Accepted Manuscript Identification of desiccation tolerance transcripts potentially involved in rape (Brassica napus L.) seeds development and germination Sirui Lang, Xiaoxia Liu, Gang Ma, QinYing. Lan, Xiaofeng Wang PII:

S0981-9428(14)00248-4

DOI:

10.1016/j.plaphy.2014.08.001

Reference:

PLAPHY 4021

To appear in:

Plant Physiology and Biochemistry

Received Date: 3 April 2014 Accepted Date: 3 August 2014

Please cite this article as: S. Lang, X. Liu, G. Ma, Q. Lan, X. Wang, Identification of desiccation tolerance transcripts potentially involved in rape (Brassica napus L.) seeds development and germination, Plant Physiology and Biochemistry (2014), doi: 10.1016/j.plaphy.2014.08.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Identification of desiccation tolerance transcripts potentially involved in Rape (Brassica napus L.) seeds

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development and germination

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Sirui Lang1†

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Email: [email protected]

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Xiaoxia Liu1†

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Email: [email protected]

Gang Ma1

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Email: [email protected]

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QinYing. Lan2

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Email: [email protected]

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Xiaofeng Wang1*

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Email: [email protected]

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Forestry University, Tsinghua East Road 35, Haidian District, Beijing 100083, P. R. China

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National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing

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Sciences, Germplasm Bank, Mengla, 666303 Yunnan, P. R. China

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Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of

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Corresponding author.

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Equal contributors

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ACCEPTED MANUSCRIPT Abstract

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To investigate regulatory processes and protective mechanisms leading to desiccation tolerance (DT) in seeds,

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cDNA amplified fragment length polymorphism (cDNA-AFLP) in conjunction with 128 primer combinations

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was used to detect differential gene expression in rape seeds in response to DT during seed development and

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germination. We obtained approximately 8,000 transcript-derived fragments (TDFs), of which 394 TDFs with

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differential expression patterns (“sustained expression”, “up-regulated”, “couple with seed DT”, and

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“down-regulated”) were excised from gels and re-amplified by polymerase chain reaction (PCR). After

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sequencing and comparison with the National Center for Biotechnology Information database, 176 TDFs

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presented significant similarity with known genes that could be classified into the following categories:

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metabolism and energy, stress resistance and defense, storage, signal transduction, and other functional

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categories. Using semiquantitative reverse-transcription PCR and real-time PCR approaches, the significance

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of the differences was further confirmed in fresh seeds and dehydrated seeds. The genes that encode

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superoxide dismutase, peroxiredoxin, caleosin, oleosin S3, steroleosin, late embryogenesis abundant protein,

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glutathione reductase, β-glucosidase, S23 transcriptional repressor, and some heat-shock proteins could be

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associated with DT. The results of this study will aid in the identification of candidate genes for future

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experiments that seek to understand seed DT.

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Key words: Rape seed; Desiccation tolerance; cDNA-AFLP; Seed development; Seed germination

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ACCEPTED MANUSCRIPT Abbreviations

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DT, desiccation tolerance; cDNA-AFLP, cDNA-amplified fragment length polymorphism; TDF,

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transcript-derived fragments; LEA, late embryogenesis abundant; DAP, day after pollination; MC, moisture

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content; ROS, reactive oxygen species; DW, dry weight; HSP, heat-shock protein; ZEP, zeaxanthin epoxidase;

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ABA, abscisic acid; GR, gutathione reductase; PRX, peroxiredoxin; SOD, superoxide dismutase

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1.

Introduction Seeds were divided into orthodox seeds and recalcitrant seeds in the early 1970s by Roberts [1].

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Orthodox seeds endure maturation drying and acquire desiccation tolerance (DT) during development with a

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low moisture content (MC) and survival against various environmental stresses. However, DT is promptly

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lost during germination after only a few hours of imbibition or when the radicle is exposed through the seed

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coat [2, 3]. Compared with orthodox seeds, recalcitrant seeds which have active metabolism should begin

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their new life cycle after dropped from the mother plant, are desiccation sensitive [1]. A number of processes

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or mechanisms are correlated with DT in orthodox seeds including the accumulation of oligosaccharides [4],

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synthesis of storage and protect proteins such as LEA proteins and heat-shock proteins [5, 6], the presence of

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antioxidant system such as glutathione [7] and antioxidant enzyme [8-10].

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Transcriptomic, proteomic, and metabolomic studies have been performed to describe the changes

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associated with seed development and germination [11-13]. Former studies found that most of the genes

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during seed development were involved in lipid, amino acid, and carbohydrate metabolisms, and some other

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genes were involved in post-translational modification, protein folding, molecular chaperone, cell signaling

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and other physiological and biochemical processes. Similar results are also reported in rape cultivars seeds

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[12, 14]. These studies have led to a significant increase in the knowledge of molecular mechanisms in seed

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maturation and germination. However, progress in elucidating signal transduction pathways for the

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acquisition of DT has been impeded by difficulty in discriminating such pathways from those involved in

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other overlapping developmental programs, such as reserve accumulation and the acquisition of germinability.

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Additionally, progress in elucidating the molecular mechanisms that control DT has come from the isolation

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and analysis of mutants that exhibit impairment in embryogenesis and maturation. Loss-of-function mutants

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of abi3 and lec1 of Arabidopsis produce seeds that lose their viability during desiccation or during the first

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few weeks after harvest. Nevertheless, abi3 and lec1 mutants are affected in multiple maturation processes,

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including storage accumulation and dormancy [15].‐‐ Previous studies on DT have been conducted on orthodox or recalcitrant seeds [16, 17]. Actually,

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comparing their different responses to desiccation is an essential way to understand the mechanism of DT.

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Due to the different genetic background, it is difficult to analysis the DT of seeds from two different species.

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Berjak [10] reported that imbibed orthodox seeds have a high degree of subcellular development and

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metabolic activity, which can be studied as desiccation-sensitive seeds instead of recalcitrant seeds. Therefore,

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comparing DT in orthodox and recalcitrant seeds with identical genetic background by a combination of

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development with germination is feasible for understanding the mechanism of DT.

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The cruciferae rape (Brassica napus L.) seed is the world’s second most widely planted oil crop and has

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strong resistance to desiccation. When the seed MC drops below 1 %, seed vigor is unaffected, but storability

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is improved (date not shown). In the present study, we analyzed differentially expressed genes during seed

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development and germination in fresh seeds and dehydrated seeds by cDNA amplified fragment length

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polymorphism (cDNA-AFLP) mechanic, and to identify specific genes that were directly involved in DT.

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The purpose of this study was to analyze the transcriptional response to DT and to highlight candidate

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genes for future research.

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2.

Materials and Methods

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2.1 Plant materials

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Rape (Brassica napus L.) seeds (Precocity Cultivar), kindly provided by Qinghai Academy of

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Agricultural Sciences, were used in this study. The original germination was 98 %, and the MC was 7 % (Dry

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weight, DW). 5

ACCEPTED MANUSCRIPT To analyze the acquisition of DT during development, the rape seeds were grown in the research field at

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Beijing Forestry University in April 2010. To collect seeds at defined developmental stages, individual

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flowers were tagged every day, and the seeds were collected from siliqua at the base of rachis 14, 18, 24, 28,

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36, and 45 days after pollination (DAP).

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Seed dehydration was rapidly achieved by first placing them on filter paper and then placing them over

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2.2 Seed dehydration

activated silica gel for different times to a MC of 7 % (DW) within closed desiccators at 25 °C.

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The fresh seeds, which were gathered from mother plants on different days after pollination (DAP),

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were frozen rapidly in liquid nitrogen and preserved at -80 ºC for further analyses. Other fresh seeds were

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dehydrated to a MC of 7 % and then frozen rapidly in liquid nitrogen. These seeds were named “dehydrated

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seeds”.

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To analyze the loss of DT during germination, mature rape seeds were surface-sterilized with 75 %

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ethyl alcohol for 1 min and then 15 % H2O2 for 3 min, rinsed three times in sterilized water, and imbibed in 9

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cm Petri dishes on two layers of filter paper with 6 ml of distilled water at 25 ºC in the dark. The seeds were

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removed at different intervals of imbibition. Parts of these imbibed seeds were frozen rapidly in liquid

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nitrogen for subsequent analyses as fresh seeds. Other seeds were dehydrated to a MC of 7 %, frozen rapidly

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in liquid nitrogen, and stored at -80 ºC as dehydrated seeds.

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2.3 Determination of H2O2 content The H2O2 content was determined according to a method described previously [18].

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2.4 Determination of moisture content The MC was assessed gravimetrically for four independent replicates of 50 seeds by determining the

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fresh weight and subsequent dry weight after 17 h at 103 ºC. The MC is expressed based on dry weight (g·g-1

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DW).

