Soluble Sugar Accumulation Can Influence Seed Size via AN3âYDA

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Soluble Sugar Accumulation Can Influence Seed Size via AN3–YDA Gene Cascade Lai-Sheng Meng, meng-ke xu, Dan Li, Ming-Ming Zhou, and Jihong Jiang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 11, 2017

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Journal of Agricultural and Food Chemistry

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Soluble Sugar Accumulation Can Influence Seed Size via

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AN3–YDA Gene Cascade

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Runing title: soluble sugar accumulation controls seed size

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Lai-Sheng Meng1＊,Meng-Ke Xu1 , Dan Li1, Ming-Ming Zhou1 and Ji-Hong Jiang1＊

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1. The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province,

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School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116,

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People’s Republic of China.

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＊Corresponding author: e-mail: [email protected]; [email protected];

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ABSTRACT

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In higher plants, seed size is central to a lot of aspects in evolutionary fitness and is a

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crucial agricultural trait. In this study, Arabidopsis an3 (angustifolia3) mutants

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present with increased seed size. Target-gene analysis revealed that YDA, which

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encodes a mitogen-activated protein kinase kinase kinase, is a target gene of AN3.

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Indeed, the loss of YDA function decreases seed size. Furthermore, AN3 and YDA

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mutations both disrupt normal sucrose and glucose contents and cause altered seed

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size in an3 or yda mutants. With these results, we provide a molecular model where

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soluble sugar accumulation might affect seed size regulation via the AN3–YDA gene

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cascade. Our findings aid to guide the synthesis of a model that predicts the

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integration of soluble sugar accumulation at AN3 to control the establishment of seed

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size. 1

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KEY WORDS: Arabidopsis, ANGUSTIFOLIA3 (AN3), Seed Size, YODA (YDA),

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Sugar Content.

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INTRODUCTION

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Seed size is regulated by three main components, namely, the seed coat, the

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endosperm and the embryo, which all originate from distinct the ovule cells. They

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have obviously different complements of paternal and maternal genomes.

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Development of seeds shows a double-fertilisation process in angiosperms, wherein

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one sperm nucleus and one egg cell fuse for producing a diploid embryo. By contrast,

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the nucleus of the other sperm combines with two polar nuclei for producing the

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triploid endosperm1. Mature Arabidopsis seeds have only one layer cell of endosperm

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that surrounds the embryo. Additionally, maternal integument produces the seed coat

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that surrounds the endosperm. Thus, the coordinated growths of zygotic tissues and

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maternal sporophytic determine the seed size.

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Mutation with the triple cytokinin receptor causes seeds to have twice wild type

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the mass, and cytokinin might modulate embryo mass through a endospermal and/or

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maternal mechanism2,3. Enhancing both embryonic cell number and size increases the

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seed size of the apetala2 (ap2) and auxin response factor2 mutants; moreover, seed

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properties are determined by the maternal sporophyte and endosperm genomes3–5.

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SHORT HYPOCOTYL UNDER BLUE1 (SHB1) is recruited to HAIKU2 (IKU2) and

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the MINISEED3 (MINI3) promoters via an unknown transcription factor regulating

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endosperm development; therefore, the shb1-D mutant has increased seed size,

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whereas the shb1-KO mutant has a slightly reduced seed size6.

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In this work, Arabidopsis an3 has a large seed size. Further analysis revealed that

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AN3 is associated with the promoter of YDA (a MAPKKK gene) during seed

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development. Indeed, we found that yda mutants have reduced seed size. At the

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cellular level, the changes in embryo sizes in an3 and yda are due to the alternations

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of their embryo cell sizes instead of cell number. And genetic analysis indicated that

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AN3–YDA forms a sugar-mediated gene cascade for seed size regulation. With these

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results, we proposed a model where normal sugar content controls normal cell

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elongation via a sugar-specific AN3–YDA gene cascade. Conversely, the lack of AN3

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induces the alternation of the sugar content, which causes the change of cell

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elongation, and finally leads to the change of seed size. Collectively, these insights

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into the molecular mechanism support new findings for various interests for cell

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biologists, ecologists, evolutionary biologists, agronomists and molecular biologists.

