Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

Plant Physiology Preview. Published on March 3, 2015, as DOI:10.1104/pp.15.00045

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Running head: Proteomic analysis of lettuce seed germination

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Corresponding author:

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Dr. Song-Quan Song

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Key Laboratory of Plant Resources and Beijing Botanical Garden,

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Institute of Botany, Chinese Academy of Sciences,

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Beijing, 100093, China

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Tel. 86 10 6283 6484

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E-mail address: [email protected]

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Research area: Cell Biology

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Copyright 2015 by the American Society of Plant Biologists

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Proteomic Analysis of Lactuca sativa Seed Germination and Thermoinhibition by

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Sampling of Individual Seeds at Germination and Removal of Storage Proteins by PEG

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Fractionation

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Wei-Qing Wang1, Bin-Yan Song1,3, Zhi-Jun Deng1,4, Yue Wang1, Shu-Jun Liu1, Ian Max

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Møller2, Song-Quan Song1∗

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1

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Chinese Academy of Sciences, Beijing 100093, China

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2

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DK-4200 Slagelse, Denmark

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3

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416000, China

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Key Laboratory of Plant Resources and Beijing Botanical Garden, Institute of Botany,

Department of Molecular Biology and Genetics, Aarhus University, Flakkebjerg,

College of Biology Resources and Environmental Sciences, Jishou University, Jishou

College of Forestry and Horticulture, Hubei Minzu University, Enshi 445000, China

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One-sentence summary:

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Methionine metabolism, ethylene production and isoprenoid biosynthesis were revealed

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to be involved in seed germination and thermoinhibition by proteomic and gene

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expression analyses.

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Footnote:

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This work was supported by the National Sciences and Technology Support Program

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(2012BAC01B05) and the National Natural Sciences Foundation of China (31171624,

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81102757). IMM was supported by a Chinese Academy of Sciences Visiting

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Professorship for senior international scientists.

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Experiment institution: Institute of Botany, Chinese Academy of Sciences

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Corresponding author:

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Song-Quan Song, email: [email protected]

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ABSTRACT

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Germination and thermoinhibition in lettuce (Lactuca sativa L. cv. ‘Jianyexianfeng No.

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1’) seeds was investigated by a proteomic comparison among dry seeds, germinated

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seeds at 15oC (15G), at 15oC after imbibition at 25oC for 48 h (25/15G) or at 25oC in

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KNO3 (25nG) (all sampled individually at germination) and ungerminated seeds at 25oC,

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a thermoinhibitory temperature (25G). Before two-dimensional (2-D) gel

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electrophoresis analysis, storage proteins (>50% of total extractable protein) were

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removed by PEG precipitation, which significantly improved detection of less abundant

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proteins in 2-D gels. A total of 108 protein spots were identified to change more than

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2-fold (P24’ on the horizontal axis) were sampled for the

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qRT-PCR analysis (Fig. 4).

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The differentially accumulated spots 682 and 707 were found to be homologous to

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a same SAM, but they, in fact, are encoded by two different genes (named SAM1a and

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SAM1b for spot 682 and 707, respectively) according to their best matched EST (Fig. 4).

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Among the three SAM-encoding genes, only expression of the SAM1b correlated with

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the protein accumulation (Fig. 4B). SAM1a increased gradually in the mRNA level in

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15G, 25G and 25nG seeds, and was expressed most in 25G seeds. Expression of this

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gene decreased in 25/15G seeds (Fig. 4A). Abundance of SAM2 transcripts decreased in

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15G, 25/15G and 25nG seeds and was maintained relatively constant in 25G seeds at the

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early stages and then increased 2-fold at 48 HI (Fig. 4C). Transcript levels of 12 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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lipoxygenase 2 (LOX2) and ICL genes, LOX2 (Fig. 4D) and ICL(Fig. 4G) increased

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continuously with time in all of the germination treatments, but accumulated much

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higher in germinated 15G, 25/15G and 25nG seeds than in ungerminated 25G seeds.

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Expressions of genes encoding acetyl-CoA acetyltransferase (AACT1, Fig. 4E),

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2,3-bisphosphoglycerate- independent phosphoglycerate mutase (BIPM, Fig. 4F),

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aldehyde dehydrogenase 1 (ADH1, Fig. 4L), ANN1 (ANN1, Fig. 4N) and CVS (CVS,

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Fig. 4P) were completely repressed or decreased slightly in 25G seeds, but increased

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greatly in 15G, 25/15G and 25nG seeds (Fig. 4L).

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The Mascot search for the PMF of spot 1070 did not find any matched EST in L.

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sativa, but found the best fit (DW064597) in a same genus, L. saligna (Table 1). The

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translated protein sequence for DW064597 was highly homologous to ACO1 in Morus

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notabilis (Table 1). Therefore, this matched EST was used to design primers to amplify

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the potential ACO gene in L. sativa (termed ACOx). When the designed primer was

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blasted against NCBI EST or nucleotide database using L. sativa as genus, no EST or

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gene sequences matched significantly to this primer. However, qRT-PCR results showed

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that a gene in L. sativa had been amplified by this primer (Supplemental Fig. S3). This

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indicates that ACOx is a possible ACO gene in L. sativa. Expression of ACOx increased

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greatly (more than 40-fold) in 15G, 25/15G and 25nG seeds when germinated, and

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increased initially and then decreased to a very low level at 48 HI in 25G seeds (Fig.

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4I).

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The abundances of mRNA for genes encoding the predicted

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alpha-1,4-glucan-protein synthase [UDP-forming] 2 (GPS2, Fig. 4J), ADF2 (ADF2, Fig.

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4K) and ATF (ATF, Fig. 4O) declined initially in 15G, 25G and 25nG seeds and then

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kept relatively constant in 25G seeds, but increased in 15G and 25nG seeds after 12 HI.

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The expression of these genes increased continuously in 25/15G seeds with extended

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time (Fig. 4, J, K and O). Transcripts of genes encoding CPN21 (CPN21, Fig. 4H) and

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predicted: probable aldo-keto reductase 2-like (AKR2, Fig. 4M) showed no significant

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changes in both germinated and ungerminated seeds.

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Expression of many genes, including SAM1b (Fig. 1B), AACT1 (Fig. 1D), BIPM (Fig. 1F), GPS2 (Fig. 1J), ADF2 (Fig. 1K), ADH1 (Fig. 1L), ANN1 (Fig. 1N), ATF (Fig. 13 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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1O) and CVS (Fig. 1P) were initiated at 6 or 12 HI in 25/15G seeds and much earlier

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than in the 15G and 25nG seeds. Among these genes, AACT1 (Fig. 4D), BIPM (Fig. 4F)

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GPS2 (Fig. 4J) and CVS (Fig. 4P) were also expressed much earlier in 15G seeds than

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25nG seeds. These results are consistent with the difference in germination rate among

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25/15G, 15G and 25nG seeds (Fig. 1).

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In conclusion, the expression of 12 genes, SAM1b (Fig. 1B), LOX2 (Fig. 1D),

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AACT1 (Fig. 1D), BIPM (Fig. 1F), ICL (Fig. 1G), ACOx (Fig. 1I), GPS2 (Fig. 1J),

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ADF2 (Fig. 1K), ADH1 (Fig. 1L), ANN1 (Fig. 1N), ATF (Fig. 1O) and CVS (Fig. 1P)

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correlates well with their protein accumulation. Transcript levels of these genes were

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much higher in 15G, 25/15G and 25nG seeds, all of which could germinated well, than

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in 25G seeds, in which very poor germination was observed.

