (stage V 3.1 and following, Fig. 1b) at

4 weeks of growth.

Cold treatment

In the final 2 h of the last dark period, hydroponic trays

with 4-week-old plantlets were transferred to a pre-

cooled tank of Hoagland’s solution in a growth chamber

at -2 �C air temperature and kept for 2 h in the dark. In

these conditions, air temperature (to which leaves were

exposed) and the Hoagland’s solution temperature (to

which roots were exposed) were slightly different (-2

and 0 �C, respectively). These temperatures are physio-

logically relevant, as during freezing nights, the soil

temperature usually remains a few degrees above air

temperature. Air temperature was selected on the basis

of preliminary survival tests (Pacifico et al. 2011).

About 10 % of the plantlets showed permanent damage;

therefore, tissues for downstream analyses were sampled

only from surviving plants. After cold treatment, leaves

and rootlets from triplicate pools of 30 surviving plants

were separately collected in the dark and immediately

frozen in liquid nitrogen. At the same time, leaves and

rootlets were collected from triplicate pools of 30 con-

trol plants.

RNA extraction and quantification

Total RNA was isolated from 200 mg of leaf or root tissue

using the Plant RNAeasy Mini kit (Qiagen). RNA samples

were treated with DNase (Life Technologies) to remove

any contaminating DNA. Purity and concentration of the

samples were checked using the spectrophotometer Nano-

quant Infinite M200 PRO (Tecan, Mannedorf, Switzer-

land). Samples with an OD 260/280 C2.0 were further

evaluated for RNA integrity on an RNA 6000 Nano Lab-

Chip using Agilent 2100 Bioanalyzer (Agilent Technolo-

gies, Santa Clara, CA, USA).

cDNA libraries preparation and sequencing

Only RNA samples with an RIN C8 for leaves, and C9 for

roots were used for library preparation. The Illumina

TruSeq RNA sample preparation kit (low-throughput) was

used for library preparation (3 lg of total RNA).

Fig. 1 a Germination of cv. Bianca seeds, about 7 days after

imbibition in Petri dishes, at the stage they were moved to hydroponic

trays. b Plantlets at the cotyledon stage, 8 days after transferring the

seedling in hydroponic condition. c Plantlets at the four-leaf stage,

28 days after transferring in hydroponic condition and 35 days after

sowing in Petri dishes. The cylindrical structure visible in c is the

rock-wool pad supporting the plantlet’s growth

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Messenger RNA fragmentation was carried out for 6 min

and 30 s at 94 �C, to maximize the production of 150–160

base pair (bp) fragments. Libraries were validated on

Agilent DNA-1000 chip (2100 Bioanalyzer, Agilent

Technologies) and size-selected on agarose gel; the

298–300 bp inserts obtained were then quantified by qPCR

in accordance with the Illumina qPCR quantification pro-

tocol, diluted and pooled, and multiplexed (three per flow-

cell lane). A 75 bp paired-end run was performed on the

Illumina Genome Analyzer II (GAXII).

De novo assembly

Low-quality reads ([50 bases with quality\7 or[10 %

undetermined bases) and putative PCR duplicates were

removed. Illumina TruSeq adapter sequences were clipped

with Scythe (https://github.com/vsbuffalo/scythe). Low-

quality bases at read ends were trimmed (minimum quality

20 on a 20 bp window, minimum read length 20 bp) with

Sickle (https://github.com/najoshi/sickle).

The transcriptome was assembled de novo using Tri-

nity (Grabherr et al. 2011), version 2012-06-08, with

default parameters. Reads from 12 samples (control and

stressed leaves, control and stressed roots, each in trip-

licate) and from a further 12 samples deriving from a

parallel sulphur deprivation experiment (not reported in

this paper ) were combined into a single transcriptome

assembly.

To assess the expression profiles, reads from each

sample were mapped against Trinity contigs and expres-

sion abundances were quantified using RSEM (version

1.1.21) with default settings (Li and Dewey 2011). Only

contigs with more than 100 aligned reads in at least one

condition were considered. The R package DESeq (Anders

and Huber 2010) was used to identify differentially ex-

pressed genes (FDR B0.05). Based on RSEM counts, the

FPKM (fragments per kilobase of exon per million frag-

ments mapped; Trapnell et al. 2010) expression values

were calculated for Trinity contigs in each condition. Tri-

nity contigs were functionally annotated on the GOslim

ontology with Blast2GO (Conesa et al. 2005).

Contigs with FPKM \0.1 in both control (A) and

stressed (B) samples were considered as not expressed.

Contigs with FPKM A \0.1 and FPKM B C0.1 were

indicated as ‘induced’; contigs with FKPM A C0.1 and

FPKM B \0.1 were defined as ‘repressed’. Sequences

with a |log2(FPKM B/FPKM A)| C 1 were considered as

significantly differentially expressed (DE) and further

classified into functional groups using the Functional

Catalogue (FunCat) at http://mips.helmholtz-muenchen.

de/proj/funcatDB/search_main_frame.html (Ruepp et al.

2004) with manual adjustments following database

searches.

Results

Analysis of RNA-Seq datasets

In total, approximately 200 million of Illumina paired-end

reads were obtained and 20,927 unique contigs with a read

count of at least 100 reads per contig were assembled, with

an average length of 2161 bp; the main sequencing statis-

tics are reported in Table 1. Most contigs had FPKM val-

ues between 0.1 and 5 both in leaves (70 % of the total

contigs) and roots (76 %) (Suppl. Material S1).