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MATERIALS AND METHODS

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Plant materials and growth conditions

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an3-4, an3-1, yda-1, emb71, and yda-107-10mutants, and 35S:AN3:3XGFP

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transgenic plants in Col-0 background have been described previously. yda-1, an3-1,

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ap2 (SALK_071140), iku2-4 (SALK_073260) and shb1 (SALK_128406) were gained

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from Ohio State University and obtained mutants (an3-4, 35S:AN3:3XGFP, and yda-

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10) have been validated, and their homozygous mutants were gained via using

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herbicide selection for a few generations and analyzing segregation ratios). The seeds

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of 35S:AN3:3XGFP lines and an3-4 mutant lines were provided via Professors G.

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Horiguchi (Rikkyo University, Japan) and H.G Nam (DGSIT, Korea), respectively.

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The plasmid of pHB-YDA:GFP and the seed of yda-10 mutant lines were provided via

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Prof H.Q Yang (Shanghai Jiao Tong University).

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The mutant of an3yda was obtained from F2 seedlings of yda-10 X an3-4 . This

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double mutant is screened based on mature stomata of in the 8-day-old cotyledons are

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developed constitutively and produced in clusters in the dark9 and had narrow rosette

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leaf blades of seedlings grown on white light (16 light/ 8 dark)7. This method has been

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described by9. Transgenic seedlings were obtained via using the floral dip method

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mediated by Agrobacterium tumefaciens12.

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Seedlings presenting the an3-4 phenotype (narrow rosette leaf blades of

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seedlings grown on white light [16 light/ 8 dark]7 in the F2 populations) were

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screened for ProYDA:GUS expression in roots. Seeds of F3 lines were obtained from

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those showing expression, and seedlings expressing GUS in all F3 were subsequently

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analyzed, as described by13. Condition of Arabidopsis plant growth was described

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by14,15.

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Plasmid constructs

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Plasmid Constructs AN3 (At5g28640) and YDA (At1G63700) were described

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previously13.

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GUS assay

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GUS assay was described by14, 15. By using a buffer mix (60 mM NaPO4 buffer, 1

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mM X-gluc, 0.4 mM of K3Fe(CN)6/K4Fe(CN)6) and 0.1% (v/v) Triton X-100, the

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samples were stained and then incubated at 36-38 ° C for 7-8h. Before GUS assay,

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chlorophyll was gradually separated from materials by washes of 30%, 50%, 70%,

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90% and 100% ethanol for about 25-35 min each. GUS staining was previously

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described by14, 15.

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ChIP-PCR

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The transgenic lines containing 35S:AN3:3XGFP in an3-4 mutants were used in this

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experiment. ChIP was performed using developing siliques (13-15 day-old after

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pollination) as materials by using similar methods in13. GFP and HA (a negative

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control)are used in this research, and their uses were described in13. Obtained DNA

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was analyzed via qPCR by five primer pairs, which were synthesized for amplifying

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fragments of about 300 bp DNA in the CDS region and promoter of YDA. The

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sequences of primers were P8- act aat ttt gat tat aac cga taa tt, P7-caa gca aat taa tct

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caa aat gtt, P6-tca aaa gca atc gaa gaa tcc aa, P5- tac aaa gat taa cgc acc aaa gg, P4-

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tca atg tga tct tca acc ta, P3-gct ttc gat ttg att cca ttt caa, P2-gaa aac cct aag tag aac aac,

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and P1-tgt gtc act aac tca ctt cac. The sequences of primers were 5'-ACC GCC ACC

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ACC ACT TCC CA-3' and 5'- GCA GCA AGA TCG GTC GCG GA-3' for CDS

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region of YDA.