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Expression of genes encoding enzymes of the MVA pathway

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Besides AACT1, abundances of another two proteins, 3-hydroxy-3-methylglutaryl-CoA

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(HMG-CoA) synthase 2 (HMGS2, spot 650) and mevalonate decarboxylase (MDPC2,

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spot 632) involved in the MVA pathway for isopronoid biosynthesis were significantly

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higher (more than 1.5-fold higher) in germinated 15G, 25/15G and 25nG seeds than in

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both dry and ungerminated 25G seeds (Supplemental Table S5, Fig. 5C and H). These

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results suggest a potential role of the MVA pathway for isopronoid biosynthesis in

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regulating seed germination and thermoinhibition. To elucidate this role further, we

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measured the expression of all the candidate genes encoding the enzymes of this

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pathway (Fig. 5). We could not find the full-length clones for those genes in lettuce.

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Therefore, we used the genes that have been reported in Arabidopsis (Vranová et al.,

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2013) to identify the homologous genes in L. sativa. Alleles of a give gene were named

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numerically according to the numerical order of their unigene accession number except

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for above mentioned AACT1.

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The initial reaction of the MVA pathway is the condensation of two molecules of

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acetyl-CoA to acetoacetyl-CoA catalyzed by AACT (Fig. 5). Three AACT alleles in

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lettuce (termed AACT1, AACT2 and AACT3) were identified to be highly homologous to

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Arabidopsis AACT (AtAACT1 and AtAACT2). AACT1 was more highly expressed in 14 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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15G, 25/15G and 25nG seeds than 25G seeds, while AACT2 and AACT3 had highest

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expression in 25G seeds (Fig. 5B).

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In the following reaction, HMGS converts the acetoacetyl-CoA to HMG-CoA. In

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lettuce, genes encoding this enzyme had two homologs, termed HMGS1 and HMGS2.

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There was a higher HMGS1 transcript level in 25G, 15G and 25/15G seeds than in

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25nG seeds at 24 HI, but a similar level in all of these seeds during the following

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imbibition (Fig. 5D). The mRNA level for HMGS2 was much higher in 15G and

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25/15G seeds both at 24 HI and >24HI and in 25nG seeds at >24HI than in 25G seeds

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(Fig. 5D).

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In the next reaction, HMG-CoA is converted to MVA by HMG reductase (HMGR).

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In Arabidopsis, genes encoding this enzyme have two alleles (AtHMGR1 and

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AtHMGR2) (Vranová et al. 2013), while three genes in lettuce (named HMGR1,

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HMGR2 and HMGR3) were highly homologous to these two alleles (Fig. 5). Transcripts

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of HMGR1 and HMGR2 accumulated abundantly in 25G seeds compared to 15G,

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25/15G and 25nG seeds, while expression of HMGR3 was lowest in 25G seeds, higher

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in 15G seeds and highest in 25/15G and 25nG seeds (Fig. 5E).

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In the following reaction of the MVA pathway, MVA undergoes two

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phophorylation reactions, catalyzed sequentially by MVA kinase (MK) and

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phospho-MVA kinase (PMK) to form MVA 5-pyrophosphate (Fig. 5). MK was

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identified to be encoded by one gene in lettuce. The amount of mRNA for MK was

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higher in 15G, 25/15G and 25nG seeds at 24 HI and much higher (more than 4-fold)

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when germinated (>24 HI) than in 25G seeds (Fig. F). Genes of PMK in lettuce had two

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alleles (termed PMK1 and PMK2) homologous to those in Arabidopsis (Fig. 5).

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Transcript for PMK1 had little difference among all the germination treatments, while

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for PMK2 was much higher in 15G, 25/15G and 25nG seeds than 25G seeds both at 24

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HI and >24 HI (Fig. 5G).

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In the final reaction of the MVA pathway, the mevalonate 5-pyrophosphate is

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decarboxylated to isopentenyl diphosphate, the universal precursor of all isoprenoids

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(Nagegowda, 2010), by MDPC (Fig. 5). Two genes (termed MDPC1 and MDPC2)

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homologous to Arabidopsis genes were identified in lettuce. In contrast to protein 15 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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accumulation, expression of the MDPC genes was greater in 25G seeds than in 15G,

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25/15G and 25nG seeds (Fig. 5I).

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Effect of lovastatin on seed germination

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Lovastatin is highly potent competitive inhibitor of HMGR not only in animals and

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micro-organisms, but also in plants (Alberts et al., 1980; Bach and Lichtenthaler, 1983).

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Inhibition of HMG-CoA reductase with this compound prevents sterol biosynthesis and

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the prenylation of proteins, and reduces plant growth (Randall et al., 1993; Josekutty,

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1998). We imbibed the lettuce seeds using a series of concentrations of lovastatin to

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examine its effect on seed germination (Fig. 6A). Lovastatin did not influence the final

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percentage of seed germination and seedling growth (data not shown), but delayed the

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germination (Fig. 6A). The seed germination rate decreased as the concentration of

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lovastatin increased (Fig. 6, A and C). Lovastatin also increased the sensitivity of

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germination to ABA (Fig. 6B). Though 10 μM lovastatin itself did not affect the

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germinate rate, it promoted the effect of ABA on seed germination (Fig. 6B). Lovastatin

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at 100 μM plus ABA strongly decreased the seed germination rate and delayed full

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germination by about 24 h (Fig. 6B).

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The effect of a number of MVA pathway products was tested to see if they could

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reverse the lovastatin inhibition of seed germination and release thermoinhibition of

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lettuce seed germination at 25oC. Three cytokinins, kinetin (KT), 6-benzylaminopurine

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(6-BA) and zeatin (ZT) partially reversed the inhibition of germination by 100 μM

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lovastatin (Fig. 6 C). KT, 6-BA and ZT promoted seed germination at the

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thermoinhibitory temperature 25oC from about 10% to 78%, 90% and 90%, respectively

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(Fig. 6D). Application of the cytokinin of thidiazuron (TDZ), three phytosterols,

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stigmasterol, β-sitosterol and campesterol, and brassinolide, a brassinosteroid (BR), had

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no effect on seed germination at 15oC in the presence of 100 μM lovastatin or at 25oC

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(data not shown).

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DISCUSSION

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Seed germination and thermoinhibition 16 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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Lettuce seeds are reported to exhibit thermoinhibition at 25 to 30oC depending on

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variety and cultivation environment (Guzman et al., 1992; Kozarewa et al., 2006;

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Contreras et al., 2009; Huo et al., 2013). In L. sativa cv. ‘Jianyexianfeng No. 1’, seed

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germination started to be inhibited at 23oC (Deng and Song, 2012), only had 10%

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germination at 25 oC (Fig. 1) and were fully inhibited at 27 oC (Deng and Song, 2012).

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This inhibition is not due to the death of the seeds or the induction of dormancy. When

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the ungerminateds seeds were transferred to 15oC after imbibition at 25oC for 48 h, all

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the seeds germinated (Fig. 1). Thermoinhibition of lettuce seed germination could be

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alleviated by exogenous application of KNO3 (Fig. 1). Application of SNP, Fe(III)CN,

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nitrite and nitrate also promoted lettuce seed germination at the thermoinhibitory

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temperatures (Deng and Song, 2012). Nitrate is a natural product and is also an

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important source of nitrogen for plant growth. Therefore, its use in the alleviation of

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lettuce seed thermoinhibition will be important for crop production.

