Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds

Accepted Manuscript Title: Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds Authors: Yin Jing, Sirui Lang, Dongmei Wang, Hua Xue, Xiao-Feng Wang PII: DOI: Reference:

S0176-1617(18)30702-8 https://doi.org/10.1016/j.jplph.2018.10.011 JPLPH 52866

To appear in: Received date: Revised date: Accepted date:

8-12-2017 10-10-2018 10-10-2018

Please cite this article as: Jing Y, Lang S, Wang D, Xue H, Xiao-Feng W, Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds, Journal of Plant Physiology (2018), https://doi.org/10.1016/j.jplph.2018.10.011 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.

Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds

Running title: GS and RS involved in DT in Arabidopsis seeds

Authors: Yin Jing1*; Sirui Lang1*; Dongmei Wang2; Hua Xue1† and Xiao-Feng

National Engineering Laboratory for Tree Breeding, College of Biological Sciences

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Wang1†

and Technology, Beijing Forestry University, Tsinghua East Road 35, Haidian District, Beijing 100083, China

Key Laboratory of Soil and Water Conservation and Desertification Combating，

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Ministry of Education，School of Soil ＆ Water Conservation，Beijing Forestry

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University, Tsinghua East Road 35, Haidian District, Beijing 100083, China

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Corresponding author: [email protected] and [email protected]

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These authors contributed equally to this work.

Functional characterization of GALACTINOL SYNTHASE and RAFFINOSE

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Abstract

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SYNTHASE in desiccation tolerance acquisition in developing Arabidopsis seeds

Raffinose family oligosaccharides (RFOs) accumulate during seed development, and have been thought to be associated with the acquisition of desiccation tolerance (DT)

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by seeds. Here, comprehensive approaches were adopted to evaluate the changes of DT

in

developing

Arabidopsis

seeds

of

wild

type,

overexpression

(OX-AtGS1/GS2/RS5), and mutant lines by manipulating the expression levels of the GALACTINOL SYNTHASE (GS) and RAFFINOSE SYNTHASE (RS) genes. Our results indicate that seeds of the double mutant (gs1, gs2) and rs5 delayed the timing of DT acquisition as compared to wild type. Subsequent detection confirmed that

seeds from OX-AtGS1/GS2 plants with high levels of galactinol, raffinose, and stachyose, and OX-AtRS5 plants possess more raffinose and stachyose but less galactinol compared to wild type. These lines all showed greater germination percentage and shorter time to 50% germination after desiccation treatment at 11 and 15 days after flower (DAF). Further analysis revealed that the role of RFOs is time limited and mainly affects the middle stage (9-16 DAF) of seed development by

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enhancing seed viability and the ratio of GSH to GSSH in cells, but there is no

significant difference in DT of mature seeds. In addition, RFOs could reduce damage

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to seeds caused by oxidative stress. We conclude that GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE play important roles in DT acquisition during Arabidopsis

seed development, and that galactinol and RFOs are crucial protective compounds in

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the response of seeds to desiccation stress.

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Keywords: Arabidopsis thaliana, desiccation tolerance, galactinol synthase, raffinose

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synthase, galactinol and raffinose-family oligosaccharides (RFOs).

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Introduction

The seeds of most plants (so-called orthodox seeds) must undergo a maturation process of dehydration during late development. During this time, the moisture

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content (MC) of seeds decreases significantly (typically ≤ 5% of fresh weight), and therefore desiccation tolerance (DT) is critical for survival. DT refers to the ability of

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seeds to withstand dehydration, to reduce the harmful effects of dehydration, to slow their metabolic activity, and to survive after rehydration (Kermode, 1997; Leprince and Buitink, 2010; Leprince et al., 1996, 2016). In orthodox seeds, DT is acquired

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during development and gradually lost after germination (Hong and Ellis, 1990; Angelovici et al., 2010; Dekkers et al., 2015). Considering that DT is obtained during seed development, this capacity is very likely to depend on substances accumulated during this stage (Vertucci and Farrant, 1995). Raffinose-family oligosaccharides (RFOs), including raffinose, stachyose, and verbascose, are important soluble sugars that accumulate during seed development,

and they have been thought to be associated with DT (Koster and Leopold, 1988; Leprince et al., 1993; Obendorf et al., 1997, 1998; Van den Ende, 2013; González-Morales et al., 2016; Leprince et al., 2016). Caffrey et al. (1988) found that oligosaccharides can assist in the protection of membranes by restricting or preventing the crystallization of sucrose. These oligosaccharides and sucrose are consistently present in the axes during the DT stage and disappear in the intolerance

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stages (Koster and Leopold, 1988). Accumulation of RFOs has been shown to coincide with the onset of desiccation tolerance in soybean, pea axes, and wheat

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embryos during seed maturation (Koster and Leopold, 1988; Blackman et al., 1992). In maize seeds, DT is not achieved until a threshold level of RFOs is reached within

the embryo (Brenac et al., 1997a, b). RFOs abundantly accumulate during

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dehydration in many resurrection plants and may have prominent roles in protecting

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the tissues via water replacement and vitrification (Farrant et al., 2007, 2009; Oliver et

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al., 2011). RFOs are thought to maintain cellular integrity by interacting with membrane phospholipids to serve as protectors of biomembranes during dehydration

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(Leopold et al., 1994; Obendorf, 1997; Hincha et al., 2003, 2006). In Medicago

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truncatula, RFOs levels increase and are correlated with longevity during seed maturation (Verdier et al., 2013). De Souza Vidigal et al. (2016) reported that the galactinol content of mature dry seed can be used as a biomarker of seed longevity in

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Brassicaceae and tomato.

Despite the positive correlation between RFOs and DT, some studies have shown

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that RFOs are not required for the development of DT or seed longevity. For example, it was found that high-level accumulation of RFOs does not appear to be essential for DT in cauliflower seeds (Hoekstra et al., 1994, Ooms et al., 1994). Wolkers et al.