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Quantitative RT-PCR analysis

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Total RNA products were extracted from a few tissues via the TRIZOL reagent

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(Invitrogen) described by13. SYBR green was utilized for monitoring the kinetics of

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PCR with the real-time RT-PCR, as has been described by13. For analysizing YDA,

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AP2, SHB1 and IKU2 expressions in an3-4 developing siliques (13-15 day-old after

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pollination), primers below were used. Used primers for AP2 are 5'-ATG TG G GAT

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CTA AAC GAC GC-3' and 5'-ACA AAA CTT AAC ACC AAA CCA GT-3'. Used

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primers for SHB1 are 5'-CAT CCA AGC TTC CCG GAA TAG GTC A-3' and 5'-CCG

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CCG TCT CGA GCC CTT CT-3' . Used primers for IKU2 are 5' -GGT GTC CGG

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AGA GTT CCC ACG A -3' and 5'-CGC TCA TGC AGC TGC TCC CA-3'. Used

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primers for YDA are 5'- ACC GGG TCT CAG GTC GAG GG-3' and 5'-GCA GCA

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AGA TCG GTC GCG GA-3'. To analysis AN3 expression in yda-1,ap2

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(SALK_071140), iku2-4 (SALK_073260) and shb1(SALK_ 128406) developing

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siliques, used primers for AN3 are 5'-GCC TCA GCC ACC AAG TGT GCA T-3' and

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5'-ACCGCC ACCACCACTTCCCA-3'.

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Assay of sugar metabolites

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Metabolites were analyzed as has been described via40, with some revision. Using

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liquid nitrogen, developing seeds at 5, 8 and 11 DAP were pestled, and these powders

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was extracted in 1.0 ml of 80-85% ethanol for about 1-1.5 h. Obtained extract liquid

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was centrifuged at 12,000-14,000 g for 10-12 min. The epipelagic products along with

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relative buffer were transferred to a tube and evaporated under vacuum for dryness for

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50-60 min, and these materials re-dissolved in 600 µl of ddH2O and placed at 65-

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70°C for 15 min.

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Utilizing isoamyl alcohol:chloroform (1:24, v/v), the above aqueous section was

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extracted 2-3 times before HPLC (High Performance Liquid Chromatography) assay.

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Sugars were identified and quantified by chromatography on an Agilent carbohydrate

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column (4.6 × 250 mm, 5 μm) and tested with a refractive index detector (Altex

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156, Altex ScientiWc Inc, CA, USA). Concentrations were measured from peak

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heights utilizing sucrose, fructose and glucose (20 mg/ml) as standard samples16.

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Protein assay

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Protein extracts from 15 mature seeds (per bio-replicates) of an3-4 mutant, wild-type,

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and 35S:AN3:3XGFP transgenic plants in an3-4 mutant background were separated

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on 10% SDS-PAGE and then dyed using Coomassie Brilliant Blue as described by15.

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Cytological experiments

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The seed embryos was performed overnight in buffer (10 mM EDTA, 1% Triton X-

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100, pH 7.0, 1% DMSO and 30 mM sodium phosphate) at 36-38°C, fixed for 1-1.5 h

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in buffer (0.01% Triton X-100, 5% acetic acid, 45% ethanol, and FAA with 10%

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formalin) and, and dehydrated with a series of ethanol, as has been described by15.

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And then the seed embryos were performed for 2 hour in Hoyer’s buffer (0.4:0.8:3 of

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glycerol: water: chloral hydrate). By a HIROX three-dimensional video microscope,

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we observed the treated embryos under relevant magnification. By using Image J

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software, average epidermal cell size and seed or embryo area in the central region of

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embryos were assayed, as has been described by15.