A plot of the log2 (FPKM) values of all transcripts in

leaves and roots of untreated 28-day-old sugar beet plant-

lets is shown in Fig. 2. The distribution of the FPKM

values is roughly centred on the origin of the axes and is

relatively symmetric. Data points distributed along the di-

agonals corresponded to transcripts (approximately 11,000)

with a very similar value of FPKM in leaves and roots.

These genes, which can be considered as ‘housekeeping

genes’ in our experimental conditions, exhibited a wide

range of expression levels (from as high as 300 FPKM

down to 0.0014), suggesting that mRNA extraction and

normalization were of good quality and therefore the ob-

tained libraries adequately represented the sugar beet

transcriptome in the specific organs and developmental

stages.

A total of 8441 contigs were found to be significantly

differentially expressed between leaves and roots of

4-week-old hydroponically grown sugar beet plants

(Table 2), among which 74 % were similar to genes coding

for known or unknown proteins available in public

databases. One-thousand and fifty-five sequences were

found only in root transcriptome, while 954 were leaf

specific; 2495 sequences were transcribed at higher values

in leaves (Table 2), while 3137 were expressed more in

roots. Gene Ontology analysis for cellular components

assigned 34 % of root-specific genes to membrane com-

ponents, 13 % to cytoplasm, and 9 % to plastids. In con-

trast, plastid and thylakoid components accounted for 33 %

of the leaf-specific genes, while 21 % were assigned to the

Table 1 Sequencing statistics and data

No. of libraries 24

No. of reads *200,000,000

No. of contigs 168,342

No. of contigs (read count[100) 20,927

Av. length of contigs 2161 bp

Minimum length of contigs 204

Maximum length of contigs 17,030

No. of transcripts in 28-day-old leaves 19,642

No. of transcripts in 28-day-old roots 20,304

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membrane compartment, 11 % to the cytoplasm and 7 % to

the vacuole. Only a minority (less than 3 %) of organ-

specific genes were assigned by Blast2GO to the tran-

scription factors category (data not shown).

Differentially expressed sequences involved in low-

temperature response

When comparing the transcriptome of control and low-

temperature-treated sugar beet plants, a total of 549 (451

positively and 98 negatively regulated) DE sequences in

leaves and 656 (555 positively and 101 negatively) in roots

were retrieved, showing either a complete induction or

repression upon cold treatment or a log2fold-

change|FPKMstressed/FPKMcontrol|[ 1. For a general un-

derstanding of genes involved in the early response of

sugar beet to cold, functional classes of DE sequences were

firstly determined using gene ontology analysis by the

Blast2GO software. The distribution of the GO functions

(Fig. 3a–c) revealed that ‘‘Cellular processes’’

(GO:0009987; 11–13 % in leaves and roots, respectively)

and ‘‘Response to stress’’ (GO:0006950; 7–8 %) were the

most represented biological process categories in both or-

gans, followed by ‘‘Metabolic process’’ (GO:0008152;

5–6 %) and ‘‘Response to abiotic stimuli’’ (GO:0009628;

5 %), which may reflect an active response to cold

(Fig. 4a). In the category ‘‘Molecular Function’’, a higher

proportion of genes involved in binding, catalytic, hydro-

lase, transferase, kinase and transporter activity were

identified in both tissues. Transcription factor activity is

also well represented (4 %) and, together with DNA

binding (4–5 %), includes many putative transcription

factors. An important number of ‘‘Cell compartment’’ GO

terms associated with cytoplasm (16–18 %), plasma

membranes (15 %), membranes (12–13 %) and plastids

Fig. 2 Logarithmic scatter plot of the FPKM expression data in

leaves and roots of control plantlets of sugar beet

Table 2 Number of sequences differentially expressed between

leaves and roots of control sugar beet plants (28 days old)

DE sequences Total Annotated

Leaf specific 954 712

Up-regulated in leaves 2495 2028

Root specific 1855 1200

Up-regulated in roots 3137 2276

Total significantly regulated genes 8441 6216

Fig. 3 Distribution of annotated DE sequences within the GO

categories of ‘‘Biological Process’’ (a), ‘‘Molecular Function’’

(b) and ‘‘Cellular Compartment’’ (c) for leaves (green bars) and

roots (yellow bars) of cold-treated sugar beet plantlets. Percentages

are calculated of the total number of GO terms retrieved for each

category. Less represented GO terms (42 for ‘‘Biological Process’’, 31

for ‘‘Molecular Function’’ and 11 for ‘‘Cellular Compartment’’ were

grouped as ‘‘Others’’. For category ‘‘Cellular Compartment’’, GO

terms cytosol and cytoplasm were grouped as ‘‘Cytoplasm’’, and GO

terms nucleus, nuclear envelope, nucleoplasm, nucleolus as

‘‘Nucleus’’

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(11–13 %) were retrieved. Generally, negligible differ-

ences were found between leaves and roots as for the GOs

involved in the cold response.

Functional categorization

The DE sequences were analysed and categorized in 19

functional groups according to FunCat (Table 3). For a

large number of sequences (41 % in leaves, 44 % in roots),

it was not possible to assign any putative function. These

sequences were grouped into categories ‘‘Classification not

yet clear’’, ‘‘Unclassified proteins’’ and ‘‘No similarity

found’’. Among non-annotated DE sequences, 91 in leaves

and 127 in roots encoded for hypothetical proteins or

proteins with unknown functions, and those remaining (126

in leaves, 157 in roots) did not match any sequence in the

database and are therefore likely to be new cold response-

associated sequences, previously unidentified.