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(1998) suggested that RFO accumulation is simply a sign of late embryonic development instead of an element essential for the development of DT. Dierking and Bilyeu (2008) reported that RFOs were not required for proper germination in some plants, such as soybean. Given this conflicting evidence, the exact relationship between RFOs and seed DT needs to be fully elucidated. GALACTINOL SYNTHASE (GolS, GS; EC2.4.1.123), participates in the first

committed step of RFO biosynthetic pathways to synthesize galactinol from UDP-Gal and myo-inositol (Fig. 1A). As the sole galactosyl donor, the levels of galactinol directly influence the total amount of RFOs in the plant (Saravitz et al., 1987; Downie et al., 2003; Zhao et al., 2004). The transcript levels of GS are induced differentially in response to abiotic stresses, including drought, high salinity, cold, and oxidative stress (Taji et al., 2002; Panikulangara et al., 2004; dos Santos et al., 2011; Zhuo et al.,

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2013; Lahuta et al., 2014). In addition, the expression of GS is associated with DT in developing Brassica napus seeds (Li et al., 2011). In Arabidopsis thaliana, seven GS

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genes have been identified. AtGS1 is induced by drought, heat, oxidative, and salinity stress; AtGS2 is induced by drought, oxidative, and salinity stress; while AtGS3 is only induced by cold and oxidative stress (Taji et al., 2002; Nishizawa et al., 2006,

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2008).

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Raffinose synthase (RS; EC2.4.1.82) catalyses the synthesis of raffinose from

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sucrose and galactinol, the second step in the biosynthesis of RFOs (Fig. 1A). There are a few reports regarding RS in cucumber, soybean, peas, Arabidopsis, and so on

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(Peterbauer et al., 2002; Dierking and Bilyeu, 2008; Sui et al., 2012; Egert et al.,

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2013). However, we know relatively less about the physiological and biochemical features of RS enzymes compared to GS, especially the potential changes in expression or function in response to abiotic stress. To date, six putative RS genes

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have been identified in Arabidopsis (Nishizawa et al., 2008). AtRS2 seems not to be a raffinose synthase but an α-galactosidase that catalyses the breakdown of raffinose

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into galactose and sucrose (Peters et al., 2010). AtRS5 has been confirmed to be a raffinose synthase and is induced during low-temperature acclimation (Zuther et al., 2004; Egert et al., 2013). A concurrent study on AtRS4 showed that it functions as a

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raffinose synthase and a high-affinity stachyose synthase, as well as a raffinose and galactinol-specific galactosyl hydrolase (Gangl et al., 2015), suggesting that this enzyme is multifunctional and plays a key regulatory role in RFO mechanisms. The actual number of AtRS genes in Arabidopsis is currently thought to be either one or two (Egert et al., 2013). In this study, we used AtGS/AtRS overexpressed or mutated to demonstrate the

relationship between these genes and DT in developing Arabidopsis seeds. By comparing wild-type, mutant, and transgenic seeds that accumulated different amounts of RFO, we were able to show that RFOs, especially raffinose and stachyose, affect DT during seed development and that they may also reduce damage to seeds caused by oxidative stress or ageing. Our findings showed that galactinol synthase and raffinose synthase are essential for protecting developing Arabidopsis seeds from

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dehydration and that galactinol and RFOs are crucial protective compounds in the

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response of seeds to desiccation stress.

Materials and methods Plant material and growth conditions The Arabidopsis thaliana accession Columbia (Col-0) was used in this study. The mutant and transgenic lines were generated in this background. Three mutants with a T-DNA insertion in AtGS1, AtGS2, or AtRS5 were obtained from the Arabidopsis Biological

Resource

Center

(ABRC).

The

three

mutant

lines

atgs1-1

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(SALK_121059C), atgs2-1 (SALK_101144C), and atrs5-1 (SALK_049583C) were

confirmed to be homozygous in three paired reactions (Fig. S1, Table S2). The mutant

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gs1, gs2 was constructed by crossing SALK_121059C with SALK_101144C. The T1

lines were self-pollinated, and the double homozygotes in the T2 lines were identified using PCR methods. T-DNA insertion sites were obtained through the SIGnAL iSect

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(http://signal.salk.edu/isect.2.html).

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Developing Arabidopsis seeds were noted by labelling flowers on their day of anthesis, and the siliques were collected each day from 7 to 24 DAF. Some of the

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fresh seeds were frozen rapidly in liquid nitrogen and preserved at −80 °C. All seeds

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were sterilized and placed on petri dishes containing Murashige and Skoog basal salts

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with 3% sucrose and 0.5% (w/v) agar. The dishes were chilled at 4 °C in the dark for 48 h and transferred to a plant growth incubator at 22°C under controlled conditions (16 h light/8 h darkness cycle) for 14 days. The seedlings were then transplanted to

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soil and grown under the same conditions. Bioinformatics analyses

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The phylogenetic trees of AtGS and AtRS were built with MEGA 5.0 using the

maximum-likelihood method. The reliability of internal branches was assessed using the bootstrap test (500 replicates), and the nodes were marked. The amino acid

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sequences of homologous proteins were aligned using ClustalX under default parameters. Determination of seed dry weight and water content Pre-weighed fresh seeds (100 seeds per replicate) were dried for 17 h at 105 °C in three replicates. Samples were weighed again after cooling. Water content and dry weight of seeds were measured. Final water content was expressed as a percentage of

water loss per gram of original seed weight. Testing of seed germination, DT, and seed vigour One hundred seeds were sown in each Petri dish containing sterile water-saturated filter paper in three independent biological replicates. After two days of chilling at 4 °C, germination assays were performed in a continuous light growth chamber at

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22 °C. Seed germination was scored daily, and the final number of germinated seeds was determined after six days. Time to 50% germination (T50) was scored every six hours and calculated according to the formulae given by Coolbear et al. (1984).