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Enzymatic assay of invertase

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Invertase in Arabidopsis seedlings was extracted and purified based on17. ELISA

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(Enzyme-Linked Immuno Sorbent Assay) was used to assay invertase activity, as has

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described by17. Briefly, we used the plant N invertase activity assay Kit (Sangon,

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Shanghai, China). Based on the manufacturer's protocols, in testing medium (48-well

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plates) with N invertase-antigen, assayed sample (antibody) and normal sample

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were supplemented and incubated in 37°C for 25-30 min. These mixture was swashed

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five times utilizing cleaning solution, and HRP (invertase label) was applied and

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incubated at 37°C for 25-30 min for forming antibody-HRP-antigen complexes. With

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reaction, these mixtures were swashed 5 times utilizing cleaning solution; and then

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TMB -B and TMB -A were supplemented and incubated at 37°C for 8-10 min for

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dyeing. These TMB was catalyzed via HRP, then became blue. With termination

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buffer used, these mixtures became yellow. By using enzyme mark instrument, OD

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values (450nm) was assayed. Finally, compared with a normal sample, the invertase

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activity in assayed samples was measured.

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Localization of invertase activities

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To localize invertase activity on seedlings of Arabidopsis, the method of

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histochemical staining is used in terms of a series of coupled redox reactions, and

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finally produces a precipitate of nonsoluble blue formazan, as we referenced the

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methods of [18] with a few modifications. In 4% formalin (pH 7.0-7.5), 5-day-old

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seedlings of Arabidopsis were fixed for 1 hour. And these above seedlings were

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washed a few times in a few changes of water and overnight for removing entire

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endogenous sugars. With 37°C and under darkness, the washed seedlings were placed

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in a mix buffer (for reaction), which contains 0.015% (wt/vol) phenazine methosulfate,

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15 units of glucose oxidase (TransgeneSA, lllkirch, France), 1% sucrose, and 0.38 M

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disodium hydrogen phosphate (pH 6.0), 0.030% (wt/vol) nitroblue tetrazolium. In

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control reactions, mix buffer did not contain sucrose. After 2 -3 h, the reaction was

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terminated via water washing an doipost, and then they were fixed in 4% formalin for

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30 min.

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RESULTS

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The an3 mutant presented increased seed size

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During our investigation of drought tolerance of an3-4 plants, we found that this plant

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has bigger cotyledons compared with the wild type (Col-0). while it was suggested

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that the an3-4 lines showed bigger embryos and cotyledons than did Col-019 ， 20, the

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influence in seed size was not mentioned. Moreover, these findings only suggest that

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an3-4 lines show bigger cotyledons than do the Col-0. Naturally, large an3-4

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cotyledons should have large seeds because cotyledons are derived from seed

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embryos.

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Both an3-1 and an3-4 are X-ray-induced mutations in the S96 and Col-0

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backgrounds, respectively. These two mutants are large deletions that eliminate the

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AN3 locus. We assayed the seeds of self-pollinated homozygous an3-1 and an3-4

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mutant plants and found that they are about 58% heavier than wild-type control

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(Figures 1A, C, S1A and B). Consistent with seed weight, both length and width in

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the an3-4 seeds are enhanced (Figures 1D and E). To determine whether these large

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seeds are caused by AN3 deletion, we obtained the an3-4 complementary line (the

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35S:AN3:3XGFP transgenic plants in the an3-4 mutant background), which largely

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restores seed size in an3-4 plants (Figures 1A and C–E). The above results indicated

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that AN3 might control seed size. However, the levels of seed defects that are detected

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in an3-4 due to abnormal/no pollination or inherent to the gametophyte or maternal

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tissues need be clarified.

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The an3-4 large seed size is not entirely due to reduced fertility

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Exploring the molecular mechanism underlining seed size control may aid to guide

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crop yield improvements in the future. A big seed size is always relative to changes in

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seed yield; such as, ap2 seedlings show enhanced seed size but have low seed yield5.