In Table 3, the number of DE sequences for the different

functional categories is reported for leaves and roots which

were subjected to low-temperature stress. A total of 85

sequences in leaves and 80 in roots were only present in

stressed plants and, therefore, are considered as induced by

cold stress; on the contrary, 35 sequences in leaves and 39 in

roots were completely repressed upon stress. In general, at

transcriptional level, most DE sequences were up- rather

than down-regulated (82 % in leaves, and 85 % in roots).

This result suggests that early cold-regulated gene expres-

sion is mainly controlled by transcription and this is con-

firmed by the presence of a high number of transcription

factors in the induced/up-regulated groups. In fact, one of

the most represented FunCat categories in both sugar beet

organs is ‘‘Transcription’’ together with ‘‘Metabolism’’

followed by ‘‘Cellular communication/signal transduction’’

and ‘‘Cell rescue, defence and virulence’’. The complete list

of DE sequences is reported in Suppl. Material S2.

Stress-regulated DE sequences common to leaves and roots

There are similarities and specificities in the sequences

differentially regulated in leaves and roots. As shown in

Fig. 4a, 210 sequences were concordantly up-regulated,

while only 14 were concordantly down-regulated in the

two organs. Figure 4b presents the proportion of organ-

specific and shared down- and up-regulated sequences for

the different FunCat categories.

Fig. 4 Venn diagrams (a) and functional categories (b) of genes with decreased or increased expression upon low-temperature stress in leaves

(yellow), roots (blue) and common to the two organs (green)

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Table 3 Sequences differentially expressed in leaves and roots upon low-temperature stress, divided according to FunCat codes and definitions

FunCat category Induced Up-regulated Repressed Down-regulated

Leaves

01. Metabolism 6 40 7 6

02. Energy 3 4 – 2

04. Storage protein – 1 – –

10. Cell cycle and DNA processing 1 3 1 1

11. Transcription 5 38 6 9

12. Protein synthesis – 2 – –

14. Protein fate 4 23 – 2

16. Protein with binding function or cofactor requirements 3 9 2 8

18. Regulation of metabolism and protein function – 3 1 –

20. Cellular transport, transport facilities and transport routes 1 16 3 3

30. Cellular communication/signal transduction mechanisms 7 34 2 4

32. Cell rescue, defense and virulence 8 39 1 2

34. Interaction with environment 1 2 – –

38. Transposable elements, viral and plasmid proteins 1 3 – –

70. Subcellular localization 2 5 – 2

77. Organ localization 1 – – –

98. Classification not yet clear 1 4 – –

99. Unclassified protein 20 59 4 8

No similarity found 21 81 8 16

Total 85 366 35 63

451 98

549

Roots

01. Metabolism 10 57 3 4

02. Energy 5 4 1 –

04. Storage protein – 1 – –

10. Cell cycle and DNA processing – 1 1 1

11. Transcription 3 47 6 11

12. Protein synthesis – 3 – –

14. Protein fate 6 24 1 –

16. Protein with binding function or cofactor requirements 2 19 1 3

18. Regulation of metabolism and protein function 2 3 2 1

20. Cellular transport, transport facilities and transport routes 4 11 1 6

30. Cellular communication/signal transduction mechanisms 6 50 – 2

32. Cell rescue, defence and virulence 2 28 3 4

34. Interaction with environment 1 3 – –

38. Transposable elements, viral and plasmid proteins 2 4 1 –

70. Subcellular localization 2 9 1 1

75. Tissue localization – 1 – –

77. Organ localization – 1 – –

98. Classification not yet clear – 6 1 –

99. Unclassified protein 19 92 7 9

No similarity found 16 111 10 20

Total 80 475 39 62

555 101

656

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In most cases, the stress-induced fold change (either

positive or negative) recorded in the two organs was

comparable for each sequence. Only six sequences were

found divergent in their regulation upon low-temperature

stress in leaves and roots: two annotated sequences (a

germin-like protein, strongly up-regulated in leaves but

down-regulated in roots, and a putative myb family tran-

scription factor, down-regulated in leaves and induced in

roots), three coding for unclassified proteins and one with

no similarity to other known sequences.

Among the DE sequences with the highest positive fold

change and consistent direction of regulation, 31 (out of 36)

were grouped in the ‘‘Transcription’’ functional category

(Suppl. Material S3). Many putative AP2/ERF domain-con-

taining transcription factors were found, either fully induced

or strongly up-regulated upon stress; CBF/DREB1 tran-

scriptional activators contain these domains and are known to

trigger the initial steps of response to a number of events,

among which is the activation of COR (cold responsive)

genes (Yamaguchi-Shinozaki and Shinozaki 1994; Thoma-

show 1999). Other transcription factors were found among

the concordantly induced or up-regulated sequences, con-

taining myb, nac, wrky, zinc finger or gras domains. A pu-

tative serine threonine protein kinase-like protein ccr4 and a

ccr4-associated factor, known to be the major players of

deadenylation in mRNA decay (Chou et al. 2014), were

strongly up-regulated in both leaves and roots.