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For DT testing, freshly harvested seeds were dehydrated in closed desiccators

above a saturated solution of LiCl (relative humidity 11.3 ± 0.3%) at 25°C. After four days of drying, seeds were rehydrated for one day in an atmosphere of 30% RH at

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25 °C. After further rehydration for one day in an atmosphere of 78% RH at 25 °C,

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germination was tested as described before. This protocol will be referred to as

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“desiccation treatment”.

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Seed artificial ageing was achieved by controlled deterioration treatment (CDT), and it was performed as previously described by Tesnier et al. (2002). The survival of

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seeds (as percentage of viable seedlings) was scored after 10 days of germination. RNA isolation and quantitative real-time PCR analysis Plant tissues were collected and immediately frozen in liquid nitrogen. Total RNA

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samples were extracted from Arabidopsis tissues using an RN38 EASY Spin Plus

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Plant RNA kit (Aidlab Biotech, Beijing, China). For reverse transcription PCR, 2 μg of total RNA after digestion with RQ1 RNase-Free DNase (Promega) was used with M-MLV Reverse Transcriptase (Promega), following the manufacturer’s instructions. For the qRT-PCR experiments, GoTaq qPCR Master Mix was used according to the

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manufacturer`s instructions (Promega) using Bio-Rad Opticon 2. The experiments were carried out in three replicates. The specific primers used in this study are listed in Table S2. The relative expression level of each gene was calculated using the 2 −ΔΔ CT

method. The efficiency of qRT-PCR primers was determined using LinRegPCR

software (Ramakers et al., 2003) with values of at least 90%.

Cloning procedures, plasmid construction, and plant transformation The pCAMBIA1300 vector was modified by inserting a double CaMV35S promoter with a tobacco mosaic virus (TMV) enhancer (synthesized by Sangon Company, Shanghai, China) into the multi-cloning site between EcoR I and Sac I. Then, the NOS terminator was cloned from the pBI121 vector and inserted into the multi-cloning site between Sph I and Hind III. The new vector was named pDE. The

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open reading frames of AtGS1, AtGS2, and AtRS5 genes were amplified and integrated into the restriction sites of the modified vector (Kpn I and Xba I). To

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visualize subcellular localization, the pDE-AtGS1/AtGS2/AtRS5-EGFP vectors were constructed by inserting the EGFP sequence after the GS1/GS2/RS5 genes (Fig. S2). All essential amplicons were sequenced.

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For generation of transgenic plants, the appropriate binary plasmids were

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transferred individually into Agrobacterium tumefaciens strain GV3101. Stable

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transgenic plants were obtained using the floral-dip method (Zhang et al., 2006). Southern blot hybridization

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The insertion copy number of the overexpressed AtGS1, AtGS2, and AtRS5 genes in homozygous transgenic plants was analysed by Southern blotting. The probe was

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amplified by PCR using the primer pair “GS1/GS2/RS5-blot-F/R” (Table S1). The forward primer is located on the pDE vector, and the reverse primer is designed to

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bind with the coding sequence (CDS) of each gene. The length of probes for detection of AtGS1, AtGS2, or AtRS5 transgenic genes was 851 bp, 783 bp, and 756 bp,

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respectively. Digoxigenin (DIG) labeling, hybridization, and detection were conducted using a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche).

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Determination of galactinol synthase and raffinose synthase activity Arabidopsis seeds in different developmental stages were ground into fine powder

in liquid nitrogen and then homogenized in pre-chilled extraction buffer: 50 mM HEPES-KOH, pH 7.5, 50 mM ascorbic acid, 4 mM MnCl2, 2 mM DTT, and 1 mM phenylmethylsulphonyl fluoride (PMSF). The homogenate was centrifuged for 20 min at 4 °C at 12,000 × g, and the supernatant was desalted by gel filtration through

Sephadex G-25 columns pre-equilibrated with extraction buffer. The desalted sample was used to measure the GS and RS activity as previously described by Peterbauer et al. (2001) with HPLC-PAD. HPLC analysis of soluble carbohydrate seed contents Seeds were frozen in liquid nitrogen. Water-soluble sugars were extracted and desalted as described previously (Li et al., 2011). Soluble carbohydrate contents were

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identified and quantified using HPLC-PAD. Three different batches of seeds were assayed for replication.

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Tetrazolium assay

To study seed viability, Arabidopsis seeds (after dehydration and cold treatment) were incubated in 1% (w/v) 2,3,5-triphenyltetrazolium chloride (TTC, Sigma)

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solution at 30 °C for two days in the absence of light, followed by washing twice with

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water. The seeds were imaged and analysed using a microscope (Olympus). Three

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biological replicates of seeds were analysed per line. Reduced TTC content was measured using 10 mg of developing seeds as described previously (Duncan and

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Widholm, 2004).

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Antioxidant assay

The contents of oxidized glutathione (GSSG) and reduced glutathione (GSH) were determined as previously described (Mittova et al., 2003).

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Oxidative stress was applied by incubation of 100 seeds (in three replicates) in 1 μM of methylviologen (MV) for three hours. Approximately 20 mg of seeds was used

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for estimation of the contents of H2O2 and malondialdehyde (MDA) according to the methods described by Salvi et al. (2016).

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Confocal laser scanning microscopy EGFP fluorescence was visualized using confocal laser scanning microscopy (Leica

TCS-SP8) at an excitation wavelength of 488 nm and recorded using an emission detection window from 522 to 535 nm. Statistical analyses The data were analysed using a one-variable general linear model procedure (ANOVA) with the SPSS software package (SPSS Inc., http://www.spss.com.cn).

Analysis of significance was performed using Duncan’s multiple range tests at P ≤ 0.05. Values are presented as the mean ± S.D. Correlation analysis was performed

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using two-tailed tests with Pearson’s correlation for different lines.