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Differently, da1 seedlings have a large seed size and high seed yield20. Therefore, we

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estimate the correlation between yield and increased seed size in an3-4 plants. We

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analysed the elongated silique number, silique length, seed/silique number,

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number, seed/silique weight, and total seed weight and found that the an3-4 lines has

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enhanced seed size; however, most parameters relative to the total seed yield have

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decreased (Figures S4A–F). In these parameters, the decreased elongated silique

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number of an3-4 mutant lines in relation to the number of flowers might be due to

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deficient reproductive development. The reduced an3-4 silique length and leaf and

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flower sizes might be results of AN3 deletion, because AN3 positively regulates cell

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proliferation17,

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decreased in the an3-4 mutant. Overall, in Arabidopsis plants, the AN3 deletion

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results in the formation of larger seeds but lower seed yield than in the wild type

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(Figures S4A–F).

21.

flower

Possibly due to decreased fertility, the seed number/silique was

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Extra resources are allocated for the few but larger seeds that form due to

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decreased fertility5; therefore, we investigated whether the large an3-4 seed size is due

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to extra resource allocation. Six primary inflorescence flowers from an3-4, male-

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sterile mutants (CS4002) and Col-0 were manually pollinated. Manual pollination

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assured that these siliques contained alike seed numbers. By utilizing pollen from the

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same genotype, these flowers were pollinated; whereas in male-sterile plants, Col-0

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pollen was utilized as the donor. With maturity, every male-sterile lines formed six

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siliques. The seed weight of the male-sterile plants was, on average, ~1.2 times than

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that of maternal plants in Col-0 (Figure 1C), implying that reduced fertility increases

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seed yield. By contrast, the seed weight in the an3-4 lines was, on average, ~1.6 times

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higher than that of Col-0 (Figure 1C), revealing that the increase in an3-4 seed size

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was not mainly due to reduced fertility and that AN3 negatively controls the seed size.

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an3-4 large seeds are due to the large embryo caused by embryo cell elongation

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The embryo forms the primary bulk of mature dry seeds in Arabidopsis. To assay

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whether the an3-4 influence on seed mass reflects an enhancement of embryo mass,

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mature embryos from an3-4 mutant and Col-0 seeds were fixed, isolated and

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visualised using the methods of5 . The an3-4 embryo was bigger than that of Col-0

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(Figures 1B and G). Moreover, cytological experiments revealed that the area of

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cotyledon embryo in the an3-4 mutant lines is, on average, ~1.38 times larger than

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that of Col-0 (Figures 1B and G). Embryo size is measured via embryo cell size and

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number. Therefore, epidermal cells were visually examined in the central regions of

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the an3-4 mutant and Col-0 cotyledons. The embryo cells of an3-4 were larger

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(Figure 1B) and the epidermal cell area of an3-4 lines was, on average, ~1.72 times

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larger than that of Col-0 (Figure 1H). Moreover, the cell number in the an3-4

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embryonic axis was lower than that in the Col-0 (Figures 1F and I). Based on these

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findings (i.e., 1.38/1.72 = 0.84), we concluded that this average number of an3-4

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embryo cells was decreased, and the increased an3-4 embryo size was due to the

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increased embryo cell size caused by cell elongation. Therefore, the knockout of AN3

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gene resulted in larger embryo cells and lower cell numbers relative to the wild type.

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The enhanced embryo cell mass in the seed mature phase increased the storage

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reserve accumulation; therefore, we analyzed the two major storage protein levels

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(12S and 2S)5 in mature Arabidopsis seeds. Protein extracts of fifteen seeds each from

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the Col-0, an3-4 + 35S-AN3-3X-GFP, and an3-4 were performed, and 12S and 2S

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storage protein levels were assayed using SDS-PAGE. We found that 12S and 2S

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levels were remarkably higher in an3-4 mutant seeds than that in the Col-0, whereas

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levels of both proteins were mostly restored in an3-4 + 35S-AN3-3X-GFP lines

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(Figure S2). Therefore, overall, the proportion in individual proteins was not

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influenced by the lack of AN3.