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2.5 cDNA-AFLP

Total RNA was extracted according to the sodium dodecyl sulfate (SDS)-LiCl protocol. Each sample

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was homogenized and mixed with 550 µl RNA extraction buffer (0.2 M Tris-HCl; pH 8.0), 25 mM EDTA (pH

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8.0), 1 % SDS, 2 % β-mercapto-ethanol, and 550 µl chloroform : isoamyl alcohol (24:1). After incubation for

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5 min, the samples were centrifuged at 12,000 rotations per minute (rpm) for 3 min at 4 ºC. Water-saturated

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phenol (500 µl) was added to the supernatant, followed by the addition of 200 µl chloroform : isoamyl alcohol

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(24:1) and centrifugation at 12,000 rpm for 3 min at 4 ºC. Ice-cold 8 M LiCl was then added to the

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supernatant to a final concentration of 2 M, and the tubes were incubated overnight at -20 ºC. After

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centrifugation at 12,000 rpm for 30 min at 4 ºC, the precipitate was dissolved in 500 µl of

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diethylpyrocarbonate (DEPC)-treated water, 7 µl of 3 M NaAc (pH 5.2), and 250 µl ethanol. The samples

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were then centrifuged at 12,000 rpm for 10 min at 4 ºC. The supernatant was transferred to a new tube,

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followed by the addition of 43 µl of 3 M NaAc (pH 5.2) and 750 µl ethanol, which was mixed well and

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incubated for 1 h at -20 ºC. Finally, after centrifugation at 12,000 rpm for 20 min at 4 ºC, the supernatant was

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discarded, followed by washing the precipitate with 75 % ethanol and resuspension in 100 µl of

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DEPC-treated water. The efficiency of the extraction was checked by agarose gel electrophoresis, and the

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concentration was determined spectrophotometrically at 260 nm.

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After RQ-DNase1 treatment, first-strand cDNA was synthesized using oligo dT (15) (Promega, USA) 7

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synthesized at 16 ºC for 2 h with DNA Polymerase I and Escherichia coli Ligase in the presence of RNase H.

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The resulting double-stranded cDNA was phenol chloroform-extracted, ethanol-precipitated, and resuspended

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in 30 µl of DEPC-treated water. Half of this volume was checked using agarose gel electrophoresis to observe

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an expected smear between 100 and 3,000 base pairs (bp). The remainder of the cDNA was subjected to

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AFLP template production.

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cDNA-AFLP analysis was performed according to a previous report [19]. Double-stranded cDNA was

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digested with the restriction enzymes BstY I (2 h at 60 ºC) and Mse I (2 h at 37 ºC). BstY Ad (50 µM) and Mse

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Ad (50 µM) adaptors were annealed from oligonucleotides BstY-ad1, BstY-ad2 and Mse-ad1, Mse-ad2,

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respectively. The adaptors were ligated with T4-DNA ligase overnight at 16 ºC, and one-tenth of this reaction

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volume was used for pre-amplification with BstY-P and Mse-P primers (Table 4) in a 50 µl reaction volume.

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Pre-amplification was initiated at 94 ºC for 5 min, followed by 25 cycles at 94 ºC for 30 s, 56 ºC for 30 s, and

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72 ºC for 1 min and termination at 72 ºC for 10 min. The pre-amplification products were checked by agarose

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gel electrophoresis (expected smear between 100 bp to 1000 bp), and their concentrations were determined

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spectrophotometrically at 260 nm. The products were diluted to obtain a final concentration of 10 ng/µl. The

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AFLP reactions were made with 5 µl of diluted pre-amplified solutions (BstY-X [50 ng] and Mse-Y [10 ng]

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primers). After initial denaturation, 10 cycles were performed with touchdown annealing (94 ºC for 30 s, 62

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ºC to 55 ºC in - 0.7 ºC‐/steps, for 30 s, 72 ºC for 1 min), followed by 24 cycles (94 ºC for 30 s, 55 ºC for 30 s,

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72 ºC for 1 min) and final extension (72 ºC for 10 min). All of the oligonucleotides were obtained from

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Primer Premier 6.0, and the amplifications were performed using a personal mastercycler.

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Fragments were separated on sequencing polyacrylamide gel (6 % bis-acrylamide, 7 M urea, 1 × TBE)

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using the Sequigen system (Bio-Rad, Hercules, CA, USA). The glass plates were treated with Plus One 8

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Repel-Silane (Amersham Biosciences, Little Chalfont, UK) and c-methacryloxypropyl-trimethoxysilane

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(Sigma) according to the manufacturer’s instructions. After 4 h of migration (2 kV, 50 ºC) in 1×TBE buffer,

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the fragments were visualized by silver staining according to a previous report [20].

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2.6 Isolation, cloning, and sequence analysis of transcript-derived fragments

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Based on their intensity of up- and down-regulation, the bands that were obviously changed were

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selected and cut from the gels with a surgical blade, dissolved in 100 µl sterile water with vigorous vortexing

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for 1 min, and incubated in boiling water for 30 min. A 5 µl portion of each dissolved band was re-amplified

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using the procedure described for the AFLP reactions with the specific selective nucleotide primers. The

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re-amplified fragments were then extracted from 1 % agarose gels, subsequently cloned into a pMD19-T

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Vector (Takara, Dalian, China), and sequenced. National Center for Biotechnology Information database

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searches were performed using the Basic Local Alignment Search Tool (BLAST) network service. Each TDF

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sequence was compared against all of the sequences in the non-redundant database using the BLASTX

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program. Default parameters were used for all of the analyses. Nucleotide sequences were also analyzed

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using Blast 2 Go (Version 2.4.4) for gene annotation, followed by assignment of the DNA sequences to

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functional categories (e.g., biological process, molecular function, cellular component).

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2.7 Semi-quantitative RT-PCR

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Semi-quantitative RT-PCR (SqRT-PCR) was used to characterize the expression of 24 TDFs with

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known functions. The RNA that was used for SqRT-PCR was obtained from three independent experiments.

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After treatment with RQ-DNase1, the purified total RNA was quantified using a NanoDrop ND 1000

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spectrophotometer (USA). Two micrograms of these RNA was used for first-strand cDNA synthesis using 9

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the first-strand cDNA was checked by agarose gel electrophoresis. The amplifications were performed using

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2 µl of 4× diluted cDNA solution. The primer design was performed using Primer Premier 6.0 software from

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identified rape TDF sequences or from available Arabidopsis sequences for the reference gene and all other

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genes of interest. A fragment of Brassica napus L. seeds, β-actin (AF111812) [21], was used as the reference

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gene for data normalization.

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SqRT-PCR was performed in a total volume of 20 µl that contained 2 µl cDNA, 1 µl (0.5 µM) of each

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primer, 6 µl ddH2O, and 10 µl of 2 × Tag PCR Master Mix. The amplification reactions were performed using

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a personal mastercycler under the following conditions: 5 min at 94 ºC, 25-28 cycles at 94 ºC for 30 s,

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annealing for 30 s at 72 ºC for 1 min, and termination at 72 ºC for 10 min. The PCR products were checked

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by 1 % agarose gel electrophoresis to detect changes in gene expression. All of the amplification reactions

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were repeated three times under identical conditions. To ensure that the PCR products were generated from

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cDNA and not genomic DNA, proper control reactions were performed.

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According to our SqRT-PCR results, we chose 12 TDFs for further expression analysis using real-time

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PCR. The cDNA templates and primers used for real-time PCR were obtained as described in Table 5. All of

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the primers were optimized for use at an annealing temperature of 60 ºC during PCR, producing a single

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amplicon. Relative real-time PCR was performed in a total volume of 25 µl that contained 2 µl cDNA, 12.5 µl

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of 1× SYBR Green I Taq Mix (Takara, Dalian, China), 0.5 µl (0.2 µM) of each primer, and 9.5 µl ddH2O. The

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amplification reactions were performed in a Line Gene K thermal cycler (Bioer, China) under the following

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conditions: 1 min at 94 ºC, followed by 40 cycles at 94 ºC for 10 s, 60 ºC for 30 s, and 72 ºC for 30 s. After 40

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specificity. All of the amplification reactions were repeated three times under identical conditions and

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included a negative control. For the quantitative real-time PCR data, the relative expression of the genes of

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interest was calculated using the threshold cycle (CT) method. The CT for each sample was calculated using

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Line-Gene K software and the method of Larionov et al. [22]. The amount of expression of target mRNAs

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compared with reference values was calculated using the equation 2-△△CT [23], where ∆CT is determined by

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subtracting the corresponding β-actin CT value (reference gene) from the specific CT of the target (gene of

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interest), and ∆∆CT is obtained by subtracting the ∆CT of each experimental sample from the calibrator

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sample (14 DAP).

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Results

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3.1 Physiological and morphological changes during seed development and germination

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During the development of rape seeds, their morphology changes along with the maturation and

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dehydration of the seeds. During early development at 14 DAP, the seeds were small and light green with a

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1-1.5 mm diameter. The seed size increased with time until 28 DAP, and the color of the seed coat darkened

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concurrently. After 28 DAP, the seed coat began to turn brown (data not shown).

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The ability of developing rape seeds to germinate appeared at 14 DAP and gradually increased

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thereafter, reaching a level of approximately 80 % at 26 DAP (Fig. 1 a). Seeds at 14 were unable to survive

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desiccation. Although seed DT increased according to seed maturation, germination of the dehydrated seeds

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was still lower than the freshly harvested seeds (Fig. 1 a).