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Sampling seed material for proteomic and genetic analysis

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It is very difficult to identify physiological changes in a single seed during germination,

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so seed batches are normally used to study germination. However, the physiological

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state differs between individual seeds in a population, e.g. germination always occurs

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over a period of time. Lettuce seeds incubated at 15oC started to germinate at 24 HI and

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finished at about 48 HI (Fig. 1). When the seeds were imbibed in KNO3 at 25oC, this

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interval was extended to nearly 50 h (Fig. 1). Thus, if we had sampled the entire seed

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batch when the first seed had germinated, as is the standard for germination sensu

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stricto, we would have been studying seeds differing in developmental stage by 24-48 h.

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Instead we removed and froze germinated seeds (15G, 25/15G and 25nG) every 2 h and

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pooled them afterwards for each batch. For the 25G seeds, which germinated very

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poorly, the few germinated seeds (10%) were discarded and only the ungerminated

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seeds were sampled. In this way we ensured that all the seeds analyzed were at precisely

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the same germination stage – newly germination sensu stricto.

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PEG precipitation increases the number of detectable protein spots on 2-D gels 17 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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Lettuce seeds contain many abundant storage proteins, accounting for more than 50% of

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total extractable protein (Fig. 2, Supplemental Table S2). Storage proteins play an

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essential role in providing nutrition and energy for seed germination and seedling

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growth (Tan-Wilson and Wilson, 2012; Rajjou et al., 2012). However, they can

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complicate proteomic studies, especially when the 2-DE approach is used. As shown in

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Fig. 2D and E, many proteins were masked in the region of 2-D gel where the storage

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proteins dominated. This situation occurs also in other plant tissues, such as leaf in

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which the RuBisCO is highly abundant (Ashan et al., 2007; Acquadro et al., 2009). PEG

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fractionation is recognized as a convenient and reproducible method for the removal of

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RuBisCO and to increase the resolution of low-abundance proteins in leaf extracts of

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many species (Xi et al., 2006; Ashan et al., 2007; Lee et al., 2007; Acquadro et al.,

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2009). This method is clearly also applicable to seed samples (Fig. 2; Supplemental

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Tables S2, S3). PEG precipitation increased the number of detectable protein spots on

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the 2-D gels (Fig. 2, C and D, Supplemental Table S3). Because of this fractionation, we

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could identify several protein spots in the storage protein-dominated region, e.g. ANN1

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(spot 981) and ACO 1 (spot 1070), that are potentially important for seed germination

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(Figs. 2 and 3, Table 1).

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Storage proteins are the most abundant proteins precipitated by PEG in lettuce

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seeds. It is a potential concern that differences in the amount of storage protein would

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lead to differential precipitation of protein between treatments, and result in unreliable

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comparisons. We observed that TP from 100 seeds was constant in dry, germinated 15G,

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25/15G and 25nG and ungerminated 25G seeds (Supplementary Table S4). The yield of

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PEG precipitation was also constant in all seed samples (Supplemental Table S4). It

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therefore seems reasonable to assume that the same percentage of storage protein was

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present in different seed samples and precipitated by the PEG. This was validated by

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1-D gel comparisons of protein samples of all seed types before and after PEG

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precipitation (Supplemental Fig. S4). PEG precipitation can not lead to false positives,

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but only to false negatives (see Supplemental discussion). P EG fractionation

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significantly increased the number of spots detected on 2-D gels (Fig. 2, Table S3) as

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well as allowed a twice as high loading of other proteins, both of which would allow 18 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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detection of more differentially accumulated non-storage proteins. We cannot exclude

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the possibility that a few differentially accumulated proteins were lost as a result of PEG

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precipitation. However, on balance we conclude that PEG precipitation is a reliable and

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useful approach in proteomic analyses of lettuce seed germination and thermoinhibition.

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Proteins increased in abundance differ greatly between the germinated and

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ungerminated seeds, but proteins decreased in abundance are mostly similar

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Seed germination is a complex process and requires activation of a series of

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physiological and metabolic processes, such as amino acid metabolism, energy

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production, protein synthesis, mobilization of storage reserve, enzymatic weakening of

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the surrounding structure and cell and embryo expansion (Nonogaki et al., 2010;

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Weitbrecht et al., 2011; Bewley et al., 2013). Proteomic analysis of lettuce seed

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germination at the optimal temperature identified several proteins participating in these

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processes.

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SAM (spots 682, 707 and 713) is involved in methionine metabolism, LOX (spot

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126) and ICL (spot 290) are both related to lipid degradation, and predicted

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peptidyl-prolyl cis-rans isomerase (PPIase) CYP40-like isoform X1 (spot 848) and

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chloroplast chaperonin 21(CPN21, spot 1243) are both involved in protein folding and

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thus assist in protein synthesis (Fig. 3B, Supplemental Table S8). However, not all of

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these identified proteins are involved in seed germination. Some proteins, such as

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PPIase CYP40-like isoform (spot 848), predicted proteasome subunit beta type-3-A-like

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(spot 1273) and beta-hydroxyacyl-ACP dehydratase (spot 1395) accumulated

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differentially in seeds imbibed both at the optimal and the thermoinhibitory

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temperatures (Supplemental Tables S8 and S9).

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It appears that a thermoinhibitory temperature regulates seed germination more by

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affecting protein synthesis, and less by affecting degradation. The number of protein

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spots increasing in abundance during imbibition were fewer in ungerminated 25G seeds

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than in germinated 15G and 25nG seeds, but the number decreased in abundance were

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approximate the same in the ungerminated and germinated seeds (Fig. 3B). In addition,

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when the ungerminated 25G seeds were transferred to 15oC, germination was initiated 19 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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and many proteins increased in abundance but few decreased (Fig. 3B). Most of the

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proteins, whose abundance increased during imbibition, were found in all germinated

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15G, 25/15G and 25nG seeds, but not in ungerminated 25G seeds. In contrast, the

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majority of the proteins decreasing in abundance were found in both germinated 15G

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and 25nG and ungerminated 25G seeds (Fig. 3C). These results are consistent with the

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role of protein synthesis in seed germination that has been proposed in Arabidopsis

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(Rajjou et al., 2004; Galland et al., 2014) and rice (Sano et al., 2012).

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The abundance of 12 proteins and their mRNA correlated positively with the

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ability of lettuce seeds to germinate

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Twenty-two protein spots belonging to 20 gene products were identified to play a

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potential role in regulating seed germination and thermoinhibition due to their higher or

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lower accumulation in germinated 15G, 25/15G and 25nG seeds than both dry and

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ungerminated 25G seeds. Four storage proteins following this accumulation pattern had

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an experimental mass lower than their theoretical mass (Supplemental Table S6). They

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may have been generated by the initiation of seed storage mobilization, which is a

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common phenomenon found during seed germination (Bewley et al., 2013; Wang et al.

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2015). Similarly, dehydrin 2 with a range of different apparent sizes (21–34 kDa) and pI

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(6.7–7.5) were found in lettuce seeds imbibed under different conditions (Supplemental

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Tables S6), indicating that it had been modified by degradation or other

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post-translational modifications. It is clear that the accumulation of storage proteins and

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dehydrin cannot simply be explained by gene expression, which was therefore not

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monitored in the present study. Of 16 selected protein-encoding genes, 12 had

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expression levels consistent with the protein accumulation (Fig. 4). They may be

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particularly important for seed germination and thermoinhibition.