Eighteen DE sequences assigned to the ‘‘Cellular com-

munication/signal transduction’’ category were concor-

dantly modulated in leaves and roots. Among these, a

mitogen-activated protein kinase kinase (MAPKK known

to trigger stress and extracellular signals, Jonak et al.

1996), and protein phosphatases type 2c (proposed to

counteract the MAP kinase pathway, Fuchs et al. 2013),

were found to be strongly up-regulated, suggesting a

change in the phosphorylation status induced by low tem-

perature. Besides, two sequences for putative hydrolases

cleaving the 1-phosphatidylinositol-bisphosphate were also

up-regulated; these genes are part of the pathway of cold-

induced generation of diacylglycerol and of inositol 1,4,5

triphosphate, key second messengers in the cold signal

transduction pathway (Arisz et al. 2013). Probably related

to the downstream action of these second messengers are

also a number of up-regulated sequences, identified as

calcium- or calmodulin-binding or related proteins. These

paths seem therefore to be present and inducible in both

leaves and roots of sugar beet.

The retrieval of 15 genes for putative protein-modifying

enzymes suggests that protein turnover is a major event

upon stress in both organs. Among them, aspartyl protease

sequences (coding for U-box containing proteins and other

factors putatively involved in the degradation of proteins

through the proteasome) were strongly up-regulated in both

organs.

Several sequences for proteins involved in the FunCat

‘‘Metabolism’’ category (23 common DE sequences) were

up-regulated in both leaves and roots. Within this category,

the regulation of genes for carbohydrate and lipid meta-

bolism appeared to be particularly relevant. A glycosyl-

transferase belonging to the family 61 (GT61), involved in

the branching of the xylan backbone in secondary cell wall

(Jensen et al. 2013), and few sequences for xyloglucan

endotransglucosylase/hydrolase (XTH) were found. As for

lipid metabolism, a choline kinase 2p-like protein, puta-

tively involved in the synthesis of phosphatidylcholine, and

the delta-8 sphingolipid desaturase, involved in the

modification of membrane components, were found.

A putative arogenate prephenate dehydratase (biosyn-

thesis of aromatic amino acids) and an ACC-synthase,

possibly involved either in the stimulation of ethylene

biosynthesis by low-temperature treatment, or the recruit-

ment of ACC as a signalling molecule (Yoon and Kleber

2013), were strongly up-regulated in both organs.

Fourteen sequences were concordantly down-regulated

in leaves and roots. A six- to seven-fold repression was

observed for the transcriptional co-activator ADA2b, a

protein contributing to chromatin occupancy regulation,

along with gcn5-related N-acetyltransferase-like protein. In

Arabidopsis, ADA2b co-activator was shown to interact

with CBF1 in generating the transcriptional response to

low temperatures (Mao et al. 2006).

Some stress-induced sequences were absent in the un-

treated leaves and roots, and their induction in treated or-

gans suggests their specific involvement in early response

to low temperature. Among them, a putative serine

palmitoyltransferase, key enzyme of sphingolipid metabo-

lism, was found. Only few sequences were concordantly

fully repressed by the stress treatment in both organs

(Suppl. Material S2).

Stress-regulated DE sequences specific to leaves and roots

In total, 319 sequences were regulated specifically in leaves

upon low-temperature exposure (238 up- and 81 down-,

Fig. 4a) and 426 in roots (342 up- and 84 down-regulated).

Sequences grouped as ‘‘Transcription’’ were mostly

shared between leaves and roots (Fig. 4b), even though a

significant number of leaf- and root-selectively expressed

transcription factors or regulators were also found. Among

them, a CBF3 orthologue sequence was found to be up-

regulated only in roots (55-fold) upon exposure to a low

temperature. Also, the co-repressor TOPLESS-related

protein was found up-regulated only in roots, thus

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suggesting for at least some of the modulated AP2/ERF

factors (Licausi et al. 2013) a repressor function.

Sequences related to the ‘‘Metabolism’’ category were

more represented in root-specific than in leaf-specific DE

gene set (36 leaf specific, 51 root specific). Among them,

putative genes involved in cell wall remodelling were all

up-regulated in roots, but not in leaves. Also, the secondary

metabolism-related transcripts were differently represented

in the two organs, with up-regulation of genes of the

biosynthesis of isoflavonoids, phenolics and volatile com-

pounds in leaves, while a strong up-regulation of genes

involved in betacyanin glycosylation was observed in

roots. It should be pointed out that betacyanins accumulate

in response to drought stress in several plants and have a

protective role against osmotic-like stress and oxidative

damage (Casique-Arroyo et al. 2014).

The metabolism of intracellular mediators turned out to

be significantly modified upon cold stress application, be-

ing represented by 13 sequences, mostly up-regulated.

Among them, only two were common to leaves and roots.

As regards the ‘‘Cellular communication/Signal trans-

duction’’ category, at least nine sequences coding for en-

zymes of the phosphatidic acid signalling pathways were

found either up-regulated or induced in roots, together with

a plethora of kinases, phosphatases and calcium/calmod-

ulin-binding proteins. In leaves, sequences involved in PA

signalling were less represented (two of them were down-

regulated), while sequences related to brassinosteroid

(brassinosteroid lrr receptor kinase and brassinosteroid in-

sensitive 1-associated receptor kinase 1) and auxin sig-

nalling (saur family protein) were all up-regulated.