Results Isolation and characterization of AtGS and AtRS from Arabidopsis seeds In the present study, seven GS genes (AtGS1 to AtGS7), the AtRS5 gene, and three putative RS genes (AtRS1, AtRS4, and AtRS6) were isolated. Quantitative PCR was performed to examine the relative expression levels of all these genes in Arabidopsis seeds. A higher level of AtGS1, 2 and AtRS4, 5 and 6 transcripts, along with lower

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expression of AtGS3, 5 and AtRS1, was detected in dry mature seeds, whereas AtGS4, 6, and 7 mRNA were barely detectable under the same conditions (Fig. 1B). AtGS1, 2

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(Fig. 1C) and AtRS4, 5, and 6 (Fig. 1D) mRNA, while present in greater abundance in

dry seeds, were in lower abundance or absent in leaves, flowers, and roots of wild-type Arabidopsis using real-time PCR. These data suggest a putative role for

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these genes in seed physiology, which was further investigated.

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Multiple sequence alignment of the AtGS and AtRS proteins was undertaken (Fig.

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2). Seven Arabidopsis GS isoforms were positioned in three groups by phylogenetic analysis. The first group contained AtGS1, 2, and 3, which are closely related to each

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other when compared to the other four AtGS proteins. The second group consisted of

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AtGS4 and AtGS7, while the third group was composed of AtGS5 and AtGS6 (Fig. 2A). The amino acid sequences of AtGS1, 2, and 3 were aligned to compare consensus sequences with known GS proteins (Fig. 2B). A conserved serine (S) at site

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271 in AtGS1 was found in all of the known GS from other species, but it was absent in AtGS2 and 3. Furthermore, all known GS proteins have a characteristic

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hydrophobic pentapeptide region located in the carboxy-terminus (APSAA). Phylogenetic analysis of AtRS isoforms revealed that AtRS4 and 5 are only slightly homologous to AtRS1 and 6 (Fig. 2C). The alignment of AtRS4, 5, and 6 amino acid

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sequences with other known RS proteins from various species revealed the presence of a conserved aspartic acid residue in the KxD and RxxxD motif, which acts as a catalytic nucleophile or plays an acid/base role, respectively (Fig. 2D). Construction of the mutants and AtGS/AtRS-overexpressing transgenic plants To investigate the function of AtGS and AtRS, transgenic Arabidopsis plants over-expressing AtGS1, 2 or AtRS5 were generated, respectively. Three T-DNA

insertion mutants lines: atgs1-1 (SALK_121059C), atgs2-1 (SALK_101144C), and atrs5-1 (SALK_049583C) (Fig. S1) were obtained from ABRC and confirmed to be homozygous using three paired reactions and the SALK T-DNA PrimerDesign tool (http://signal.salk.edu/tdnaprimers.2.html). Among three vectors with different promoters (Fig. S2 A-C), the double-CaMV35S promoter with a TMV enhancer was superior to single CaMV35S or napin promoters, so it was used in further research

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and named pDE vector.

As no obvious difference in DT was found in the atgs1-1 or atgs2-1 single mutant

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line (Fig. S3), a double mutant (gs1, gs2) was constructed. Six overexpressing lines of AtGS1, AtGS2, and AtRS5 were obtained (Fig. 3A), and two single-copy insertion lines of each gene (verified by Southern blot) were used in this work (Fig. 3B).

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Phenotype of the overexpression and mutant lines in acquisition of DT during

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seed development

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To determine the involvement of the AtGS and AtRS in DT during seed development, the dry mass and water content of different lines were measured. The

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results showed the dry weight of seeds increased to a maximum at 18 DAF and

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remained there until 20 DAF. The water content decreased during seed development from approximately 56% at 10 DAF to 8% at 19 DAF and showed no obvious difference between different lines (Fig. 3C, D). Further results show there were no

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significant differences in the germination percentage of fresh seeds (without dehydration) for all lines during development (Fig. 3E, F).

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The seeds at different developmental stages were dehydrated above a lithium

chloride-saturated solution for four days (13% RH, 25 °C) and rehydrated, and then, the DT of the seeds was analysed by measuring their germination percentage. We

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found that wild-type seeds acquired DT initially by 11 DAF (germination 10.18%), while the gs1, gs2 line postponed this time to 13 DAF (germination 18.51%). The wild-type seeds reached 100% DT at 17 DAF. However, the mutant line acquired DT completely by 19 DAF. The OX-GS1 and OX-GS2 lines showed a greater DT than the wild-type and the gs1, gs2, particularly at 11 DAF (Fig. 3G). These results indicate that the acquisition of DT in developing Arabidopsis seeds may be affected

by AtGS1 and AtGS2. The DT of the atrs5-1 line was weaker than that in the wild-type but not as severe as in the gs1, gs2 line. OX-RS5 lines exhibited greater DT than the wild-type during seed development, with statistical significance (Fig. 3H). These results suggest that AtRS5 may also affect the DT during seed development. However, single mutants of rs5 had a weaker effect on DT acquisition compared with the double mutant of gs1,

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gs2.

Then, seeds collected at 11 DAF (the onset of DT acquisition), 15 DAF (the

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midpoint of DT acquisition), and 19 DAF (DT acquisition complete) during seed

development were chosen for further experiments. The time to 50% germination (T50) differed among lines at 11 DAF and 15 DAF after desiccation and rehydration

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treatment (Fig. 3I). Seeds of OX-GS1 and OX-GS2 had a shorter T50 (55.8 h and 57.3

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h on average) than wild-type (67.2 h and 69.6 h) at 11 DAF and 15 DAF. However,

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seeds of the gs1, gs2 and rs5 extended this time to 84.6 h and 77.2 h at 15 DAF, respectively. It is noteworthy that a significantly shorter T50 was observed for seeds

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from OX-RS5 lines (average 42.4 h) compared to wild-type and OX-RS lines at 15

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DAF. When all seeds matured and dehydrated naturally at 19 DAF, T50 was shortened, and there were no obvious differences among the lines. The results showed that the seed germination was not affected by the overexpression or deletion of GS and RS

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genes under normal conditions but that the vigour of seeds was affected under dehydration stress, indicating that GS1, GS2, and RS5 may be involved in DT.