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The abundances of three SAMs were low or undetectable in dry seeds and

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increased greatly in germinated 15G, 25/15G and 25nG seeds, but did not change, or

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even decreased, in ungerminated 25G seeds (Table 1). Among the SAM family

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members, SAM1b might play the primary role in lettuce seed germination and

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thermoinhibition. Expression of SAM1b increased only in the germinated seeds, while it 20 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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was constant in ungerminated seeds (Fig. 4B), which accords with the change in

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abundance of its encoded protein. SAM catalyzes the synthesis of

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S-adenosylmethionine (AdoMet) from methionine and ATP. AdoMet supplies the

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methyl group for many specific transmethylation reactions (Hanson and Roje, 2001).

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Some of AdoMet-dependent methyltransferases, such as protein L-isoaspartyl

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methyltransferase and the DNA methyltransferases have been proposed to play roles in

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seed germination and seedling growth (Rajjou et al., 2012). In addition, AdoMet is a

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precursor for biosynthesis of polyamines, ethylene and biotin (Roje, 2006; Rajjou et al.,

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2012). Seed germination is blocked when 7-keto-8-aminopelargonic acid synthase, an

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enzyme in the early step of the biotin biosynthesis, is inhibited by triphenyltin acetate,

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and this block is reversed by biotin (Hwang et al., 2010).

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Ethylene is an important signal molecule involving in seed germination (Linkies

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and Leubner-Metzger, 2012). It promotes seed germination by inhibiting ABA signaling

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(Linkies and Leubner-Metzger, 2012). Ethylene alleviates the thermoinhibition of

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lettuce seed germination especially when combined with GA or a cytokinin (Abeles,

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1986; Saini et al., 1986; Khan and Prusinski, 1989), and ethylene production is reduced

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in seeds germinated at thermoinhibitory temperature (Prusinski and Khan, 1990;

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Nascimento et al., 2000). Expressions of ACS1 and ACO2, two ethylene biosynthetic

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genes, are tightly related to the thermoinhibition of lettuce seed germination (Agris et al.,

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2008; Huo et al., 2013). Here, we found that a potential ACO enzyme identified in L.

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saligna (ACOx, spot 1070) increased in protein abundance in 15G, 25/15G and 25nG

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seeds, but was constant in 25G seeds compared to the dry seeds (Table 1). Transcripts

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of ACOx-encoding genes were more than 40 fold higher in abundance in 15G, 25/15G

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and 25nG seeds at >24HI than in 25G seeds (Fig. 4I). The accumulation of SAM and

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ACO at both the protein and gene expression level is consistent with the important role

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of ethylene in modulation of seed germination and thermoinhibition. This role is further

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underlined by our finding that application of the ethylene producer, ethephon, promoted

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the germination of all seeds at 25oC and application of an inhibitor of ethylene action

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(2,5-norbornadiene), but not of ethylene production (CoCl2), inhibited lettuce seed

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germination at 15oC (Supplemental Fig. S5). 21 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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Lipid is one of the storage reserves in lettuce seeds (Ouellette and Bewley, 1986).

620

Mobilization of lipid provides the metabolic building blocks and energy for seed

621

germination and seedling growth (Graham, 2008; Kelly et al., 2011). We found that the

622

abundance of LOX2 (spot 126) and ICL (spot 290), both participating in the

623

mobilization of lipid, were higher in germinated 15G, 25/15G and 25nG seeds than both

624

dry and ungerminated 25G seeds (Table 1). Gene expression of LOX2 and ICL was also

625

much greater in the germinated seeds than in the ungerminated seeds (Fig. 4, D and G).

626

LOXs, belonging to the nonheme iron-containing dioxygenases, catalyze the addition of

627

molecular oxygen to polyunsaturated fatty acids containing a (Z, Z)-1,4-pentadiene

628

system to produce an unsaturated fatty acid hydroperoxide (Porta and Rocha-Sosa,

629

2002). In seeds, LOXs may function as storage proteins, assist membrane retailoring

630

during germination and play a role in pathogen resistance (Porta and Rocha-Sosa, 2002;

631

Mo et al., 2006). In addition, LOXs have been observed to localize to the oil bodies

632

(Feussner et al., 2001; Zienkiewicz et al., 2014) and are thought to play an important

633

role in regulating lipid degradation during seed germination (Feussner et al., 2001;

634

Rudolph et al., 2011). After oxygenation by LOXs, lipid is preferentially cleaved by

635

specific triacylglycerol lipases from oil bodies (Feussner et al., 1995, 2001).

636

ICL is an enzyme in the glyoxylate cycle, which is one of main pathways involved

637

in lipid mobilization (Graham, 2008). This cycle allows the acetyl-CoA derived from

638

the breakdown of fatty acids to be used for the synthesis of carbohydrates

639

(gluconeogenesis) that are used to support seedling growth (Graham, 2008). ICL may

640

play a major role during seedling growth. In oilseeds, there is a strong correlation

641

between the expression of ICL and degradation of lipid during post-germinative growth

642

(Eastmond and Graham, 2001). Mutants of two ICL alleles, icl1 and icl2 showed no

643

difference in germination and seedling establishment under continuous light, but

644

exhibited a gradual decrease in seedling establishment with decreasing light intensity

645

compared to wild type (Eastmond et al., 2000). The moment that seeds have completed

646

germination sensu stricto is by definition the time that seedling growth commences.

647

Therefore, it is not surprising to find that ICL amount and gene expression were highly

648

abundant in the germinated lettuce seeds. However, whether ICL plays a role in lettuce 22 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

649

seed germination or seedling growth or both requires further investigation.

650

In many species, embryo cell elongation, but not cell division, is thought to be

651

essential for the completion of seed germination (Barroco et al., 2005; Gimeno-Gilles et

652

al., 2009; Sliwinska et al., 2009). Several proteins involved in this process including

653

ADF2 (spot 1431), GPS2 (spot 807) and ANN1 (spot 981) increased greatly in

654

abundance in germinated 15G, 25/15G and 25nG seeds but much less in ungerminated

655

25G seeds (Table 1). Expression of the genes encoding these proteins exhibited the

656

same pattern (Fig. 4, J, K and N). Actin is one of the central components of the

657

cytoskeleton. The actin cytoskeleton constantly undergoes dynamic assembly and

658

disassembly, which allows the cell to reorganize the cellular structure that rapidly

659

responds to multiple cellular processes, such as cell motility, cell elongation,

660

intracellular trafficking and cytokinesis (Staiger, 2000; Pollard et al., 2009; Staiger et al.,

661

2010). ADF is one of the modulators of this process (Augustine et al., 2008; Ketelaar,

662

2013). In tomato (Sheoran et al., 2005) and pea (Wang et al., 2012), this protein

663

increases also in abundance during seed germination. One would expect cell elongation

664

to require a dynamic change in the cytoskeleton, and the ADF2 may have this role. In

665

addition, during cell elongation the cell wall extension may require newly synthesized

666

cell wall components, and here GPS2 could have a role, since it is an enzyme

667

responsible for the biosynthesis of cellulose, one of the main components of plant cell

668

wall (Bewley et al., 2013).

669

Annexins are multifunctional lipid-binding proteins and play important roles in

670

various biological processes (Mortimer et al., 2008; Laohavisit and Davies, 2011).

671

Many evidences have pointed toward a role of annexins in cell elongation. Expression

672

of annexins has been detected in the root elongation zone of maize (Carroll et al., 1998)

673

and Arabidopsis (Clark et al., 2005a, b) and is associated with cotton fibre elongation

674

(Yang et al., 2008) and expansion of the tomato pericarp (Faurobert et al., 2007) and

675

tobacco cells (Seals and Randall, 1997). Hayes et al (2006) found that annexin 2

676

interacting with actin plays a role in regulating acitin filament dynamics. Annexins have

677

also been proposed to function in the Golgi-mediated secretion of cell wall and plasma

678

membrane materials (Mortimer et al., 2008; Laohavisit and Davies, 2011). Other 23 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

679

important functions of annexins include peroxidase activity and ion transport and Ca2+

680

signalling (Mortimer et al., 2008; Laohavisit and Davies, 2011). In view of these roles,

681

it seems reasonable that it is important for seed germination. Consistent with this view,

682

Lee et al (2004) reported that mutants of AtAnn 1 and AtAnn 4 display delayed

683

germination and hypersensitivity to osmotic stress and ABA during germination.