A total of 75 sequences were assigned to the ‘‘Cell

rescue, defence and virulence’’ category, among which 38

were leaf specific and 25 were root specific. Sequences

were then further classified as ‘‘Stress response, disease,

virulence and defence’’ and ‘‘Detoxification’’. In leaves,

most sequences were related to defence (18) and detoxifi-

cation (14), while in roots most sequences (11) were related

to stress response.

Finally, while the differentially expressed genes related

to the ‘‘Cellular transport, transport facilities and transport

routes’’ category in leaves were mostly related to carbo-

hydrate translocation and transport, in roots the picture was

more complex, including some ion channels and trans-

porters for Ca??, Na?, NH4?, a bile acid:Na? symporter

protein and an inositol transporter.

Discussion

Sugar beet is known to be a relatively resistant crop to low

temperatures, as testified by its increasingly widespread

cultivation as winter crop. Variety specialization is mainly

focused on resistance to early bolting upon cold period

exposure, rather than on resistance to low temperature it-

self. However, its capability of withstanding low tem-

peratures was demonstrated to be strictly dependent upon

the growth stage and other environmental conditions

(Reinsdorf et al. 2013; Loel and Hoffmann 2014). An

improved knowledge of the plant response to low or

freezing temperature exposure is important for both winter

and spring sugar beet varieties, though the growth stages at

which the plants experience this kind of stress are very

different in the frame of the two agronomical practices.

The controlled experimental conditions used in this work

simulated the frequently occurring exposition of sugar beet

plantlets to night freezing temperatures early after emer-

gence, as occurring during late winter/early spring in sev-

eral sugar beet growing areas, e.g. in plains in Northern

Italy. These conditions, depending on the temperature

reached, frequently lead to a significant crop loss and to the

necessity of re-sowing the stand, partially or entirely, with

significant economic losses.

In our controlled environment tests, the short exposure

(2 h) of four-leaf stage sugar beet plants to a marked and

sudden drop in air temperature to -2 �C only caused a

small number of plants to die, at least in the hydroponic

conditions used for the experiment. Upon equilibration of

the hydroponic tank in a temperature-controlled cabinet,

the air temperature reached is, however, slightly different

from the medium temperature, that remains around 0 �Cand in an incipient freezing state of the liquid surface.

Therefore, in our system, leaves and roots were exposed to

different temperatures, mimicking the real situation expe-

rienced by the sugar beet plantlets in the field during early

spring’s freezing nights, when air temperature may still

drop a few degrees below zero, but the temperature of the

superficial portion of the soil is always higher, around

0 �C.As a consequence, the experimental design adopted has

some major implications: (1) only early transcriptional

events, or events that are not exhausted within a 2-h period,

can be detected. The application of a short period of low

temperatures has all the disadvantages underlined by

Winfield et al. (2010) as it does not enable the identifica-

tion of all the long-term consequences of exposure to low

temperatures; however, this design has the advantage of

taking a significant picture of the earliest events of tem-

perature sensing, as demonstrated by the high proportion of

transcription factors identified. (2) It has been possible to

highlight differences in the response of foliar and root

tissues of young sugar beet plantlets early after stress; in-

deed, it is becoming increasingly clear that organ and tissue

specificity in stress responses can occur (Ganeshan et al.

2008; Qi et al. 2014); to our knowledge, this is the first

report describing differences in the transcriptional response

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of leaf and root organs subjected to low temperatures,

applied in an experimental setup mimicking actual field

conditions. (3) Because the cold treatment was applied in

the final 2 h of the last dark period, it was expected that the

response observed would largely exclude those due to the

interaction between light and cold (Crosatti et al. 1999;

Franklin and Whitelam 2007), though for some genes a

circadian gating was reported (Fowler et al. 2005), which

may well have impacted the observed response (see below

the case of CBF3). Among the results of such interactions,

a dramatic rise in reactive oxygen species (ROS) due to

transfer of reducing power from photosystems to oxygen

has been shown to amplify the transcriptional response to

the low-temperature treatment in Arabidopsis (Soitamo

et al. 2008).

Response to low temperature has frequently been di-

vided into avoidance and tolerance (Levitt 1980). Though

the underlying mechanisms are not mutually exclusive, the

short duration of the stress imposed in our experiments is

likely to mobilize ‘‘first emergence’’ cellular and molecular

events contributing to both mechanisms (Gusta and Wis-

niewski 2013).

Transcriptional studies of cold-regulated genes have

been carried out by several authors by microarray and

whole transcriptome sequencing technologies. In Ara-

bidopsis, the number of genes regulated by a short expo-

sure to low temperatures was between 4 and 20 % (Hannah

et al. 2005; Lee et al. 2005), while in our experiments, out

of 20,927 transcripts identified, 549 (2.7 %) and 656

(3.3 %) were meaningfully regulated upon cold stress.

Another feature of our data was that induced and up-

regulated sequences accounted for a much higher fraction

(82 % of the regulated sequences in leaves and 78.5 % in

roots) compared to those completely shut off and down-

regulated. The preferential up-regulation of DE sequences

as a consequence of cold stress has also been observed by

other authors using either microarrays or RNA-Seq (Fowler

and Thomashow 2002; Matsui et al. 2008). Besides, the

number of significant DE sequences was comparable in

leaves and roots; therefore, even if exposed to two different

temperatures (leaves at -2 �C; roots at 0 �C), these data

suggest the involvement of both sugar beet organs in the

early sensing and response to cold stress and their likely

coordination at transcriptional level, also confirmed by the

high proportion of shared DE sequences of the total (42 %

in leaves and 35 % in roots).