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Expression of AtGS and AtRS genes and enzyme activity after desiccation of developing Arabidopsis seeds As GS1, GS2, and RS5 may be involved in DT, we examined the expression level

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of these genes in different lines after desiccation treatment. The results showed that the expression of both GS1 and GS2 were not detected in the gs1, gs2 mutant (Fig. 4A, B), and the expression of RS5 was almost abolished in rs5 lines (Fig. 4C). The expression level of AtGS1 was approximately 9.9- and 12.4-fold greater in OX-GS1-19 and OX-GS1-22 lines, respectively, at 11 DAF, and it sharply increased by 82.9 and 101.8-fold at 15 DAF compared with wild-type seeds, followed by a

slight decrease at 19 DAF (Fig. 4A). A similar expression pattern of GS2 (Fig. 4B) and RS5 (Fig. 4C) was observed in OX-GS2 and OX-RS5 lines. These results indicate that the expressions of the AtGS and AtRS genes were significantly increased in the overexpression lines under desiccation stress and that they were more strongly induced at 15 and 19 DAF when DT was partially/completely acquired. After desiccation treatment, the total GS enzyme activity in the seeds of the gs1,

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gs2 at 11, 15, and 19 DAF (23.08 ± 5.11, 131.62 ± 20.15, and 86.32 ± 5.33 nmol·min-1 mg-1 protein) was much lower than that of the wild-type (94.27 ± 17.10,

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291.08 ± 28.45, and 188.6 ± 12.31 nmol·min-1 mg-1 protein, respectively). OX-GS1 and OX-GS2 showed higher enzymatic activity among the three development stages compared to wild-type, with a peak at 15 DAF (Fig. 4D). The total RS isoenzymes

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activity also increased dramatically in OX-RS5 and was reduced in rs5 seeds

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compared with that of wild-type (Fig. 4E).

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Accumulation of soluble sugars during desiccation

As shown in Fig. 5A, galactinol accumulated in both OX-GS1 and OX-GS2 lines

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compared to wild-type was reduced in the gs1, gs2 line at 11, 15, and 19 DAF after

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desiccation treatment. In addition, the galactinol content was higher in rs5-1 and slightly lower in OX-RS5 lines, whether at 11, 15, and 19 DAF, consistent with its role as a substrate of raffinose synthase (Fig. 5A). More raffinose accumulated in GS

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and RS overexpression lines compared with the gs1, gs2 or rs5 mutant lines with the maximum difference at 15 DAF (Fig. 5B). Stachyose was not detected in seeds at 11

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DAF. However, in seeds collected at 15 and 19 DAF, the concentration of stachyose was increased in the OX-GS1, GS2, and RS5 lines, whereas it was decreased in the mutant lines compared to wild-type (Fig. 5C). Thus, overexpression of GS and RS

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seems to promote the downstream accumulation of stachyose. The correlation analysis illustrated that DT is related to the content of galactinol and raffinose at 11 and 15 DAF, and no obvious correlation was found between DT and RFO content at 19 DAF (Table 1). As shown in Fig. 5D, the sucrose concentration exhibited no obvious difference between these lines, indicating that the sucrose level was not influenced by GS and RS.

Overexpression of AtGS1 and AtRS5 enhanced seed viability after desiccation treatment Triphenyl tetrazolium chloride (TTC) staining is a standard test of seed viability (Debeaujon et al., 2000, Kai et al., 2010). Compared with wild-type, overexpression of AtGS1, AtGS2, and AtRS5 resulted in more intense TTC staining at 11 and 15 DAF. Similar to seed vigour reflected from T50 data at 15 DAF (Fig. 3I), the atrs5-1 seeds

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were stained weaker compared to wild type, and OX-RS5 seeds showed more intense

TTC staining than wild-type and OX-GS lines at 15 DAF (Fig. 6A, B).

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Spectrophotometric analysis of TTC staining gave quantitative evidence that generally the content of TTC was decreased in mutant lines and increased in overexpression lines (Fig. 6B).

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The seed vigour was further tested by ageing the seeds of different lines under KCl

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saturated solution (85% RH) at 40 °C for up to eight days. The results indicated that

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the seed vigour of the wild-type declined progressively from the second day onward, reduced to 50% at four to five days and to less than 10% at eight days of ageing.

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Compared with the wild-type seeds, the gs1, gs2 and rs5 mutant seeds were more

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sensitive to deterioration treatment and completely lost vitality at eight days after ageing, while the overexpression lines of GS1, GS2, and RS5 exhibited stronger tolerance to deterioration (Fig. 6C, D).

seeds

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Accumulation of galactinol and RFOs can strengthen antioxidant capacity in

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Glutathione status (GSH/GSSG ratio) is an indicator of intracellular redox state

(Colville and Kranner, 2010). In gs1, gs2 and atrs5-1 lines, the ratio of GSH to GSSG significantly decreased among all of the three development stages after desiccation

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treatment. When GS or RS was overexpressed, the GSH/GSSG ratio significantly increased in the OX-GS seeds at both 15 and 19 DAF, and it was higher in OX-RS seeds at 15 DAF, with statistical significance (Fig. 7A). These results revealed that glutathione status was affected by expression of GS and RS genes during acquisition of DT. To further examine changes in redox state, the ROS level and its influence on lipid

peroxidation and MDA contents were analysed in 19 DAF seeds before and after desiccation. The content of H2O2 in the wild-type seeds was approximately 30 nmol·g-1 FW, with no obvious difference detected among the lines before dehydration. After desiccation, the H2O2 levels were raised in all lines. It increased by 48.4% in gs1, gs2 (82.5 nmol·g-1 FW) and by 29.9% in rs5 (72.2 nmol·g-1 FW) mutants compared with wild-type (55.6 nmol·g-1 FW) (Fig. 7B) and showed a mild but

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significant decrease in OX-GS1/GS2 and RS5 transgenic seeds. Similarly, the MDA content was lower in OX-GS1/GS2 and RS5 transgenic seeds and higher in the gs1,

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gs2 and rs5 mutant lines after dehydration compared to the wild-type control (Fig. 7C).