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AN3 modulates negatively YDA transcripts with reproductive growth

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AN3 transrepresses YDA during vegetative growth13; therefore, determining whether

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AN3 transrepresses YDA during reproductive growth is needed. We assayed the

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expression levels of YDA and those genetic factors involved in modulating seed mass

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using Q-RT-PCR (quantitative reverse transcription polymerase chain reaction) in the

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developing siliques of Col-0 and an3-4 mutant plants. YDA transcript levels were

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enhanced in an3-4 compared with Col-0 developing siliques (Figure 2A). However,

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IKU2, AP2 and SHB1 expression levels in an3-4 were alike with those in Col-0

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seedlings (Figure 2A). Moreover, AN3 expression was not obviously distinct in the

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wild type and iku2, ap2, yda mutants (Figure 2B). AN3 is strongly expressed in the

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mesophyll of hypocotyls or cotyledons but not in the epidermis in the wild-type

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background22; whereas YDA was obviously expressed in the epidermis of cotyledons

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and hypocotyls in the wild-type background [Figure 2C(a); 8]. However, in the an3-4

268

background, YDA was strongly expressed in the epidermis of cotyledons and

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hypocotyls [Figure 2C(b)]. Moreover, during reproductive growth, ProAN3:GUS was

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weakly expressed in flowers, siliques, mature pollen and embryo (Figures S3A, C, F

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and H); whereas ProYDA:GUS was strongly expressed in flowers, siliques, mature

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pollen, ovule and embryo (Figures S3B, D, E, G and I). The above results revealed

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AN3 regulates negatively YDA expression at the transcriptional levels.

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AN3 associates with YDA promoter in vivo

275

A study for motifs (AN3 Binding Sites in Genome-Wide Determination) revealed the

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identities of two mainly enriched motifs, including the tgaCACGTGgca motif

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containing the TCTC motif (TCTCTCTC) and the core G-box sequence (CACGTG)13,

278

in

279

TCTCTCTCTCTCTCTC) exist between 850 and 950 bp in the promoter of YDA

280

(Figure 2D). Moreover, several amplicons in the promoter of YDA utilized for ChIP

281

(chromatin immunoprecipitation) analysis were exhibited (Figure 2D). To assess this,

282

developing siliques that expressed the 35S:AN3:3XGFP construct were used for a

283

ChIP analysis. Interaction of AN3 and the YDA promoter was assayed in vivo. The

284

antibody of anti-GFP was immunoprecipitated with chromatin relative to AN3-GFP,

285

and Q-RT-PCR assay was measured using specific primers for different regions of the

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YDA promoter (Figure 2D). Anti-HA was utilised as the negative control, and regions

the

peak

sequences.

Two

TCTC

motifs

(TCTCTCTCTC

and

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of Y2 (388–680 bp), Y3 (688–984 bp) and Y4 (1000–1322 bp) primers formed

288

numerous PCR products; whereas no PCR product was detected in the regions of Y1

289

(3–310 bp) and CDS primers (Figure 2E). We observed that Y3 (689–985 bp)

290

contained two TCTC motifs (TCTCTCTCTC and TCTCTCTCTCTCTCTC) in the

291

YDA promoter (Figure 2D;

292

1322 bp) were nearby with, but do not include, TCTC motifs; and regions of the Y1

293

and CDS primers were distal to two TCTC motifs (Figure 2D). As consequence, Y3

294

primer regions lead to the highest PCR product, and the amplification product of PCR

295

was declined with for Y2 or Y4 primer regions, and even more so with those for Y1

296

and CDS primer regions (Figure 2E). The above findings confirmed AN3 to be a main

297

factor to suppress the YDA promoter. AN3 encodes a homolog of the human

298

transcription coactivator SYT and is a putative transcription coactivator7. Therefore,