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During the imbibition of mature rape seeds, the MC increased rapidly during the early phase until 2 h,

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followed by a short period with a slower increase. Afterward, a further more rapid increase to 1.232 g·g-1 was 11

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observed after imbibition for 20 h (Fig. 1 b). After 10 h of imbibition (radicle emergence), the rape seeds

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became vulnerable to desiccation. The survival ability decreased rapidly to 40 % after 14 h of imbibition,

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indicating that the seeds were desiccation-sensitive (Fig. 1 c). The H2O2 content of the rape seeds was high in immature seeds, decreased gradually with seed

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development, and reached 2.9 µ mol·g-1 dry weight at 45 DAP, in parallel with passage of the seeds into a

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metabolically inactive stage and DT. During seed imbibitions, H2O2 content continued to increase during the 24

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h of imbibition, reaching a value 1.5-times higher than control seeds because germinating tissues exhibit active

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metabolism during this period (Fig. 1 d).

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Based on the results above, changes in transcription were followed at eight stages throughout seed development (14, 18, 24, 28, 36 and 45 DAP) and imbibitions (14 h and 20 h) of fresh seed.

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3.2 Identification and classification of desiccation tolerant-related transcripts by cDNA-AFLP

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Gene expression analyzed by cDNA-AFLP was performed using fresh seeds during development and

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germination. With the 128 primer combinations tested, a total of approximately 8,000 TDFs were generated

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and visualized on 6 % denaturing polyacrylamide gel, of which 394 clear TDFs were excised from the gels,

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re-amplified, and sub-cloned into T-vector for sequencing. The sequence obtained for each TDF was

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compared with the GenBank non-redundant public sequence database using the BLASTX program [24]. The

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results showed that 176 TDFs had encoding sequences or homologous sequences, 218 clones had no

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significant homologous sequences in NCBI database. In Brassica Database (http://brassicadb.org/brad/) we

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identified these 218 TDFs and find they were homologous to the different fragment in “Brassica rapa

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choromosome V1.5” but without functional annotations (data not shown). The temporal expression patterns

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of these 394 TDFs from the cDNA-AFLP experiment were classified as “sustained expression” (Class A),

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Transcript-derived fragments that belonged to the “sustained expression” category with no significant

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changes during seed development and germination may be involved in the growth and development of the

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seeds whose expression patterns were not subjected to or less influenced by dehydration. “Up-regulated”

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TDFs had little or no expression during the early stage of development and were up-regulated during

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postembryonic development and germination. Transcript-derived fragments that belonged to the “couple with

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seed DT” category were up-regulated during seed development and down-regulated during germination.

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Transcript-derived fragments that belonged to the “down-regulated” category had high expression levels

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during the initial stage but were down-regulated compared with seed development.

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The 176 TDFs with known or putative functions are shown in Supplementary Table 1. Many of them

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were never characterized either in seed development or seed-specific expression. The new expression data

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indicate that the numerous mechanisms involved in seed dehydration are still unknown. To validate the

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information contained in the database described herein and highlight the respective roles, we used Blast 2 Go

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(version 2.4.4) for gene annotation and assigned DNA sequences to functional categories (Fig. 2). The

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findings revealed that the acquisition and loss of DT during seed development and germination did not occur

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through the regulation of a single gene but rather through synergistic interactions between multiple processes.

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3.3 Expression patterns of genes detected by SqRT-PCR

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After identification by cDNA-AFLP, gene expression was further studied for 24 TDFs (Table 2) using

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SqRT-PCR with gene-specific primers (Table 3). These TDFs of interest were selected based on the

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expression intensity and differential patterns of expression in the cDNA-AFLP experiment (mainly Class C).

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According to the results, the expression patterns of most of the cases were confirmed, indicating that the 13

ACCEPTED MANUSCRIPT accuracy of the cDNA-AFLP reliably and efficiently identified the different TDFs during seed development

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and germination. The selected TDFs included nine TDFs that encode metabolism- and energy-related genes

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(82-12, 83-54, 85-71, 91-32, 85-31, 85-41, 85-51, 88-63, 90-41), five TDFs that encode gene expression

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regulation-related genes (79-32, 84-12, 84-21, 94-12, 81-74), four TDFs that encode cell fate-, rescue-, and

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defense-related genes (80-12, 83-61, 88-11, 89-63), four TDFs that encode storage-related genes (80-61,

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88-65, 91-51, 81-82), and two TDFs (79-11, 79-61) that encode protein synthesis fate- and binding-related

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genes, respectively.

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Among the nine TDFs that were considered to be related to metabolism and energy, 82-12, 83-54, 85-71,

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and 91-32 that encode Succinyl-CoA ligase, arginine-tRNA protein transferase 1, cytochrome P450, and

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glutathione reductase, respectively, were increased during the entire process of development and germination,

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whereas 85-31, 85-41, 85-51, 88-63, and 90-41 that encode β-glucosidase, superoxide dismutase,

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myrosinase-binding protein, zeaxanthin epoxidase, and peroxiredoxin antioxidant were up-regulated during

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the seed development and down-regulated during germination (Fig. 3). Four TDFs that encode gene

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expression regulation-related genes (79-32, 84-12, 84-21, 94-12) were gradually up-regulated. In contrast, the

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81-74 TDF that encodes S23 transcriptional repressor decreased from 28 DAP during seed development. The

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expression of genes that are related to cell fate, rescue, and defense, such as 80-12, 83-61, 88-11, and 89-63,

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was rapidly induced within 45 DAP and then declined during germination. Genes that are related to storage,

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such as 80-61 that encodes caleosin, 88-65 that encodes steroleosin, and 91-51 that encodes oleosin, were

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changed with the same pattern as seed DT, and 81-82 that encodes cru4 was down-regulated during seed

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germination such that there were enough small-molecule precursors used for metabolism. Another two TDFs

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that encode genes related to protein synthesis fate (79-11) and binding (79-61) were both up-regulated,

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indicating their participation in seed maturation (Fig. 3).

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3.4 Dehydration treatment to detect genes associated with desiccation tolerance To confirm that the genes we identified are associated with DT rather than development and

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germination, the fresh seeds gathered from mother plants on different days after pollination (DAP) were

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dehydrated to a MC of 7 % (as describe in Materials and Methods). Compared with fresh seeds, these seeds

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were named “dehydrated seeds”. We used SqRT-PCR to compare gene expression between fresh seeds and

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dehydrated seeds. Because the 14 DAP seeds were filled with liquid, RNA could not be extracted when the

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seeds were dried using silica gel. Therefore, we further compared the gene expression of 18 DAP, 24 DAP, 36

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DAP, and 45 DAP seeds with 14 and 20 h imbibition between fresh seeds and dehydrated seeds.

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As shown in Fig. 4, we selected 16 TDFs that had similar expression patterns coupled with DT in fresh

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seeds and then tested the expression patterns of these 16 TDFs in dehydrated seeds. The results showed that

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the expression of 16 TDFs was consistent in both fresh seeds and dehydrated seeds. The expression of 80-61,

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83-61, 85-31, 85-41, 88-11, 88-63, 88-65, 89-63, 90-41, and 91-51 was consistent with the acquisition of DT,

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suggesting that they are directly involved in DT.

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3.5 Validation of the expression of 12 transcript-derived fragments by real-time PCR

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To further confirm the results obtained by SqRT-PCR and quantify the expression levels of candidate

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genes, 12 TDFs were chosen to conduct real-time PCR (Fig. 5). As shown in Fig. 5, the expression of TDFs

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85-41, 88-65, and 90-41 (Fig. 5 d, g, h) was up-regulated during seed development but decreased upon

312

imbibition. Transcript-derived fragments 85-31, 88-63, and 88-11 (Fig. 5 c, f, e) were up-regulated from 18

313

DAP to 36 DAP and then down-regulated at 45 DAP, but they exhibited very low expression during

314

germination. Transcript-derived fragments 80-61 and 91-51 (Fig. 5 a, j) exhibited an increase in expression as

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the fresh weight decreased, peaking at 28 DAP followed by down-regulation. Transcript-derived fragment

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ACCEPTED MANUSCRIPT 83-61 (Fig. 5 b) was similar to 80-61 and 91-51, with the exception of 28 DAP when its expression increased

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to a five-times higher level compared with 24 DAP, indicating that the HSP22 gene may play another

318

important role during this developmental phase. The expression pattern of TDF 81-74 (Fig. 5 i) was different

319

from the other TDFs, in which its expression began at a high level at 14 DAP and decreased during seed

320

development and germination. Moreover, TDF 91-32 (Fig. 5 l) showed persistent expression throughout

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development and germination. These results indicate that almost all of the genes chosen for RT-PCR were

322

DT-related, and their expression levels were obviously affected by the development phase and seed

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imbibition.

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For each gene, a graph that shows its kinetically assessed expression by real-time PCR is placed above

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its SqRT-PCR pattern. The level of expression of each gene was normalized to β-actin (AF111812) [21] and is

326

expressed with a value relative to the gene expression level at 14 DAP. The real-time PCR data show the

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mean values obtained from three independent amplification reactions, and the error bars indicate the standard

328

deviation.

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4.

Discussion

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In the present study, rape seeds acquired DT during development but gradually lost this feature during

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germination (Fig. 1 a-c), which is consistent with previous studies [13, 25]. After sequencing and comparison

333

with the National Center for Biotechnology Information database, 176 TDFs presented significant similarity

334

with known genes that could be classified into the following categories: metabolism and energy, stress

335

resistance and defense, storage, signal transduction, and other functional categories. The diverse function of

336

DT-regulated genes found in our study revealed the complexity and variety of pathway involved in DT

337

response in rape seed, as shown for other results [12-14]. We identified 12 TDFs as potential candidates

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according to their expression profile, putative function, and previously reported literature.