The germination ability of different lines following oxidative damage caused by

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methylviologen (MV) was further measured. The germination percentage was

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decreased in gs1, gs2 and rs5 mutant lines compared with that in wild-type seeds. In

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contrast, overexpression of GS1, GS2, and RS5 all exhibited a protective effect in resisting oxidative stress (Fig. 7D, E). These results indicate that galactinol and RFOs

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may have direct or indirect agonistic effects in response to oxidative stress.

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AtGS1 and AtRS5 exhibit different subcellular localizations In previous studies, the subcellular localization of GS varied in different species and stress conditions. According to some recent reports, GS localized to the cell

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membrane and nucleus (Keller et al., 1992; Schneider & Keller; 2009; Zhou et al., 2012). Thus, GS and RS were found to be extra-chloroplastic (probably cytosolic) in

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location (Schneider and Keller, 2009). In this study, transgenic lines stably expressing EGFP-fusion proteins (Fig. S2 D, E, F) were obtained, and the seeds containing AtGS1-EGFP or AtRS5-EGFP showed obvious EGFP fluorescence at 11 and 15 DAF

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(Fig. 8). As expected, EGFP was found in both the nucleus and the cytoplasm in wild-type seeds (Fig. 8E). The AtGS1-EGFP was found in the nucleus, cell membrane, and cytoplasm (Fig. 8J), while the AtRS5-EGFP showed most of the EGFP fluorescence in the cytoplasm (Fig. 8O). The localization of the nucleus was confirmed using DAPI staining (Fig. 8D, I, N).

Discussion Oono et al. (2003) used cDNA microarray analysis to characterize the AtGS and AtRS genes as rehydration-repressed and dehydration-inducible genes, which are related to the synthesis of osmotic regulators in Arabidopsis thaliana; similar results have been reported in recent studies on resurrection plants (Rodriguez et al., 2010; Gechev et al., 2013). Downie et al. (2003) and Zhao et al. (2004) reported that LeGS1

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and ZmGS mRNA accumulated in developing seeds concomitant with maximum dry weight deposition and DT acquisition. Our preliminary results showed that the

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expression of BnGS1 may be associated with DT in developing oilseeds and with the

formation of RFOs (Li et al., 2011). Recently, we identified BnGS1 as a key factor that was up-regulated by a heat shock transcription factor and was associated with DT

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(Lang et al., 2017). In the present study, we found AtGS1 and AtGS2 were relatively

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highly expressed in mature seeds (Fig. 1B), which is consistent with the findings of

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Taji et al. (2002).

As the downstream products of the RFO pathway, raffinose and stachyose are the

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major soluble carbohydrates in seeds, roots, and tubers of many plant species

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(Sengupta et al., 2015). Previous studies have reported that the expression of GS and RS and the intracellular accumulation of these enzymes in plant cells are closely associated with responses to environmental stress (Panikulangara et al., 2004,

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Nishizawa-Yokoi et al., 2008, Egert et al., 2013, Sun et al., 2013). Our data demonstrated that overexpression or deletion of the GS or RS genes changed the

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timing of DT acquisition (Fig. 3G, H) but did not affect germinability, water content, and dry weight during seed development (Fig. 3C–F). Further study demonstrated that overexpression of AtGS1 and AtGS2 enhanced DT in developing Arabidopsis seeds

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and that the double mutant gs1, gs2 was more sensitive to desiccation treatment, exhibiting an obvious delay in DT acquisition compared with wild-type (Fig. 3G). The results showed that the acquisition of DT in developing seeds was associated with up-regulation of GS genes (Fig. 4A, B), an increase in enzyme activity (Fig. 4D), and accumulation of galactinol, especially raffinose and stachyose (Fig. 5A–C). Our results are similar to those reported by González-Morales et al. (2016), who found

that oligosaccharides, including raffinose and stachyose, were reduced in desiccation-intolerant mutant lines, but not in wild-type controls. In addition, the transcription of genes encoding GS1, GS2, and stachyose synthase (STS) was repressed. The above results suggest a clear correlation between DT, activity of RFOs synthesis-related enzymes, and galactinol and RFO accumulation. Compared to wild-type seeds, OX-RS5 lines exhibited stronger DT in developing

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seeds and rs5 mutant seeds possessed weaker DT, as shown in Fig. 3H. Further study showed that the total RS isoenzyme activity, as well as the content of raffinose and

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stachyose, decreased but was still present in the atrs5 line (Fig. 4C, E; 5A–C),

suggesting that there are other raffinose synthase genes in Arabidopsis besides AtRS5. This result was consistent with that of Egert et al. (2013), showing that due to the

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presence of other seed-specific raffinose synthases, phenotypic differences were not

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significant in rs5. Similar results were also found in the gs1 and gs2 single mutant

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lines. There was no obvious difference between gs single mutant and wild-type seeds in germination percentages (Fig. S3). However, the gs1, gs2 double mutant line

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showed an obvious decrease in germination (Fig. 3G), indicating there is redundancy

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in GS genes.