The number of DE transcription factor-related se-

quences in cold-treated vs. untreated leaves and roots was

relatively high: about 10 % of the DE sequences were

classified as transcription factors (Suppl. Material S2 and

S3; see also Fig. 3 for GOs and Table 3 for FunCat cate-

gories). It should be pointed out that this fraction was al-

most the same in both organs (Table 3).

Sugar beet has been reported to have a lower number of

transcription factor-encoding genes than any other flower-

ing plant with already sequenced genome (Dohm et al.

2013), and therefore gene interaction networks were hy-

pothesized to have evolved differently in sugar beet com-

pared to other species. In our experiments, a short pulse of

cold stress was able to elicit a high proportion of tran-

scription-related factors, indicating that sugar beet plantlets

widely modify their metabolism to cope with the cold stress.

Moreover, among the 88 transcription-related DE se-

quences, 37 were common to both organs (36 with con-

cordant and 1 with opposite modulation), suggesting a

widely shared path of regulation of the early response to

cold stress. Therefore, it could be hypothesized that the 37

transcription-related DE sequences are those more closely

linked to the specificity of cold response and likely to be

activated by common pathways or by long-distance sig-

nalling between the two organs. FunCat attributed most of

these transcription-related sequences to RNA synthesis

(zinc fingers, AP2/ERF domain, ERF, etc.) and only a mi-

nority to RNA processing or editing (ccr4 associated fac-

tors, pentatricopeptide repeat-containing proteins, SWAP

SURP domain proteins, etc.; see Suppl. Material S3).

Among the transcription factors mediating the low-

temperature response, CBF is known to be strongly up-

regulated within a few minutes from stress application

(Chinnusamy et al. 2014) and this is one of the earliest

events responding to low-temperature exposure; however,

it has been shown in Arabidopsis that CBF accumulation

depends upon the time of day at which plants are exposed

to low temperature (Fowler et al. 2005). In our study, after

2 h at -2 �C (leaves) and 0 �C (roots), we identified an

Arabidopsis CBF3 homologue as the main up-regulated

sequence (55 fold increased), but only in roots. This sug-

gests that CBF3 transcription in stressed plants is either

maintained for a longer time, or begins earlier in roots

compared to leaves. A difference in the time scale of the

metabolic response between leaves and roots after expo-

sure to cold stress was also hypothesized as a possible

cause for the ‘‘asynchronous’’ metabolic response observed

for the two organs of Fragaria vesca (Rohloff et al. 2012).

Alternatively, the circadian-dependent time course of ac-

cumulation of CBFs observed by Fowler et al. (2005) could

hold true for sugar beet too, but in this case an ‘‘uncou-

pling’’ mechanism of CBF3 transcript accumulation in

leaves and roots should be postulated. Finally, the fact that

cold treatment was imposed under a dark period, could also

be another reason for the lack of CBF transcription in

leaves, as some of the regulatory proteins are modulated in

a light-dependent way in Arabidopsis and wheat (Badawi

et al. 2007, 2008; Franklin and Whitelam 2007).

The high number of TF-related sequences rapidly up-

regulated by low temperature could represent a

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mobilization of the ‘‘first wave’’ TFs recently identified by

Park et al. (2015) and targeting the CBF regulon along with

CBFs. The main group of transcription factor-related se-

quences found to be either activated or up-regulated in all

organs by cold stress was the AP2/ERF family, and par-

ticularly the ERF subfamily (Mizoi et al. 2012). This is an

expected result, as it is known that these TFs are rapidly

induced upon exposure to low temperatures in Arabidopsis

(Lee et al. 2005). AP2/ERF TFs are involved in the

regulation of primary and secondary metabolism and in a

number of jasmonate-mediated responses (Licausi et al.

2013). Only few AP2/ERF TFs were found to be either leaf

or root specific.

Several other transcription factor families were found to

be up- or down-regulated in leaves and roots. Virtually, all

major families of TFs were represented (Suppl. Material

S3): MYB-type, zinc fingers, WRKY, auxin-response fac-

tors and others. This variety of stress-regulated TFs sug-

gests that the short-term cold treatment elicited a wide-

spectrum range of responses involving both organs sensing

the low temperature. These TFs might be involved in

metabolic, structural and physiological rearrangements,

partly shared by the two organs and partly specific.

The fact that the majority of the TF sequences were up-

regulated or turned on, and only in a minority of cases were

they down-regulated/turned off, suggests that most of them

are directly involved in cold response and were activated to

trigger defence mechanisms by activating/repressing the

downstream genes discussed below.

In Arabidopsis ada2b mutants, it has been reported that

the lack of this transcriptional adaptor leads to an increase

of freezing tolerance by affecting nucleosome occupancy

(Vlachonasios et al. 2003). On the basis of the strong re-

pression of ada2b in our experiments, it is possible to

speculate that the same mechanisms are operating in sugar

beet, leading to chromatin remodelling and modulation of

the downstream genes and resulting in an increase of tol-

erance to stress. According to our data, also a putative

histone acetylase and a lysine-specific demethylase are

strongly up-regulated in leaves, confirming that chromatin

remodelling and modification are active.

The metabolism was especially affected in roots, as

testified not only by the amount of sequences modulated,

but also by the intensity of modulation. Moreover, many

sequences assigned to ‘‘Metabolism’’ were turned on or

shut off (26 out of 106), suggesting a deep and fast rear-

rangement of several metabolic pathways as part of the

sugar beet early response to cold stress.