339 340

4.1 Genes for dehydration-associated proteins We isolated three genes, TDFs 83-61, 88-11, and 89-63. Two of these (83-61 and 89-63) encode

342

BnHSP22 and BcHSP17.6, and one of these (88-11) is homologous to LEA genes of Arabidopsis thaliana. All

343

of these genes were up-regulated or retained the same level of expression during seed development and lower

344

expression during germination (Fig. 4 and Fig. 5), suggesting that they may encode proteins that are essential

345

for seed development but not germination. Their disappearance during imbibition likely resulted from

346

degradation during water uptake. Transgenic tobacco that over-expressed PF03760 LEA gene from the

347

resurrection plant B. hygrometrica exhibited an increase in peroxidase and SOD activity during drought stress,

348

suggesting that the accumulation of LEA proteins may have an indirect effect or synergistic effect with other

349

protective molecules by either changing osmotic adjustment or inducing signaling pathways [26]. Small HSPs,

350

which accumulate during the late stage of seed development, may help minimize the aggregation effects of

351

cytoplasm condensation by acting as molecular chaperones, thereby contributing to the stabilization of a

352

glassy state. Magdalena et al. [27] showed that HSP22 may play a major role in response to abiotic stress, but

353

the regulatory mechanism is not entirely known. The expression of TDFs 83-61 and 88-11, which encode two

354

types of HSPs, remained the same during seed development and were down-regulated during seed

355

germination, implying that their expression may also be involved in seed DT and play a general protective

356

role throughout the life cycle of the seed.

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Furthermore, we selected one TDF (81-74) that encodes S23 transcriptional repressor, which has rarely

358

been reported (Gene Bank EU 186361.1). The expression of S23 transcriptional repressor is continuously

359

down-regulated during seed development and germination (Fig. 5 i). Seed storage materials that accumulate 17

ACCEPTED MANUSCRIPT during vegetative development may be related to transcription repressors that govern seed maturation before

361

germination in Arabidopsis. Gao et al. [28] showed that ASIL1, a member of the plant-specific trihelix family

362

of DNA binding transcription factors, played a substantial role in the regulatory network that down-regulates

363

embryonic gene expression during vegetative growth. The results indicated that ABA may interact with

364

ASIL1 in the depression of seed-specific genes in plant tissues. Additionally, ABA has been verified as a vital

365

factor in the acquisition of DT or in response to water deficiency [29]. Altogether, these results suggest that

366

this decrease in transcription repressors during the two stages may relieve the inhibition of some seed-specific

367

proteins so that more proteins can protect seeds against dehydration during development and act as a

368

nutritional ingredient for seed germination.

369 370

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4.2 Genes for oligosaccharides and hormone metabolism

Previous studies showed that there would be a shift in carbohydrate accumulation from simple to more

372

complex sugars in preparation for acquiring DT. Our previous results indicated that galactinol synthase, a key

373

enzyme in raffinose family oligosaccharides (RFO) synthesis, is positively associated with DT in Brassica

374

napus L. seeds during development [18]. β-glucosidase (85-31), encodes an important component of

375

cellulose-decomposing enzymes, can hydrolyze the β-D-glucoside bond at the non-reducing end and release

376

the corresponding ligand of β-D-glucose. Kaur et al. [30] showed that β-glucosidase, which is responsible for

377

the regulation of the cellulolytic process, can produce glucose from cellobiose and reduce the inhibition of

378

cellobiose to make the endoglucanase and exoglucanase enzymes function efficiently. In the present study,

379

TDF 85-31 exhibited a significant increase in expression during seed development and a rapid decrease

380

during germination (Fig. 5 c), indicating that this gene likely regulates the release of glucose that is used for

381

RFO synthesis and further participate in the acquisition of seed DT.

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ACCEPTED MANUSCRIPT The expression of TDF 88-63 was similar to the gene that encodes Zeaxanthin epoxidase (ZEP), which

383

was found to play a crucial role in the biosynthesis of ABA. The expression of Arabidopsis AtZEP mRNA,

384

which encodes epoxidase, was reported to increase in response to osmotic stress or ABA treatment in both

385

roots and shoots [31]. ABA has been confirmed as a vital factor in the acquisition of seed DT [29]. Therefore,

386

ZEP appears to play an indirect role in the acquisition of seed DT through the regulation of ABA content.

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387 4.3 Genes for lipid metabolism

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We isolated three genes that are related to lipid metabolism—91-51 (encoding oleosin S3), 80-61

390

(encoding oleosin S3), and 88-65 (steroleosin SLO1)—whose expression patterns were all gradually

391

up-regulated during development but rapidly down-regulated when the seeds were imbibed. Their expression

392

patterns were consistent with the change in DT during seed development and germination, suggesting that

393

they are related to seed DT. Oleosin, the richest protein that combines with oil bodies in seeds, has been

394

suggested to serve as a structural protein to keep lipid storage organelles unbroken. Previous studies

395

demonstrated that the expression of oleosin is induced by the seed-specific transactivator ABI3 [32],

396

suggesting that oleosin may be involved in the acquisition of DT. Caleosin (CLO) was found on lipid bodies

397

in a similar manner as oleosin. The accumulation of caleosin begins at the early stage of seed development

398

and increases with seed maturation [33]. In Arabidopsis, seed-specific caleosin associated with oil bodies is

399

involved in the adjustment of peroxygenase activity via Ca2+ and the process of lipid degradation. AtCLO4,

400

encodes a specific membrane-bound caleosin, was significantly up-regulated when subjected to abiotic stress

401

and ABA treatment. These studies show that caleosin may participate in the Ca2+ and ABA signal transduction

402

pathways and play a role in acquisition of seed DT [34].

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Together with oleosin and caleosin, steroleosin is a distinct oil body-bound protein that is expressed 19

ACCEPTED MANUSCRIPT during seed development when lipid bodies are strongly active. Avelange et al. [35] reported that the

405

remodeling of or increase in phospholipid catabolism is an adaptive response that is common to osmotic

406

adjustment and DT. These three oil-body proteins may act as signaling molecules to induce the expression of

407

some specific genes, such as those that encode phytohormones or regulatory factors associated with DT or

408

structural proteins that aid the efficiency of lipids that participate in the process of DT (Fig. 5 a, g, j). The

409

relationship between these three oil-body proteins and seed DT requires further research.

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4.4 Genes involved in reactive oxygen species exclusion

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When rape seeds were exposed to dehydration, reactive oxygen species (ROS) are readily decreased,

413

accompanied by seed development, but increase with imbibition (Fig. 1 d). Together with the expression

414

patterns of the three genes we identified, TDFs 90-41, 85-41, and 91-32 that encode peroxiredoxin (PRX),

415

superoxide dismutase (SOD), and glutathione reductase (GR2), the SqRT-PCR results showed that the PRX

416

and SOD genes have a low level of expression during the initial stage of seed development, are up-regulated

417

during seed maturation, and are gradually down-regulated after germination, but the level of GR2 increased

418

during the entire process (Fig. 5).

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Angelovici et al. [36] showed that seeds have active metabolism and transcription mechanisms during

420

the stages of storage accumulation and desiccation. This indicates that more antioxidant is required to

421

scavenge excessive desiccation-induced ROS during seed development. In the early 1990s, Leprince et al. [37]

422

showed that SOD activity increased during the first stage of germination and that the loss of DT is a

423

consequence of the increased formation of one or more forms of activated oxygen molecules coupled with a

424

substantial decrease in the protection conferred by SOD. These results were similar to the variation in SOD

425

content during seed development and germination measured by SqRT-PCR.

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ACCEPTED MANUSCRIPT Peroxiredoxin, a ubiquitous antioxidant protein, was classified into 1-Cys Prxs and 2-Cys. Buitink et al.

427

[38] reported that a gene that encodes 1-cys peroxiredoxin (1-Cys-Prx) was accumulated during the

428

acquisition or re-induction of DT in M. truncatula seeds, suggesting that PRXs play a specific role in the

429

formation of DT. Ratajczak et al. [39] showed that the level of PRXIIF proteins remains steady in embryonic

430

axes of the Norway maple, which is desiccation-tolerant, compared with a decrease in the same protein in

431

embryo axes of the sycamore, which is desiccation-sensitive, during seed desiccation. Consistent with the

432

previous study and the present results (Fig. 1 d), the functional expression of PRX is diverse, acting as an

433

antioxidant enzyme or regulation factor in seed DT.

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A recent study demonstrated that glutathione reductase (GR) activity increased during development and

435

germination in dried maize embryos [25]. These studies are consistent with the present results, in which the

436

activity of GR increased during seed development and germination (Fig. 5 l). Pukacka et al. [7] suggested that

437

the glutathione-ascorbate cycle, a metabolic pathway, plays a crucial role in the process of H2O2

438

detoxification. This corroborates our results, in which H2O2 content continuously decreased during seed

439

development (Fig. 1 d). Thus, GR appears to be essential to protect cells against ROS damage induced by

440

water deficiency.

442

5.