The time required to reach 50% of the final germination rate (T50) was used to describe the seed vigour (Coolbear et al., 1984). We found that the T50 time was

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shortened in OX-GS1, GS2, and RS5 lines at 11 and 15 DAF after desiccation treatment and that it was longer in gs1, gs2 and rs5 (Fig. 3I), which was consistent

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with the results from other viability tests of seeds (Fig. 6). Further study indicated that the total concentration of galactinol, raffinose, and stachyose was higher in the overexpression lines and that it was lower in gs1, gs2 and rs5 (Fig. 5B). These results

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combined indicate a critical role of galactinol, raffinose, and stachyose in seed vigour (Fig. 3I). It is worth noting that the T50 of OX-RS5 seeds was pronouncedly shortened at 15 DAF compared to OX-GS lines, but it exhibited a similar DT to OX-GS lines. To interpret this conflict, we carefully analysed both the content and the composition of galactinol and RFOs. Notably, although the total oligosaccharide content (galactinol, raffinose, and stachyose) was similar, we found OX-RS5 seeds possessed

more raffinose and stachyose compared to OX-GS1/GS2 lines. Based on these analyses, we speculated that raffinose and stachyose may have a higher protective potency in response to desiccation treatment. Previous results also indicated that the accumulation of stachyose and raffinose appears to be a key factor for the acquisition of DT in Arabidopsis seeds (González-Morales et al., 2016). Other studies also showed that RFOs, especially raffinose and stachyose, accumulated under abiotic

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stresses such as cold, drought, heat, and salinity (Bachmann et al., 1994; Pennycooke et al., 2003; Peters and Keller, 2009; Peters et al., 2010). These studies give support to

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our inference.

For all lines, the seed populations showed 100% DT with different sugar contents at 19 DAF (Fig. 3G, H; Fig. 5). This might be due to the accumulation of other

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components during seed maturation, such as lipids, free radical scavengers, and LEA

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proteins that are more powerful than RFOs in DT acquisition at this stage (Wolkers et

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al., 1999; Wise and Tunnacliffe, 2004). We considered that the role of RFO in promoting DT acquisition is time limited, which is primarily important from 9–16

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DAF and less important during the later stages of seed development (17 DAF and

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onwards). Our results showed the content of sucrose increased during seed development (Fig. 5D), which was similar with previous studies (Baud et al., 2002). For the 100% DT in all lines at 19 DAF, another explanation could be that the most

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abundant soluble non-reducing sugar is sucrose (60–80% of total soluble sugars) in mature Arabidopsis seeds, and that it may play crucial protective roles and mask the

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role of galactinol and RFO (Koster and Leopold, 1988; Righetti et al., 2015; Leprince et al., 2016).

Previous studies have shown that the GSH/GSSG ratio tends to increase as seeds

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are desiccating (Kranner and Grill, 1993; Kranner and Birtić, 2005; Colville and Kranner, 2010). Nishizawa et al. (2008) also indicated that higher accumulation of galactinol and RFOs may assist in maintaining levels of GSH in plants under abiotic stress. Our results showed the GSH/GSSG ratio increased in OX-GS and OX-RS lines and decreased in mutant lines (Fig. 7A), indicating GS and RS may function to maintain the GSH/GSSG ratio to resist stress. In addition, although DT was not

affected in mature seeds, GS and RS reduced ROS accumulation and the MDA content under dehydration conditions (Fig. 7B, C) and improved the oxidation resistance to MV treatment (Fig. 7D, F). Similar antioxidative functions of RFOs were also reported previously (Sengupta et al., 2015). Seed-specific overexpression of CaGS1 and CaGS2 in Arabidopsis improved seed vigour and longevity by limiting excess ROS and lipid peroxidation (Prafull et al., 2016). Higher galactinol and

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raffinose contents improved survival of transgenic lines by acting as ROS scavengers (Taji et al., 2002; Nishizawa et al., 2008).

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Zhou et al. (2012) reported GhGS1 protein is targeted to the cell membrane of

onion epidermis cells. Salvi et al. (2016) observed that CaGS1 and CaGS2 in chickpea were predominantly in the nucleus and plasma membrane and detectable in

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the cytosol. Similar to the TaGS protein (Wang et al., 2015), we found that AtGS1

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was observed predominantly in the nucleus (Fig. 8J), while AtRS5 was in the

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cytoplasm. It has been demonstrated that a large number of functional gene products are located in the nucleus and that the expression of these genes can confer tolerance

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in plants to a variety of abiotic stresses (Qin et al., 2008; Yoshida et al., 2010). Thus,

Conclusion

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abiotic stress.

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we speculated that AtGS1 protein may play a role in the nucleus and in responses to

Taken together, our results indicate that galactinol synthase and raffinose synthase

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had no significant effect on seed development but affected the timing of DT acquisition during seed development. Overexpressing the AtGS1, AtGS2, or AtRS5 genes improved seed DT and viability after dehydration probably due to the increased

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enzyme activity, and accumulation of galactinol and RFOs improved oxidation resistance under dehydration stress. We conclude that galactinol and RFOs are important components for DT acquisition during Arabidopsis seed development, and further investigations are needed to identify the regulatory mechanism of GS and RS in DT acquisition.

Acknowledgements We would like to thank Dr. Hugh W. Pritchard, UK, for useful discussions and critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (31770700), National Training Program of Innovation and Entrepreneurship for Undergraduates (NO. 201810022036), and Beijing Municipal Training Program of Innovation and Entrepreneurship for Undergraduates

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(NO. S201810022048).

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Van den Ende, W. (2013) Multifunctional fructans and raffinose family oligosaccharides. Frontiers in Verdier, J., Lalanne, D., Pelletier, S., Torres-Jerez, I., Righetti, K., Bandyopadhyay, K., Leprince, O., Chatelain, E., Vu, B.L. and Gouzy, J. (2013) A regulatory network-based approach dissects

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Wolkers, W.F., Alberda, M., Koornneef, M., Léon-Kloosterziel, K.M. and Hoekstra, F.A. (1998) Properties of proteins and the glassy matrix in maturation-defective mutant seeds of

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22, 1775-1785. Zhuo, C., Wang, T., Lu, S., Zhao, Y., Li, X. and Guo, Z. (2013) A cold responsive galactinol synthase gene from Medicago falcata (MfGolS1) is induced by myo-inositol and confers multiple tolerances to abiotic stresses. Physiologia plantarum, 149, 67-78. Zuther, E., Büchel, K., Hundertmark, M., Stitt, M., Hincha, D.K. and Heyer, A.G. (2004) The role of

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raffinose in the cold acclimation response of Arabidopsis thaliana. FEBS letters, 576, 169-173.