684

ADH1 increased in abundance in germinated 15G, 25/15G and 25nG seeds, but not

685

in ungerminated 25G seeds (Table 1). The mRNA of ADH1 increased more than 30-fold

686

in 15G, 25/15G and 25nG seeds at >24 HI, but kept relatively constant in 25G seeds

687

(Fig. 4). ADHs catalyze the irreversible oxidation of a wide range of reactive aldehydes

688

to their corresponding carboxylic acids (Perozich et al., 1999). ADHs might play a

689

pivotal role in detoxifying the aldehydes generated by reactive oxygen species (ROS),

690

e.g. by lipid peroxidation during seed germination. ROS production is an unavoidable

691

process during seed germination (Bailly, 2004). In plant cells, the mitochondrial

692

electron transport chain is a major site of ROS production (Møller et al., 2001). During

693

seed germination, activation of mitochondrial respiration will result into the production

694

of ROS (Bailly, 2004). ADHs may also have a role in the response to hypoxia during

695

seed germination. After imbibition, the inside of the seed may become hypoxic. In pea

696

seeds, hypoxia was detected after 7 h of imbibition due to both oxygen diffusion

697

limitation caused by the seed covering layers and an increase in the rate of respiration

698

(oxygen consumption) (Rolletschek et al., 2009). Fermentation occurring during

699

hypoxia will lead to production of toxic acetaldehyde, which is converted to acetic acid

700

by ADHs. When the seeds germinated, the broken seed coat increases the oxygen

701

diffusion into the inner seeds. Reoxygenation of the hypoxia tissues will induce the

702

production of ROS and aldehydes (Magneschi and Perata, 2009). Therefore,

703

accumulation of ADH in the germinated seeds might protect the seeds from the damage

704

caused by these toxic compounds.

705

Glycolysis is activated during seed germination in many species (Weitbrecht et al.,

706

2011). In lettuce seeds, both protein accumulation and gene expression of a glycolytic

707

enzyme, BIPM (spot 298) were more abundant in well germinated 15G, 25/15G and

708

25nG seeds than in dry and poorly germinated 25G seeds (Table 1, Fig. 4F). 24 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

709

We observed that ATF and CVS with unclear functions varied greatly not only in

710

protein accumulation, but also in gene expression between the germinated and

711

ungerminated seeds (Table 1, Fig. 4, O and P). Therefore, it will be interesting to study

712

the function of these proteins and their roles in seed germination.

713 714

Involvement of the MVA pathway for isoprenoid synthesis in seed germination

715

Isoprenoids represent the functionally and structurally most diverse group of

716

metabolites, with over 50,000 natural products identified in extant organisms (Vranová

717

et al., 2013). All isoprenoids are derived from a core biosynthetic precursor isopentenyl

718

pyrophosphate (IPP), which is synthesized via two independent pathways: the MVA

719

pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Isopronoids

720

derived from the MVA pathway are responsible for production of cytokinins,

721

sesquiterpenes, triterpenes, phytosterols and BRs, which are involved in the regulation

722

of many biological processes, such as membrane biogenesis and growth and

723

development (Newman and Chappell, 1999; Vranová et al., 2013). The isoprenoids

724

farnesyl diphosphate and geranylgeranyl diphosphate are also used for protein

725

prenylation, a process that regulates several developmental processes in plants (Galichet

726

and Grulssem, 2003; Crowell and Huizinga, 2009).

727

In lettuce seeds, proteomic, genetic and physiological analyses all point towards an

728

involvement of MVA pathway for isoprenoid biosynthesis in seed germination. Three

729

proteins (AACT1, HMGS2, MDPC2) participating in this pathway were identified to

730

have significant differences in abundance between the germinated and ungerminated

731

seeds (Fig. 5). mRNA levels of AACT1 and HMGS2 consistently correlated with protein

732

accumulation, and expression of HMGR3, MK and PMK2 involved in this pathway also

733

had a positive correlation with the seed germination ability (Fig. 5). At the same time,

734

lovastatin inhibition of isoprenoid biosynthesis via the MVA pathway delayed seed

735

germination at 15oC, but it did not fully inhibit it (Fig. 6A). One possible explanation is

736

that isoprenoids, or their derived products, are not absolutely required for seed

737

germination. Another possibility is that isoprenoids from the MEP pathway compensate

738

for the decreased biosynthesis via the MVA pathway, because there is cross-talk 25 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

739

between the MVA and MEP pathways of isoprenoid biosynthesis (Laule et al., 2003). It

740

has been reported that overexpression of Brassica juncea HMGS1 in Arabidopsis

741

increases the seed germination rate (H. Wang et al., 2012). These results suggest that

742

isoprenoids from the MVA pathway may play a role in regulation the germination

743

ability of lettuce seed at different temperatures. Cytokinins produced from this pathway

744

are potentially responsible for lettuce seed germination ability at different conditions as

745

the cytokinins KT, 6-BA and ZT partially reversed the lovastatin inhibition of seed

746

germination (Fig. 6C) and released the thermoinhibition of lettuce seed germination at

747

25oC (Fig. 6D). Although BRs, one of the end products of the MVA pathway have been

748

reported to promote seed germination in many species, including Arabidoposis (Steber

749

and McCout, 2001) and tomato (Leubner-Metzger, 2001), we did not observe any effect

750

of this chemical on lettuce seed germination (data not shown).

751

Application of KT, 6-BA or ZT did not fully reverse the inhibition of germination

752

by lovastatin. It is possible that other products derived from the MVA pathway are also

753

involved in lettuce seed germination. Several studies have shown that protein

754

prenylation plays an essential role in seed germination (Culter et al., 1996; Galichet and

755

Grulssem, 2003; Crowell and Huizinga, 2009 ). Substrates for protein prenylation,

756

includes farnesyl diphosphate or geranylgeranyl diphosphate are derived from the MVA

757

pathway. Cutler et al. (1996) reported that knockout mutations in Arabidopsis ERA1, a

758

gene encoding the β-subunit of protein farnesyl transferase, cause a prolonged seed

759

dormancy due to an enhanced response to ABA. They proposed that protein

760

farnesylation may be essential for negative regulation of ABA signaling in seeds (Cutler

761

et al., 1996). Therefore, it was not unexpected to observe that lovastatin inhibition of the

762

MVA pathway increased the sensitivity of germination to ABA (Fig. 6B). ABA plays a

763

major role in preventing seeds germinating at the thermoinhibitory temperature (Gonai

764

et al., 2004; Tamura et al., 2006; Argyris et al., 2008; Huo et al., 2013). In lettuce seeds,

765

a thermoinhibitory temperature decreased the accumulation of proteins and expression

766

of the genes in the MVA pathway (Fig. 5). This might decrease the production of

767

isoprenoids and thus promote the effect of ABA on inhibiting seed germination.

768 26 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

769 770

CONCLUSIONS

771

PEG fractionation increased the number of protein spots detected on the 2-D gels,

772

especially in the areas where the storage proteins dominated. Many proteins increasing

773

in abundance were found in all of the germinated seeds, but not in the ungerminated

774

seeds, while proteins decreasing in abundance were found in both the germinated and

775

ungerminated seeds, indicating the necessity for protein synthesis in seed germination.