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Conclusion

443

In summary, the cDNA-AFLP technique was successfully used to identify genes that are differentially

444

expressed in fresh and dehydrated seeds. We identified 176 desiccation-induced genes in rape seeds during

445

development and germination. Our detailed study of the expression of 12 genes indicated cross-talk in rape

446

seed DT. Several novel desiccation-responsive genes that are expressed in rape seeds indicated particular

447

mechanisms of desiccation adaptability. Revealing the function of desiccation-inducible genes is important to 21

ACCEPTED MANUSCRIPT 448

further understand the molecular mechanisms of DT and how functions, targets, and ultimately expression

449

and translation are regulated to improve abiotic stress tolerance in crops through genetic engineering.

450 Authors’ contributions

452

Gang Ma, Sirui Lang and Xiaoxia Liu performed the research; Sirui Lang, and Xiaoxia Liu analyzed the data

453

and prepared the manuscript; Xiaofeng Wang proposed the research project and guided the research. All

454

authors read and approved the final manuscript.

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455 Acknowledgments

457

This work was supported by the Special Fund Project for the Scientific Research of the Forest Public Welfare

458

Industry (201104024), the National Natural Science Foundation of China (31271807). We thank the college

459

of biological science and technology, Beijing forestry University; and the seed used in this study was

460

kindly provided by Qinghai Academy of Agricultural Sciences. This manuscript was edited by native

461

English-speaking experts of BioMed Proofreading.

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germination at different water potential PEG-6000 in relation to oxidative process, Plant Physiology and [26] X. Liu, Z. Wang, L. Wang, R. Wu, J. Phillips, X. Deng, LEA 4 group genes from the resurrection plant Boea hygrometrica confer dehydration tolerance in transgenic tobacco, Plant Science, 176 (2009) 90-98. [27] L. Magdalena, E. Coipan, A. Vladimirescu, L. Savu, M. Costache, L. Gavrila, Downregulation of hsp22 gene expression in Drosophila melanogaster from sites located near chemical plants, Genetics and Molecular Research, 11 (2012) 739-745.

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maturation genes by a trihelix transcriptional repressor in Arabidopsis seedlings, The Plant Cell, 21 (2009) 54-71.

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ACCEPTED MANUSCRIPT [36] R. Angelovici, G. Galili, A.R. Fernie, A. Fait, Seed desiccation: a bridge between maturation and germination, Trends in plant science, 15 (2010) 211-218. [37] O. Leprince, R. Deltour, P.C. Thorpe, N.M. Atherton, G.A. Hendry, The role of free radicals and radical processing systems in loss of desiccation tolerance in germinating maize (Zea mays L.), New Phytologist, 116 (1990) 573-580. [38] J. Buitink, J.J. Leger, I. Guisle, B.L. Vu, S. Wuillème, G. Lamirault, A.L. Bars, N.L. Meur, A. Becker, H. Kü ü ster, Transcriptome profiling uncovers metabolic and regulatory processes occurring during the transition sensitive to desiccation

tolerant stages in Medicago truncatula seeds, The Plant

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Journal, 47 (2006) 735-750.

[39] E. Ratajczak, E. Ströher, M.-L. Oelze, E.M. Kalemba, K.-J. Dietz, The involvement of the mitochondrial peroxiredoxin PRXIIF in defining physiological differences between orthodox and recalcitrant seeds of two

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Acer species, Functional Plant Biology, (2013).

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Figure Legends

565 Fig. 1. Changes in germination rate in different DAP seeds (a), moisture content (b), germination rate

567

with different imbibition times (c), and H2O2 content (d) in Brassica napus L. seeds during development

568

and imbibition. (a) Germination of fresh seeds and dehydrated seeds at different times during development

569

(boxes: fresh seeds; triangles: dehydrated seeds). (b) Moisture content of fresh seeds during development and

570

germination. The developmental stage was measured based on the days after pollination (DAP), and

571

germination was measured based on hours of imbibition (boxes: seed development; triangles: seed imbibition).

572

(c) Percentage of seed germination and loss of DT during imbibition at 20 ºC. Germination and DT percentages

573

were determined as described above (boxes: fresh seeds; triangles: dehydrated seeds). (d) H2O2 content during

574

seed development and germination (boxes: seed development; triangles: seed imbibition). All of the data were

575

determined in triplicate (average ± SE)

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Fig. 2. Functional categories of genes related to DT during development and germination in Brassica

578

napus L. seeds

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Fig. 3. SqRT-PCR analysis of 24 TDFs of Brassica napus L. seeds during development and germination.

581

The expression of the first 10 TDFs (79-11, 79-32, 79-61, 82-12, 83-54, 84-12, 84-21, 85-71,91-32,94-12)

582

gradually increased during seed development (from 14 DAP to 45 DAP) and continually increased during the

583

germination stage when imbibed for 14 and 20 h. The expression patterns of the next 12 TDFs were

584

consistent with the trend of acquisition of DT during seed development and loss of DT during germination.

585

The expression patterns of the last two genes (81-74, 81-82) were generally down-regulated. The expression 26

ACCEPTED MANUSCRIPT of all three classes (A, B and C) of genes was normalized to β-actin

587

Fig. 4. SqRT-PCR detection of genes associated with DT. The figure shows comparisons between fresh

588

seeds and dehydrated seeds at different development and imbibition stages. The expression patterns of four

589

TDFs (82-12, 84-12, 85-71, 91-32) were induced at a specific stage during development and consistently

590

up-regulated. Ten TDFs (80-61, 83-61, 85-31, 85-41, 88-11, 88-63, 88-65, 89-63, 90-41, 91-51) were

591

associated with DT (up-regulated during development, and down-regulated during imbibition). Two TDFs

592

(81-74 and 81-82) exhibited low expression levels during the late development and imbibition stages. All of

593

the expression patterns of the dehydrated seeds were consistent with fresh seeds

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594

Fig. 5. Comparison of expression patterns of 12 TDFs identified by SqRT-PCR and real-time PCR. Ten

596

TDFs were associated with DT, the expression patterns of which were generally increased during seed

597

development and decreased during germination. The expression of three of these TDFs reached a peak at 28

598

DAP (a, b, j), and the other seven (c, d, e, f, g, h, k) reached a peak during the maturation period (36 DAP or 45

599

DAP). The expression patterns of two TDFs (81-74, 91-32) did not parallel DT, but the function of these two

600

genes may be correlated with the acquisition of DT. One TDF (81-74, i) decreased from 14 DAP in the

601

development stage to 20 h in the germination stage, and the other TDF (91-32, l) gradually increased from 14

602

DAP

603

Supplementary Table 1 Sequence homology analysis of 176 TDFs

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ACCEPTED MANUSCRIPT Table 1 Classification of TDFs expression patterns during development and germination in Brassica napus. L seed Class

Expression Pattern

Class A

Sustained expression

Class C

18

24

28

36

45

14h

20h

Number of TDFs (%) 19(4.8)

Up-regulated

57(14.5)

Couple with seed DT

265(67.2)

Down-regulated

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Class D

14

Gernmiantion (h)

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Class B

Development (DAP)

53(13.5)

ACCEPTED MANUSCRIPT Table 2 List of TDFs used in SqRT-PCR

1 TDFs

Length

class

No． ．

(bp)

Homologous protein (species)

GenBank Accession No.

E-value

412

Succinyl-CoA ligase (Arabidopsis thaliana)

NM_127601.3

2E-70

83-54

1343

arginine-tRNA protein transferase 1 (Arabidopsis thaliana)

NM_120652.4

1E-27

85-71

1206

cytochrome P450 (Brassica rapa subsp. pekinensis)

FJ376051.1

3E-57

91-32

611

glutathione reductase (GR2) (Brassica juncea)

AF349449.1

2E-81

85-31

479

beta-glucosidase (Brassica napus)

X82577.1

1E-142

85-41

241

superoxide dismutase (Brassica oleracea)

DQ267204.1

2E-12

85-51

301

myrosinase-binding protein (Brassica napus)

U59443.1

2E-171

88-63

916

zeaxanthin epoxidase (Brassica napus)

GU361616.1

7E-110

90-41

374

peroxiredoxin antioxidant (Brassica napus)

AF139817.1

3E-36

79-32

822

WRKY21-1 transcription factor (Brassica napus)

EU912394.1

3E-19

84-12

1201

calmodulin-binding transcription activator 5 (Arabidopsis thaliana subsp. lyrata)

XM_002870132.1

5E-77

84-21

177

plastid transcriptionally active 14 protein (Arabidopsis thaliana)

NM_118132.4

8E-96

94-12

478

transfactor-like protein (Arabidopsis thaliana)

AF370550.1

5E-52

81-74

349

S23 transcriptional repressor (Brassica rapa)

EU186361.1

2E-15

80-12

374

tryptophan-rich sensory protein-like protein (Arabidopsis thaliana)

NM_130344.2

2.00E-129

TE D

M AN U

SC

RI PT

82-12

CY-1HSP22 (Brassica napus)

JN106325.1

4.00E-169

329

low molecular weight heat-shock protein (Brassica rapa)

AF022217.1

2E-120

89-63

632

late embryogenesis abundant domain-containing protein (Arabidopsis lyrata subsp. lyrata)

XM_002869867.1

4E-57

80-61

769

caleosin CLO1 (Brassica napus)

EU678287.1

3E-129

88-65

822

steroleosin SLO1 (Brassica napus)

EU678275.1

3E-40

91-51

225

oleosin S3-3 (Brassica napus)

EU678267.1

5E-168

81-82

513

cru4 (Brassica napus)

X57850.1

3E-88

79-11

1062

ubiquitin 10.1 (Brassica napus)

FJ529184.1

2E-90

79-61

852

RNA-binding protein 47B(Arabidopsis thaliana)

NM_112800.3

6E-108

EP

306

88-11

fate

83-61

AC C

regulation defense

expression rescue and synthesis binding

protein

storage

cell fate,

gene

metabolism and energy

Function

ACCEPTED MANUSCRIPT Table 3 Primers of the selected differentially expressed genes verified by SqRT-PCR

2

Primer sequence 5’-3’

TDFs No.