Figure legends: Fig. 1. Expression of the GS and RS genes in Arabidopsis. (A) Overview of the galactinol, raffinose, and stachyose biosynthetic pathway. (B) Expression of AtGS and AtRS genes in mature wild-type seeds. Expression of AtGS (C) and AtRS (D) genes in various organs of Arabidopsis. Relative amounts were calculated and normalized with Actin2 (At3G18780). The data are expressed as the

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mean ± S.D. of three independent experiments. Different letters above the error bars

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indicate groups with significant differences (P ≤ 0.05).

Fig. 2. Amino acid sequence comparison of the AtGS and AtRS proteins.

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(A) The phylogenetic tree of GS family proteins in Arabidopsis. (B) Alignment of the amino acid sequences of AtGS with GS from other species. Sequences of AtGS1,

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BnGS1, AtGS2, AtGS3, PtGS3, CmGS, XvGS, and ArGS1 were used. Asterisks indicate conserved serine. The bar displays a characteristic hydrophobic pentapeptide (APSAA). (C) The phylogenetic tree of RS family proteins in Arabidopsis. (D)

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Alignment of amino acid sequences of AtRS with RS from other species. Sequences of PsRS, GmRS2, GmRS3, BhRS, CsRS, AtRS5, AtRS4, and AtRS6 were used. The bar displays the KxD and RxxxD motifs.

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Fig. 3. Construction of transgenic plants and the growth parameters of developing Arabidopsis seeds. (A) Construct of the AtGS1 or AtRS5 expression vector containing a double-CaMV35S promoter with a TMV enhancer. (B) Southern blot analysis of wild-type and transgenic plants. Water content (active lines) and dry weight (dash lines) of GS (C) and RS (D) mutants and transgenic seeds during development. The

data are expressed as the mean ± S.D. of three independent experiments. Germination (without dehydration) of the GS (E) or RS (F) mutants and transgenic seeds. Germination (after desiccation treatment) of the GS (G) or RS (H) mutants and transgenic seeds. (I) Time to 50% DT acquisition (T50) of Arabidopsis seeds at 11, 15, and 19 DAF. The data are expressed as the mean ± S.D. of three independent experiments. Different letters above the error bars indicate groups with significant

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differences (P ≤ 0.05).

IP T SC R U N A M ED PT CC E A Fig. 4. The transcription levels of AtGS and AtRS genes and GS and RS activities in developing seeds of various lines after desiccation treatment. Expression of AtGS1

(A), AtGS2 (B), and AtRS5 (C) in seeds at 11, 15, and 19 DAF. Relative amounts were calculated and normalized to actin2. Total activity of GS (D) and RS (E) in 11, 15, and 19 DAF seeds. The data are expressed as the mean ± S.D. of three independent experiments. Different letters above the error bars indicate groups with significant

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differences (P ≤ 0.05).

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Fig. 5. Soluble sugar content in the seeds of various lines after desiccation treatment. galactinol (A), raffinose (B), stachyose (C), sucrose (D), and total RFO (E) content in

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11 DAF, 15 DAF, and 19 DAF seeds. The data are expressed as the mean ± S.D. of three independent experiments. Different letters above the error bars indicate groups

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with significant differences (P ≤ 0.05).

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Fig. 6. Viability tests of seeds after desiccation treatment. Seeds of different lines

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were stained in 1% TTC at 30 °C for 2 d in the absence of light. (A) Seed viability

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was confirmed by TTC staining. Scale bar = 1 mm. (B) Reduced TTC levels of different lines. Embryos were dissected from the seed coat. Dark-red staining indicates that seeds are highly vigorous. Seeds stained lightly were characterized as

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“low vigour” seeds. Influence of accelerated ageing in the seeds of different GS (C) or RS (D) lines. Different letters above the error bars indicate groups with significant

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differences (P ≤ 0.05).

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Fig. 7. Effect of desiccation treatment on RFOs in resistance to ROS in the seeds of different lines. (A) The ratio of GSH to GSSG (GSH/GSSG) in seeds at 11/15/19 DAF.

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MDA (B) and H2O2 (C) content of different seeds before and after desiccation treatment. Germination rates of different GS (D) or RS (E) lines after 1 μM MV treatment for 3 h. The results shown are a representative experiment that was performed in triplicate. Different letters above the error bars indicate groups with significant differences (P ≤ 0.05).

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Fig. 8. Subcellular localization of AtGS1 and AtRS5 in developing seeds of the transgenic Arabidopsis. Localization of EGFP (A–E), AtGS1-EGFP (F–J) or

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AtRS5-EGFP (K–O) in seeds at 11 (A, F, K) and 15 (B, G, L) DAF. Fluorescence images were obtained by confocal-laser scanning microscope. The nucleus was stained by 4, 6-diamidino-2-phenylindole (DAPI) (D, I, N).

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Table 1.Correlation analysis between DT and galactinol/raffinose/stachyose content at 11, 15 and 19DAF.

11DAF DT

Pearson

Gal 1

Significance

Raf

Sta

total

.797*

.888**

.a

.010

.001

.

(bilateral test) 9

9

9

Pearson

1

.452

.852**

.872**

.222

.004

Significance

.007

9

9

.427

.325

.296

.252

.394

9

9

9

9

9

Pearson

1

-.056

.393

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Significance

.887

(bilateral test) 9

9

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.820**

.002

(bilateral test) 19DAF DT

9

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9

.003

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15DAF DT

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.853**

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DT

9