The metabolic pathway most affected by low tem-

perature was the carbohydrate metabolism. The majority of

root-specific and concordantly modulated DE sequences

were related to cell wall modification and rearrangement

(xyloglucan endotransglucosylase hydrolase, cellulose

synthase, glucan endo-1,4-beta-glucosidase, etc.). Many

leaf-specific DE sequences were involved in sugar meta-

bolism and transport (hexokinase, sugar isomerase, alpha

amylase). Therefore, it can be speculated that, upon ex-

posure to low temperature, while sugar beet leaves tend to

cope with the freezing stress by adjusting their role of

source organ, roots are mainly engaged in the repair or re-

organization of structural elements. The plant cell wall is a

key determinant of plant response to ambient stress. The

major polysaccharides in the primary cell wall are cellu-

lose, hemicellulose and pectins. Putative cellulose syn-

thases and cellulose synthase-like (CSL) genes, belonging

to the glycosyltransferase family, have been found to be

up-regulated to different extents in sugar beet roots; a CSL

protein was recently found to be involved in the osmotic

stress response in Arabidopsis (Zhu et al. 2010a). A puta-

tive b-expansin was also up-regulated in roots, and this

enzyme is reported to play a role in root elongation (Cos-

grove 2000). Many sequences similar to XHTs were found

to be strongly up-regulated upon cold exposure; these re-

sponses have previously been reported in relation to

drought rather than to cold stress (Sasidharan et al. 2011).

The most likely candidates regulating the expression of

expansins and XHTs at the onset of drought appeared to be

phytohormones such as ABA, cytokinins and ethylene,

and, according to our data, the same seems to hold true also

in response to cold stress.

It is well established that the major factor in the per-

ception of the cold stimulus are changes in the flu-

idity/rigidity of the membranes. In our biological system, it

seems that, early after cold perception, a wide rearrange-

ment of the lipid and fatty acid metabolism follows back,

probably targeted either at the adaptive modification of the

membrane itself or at the generation of second messengers

involved in lipid-mediated signalling pathways. The in-

volvement of lipid metabolism was indeed well represented

by several sequences mostly up-regulated or induced upon

stress, in leaves, roots or in both organs. Only four leaf-

specific sequences were either down-regulated (a class 3

lipase) or repressed (three sequences for 4-hydroxy-3-

methylbut-2-enyl diphosphate reductase, HMDR). HMDR

is the last enzyme of the chloroplastic non-mevalonate

pathway for isoprenoids biosynthesis, independent from

that of the cytosolic mevalonate (Pulido et al. 2012). The

fact that all these sequences are repressed suggests a switch

in the regulation of isoprenoid metabolism, in favour of the

mevalonate pathway.

Among the sequences retrieved in both organs, a puta-

tive serine palmitoyltransferase was induced, and a delta-8

sphingolipid desaturase was up-regulated by cold stress.

This enzyme is involved in the sphingolipids biosynthesis;

it has been reported that there are differences in membrane

sphingolipid abundance/composition in acclimated vs. non

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acclimated plants and cold-tolerant vs. cold sensitive spe-

cies (Cantrel et al. 2011; Chen et al. 2012). Moreover, the

retrieval of 12 DE sequences putatively involved in plant

PA signalling (like phospholipases and phosphodiesteras-

es) suggests that phospholipid second messengers are an

important part in early cold signalling also in Beta vulgaris.

They were mostly found in roots.

Change in the transcription of secondary metabolism

genes is a feature commonly reported upon application of

various types of stress in plants; in our experimental sys-

tem, we found 13 DE sequences related to this functional

category, none of which were common to leaves and roots.

The high degree of organ specificity probably highlights a

differentiated repertoire of compounds synthesized by the

two organs upon stress.

The observed strong modulation at transcriptional level

of sequences putatively involved in ethylene biosynthesis

(ACO and ACS, of which different sequences were iden-

tified) suggests that this plant growth regulator plays a role

during the early stages of the response to cold. Indeed,

ethylene signalling is known to cross talk with other hor-

mones in response to abiotic stress (Harrison 2012). In fact,

sequences correlated to genes of hormone homoeostasis

pathways (cytokinins, ABA, auxins, gibberellins) were

modulated in roots, suggesting a high cold sensitivity at the

time chosen for the analysis. The most up-regulated one

was a putative cytokinin oxidase, the major gene involved

in oxidative cytokinin degradation in plants (Bilyeu et al.

2001). In leaves, only ethylene, auxin and jasmonate-re-

lated genes were regulated upon stress in our experimental

conditions, thus suggesting the involvement of different

growth regulators in the two organs.

Previous studies reported that ABA biosynthesis was not

a major event in cold stress (Lee et al. 2005). However, in

our work, a 9-cis-epoxycarotenoid dioxygenase, NCED

(the rate-limiting enzyme in the ABA biosynthesis) and an

abscisic acid-8-hydroxylase (involved in ABA degradation

to phaseic acid) are both up-regulated (the first in roots and

the second in both organs), suggesting for ABA the onset

of differential turnover pathways in leaves and roots.

Brassinosteroids (BR) are involved in responses to bi-

otic and abiotic environmental stress, and a possible cross

talk with ethylene and ABA signalling pathways has been

proposed (Divi et al. 2010). It has been suggested that these

plant hormones increase plant tolerance to low temperature

by promoting growth (Yang et al. 2011). In this work, we

found genes putatively involved in BR signalling that are

all up-regulated, either in both organs or specifically in

roots or leaves.