Forward

Reverse

Size/bp

ACAATGTCAAAGCCAAGA

TTAGAAAGCACAATAGGGA

79-32

TTTACTATCTGGGTCTTTCC;

TTAGAGCAGTGGCATCG

822

79-61

TCTCAATCAGAAGGGTATGG

GTTGGAGTCGTGGTGGT

852

80-12

GAGGGCGATGGCGAAAC

CCTTGTAGCATCCGAATAA

374

80-61

TGAGTACGGCGACTGAGAT

TGTTGGGAGGAGGATGAGA

769

81-74

ACTTCACTCCCTAAACCG

GCAACCCTCTACTCCTCAA

349

81-82

CTTTACTTGCCCACTTTCTT

GTTGCCACGGTTGACTT

513

82-12

AATGGCAGAACCAACAG

GGTCCAGCAGATTTACG

412

83-54

TGCGGCTACTGTAAATC

GGAACCCACTGGAAACG

1343

83-61

GCCATACTTCCCTCTGTT

CGATTCTCCTCCACCTC

306

84-12

ATTTGTGCGAAGGTGTTA

AGTCCTGCTGGCGAGTT

1201

84-21

GGCTTGCTGAAGATGCT

TGGGACGCCAATGTAGG

174

85-31

TGCTCGTAACTCTTGTCG

AAAGCCACCGTACTCATC

479

85-41

CGAAGGTGCTGCTGTGC

TATGTCCGAAGCGTATT

241

85-51

GACAAAGGAAGGACCCG

GGCTGCTGGAGCGATAT

301

85-71

CCCAACACTTCCTCTTTC

CTACCATTGCGACTCCA

1206

88-11

TAACGCCTTCAGGGATG

CACCGTGACCGACAACA

329

88-63

CTCTGGCACTTGGTATGTC

CGGATGCGGTACTCTAA

916

88-65

GCCCGAAGGAAGAACCG

ACTCTACTCCGACTTGATTT

822

89-63

CGTCTGCTTCGCCTCCT

CTTCCACGCCTTTATCG

632

90-41

CATACAGTCGCACAAAGAT

TGAAACCCTGAGGAAACA

374

91-32

GGACCGCCACTACGACT

CTTCACTGCCAAGACCAT

611

91-51

CTCTGACTGTTGCGACTC

TATCCTGAACTTTGCCTC

225

94-12

TTCGTTGATGCCGTTGC

CTGGTCCACAGTCCTTGC

478

β-Actin

CGCCGCTTAACCCTAAGGCTAACAG

TTCTCTTTAATGTCACGGACGATTT

322

SC

M AN U

TE D

EP AC C

3

RI PT

79-11

1062

ACCEPTED MANUSCRIPT Table 4 Primers used in cDNA-AFLP analysis

4

Primer sequence 5’-3’

TDFs No.

Forward

Reverse

Size/bp

TACGGCGACTGAGATAATGGAGA

TGACAACAACTAGGATGCAAGAGAA

150

81-74

CCGCTAGTTTCGACACTTCACCAGG

GGGTTACTCCCGCAGAGCCAACAT

282

81-82

GCTCTTCGTGGCTCCATCCATAACA

TCTGTGCGTTGTCGTTGCTCTTGA

250

83-61

GCTTGCTATCCGATCTCTGGCTAGA

TCAACTCTGTGCCACTGATCTCCTT

274

85-31

GCTCGTAACTCTTGTCGGCTCTCC

GGCTTGGTCCTCTGCAACCTTCAT

167

85-41

TCTGGATGGGTGTGGTTTGGACTA

TCTCGGTTGTGCTTCACGAATATGT

239

85-71

TGGTAGCAGTCGCAGCCTTCTTC

TCAGGAGAGGACGAGCGGTGAA

273

88-11

GCGAAAGTGGACTGGAGGGAGACA

TCTTAGGCACCGTGACCGACAACA

280

88-63

GGCTCAACTCCCTTCTGCTACTCAA

GTTACTGTCTCCCGCTTCTCCTCTT

227

88-65

GGTTCTCATCACTGGTGCTTCCTC

CAGCGTGAACTGTGACAACATTAGG

167

90-41

TCCATGACTACTTCGCCGATTCTT

GCTTCCAGGAGTAAATGCCTCAATG

206

91-32

CCGACCGCAATCAACCTACCATCTT

CGAGAGGAGGAGGAGGAGGAGTAGT

134

β-Actin

TCTTCCTCACGCTATCCTCCG

AC C

EP

SC

M AN U

TE D

5

RI PT

80-61

AGCCGTCTCCAGCTCTTGC

181

ACCEPTED MANUSCRIPT 6 7

Table 5 Primers of the selected differentially expressed genes verified by Real time-PCR

Name Adaptor

Sequences BstY I adaptor

sequences

5’- CTCGTAGACTGCGTAGT -3’ 5’- GATCACTACGCAGTCTAC -3’

Mse I adaptor

5’- GACGATGAGTCCTGAG -3’

Pre-amplified

Pre-amplified primer of BstY I

primers

5’ - GACTGCGTAGTGATCT -3’

5’ - GACTGCGTAGTGATCC -3’

Selective-amplified

Pre-amplified primer of Mse I

5’ - GATGAGTCCTGAGTAA - 3’

Selective-amplified primer of BstY I

5’– GACTGCGTAGTGATCTN -3’ 5’- GACTGCGTAGTGATCCN -3’

Selective-amplified primer of Mse I

5’- GATGAGTCCTGAGTAANN -3’

AC C

EP

TE D

M AN U

Note： N means one of A, T, C, G

SC

primers

8

RI PT

5’- TACTCAGGACTCAT -3’

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights We selected 8 stages during development and germination of rape (Brassica napus L.) seeds to identify its desiccation tolerance. We identified 176 TDFs participated in the maturation and germination of rape seeds by cDNA-AFLP. Sixteen TDFs may related to desiccation tolerance of rape seeds.

AC C

EP

TE D

M AN U

SC

RI PT

Detailed study of 12 genes expression indicates a cross-talk response of rape seeds to DT.

ACCEPTED MANUSCRIPT Supplementary Table 1 Sequence homology analysis of 176 TDFs TDFs Similar sequence

Description of similar sequence

NO.

BLAST [E] value

protein synthesis and fate ubiquitin 10.1(Brassica napus )

2.00E-90

79.12 FJ529185.1

ubiquitin 10.2(Brassica napus )

5.00E-45

79.13 FJ529185.1

ubiquitin 10.2(Brassica napus )

79.15 FJ529185.1

ubiquitin 10.2 (Brassica napus )

82.72 D10840.1

ITS1, 5.8S rRNA, ITS2, 25S rRNA and IGS(Brassica napus )

2.00E-96

86.12 D10840.1

ITS1, 5.8S rRNA, ITS2, 25S rRNA and IGS(Brassica napus )

1.00E-34

87.71 D10840.1

ITS1, 5.8S rRNA, ITS2, 25S rRNA and IGS(Brassica napus ) 4.00E-107

88.13 NM_001203345.1

40S ribosomal protein S6-2 (EMB3010)(Arabidopsis

88.61 NM_106533.2

curculin-like (mannose-binding) lectin family protein

2.00E-43

M AN U

(Arabidopsis thaliana)

6.00E-65

SC

thaliana)

RI PT

79.11 FJ529184.1

3.00E-83

1.00E-29

88.64 EF110942.1

ribosomal protein S27 (Brassica rapa )

90.11 AY293401.1

ribosomal protein S6 (RPS6) (Brassica napus)

91.21 U55032.1

aspartic protease(Brassica napus)

7.00E-74

91.22 U55032.1

aspartic protease(Brassica napus)

3.00E-72

91.23 U55032.1

aspartic protease(Brassica napus)

2.00E-54

cDNA-AFLP fragment 002BT11M21-384.5(Sesbania

2.00E-06

exprssed protein

TE D

79.23 EF415667.1

4.00E-62 3.00E-103

rostrata)

79.31 AY091708.1 79.41 DQ414510.1 79.52 AC232445.1

5.00E-28

R1-5 (Secale cereale)

1.00E-03

KBrB006F18(Brassica rapa subsp. pekinensis)

3.00E-58

KBrB080J15(Brassica rapa subsp. pekinensis)

1.00E-30

EP

79.63 AC189474.1

AT3g10020/T22K18_16(Arabidopsis thaliana)

KBrB035A14(Brassica rapa subsp. pekinensis)

7.00E-53

80.31 AC189547.2

KBrH004P05(Brassica rapa subsp. pekinensis)

9.00E-150

80.32 AC189547.2

KBrH004P05(Brassica rapa subsp. pekinensis)

4.00E-133

AC C

79.65 AC189315.2

80.34 DQ244946.1

11869 mRNA sequence(Zea mays)