776

The accumulation of 12 proteins and their mRNA correlated positively with the ability

777

of the seeds to germinate. The proteins are involved in Met metabolism, ethylene

778

production, lipid mobilization, cell elongation, glycolysis, isoprenoid biosynthesis and

779

detoxification of aldehydes, which means that these processes are directly or indirectly

780

involved in seed germination. The thermoinhibitory temperature repressed the activation

781

of these processes in lettuce seeds during imbibition, which might prevent the seeds

782

from germinating. Protein accumulation, gene expression and effect of lovastatin on

783

seed germination all suggest an involvement of the MVA pathway for isoprenoid

784

biosynthesis in seed germination and thermoinhibition. Production of cytokinins from

785

this pathway could influence the germination ability of lettuce seeds. However, the

786

MVA pathway can produce a large number of bioactive products, some of which, such

787

as farnesyl diphosphate and geranylgeranyl diphosphate may also play the role in seed

788

germination. In addition, there is a cross-talk between the MVA and MEP pathways

789

(Laule et al., 2003). The role of MVA pathway in seed germination is therefore quite

790

complex.

791 792 793

MATERIALS AND METHODS

794 795

Plant material

796

Lettuce (Lactuca sativa L. cv. ‘Jianyexianfeng No. 1’) seeds were kindly provided by

797

Mr. Guangji liu (Zhongdu Sichuan Co. Ltd, Chengdu, Sichuan, China). Seeds were

798

stored at –20°C in an aluminum foil bag until use for the following study. 27 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

799 800

Seed germination

801

Three replicates of 50 seeds each were placed on two layers of filter paper in closed

802

90-mm diameter Petri dishes and incubated at 15oC (15G), 25oC (25G) or 15oC after

803

imbibition at 25oC for 48 h (25/15G) in 4.5 ml distilled water and at 25oC in 4.5 ml 10

804

mM KNO3 (25nG) in 14 h light (66.3 μmol m−2 s−1) and then continuous darkness. To

805

examine the effect of lovastatin (an inhibitor of HMGR, National Standard Materials

806

LTD., Beijing, China, 98% purity), ABA and ABA plus lovastatin on seed germination,

807

seeds were imbibed in 0, 10, 50 and 100 μM lovastatin, 10 μM ABA or 10 μM ABA

808

plus 10 or 100 μM lovastatin at 15oC under conditions as above. To examine whether

809

MVA pathway derived products could reverse the inhibition of germination by

810

lovastatin, seeds were imbibed in 10 and 50 μM KT (Sigma, Wuxi, Jiangsu, China),

811

6-BA (Sigma, Jiangsu, China), ZT (Sigma, St. Louis, MO, USA) and TDZ (sigma, St.

812

Louis, MO, USA), 0.001, 0.01 and 0.1 μM stigmasterol (J&K Scientific Ltd., Beijing,

813

China), β-sitosterol (J&K Scientific Ltd., Beijing, China) and campesterol (J&K

814

Scientific Ltd., Beijing, China) and 0.001, 0.01 and 0.1 μM brassinolide (Santa Cruz

815

Biotechnology, Inc. Santa Cruz, CA, USA) plus 100 μM lovastatin at 15oC under

816

conditions as above. Seeds were also imbibed in the same concentration of above MVA

817

pathway derived products at 25oC under conditions as above. A seed was considered to

818

be germinated when protrusion of radicle from the seed coat was observed. The

819

germinated seeds were sampled within 2 h of their germination as described in Results

820

(Fig. 1, Supplemental Fig. S2). Germination rate was considered as the reverse time to

821

reach 50% germination.

822 823

PEG fractionation of total extractable proteins

824

Extraction of total protein

825

Three replicates of 200 seeds each were ground in a pre-chilled mortar and

826

homogenized in 3 ml of ice-cold protein extraction buﬀer containing 0.5 M Tris–HCl

827

(pH 7.8), 2% (v/v) NP-40, 20 mM MgCl2, 2% (v/v) β-mercaptoethanol, 1 mM

828

phenylmethylsulfonyl fluoride, 1 mM ethylenediamine tetraacetic acid and 1% (w/v) 28 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

829

Polyvinylpolypyrrolidone. The homogenate was centrifuged at 16, 000 g at 4°C for 15

830

min. After discarding the pellets, the supernatant was further centrifuged at 32, 000 g at

831

4°C for 15 min. The pellets were discarded, and the supernatant contains the total

832

extractable protein.

833

PEG fractionation

834

PEG fractionation was performed according to Acquadro et al (2009). The flow chart of

835

PEG fractionation procedure is shown in Fig. 2A. Total extractable proteins (TP) was

836

added PEG4000 up to 4% (w/v) concentration, the mixture was shaken on ice for 30

837

min, and then centrifuged at 16, 000 g at 4°C for 15 min. The pellet (P4) was saved and

838

PEG4000 up to 8% (w/v) was added to the supernatant (S4), the mixture was shaken

839

and centrifuged as above. The pellet (P8) was saved and the supernatant (S8) was

840

treated as S4 to produce the 12% PEG fractions P12 and S12 (Fig. 2A). Samples from

841

TP, from supernatants, S4, S8 and S12, and pellets, P4, P8 and P12, were precipitated

842

with five volumes of cold 10% (w/v) trichloroacetic acid/acetone at –20oC for more

843

than 6 h. The pellets were rinsed four times with ice-cold acetone containing 13 mM

844

dithiothreitol (DTT) and then lyophilized overnight at –20oC. All the these samples

845

were subjected to one-dimensional gel electrophoresis (1-DE) and the samples TP and

846

S8 were also subjected to 2-DE analysis. The protein concentration in each sample was

847

determined by the Bradford (1976) method using bovine serum albumin as the standard.

848 849

1-DE

850

For 1-DE analysis, each fraction, supernatants and pellets, was mixed with sample

851

buffer (Laemmli 1970) and heated at 95°C for 4 min. Discontinuous

852

SDS-polyacrylamide gels (12% separation gel, 5% stacking gel) were prepared and run

853

according to Laemmli (1970) using a Mini-Protein II electrophoresis cell (Bio-Rad,

854

Hercules, CA, USA).

855 856

2-DE

857

Isoelectrofocusing (IEF) was carried out using a PROTEAN i12 IEF system (Bio-Rad, 29 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

858

Hercules, Piscataway, NJ, USA) and 17 cm Immobiline Dry Strips with a linear pH

859

gradient of 5–8 (Bio-Rad). Sample, 500 μg protein, was loaded onto the gel strip. The

860

second dimension was carried out in a 12% SDS-polyacrylamide gel. Electrophoresis

861

was performed at 15ºC in SDS electrophoresis buffer (pH 8.3), containing 25 mM Tris

862

base, 192 mM glycine and 1% (w/v) SDS.

863 864

Image analysis, in-gel digestion with trypsin and protein identification by

865

MALDI-TOF-TOF mass spectrometry

866

Gels (1-D and 2-D) were stained overnight with 0.25% (w/v) CBB R-250 in 5:1:4 (v/v)

867

methanol: acetic acid: water and destained in 2:1:7 (v/v) methanol: acetic acid: water

868

solution with 3–5 changes of the solution, until a colorless background was achieved.