Cell responses to external stimuli are mediated by signal

transduction pathways transmitting information to the nu-

cleus, where transcriptional reprogramming and physio-

logical performance under a given set of conditions are

decided. Therefore, it was expected that early response to

cold stress included a high proportion of DE sequences in

both leaves and roots that was related to the functional

category ‘‘Cellular communication/signal transduction

mechanism’’; indeed, about 8.5 % of DE sequences in both

organs were assigned to this functional category.

A universal phenomenon associated with stress sig-

nalling is the increase of cytosolic Ca2? concentration.

Calcium itself and/or proteins sensing cytoplasm Ca2?

perturbations and relaying this information to downstream

molecules are important components of cold signalling

pathway (Knight and Knight 2012). Also in our ex-

periments, several sequences putatively encoding proteins

activated by Ca2? were found to be either up-regulated or

induced, confirming the role of calcium in cold-induced

signal transduction.

Another well-known mechanism implicated in signal

transduction is protein phosphorylation/de-phosphoryla-

tion; changes in the phosphorylation status induced by cold

are documented in this work by the high number of puta-

tive kinases and phosphatases retrieved among DE genes.

Even if cold treatment was, in our case, applied during

the dark period, a significant percent of DE sequences were

related to ROS scavenging and detoxification: catalases,

peroxidases and glutathione-S-transferases were all up-

regulated, especially in leaves. This pathway showed a

high degree of organ specificity, as only 2 sequences out of

20 were common to both leaves and roots.

A number of sequences related to genes known in lit-

erature as ‘‘cold responsive’’ (COR) were found up-

regulated: a low-temperature and salt-responsive protein

lti6b, a putative early-responsive to dehydration stress-re-

lated (ERD) protein, two late embryogenesis abundant

(LEA) proteins, a desiccation-related (DRE) protein and a

dehydrin.

Other stress proteins typically induced upon cold stress

are the antifreeze proteins (AFPs) that can directly interact

with ice in planta and reduce cold injury by slowing ice

growth and crystallization (Griffith et al. 2005). Most plant

AFPs are pathogenesis-related (PR) proteins. In this work,

at least 17 sequences for putative PR proteins were found,

mostly up-regulated at high levels of induction, belonging

to different recognized families of PR proteins. Moreover,

some AFPs are known to be characterized by leucine-rich

repeat (LRR) motifs and some putative LRR resistance

genes were also found both in leaves and roots with dif-

ferent modulation. Other defence-related genes not appar-

ently strictly related to cold stress response were identified,

namely, a nematode-resistant gene, three ribosome-inacti-

vating proteins, a putative polygalacturonase-inhibiting

protein (known to function against fungal infection) and

two sequences related to response to bacteria (rpm1-inter-

acting protein 4 and nhl3), indicating that different

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signalling pathways may cross talk to trigger a common set

of responses (Snider et al. 2000; Huang et al. 2010; Zhu

et al. 2010b).

Conclusions

A short pulse of sub-lethal low temperatures during the

dark period triggers a wide-spectrum response at tran-

scriptional level, both in young leaves and in rootlets of

4-week-old Beta vulgaris plants.

Such a response appears to be temporally coordinated,

as a similar number of sequences were modulated in the

two organs after only 2 h of stress exposure. About 3 % of

the total transcripts identified were modulated by stress in

both organs, representing therefore the very early events of

the transcriptional response to cold. The modulation ob-

served was mainly an up-regulation/induction of tran-

scription that accounted for over 80 % of the regulated

sequences after stress in leaves and roots. This indicates

that a wide rearrangement of plant metabolism occurs

immediately after cold perception.

Transcriptional responses were widely shared by leaves

and roots. The observed modulation mainly consisted in an

up-regulation or induction of several genes, as 38–47 % of

up-regulated/induced sequences were shared by the two

organs, while only 15 % of down-regulated/repressed se-

quences were common to both.

Most DE sequences identified related to transcription

regulation, protein phosphorylation/dephosphorylation,

protein turnover, cell wall remodelling, calcium and hor-

mone signalling, and carbohydrate and lipid metabolism, in

agreement with reports on the earliest effects of low-tem-

perature stress in other plants.

On the whole, the data presented provide a thorough

picture of transcriptome modulation in young sugar beet

plants exposed to cold stress during the night, and organ-

specificities and shared paths involved in the plants’

physiological response to low temperatures.

Author contribution VMCM and GM conceived and de-

signed the research. CO and DP conducted cold stress

experiments in hydroponic conditions. VMCM and LO

prepared the libraries and conducted RNA sequencing. CA,

AF and MD performed bioinformatics analyses. VMCM,

RP and GM elaborated and discussed the final data and

wrote the manuscript. LC critically reviewed the manu-

script. All authors read and approved the manuscript.

Acknowledgments The present work was funded by the Italian

Ministry of Agriculture and Forestry (MIPAF), in the frame of the

research project ‘‘Agronanotech, Le nuove tecnologie molecolari per

l’analisi del genoma di organismi di interesse agrario’’. The authors

wish to thank dr. Enrico Biancardi (CRA-Research Center for

Industrial Crops, Rovigo) for providing helpful discussion on sugar

beet stress physiology and genetics and for seeds of the sugar beet cv.

Bianca.

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