5.00E-96

80.35 DQ068110.1

isolate mutant Cr3529 clone Bncr2 unknown mRNA(Brassica

2.00E-80

napus)

81.52 AC189305.1

KBrB031O20(Brassica rapa subsp. pekinensis)

1.00E-137

81.53 AC189568.2

KBrH009I04(Brassica rapa subsp. pekinensis)

1.00E-18

81.83 EF110965.1

840 unknown mRNA(Brassica rapa)

5.00E-91

82.13 BT028899.1

unknown protein (At1g12845) mRNA(Arabidopsis thaliana)

3.00E-43

82.31 AC189373.2

KBrB048F07(Brassica rapa subsp. pekinensis)

4.00E-108

82.51 AC189573.1

KBrH010N11(Brassica rapa subsp. pekinensis)

2.00E-62

82.64 AC232526.1

KBrB083J11(Brassica rapa subsp. pekinensis)

4.00E-56

82.73 AC189627.2

KBrS001M03(Brassica rapa subsp. pekinensis)

5.00E-40

83.13 AC189519.2

KBrB091G04(Brassica rapa subsp. pekinensis)

2.00E-63

ACCEPTED MANUSCRIPT mitochondrial DNA(Brassica napus)

83.51 AC189500.1

KBrB086N06(Brassica rapa subsp. pekinensis)

3.00E-109

83.52 AC189500.1

KBrB086N06(Brassica rapa subsp. pekinensis)

6.00E-106

83.62 XM_002889506.1

hypothetical protein(Arabidopsis lyrata subsp. lyrata)

2.00E-75

83.64 AC232562.1

KBrH054E04(Brassica rapa subsp. pekinensis)

2.00E-37

83.81 AC232562.1

KBrH054E04(Brassica rapa subsp. pekinensis)

2.00E-37

84.31 XM_002892621.1

hypothetical protein(Arabidopsis lyrata subsp. lyrata)

6.00E-67

84.61 AC189388.2

KBrB053D06(Brassica rapa subsp. pekinensis)

8.00E-40

84.82 AC241133.1

KBrH046K16(Brassica rapa subsp. pekinensis)

2.00E-55

85.11 AP006444.1

mitochondrial DNA(Brassica napus)

3.00E-46

85.62 AC189592.2

KBrH013K13(Brassica rapa subsp. pekinensis)

2.00E-36

85.81 AC172883.2

KBrH125N23(Brassica rapa subsp. pekinensis)

3.00E-58

85.82 EU581950.1

BAC B67C16(Brassica oleracea)

1.00E-50

86.14 AC189537.2

KBrH003E13(Brassica rapa subsp. pekinensis)

3.00E-48

86.34 AC189633.2

KBrS004A14(Brassica rapa subsp. pekinensis)

4.00E-87

87.13 AC189498.2

KBrB086L12(Brassica rapa subsp. pekinensis)

4.00E-15

87.22 AF342783.1

GF14 kappa mRNA, partial cds(Brassica napus)

1.00E-93

87.43 AC232526.1

KBrB083J11(Brassica rapa subsp. pekinensis)

5.00E-86

87.51 XM_002892921.1

hypothetical protein(Arabidopsis lyrata subsp. lyrata)

3.00E-19

88.22 AC189300.2

KBrB028P01(Brassica rapa subsp. pekinensis)

3.00E-51

88.31 AC189195.2

KBrB004B12(Brassica rapa subsp. pekinensis)

1.00E-93

88.32 AC189573.1

KBrH010N11(Brassica rapa subsp. pekinensis)

3.00E-63

88.44 AC241128.1

KBrH040N18(Brassica rapa subsp. pekinensis)

1.00E-55

SC

M AN U

KBrB018D16(Brassica rapa subsp. pekinensis)

8.00E-63

uncharacterized protein(Arabidopsis thaliana)

1.00E-26

hypothetical protein(Arabidopsis lyrata subsp. lyrata)

9.00E-39

KBrB092P05(Brassica rapa subsp. pekinensis)

7.00E-55

KBrB002B23(Brassica rapa subsp. pekinensis)

9.00E-104

90.22 AF342783.1

GF14 kappa mRNA, partial cds(Brassica napus)

5.00E-92

90.32 AC232437.1

KBrB002B23(Brassica rapa subsp. pekinensis)

2.00E-23

90.34 AC232437.1

KBrB002B23(Brassica rapa subsp. pekinensis)

2.00E-23

90.42 EF471212.2

LhCa2 protein mRNA(Brassica juncea)

8.00E-47

90.52 AC189305.1

KBrB031O20(Brassica rapa subsp. pekinensis)

2.00E-59

90.61 NM_001203390.1

uncharacterized protein(Arabidopsis thaliana)

3.00E-68

90.71 AC189649.2

KBrS010I09(Brassica rapa subsp. pekinensis)

5.00E-127

90.74 AC232512.1

KBrB065B14(Brassica rapa subsp. pekinensis)

3.00E-26

91.62 AC172875.2

KBrH014P02(Brassica rapa subsp. pekinensis)

8.00E-58

91.74 AC189325.2

KBrB036M17(Brassica rapa subsp. pekinensis)

4.00E-66

91.83 AC189564.2

KBrH009B23(Brassica rapa subsp. pekinensis)

5.00E-34

92.31 AC189627.2

KBrS001M03(Brassica rapa subsp. pekinensis)

2.00E-126

92.71 AC189320.2

KBrB036H12(Brassica rapa subsp. pekinensis)

8.00E-120

93.11 AC189569.2

KBrH009I12(Brassica rapa subsp. pekinensis)

2.00E-69

93.32 AC189640.2

KBrS006L21(Brassica rapa subsp. pekinensis)

3.00E-32

94.13 AC241033.1

KBrB045E05(Brassica rapa subsp. pekinensis)

3.00E-83

89.12 NM_129137.3 89.32 XM_002879107.1 89.42 AC189529.2

AC C

EP

90.21 AC232437.1

TE D

88.82 AC232454.1

3.00E-17

RI PT

83.32 AP006444.1

ACCEPTED MANUSCRIPT 94.14 AC241033.1

KBrB045E05(Brassica rapa subsp. pekinensis)

3.00E-83

94.23 AC189506.1

KBrB088E10(Brassica rapa subsp. pekinensis)

4.00E-92

94.26 AC189394.2

KBrB055G10(Brassica rapa subsp. pekinensis)

2.00E-39

94.27 AC232449.1

KBrB008G18(Brassica rapa subsp. pekinensis)

3.00E-41

94.31 XM_002893527.1

hypothetical protein(Arabidopsis lyrata subsp. lyrata)

2.00E-92

94.32 AC189524.2

KBrB092B15(Brassica rapa subsp. pekinensis)

1.00E-76

94.44 AC155339.1

chromosome Cytogenetic chromosome 1 clone

6.00E-24

RI PT

KBrH015H17(Brassica rapa subsp. pekinensis) 94.51 AC189569.2

KBrH009I12(Brassica rapa subsp. pekinensis)

8.00E-43

94.74 AC189630.2

KBrS003G14(Brassica rapa subsp. pekinensis)

5.00E-56

94.75 DQ455378.1

cDNA-AFLP fragment BT22M22_317 sequence(Medicago

2.00E-04

truncatula)

KBrB078A03(Brassica rapa subsp. pekinensis)

94.83 AB618066.1

BrALS3 gene for acetolactate synthase(Brassica rapa subsp.

SC

94.82 AC189468.2

pekinensis)

KBrB008O16(Brassica rapa subsp. pekinensis)

gene expression regulation 79.32 EU912394.1

M AN U

94.86 AC240985.1

WRKY21-1 transcription factor (WRKY21-1) (Brassica napus)

3.00E-64

6.00E-111 3.00E-31 3.00E-19

79.72 AY496435.1

eukaryotic translation initiation factor-5A(Brassica napus)

4.00E-18

81.74 EU186361.1

transcriptional repressor mRNA(Brassica rapa)

2.00E-15

84.12 XM_002870132.1

calmodulin-binding transcription activator 5(Arabidopsis

5.00E-77

lyrata)

plastid transcriptionally active 14 protein (Arabidopsis

TE D

84.21 NM_118132.4

8.00E-96

thaliana )

86.25 XM_002881706.1

myb family transcription factor(Arabidopsis lyrata subsp.

1.00E-10

lyrata)

87.73 NM_111346.3

RNA polymerase II transcription mediator

8.00E-49

EP

(SWP)(Arabidopsis thaliana)

transfactor-like protein(Arabidopsis thaliana )

94.24 NM_001202956.1

translational initiation factor 4A-1(Arabidopsis thaliana)

6.00E-50

94.84 XM_002280565.2

RNA-binding post-transcriptional regulator csx1-like

2.00E-05

AC C

94.12 AF370550.1

5E-52

(LOC100250363)(Vitis vinifera )

binding

79.61 NM_112800.3 79.62 NM_179965.3

RNA-binding protein 47B (RBP47B) (Arabidopsis thaliana) RNA-binding KH domain-containing protein

6.00E-108 2.00E-96

(AT2G38610)(Arabidopsis thaliana)

89.73 U59443.1

myrosinase-binding protein(Brassica napus)

1.00E-33

94.21 NM_113208.3

SRPBCC ligand-binding domain-containing

1.00E-119

protein(Arabidopsis thaliana ) transporter 79.71 NM_119849

PQ-loop repeat family protein / transmembrane family

3.00E-131

protein(Arabidopsis thaliana) 80.52 NM_001203242.1

protein transport protein SEC31 (AT3G63460)(Arabidopsis

1.00E-51

ACCEPTED MANUSCRIPT thaliana) 88.62 XM_002893678.1

proton-dependent oligopeptide transport family

4.00E-93

protein(Arabidopsis lyrata subsp. lyrata) cell wall,cytoskeleton and vesicle trafficking 79.81 XM_002876533.1

pectinesterase family protein(Arabidopsis lyrata subsp.