869

The stained gel was scanned at a 300 dpi resolution with a UMAX Power Look 2100XL

870

scanner (Maxium Tech., Taipei, China). Spot detection and gel comparisons were made

871

with ImageMaster 2D Platimum, version 5.01 (GE Healthcare Bio-Science, Little

872

Chalfont, UK). After automated detection and matching, manual editing was carried out

873

to correct the mismatched and unmatched spots.

874

Well-separated gels of the four independent biological replicates for each treatment

875

were used for proteomic comparisons. Spots were considered reproducible when they

876

were well resolved in at least in three biological replicates. The normalized volume

877

(based on the total spot volumes) of each spot was assumed to represent its protein

878

abundance. When comparing spot size between groups, a difference was considered

879

significant when the change was >2.0-fold and P 20)) and

909

of matched peptides (the peptides matched by the peptide mass fingerprinting (PMF))

910

and theoretical protein mass and pI found in the databases are shown in Supplemental

911

Tables S6 and S7.

912 913

Gene expression analysis

914

The best matched EST for a given protein spot was searched against NCBI unigene

915

database to find a corresponding unigene, which was then used for primer design. For

916

genes encoding the enzymes of MVA pathway, the candidate gene sequences

917

corresponding to the best BLAST hit were identified within the NCBI Transcriptome 31 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

918

Shortgun Assembly (TSA) database through sequence homology to known genes in A.

919

thaliana (Vranová et al., 2013). Primer sequences were designed using Primer Premier

920

6 (Applied Biosystems) to amplify 80- to 250-bp PCR amplicons for qRT-PCR analyses.

921

Genes and primers used for qRT-PCR analyses are shown in Supplemental Tables S12

922

and S13.

923

Total RNA was extracted from dry or imbibed lettuce seeds using SV Total RNA

924

Isolation System (Promega, Madison, WI, USA). RNA quality was assessed by gel

925

analysis and 260:280-nm and 260:230-nm spectrophotometric absorbance ratios. Two

926

μg of total RNA was reversely transcribed using random primers (TransScript®

927

First-Strand cDNA Synthesis SuperMix, TransGen Biotech www.transgen.com.cn,

928

Beijing, China). cDNAs from housekeeping ubiquitin ligase gene and genes of interest

929

were PCR amplified in an Agilent Mx3000P Real-Time PCR System using KAPA

930

SYBR® FAST qPCR Kits (Kapa Biosystems, www.kapabiosystems.com/, Cape Town,

931

South Africa). The relative quantification of the transcript levels was performed using

932

the comparative Ct method (Livak and Schmittgen, 2001). Three biological plus two

933

technical replications were conducted for each sample.

934 935

Supplemental Data

936

Supplemental Figure S1. Protein bands in 1D gel and spots in 2D gel of 15G seed

937

subjected to identify by MALDI-TOF-TOF.

938

Supplemental Figure S2. Flow chart of proteomic analysis of lettuce seed germination

939

and thermoinhibition.

940

Supplemental Figure S3. Amplication plots, dissociation curve and standard curve for

941

RT-PCR analysis of ACOx gene expression.

942

Supplemental Figure S4. 1-D gel maps of total extractable protein and supernatant and

943

pellet after 8% PEG precipitation.

944

Supplemental Figure S5. Effect of ethephon (A, 25oC), CoCl2 (B, 15oC) and

945

2,5-norbornadiene (C, 15oC) on germination of seeds.

946

Supplemental Table S1. Identification of storage proteins in the 1-D (Fig. 1S left) and

947

2-D (Fig. 1S right) gel of 15G seeds. 32 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

948

Supplemental Table S2. Protein contents and yields of various PEG precipitation

949

fractions.

950

Supplemental Table S3. Spot numbers in 2-D gel before and after PEG precipatation.

951

Supplemental Table S4. Total protein content and protein yield after 8% (w/v) PEG

952

fractionation of total protein extracted from seeds under different germination

953

conditions.

954

Supplemental Table S5.Volume (%) of protein spots accumulated differentially under

955

different germination conditions and the ratio and associated P values of comparison.

956

Supplemental Table S6. Differentially accumulated protein spots identified to match to

957

one protein when searched in the plant EST or NCBInr database.

958

Supplemental Table S7. Protein spots identified to match to more than one protein.

959

Supplemental Table S8. Proteome changes in seeds during germination at 15oC

960

(germinated 15G seeds versus dry seeds).

961

Supplemental Table S9. Proteome changes in seeds germinated at 25oC (ungerminated

962

25G seeds versus dry seeds).

963

Supplemental Table S10. Proteome changes in seeds during germination at 15oC after

964

imbibition at 25oC for 48 hours (germinated 25/15G seeds versus ungerminated 25G

965

seeds).

966

Supplemental Table S11. Proteome changes in seeds during germination at 25oC in

967

KNO3 (germinated 25nG seeds versus dry seeds).

968

Supplemental Table S12. Primer design for genes encoding candidate proteins

969

involved in seed germination and thermoinhibition.

970

Supplemental Table S13. Primer design for genes encoding enzymes of the MVA

971

pathway for isoprenoid biosynthesis.

972 973

ACKNOWLEDGMENTS

974

We are grateful to Dr. Zhuang Lu (Institute of Botany, Chinese Academy of Sciences)

975

for doing the mass spectrometry analyses.

976 977

LITERATURE CITED 33 Downloaded from www.plantphysiol.org on March 19, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Table 1. Candidate proteins involved in lettuce seed germination and thermoinhibition. Spot ID

Best matching EST Acc.

Homologous protein

Biological process

682

DW133353

S-adenosyl-L-methionine synthetase

707

DW137691

713

aExp.

level D-25G-15G25/15G-25nG

Spot ID

Best matching EST Acc.

Homologous protein

Biological process

Amino acid metabolism

1411

DW114752

11S globulin precursor isoform 4

Storage protein

S-adenosyl-L-methionine synthetase


1414

BQ868270

PREDICTED: 11S globulin subunit beta-like

Storage protein

DW137090

S-adenosylmethionine synthase 2


1070

DW064597

1-aminocyclopropane-1carboxylate oxidase 1

Cell growth

126

BQ869880

lipoxygenase 2

Lipid & sterols metabolism

807

DY984395

Cell structure

783

DY974670

PREDICTED: acetyl-CoA acetyltransferase, cytosolic 1

Lipid& sterols metabolism

1431

DW134585

PREDICTED: alpha-1,4glucan-protein synthase [UDP-forming] 2 actin depolymerizing factor 2

298

DW129004

2,3-bisphosphoglycerate-indepen dent phosphoglycerate mutase

Glycolysis

424

DY960524

aldehyde dehydrogenase 1

Detoxification

290

BQ864080

isocitrate lyase

Glyoxylate cycle

898

DY984667

PREDICTED: probable aldo-keto reductase 2-like

Stress response

1243

BQ844772

chloroplast chaperonin 21

Protein folding

981

DW132409

annexin 1

Stress response

1012

DW139333

7S globulin

Storage protein

1069

DY979528

dehydrin 2

Stress response

1388

DW114752


Storage protein

420

BU012851

acyltransferase homolog

Unclassified

1406

BQ848945


Storage protein

800

DW127960

clavaminate synthase-like protein

Unknown


Cell structure

aExp.

level D-25G-15G25/15G-25nG

Acc: Accession number. Exp level: expression level. a: protein expression levels in dry (D), the ungerminated 25Gseeds (seed imbibed at 25oC) at 48 hours of imbibitions (HI) and germinated 15G (seed imbibed at 15oC), 25/15G (seed imbibed at 15oC after imbibition at 25oC for 48 h) and 25nG (seed imbibed at 25oC in KNO3) seeds are shown as the percentage of normalized volume ×100. The columns in the graph are arranged as following order: D-25G-15G-25/15G-25nG.


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Figure 1. Time courses of lettuce seed germination under different conditions. The seeds were imbibed in water at 15oC (●) and 25oC (○) or in 10 mM KNO3 at 25oC (♦) in 14 h light (66.3 μmol m-2 s-1) and then continuous darkness. The ungerminated seeds at 25oC for 48 h were transferred to 15oC for a further germination in 14 h light (66.3 μmol m-2 s-1) and then continuous darkness (▲). Data are mean±SE (n=3).


Figure 2. PEG precipitation of highly abundant proteins in lettuce seeds. A, Flow chart of PEG fractionation; B, 1-D SDS-PAGE of unfractionated and fractionated samples; C–E, 2-DE maps of unfractionated and fractionated samples. TP, total extractable protein; S4, S8 and S12, the supernatants of the fractionated samples with 4, 8 and 12% PEG, respectively; P4, P8 and P12, the pellets of the fractionated samples with 4, 8 and 12% PEG, respectively. The boxed areas of a and b show the regions where storage protein are dominant and where a significant change was visible after PEG precipitation. Seeds germinated at 15oC were used as material for developing the PEG fractionation method.


Figure 3. Differentially accumulated proteins during lettuce seed germination. A, Representative images of the Coomassie Brillant Blue (CBB) R-250-stained gel from 25nG seeds. A total of 600 μg of proteins was extracted and separated by 2-DGE and visualized with CBB R-250. Seeds were imbibed in water at 15oC (15G), at 25oC (25G) and at 15oC after imbibition at 25oC for 48 h (25/15G) and in 10 mM KNO3 at 25oC (25nG). The dry seeds, germinated 15G, 25/15G and 25nG seeds, ungerminated 25G seeds were subjected to 2-DGE and compared to each other. Spots (indicated by a red arrow and a number) showing a significant change of more than 2-fold (P24’ on horizontal axis) and from the germinated 15G, 25/15G and 25nG seeds (shown as ‘>24’ on horizontal axis). Selected candidate proteins (Table 1) and their encoding genes are: A-C, S-adenosyl-L-methionine synthetase (spot 682, SAM1a; spot 707, SAM1b; spot 713, SAM2); D, lipoxygenase 2 (spot 126, LOX2); E, predicted: acetyl-CoA acetyltransferase, cytosolic 1 (spot 783, AACT1); F, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (spot 298, BIPM); G, isocitrate lyase (spot 290, ICL); H, chloroplast chaperonin 21 (spot 1243, CNP21); I, 1-aminocyclopropane-1-carboxylate oxidase 1 (spot 1070, ACOx); J, predicted: alpha-1,4-glucan-protein synthase [UDP-forming] 2 (spot 807, GPS2); K, actin depolymerizing factor 2 (spot 1431, ADF2), L, aldehyde dehydrogenase 1 (spot 424, ADH1), M, predicted: probable aldo-keto reductase 2-like (spot 898, AKR2); N, annexin 1 (spot 981, ANN1); O, acyltransferase homolog (spot 420, ATF); P, clavaminate synthase-like protein (spot 800, CVS). Note that different scales are used along the y-axis in the different panels. Data are mean±SE (n=3).


Figure 5. Accumulation of proteins and expression of genes related to mevalonate (MVA) pathway for isoprenoid biosynthesis. Protein accumulation and gene expression are shown to the left and right of the MVA pathway, respectively. Dry (D), the ungerminated 25G (seed imbibed at 25oC) at 48 h of imbibition (HI) and germinated 15G (seed imbibed at 15oC), 25/15G (seed imbibed at 15oC after imbibition at 25oC for 48 h) and 25nG (seed imbibed at 25oC in KNO3) seeds were subjected to 2-DGE analysis. Three proteins in the MVA pathway, predicted: acetyl-CoA acetyltransferase, cytosolic 1 (AACT1) (A, spot 783), HMG-CoA synthase 2 (HMGS2) (C, spot 650) and mevalonate decarboxylase (MDPC2) (H, spot 632) were identified to be differentially accumulated by the 2-DGE analysis. Accumulation of those proteins in each treatment is arrayed from left to right as: D, 25G, 15G, 25/15G and 25nG. The protein accumulation level was calculated as in Table 1. Genes encoding MVA pathway enzymes, including three AACTs (B), two HMGSs (D), three HMG-CoA reductases (E, HMGR), one MVA kinase (F, MK), two phospho-MVA kinases (G, PMK) and two MDPCs (I) in lettuce were selected for gene expression analysis. mRNA were extracted from 15G, 25G, 25/15G or 25nG seeds at 24 HI (white bar), from ungerminated 25G seeds at 48 HI and from germinated 15G, 25/15G and 25nG seeds (black bar, labeled ‘>24 HI’ in the heading). Lovastatin is an inhibitor of HMGR in the MVA pathway. The data of gene expression are arrayed as: 25G, 15G, 25/15G and 25nG seeds. Data are plotted as mean±SE (n=3). Note that gene expressions of HMGR3, PMK1, PMK2 and MDPC1 were down regulated during imbibition under all conditions (c, b>c, Rx(15G/25G)=c/c=1

24

When PEG was added to protein sample, protein X would be precipitated in both

25

15G and 25G until reaching to a concentration of c mg/ml.

26

Case 2) when a>c, b=c, Rx(15G/25G)=c/c=1

27

Protein precipitation only occurred in 15G seeds.

28

Case 3) when a>c, b

Effects of salinity, temperature, and polyethylene glycol on the seed germination of sunflower (Helianthus annuus L.).

Morphactin-kinetin interaction in lettuce seed germination and seedling growth.

Lettuce seed germination: A phytochrome-mediated increase in the growth rate of lettuce seed radicles.

Germination of lettuce induced by inhibitors of protein synthesis.

Transcriptome Analysis of the Effects of Shell Removal and Exogenous Gibberellin on Germination of Zanthoxylum Seeds.

Reversal by pressure of seed germination promoted by anesthetics.

Effect of light and temperature on the germination of lettuce seeds.

Activation energy and germination times for isothermal seed germination.

A histone methyltransferase inhibits seed germination by increasing PIF1 mRNA expression in imbibed seeds.

Immediate phytochrome action in lettuce seeds and its interaction with gibberellins and other germination promoters.

Interactions between ethylene-bis-nitrourethane and Gibberellic acid during the germination of lettuce seed.

Kinetics and thermodynamics of isothermal seed germination.

Proteomic analysis reveals a role of melatonin in promoting cucumber seed germination under high salinity by regulating energy production.

In-depth proteomic analysis of rice embryo reveals its important roles in seed germination.

Germination and ultrastructural studies of seeds produced by a fast-growing, drought-resistant tree: implications for its domestication and seed storage.

An action spectrum in the blue for inhibition of germination of lettuce seed.

Dependence of gibberellic acid-induced dark germination of lettuce seed on RNA synthesis.

Effects of origin, seasons and storage under different temperatures on germination of Senecio vulgaris (Asteraceae) seeds.

Seed biology and in vitro seed germination of Cypripedium.

Hydroquinone; A Novel Bioactive Compound from Plant-Derived Smoke Can Cue Seed Germination of Lettuce.

Lipases in the storage tissues of peanut and other oil seeds during germination.

Action spectra for light-induced germination in dormant lettuce seeds : I. Red region.

The seed germination model of enzyme catalysis.

Changes in germination and respiratory potential of embryos of dormant Grand Rapids lettuce seeds during long-term imbibed storage, and related changes in the endosperm.