5.00E-54

lyrata) exocyst subunit EXO70 A1 (EXO70) (Brassica napus )

2.00E-21

79.64 U21745.1

PEP-carboxykinase(Brassica napus)

6.00E-75

79.74 Y10853.1

metallothionein-like protein (LSC210)(Brassica napus )

3.00E-58

80.73 NM_100850.2

2,3-bisphosphoglycerate-independent phosphoglycerate

5.00E-41

metabolism and energy

RI PT

80.62 GQ503256.1

SC

mutase 1 (AT1G09780) (Arabidopsis thaliana ) 81.12 NM_126589.2 82.12 NM_127601.3

aconitate hydratase 2 (ACO3)(Arabidopsis thaliana )

2.00E-44

Succinyl-CoA ligase [GDP-forming] subunit beta

2.00E-70

82.14 XM_002865639.1

M AN U

(AT2G20420)(Arabidopsis thaliana)

KAT5/PKT1/PKT2 peroxisomal 3-keto-acyl-CoA

4.00E-51

thiolase(Arabidopsis lyrata subsp. lyrata) 82.22 XM_002883920.1

NADP-malic enzyme 1 (ATNADP-ME1) (Arabidopsis lyrata subsp. lyrata)

5.00E-118

83.11 NM_129840.4

citrate synthase 3 (CSY3) (Arabidopsis thaliana)

3.00E-79

83.31 AK229614.1

pyruvate kinase(Arabidopsis thaliana)

2.00E-38

83.54 NM_120652.4

arginine-tRNA protein transferase 1 (ATE1) (Arabidopsis

1.00E-27

84.11 XM_002867774.1

TE D

thaliana)

methionine sulfoxide reductase domain-containing

8.00E-51

protein(Arabidopsis lyrata subsp. lyrata)

85.12 NM_111131.3

acyl-[acyl-carrier-protein] desaturase

1.00E-108

(AT3G02630)(Arabidopsis thaliana)

asparagine synthetase(Brassica oleracea)

5.00E-81

peptide methionine sulfoxide reductase B5 (MSRB5)

5.00E-46

EP

85.13 X84448.1

85.22 NM_116721.3

(Arabidopsis thaliana) beta-glucosidase(Brassica napus)

85.41 DQ267204.1

BoCAR2 superoxide dismutase(Brassica oleracea)

85.51 U59443.1

myrosinase-binding protein(Brassica napus)

85.61 X82577.1

beta-glucosidase(Brassica napus)

9.00E-69

85.71 FJ376051.1

cytochrome P450 83b1 (CYP83B1)(Brassica rapa)

3.00E-57

86.13 NM_148330.2

cytochrome P450, family 702, subfamily A, polypeptide 6

4.00E-52

AC C

85.31 X82577.1

1.00E-142 2.00E-12 2.00E-171

(CYP702A6)(Arabidopsis thaliana) 86.21 XM_002883042.1

dihydroorotate dehydrogenase family protein(Arabidopsis

2.00E-54

lyrata subsp. lyrata) 86.23 AJ251524.1

peroxin Pex14(Arabidopsis thaliana)

4.00E-52

87.31 Z22620.1

cold induced protein (BnC24B)(Brassica napus)

7.00E-29

87.82 NM_127683.2

Peptidyl-prolyl cis-trans isomerase CYP19-2

4.00E-27

(AT2G21130)(Arabidopsis thaliana)

ACCEPTED MANUSCRIPT 87.83 NM_130225.3

calcium-binding EF-hand-containing protein

2.00E-33

(AT2G46600)(Arabidopsis thaliana) 87.84 NM_127683.2

Peptidyl-prolyl cis-trans isomerase CYP19-2

4.00E-27

(AT2G21130)(Arabidopsis thaliana) 88.42 XM_002866385.1

exonuclease family protein(Arabidopsis lyrata subsp. lyrata)

1.00E-57

88.51 XM_002888376.1

calcium-binding EF hand family protein(Arabidopsis lyrata

2.00E-117

subsp. lyrata) zeaxanthin epoxidase (zep)(Brassica napus)

88.83 NM_118524.2

UDP-D-glucose/UDP-D-galactose 4-epimerase 2 (UGE2) (Arabidopsis thaliana)

89.64 XM_002879330.1

CTP:phosphocholine cytidylyltransferase(Arabidopsis lyrata subsp. lyrata)

7.00E-110

RI PT

88.63 GU361616.1

8.00E-26

7.00E-38

S18 superoxide dismutase(Brassica rapa)

90.41 AF139817.1

peroxiredoxin antioxidant (per1) (Brassica napus)

3.00E-36

91.25 GQ861354.1

strain ZY036 chloroplast(Brassica napus)

6.00E-64

91.32 AF349449.1

glutathione reductase (GR2) mRNA(Brassica juncea)

2.00E-81

91.71 NM_001203227.1

cytochrome p450 78a9 (CYP78A9) (Arabidopsis thaliana)

2.00E-49

tryptophan-rich sensory protein-like protein(Arabidopsis

1.00E-20

cellfate,rescue and defense 80.12 NM_130344.2

thaliana) 80.21 NM_201927.2

M AN U

SC

90.13 EU186343.1

glycine-rich protein / late embryogenesis abundant

4.00E-55

2.00E-31

protein(Arabidopsis thaliana)

CY-1 HSP22 (HSP22) (Brassica napus)

4.00E-169

83.63 JN106325.1

CY-1 HSP22 (HSP22)(Brassica napus)

4.00E-169

84.32 NM_118525.2

TE D

83.61 JN106325.1

late embryogenesis abundant hydroxyproline-rich

9.00E-44

glycoprotein (AT4G23930) (Arabidopsis thaliana)

84.81 NM_180521.2

ARM repeat protein interacting with ABF2 (ARIA)

5.00E-78

(Arabidopsis thaliana)

dehydrin Rab18 (RAB18) (Arabidopsis thaliana)

EP

86.31 NM_126038.2

low molecular weight heat-shock protein (Brassica rapa)

88.12 AF022217.1

low molecular weight heat-shock protein (BcHSP17.6)

AC C

88.11 AF022217.1

89.63 XM_002869867.1 89.65 XM_002869867.1

2.00E-16 2E-120 1.00E-118

(Brassica rapa)

late embryogenesis abundant domain-containing

4.00E-57

protein(Arabidopsis lyrata subsp. lyrata) late embryogenesis abundant domain-containing

8.00E-59

protein(Arabidopsis lyrata subsp. lyrata)

91.72 JN106325.1

CY-1 HSP22 (HSP22)(Brassica napus)

2.00E-25

92.72 FJ598132.1

heat shock protein(Ageratina adenophora)

5.00E-12

storage 80.61 EU678287.1

caleosin CLO1-10 (CLO1) (Brassica napus)

3.00E-129

81.82 X57850.1

cru4 mRNA for cruciferin(Brassica napus)

3.00E-88

83.53 NM_112743.2

oleosin / glycine-rich protein (AT3G18570)(Arabidopsis

8.00E-33

thaliana) 88.65 EU678275.1

steroleosin SLO1-2 (SLO1)(Brassica napus)

3.00E-40

ACCEPTED MANUSCRIPT 89.54 AF319771.1

CUC2-like protein and prohibitin 1-like protein and cruciferin

6.00E-29

subunit (Brassica napus) 90.81 AF319771.1

CUC2-like protein and prohibitin 1-like protein and cruciferin

4.00E-45

subunit (Brassica napus) 91.26 AF319771.1

CUC2-like protein and prohibitin 1-like protein and cruciferin

7.00E-37

subunit (Brassica napus) oleosin S3-3 (S3) (Brassica napus)

5.00E-168

91.61 EU678276.1

steroleosin SLO1-3 (SLO1) (Brassica napus)

91.84 X57850.1

cru4 (Brassica napus)

93.41 AF319771.1

CUC2-like protein and prohibitin 1-like protein and cruciferin

5.00E-96

subunit (Brassica napus) cruciferin(Brassica napus)

94.16 AB115552.1

cruciferin(Brassica napus)

94.52 AB115552.1

cruciferin(Brassica napus)

signal transduction

1.00E-92

6.00E-75 2.00E-69

SC

93.52 AB115552.1

3.00E-98

RI PT

91.51 EU678267.1

2.00E-43

SHATTERPROOF1(Brassica napus)

85.21 DQ222996.1

temperature-induced lipocalin (TIL)(Brassica napus)

4.00E-92

92.23 EF079955.1

elongation factor 1-beta(Brassica rapa)

4.00E-67

94.28 FJ529180.1

elongation factor 1 alpha (EF1alpha.1)(Brassica napus)

5.00E-28

M AN U

82.32 AF226865.1

AC C

EP

TE D

cell growth

5.00E-56