299

AN3 may act as a cofactor and be interaction with other undiscovered transcription

300

factor and gain its regulation over the transcription of YDA in the initial phase of seed

301

growth and development. A similar function was reported for numerous other proteins:

302

i.e., GIGANTEA (GI), FLOWERINGLOCUS T (FT), KELCH REPEAT, FLAVIN

303

BINDING, F-BOX1 (FKF1) and SHB16,23, 24. Therefore, AN3 is associated with the

304

YDA promoter to suppress YDA expression during reproductive growth and ultimately

305

regulates seed size.

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yda mutants display small seed size

307

AN3 negatively regulates YDA. Moreover, an3-4 mutant plants had bigger seeds than

308

the Col-0. YDA was expressed in embryo tissues, the yda zygote elongation was

). Moreover, regions Y2 (389–681 bp) and Y4 (1000–

13

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suppressed, and the yda embryos grew in a narrow niche of the seed coat8. These data

310

suggested that YDA might regulate seed size. Therefore, we assayed this possibility.

311

We gained two mutants: yda-1/+ (the protein within the catalytic domain is truncated,

312

and is a non-sense mutation) and yda-10 9,25. The seeds of self-pollinated homozygous

313

yda-10 and heterozygous yda-1 mutant plants weighed ~30% lower than the control

314

samples (Figures 3A and E). Moreover, emb71 is an EMS heterozygous mutant of

315

YDA and its homozygous seeds are dark purple (Figure S1C). The seed size of self-

316

pollinated homozygous emb71 plants was dramatically reduced to ~1/10 of wild type

317

(Figure S1C).

318

The small seeds size of yda mutants is due to small embryo cell size

319

The yda mutant zygotes have impaired elongation during embryonic development;

320

and the lengths of these mutant embryos were consistently approximately half those

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of the wild type8. Due to the lack of a suspensor, yda embryos grew in a narrow niche

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of the seed coat above the micropyle and produced a wedge-shaped group of

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isodiametric and irregularly arranged cells 8. These data implied that the small seed

324

size of yda mutants might be due to small embryos. The mature embryos from yda-10

325

and Col-0 seeds were then isolated and visualised. The yda-10 mature embryos were

326

smaller than the Col-0 (Figure 3B). Whether the yda-10 small embryos are caused by

327

embryo cell size or number was investigated. Cytological experiments revealed that

328

the average area of Col-0 cotyledon embryos is ~1.30 times larger than that of yda-10

329

(Figures 3B and C); whereas the area of Col-0 cotyledon embryo cells is, on average,

330

~1.40 times larger than that of yda-10 (Figures 3B and D). Based on these results (i.e.,

15



Page 16 of 42

331

1.40/1.30 3 1.0), we concluded that the yda-10 embryos are small due to the

332

suppressed embryo cell elongation. Concluding that YDA regulates cell size is

333

difficult when development is severely affected using yda-1 and yda-2 mutants as

334

research materials. However, the yda-10 mutant had normal development 9. Therefore,

335

based on these findings, YDA positively modulates the embryo size by regulating

336

embryo cell size.

337

AN3 genetically acts upstream of YDA in modulating seed size

338

Double-mutant analysis was performed by crossing yda-10 (small seeds) with an3-4

339

(large seeds) to assay whether AN3 acts upstream of YDA. Moreover, we selected an

340

an3-4 yda-10 mutant for further analysis. The yda-10 dramatically decreased the

341

an3-4 big seed mass (Figures 3A and F). The an3-4yda-10 mutant and yda-10 had

342

similar seed sizes (Figures 3A and F), indicating that yda-10 is epistatic to an3-4.

343

These findings confirmed that AN3 genetically acts on upstream of YDA in

344

modulating seed size.

345

an3 and yda presented different responses to high sugar concentration

346

Before bolting, the rosette leaf number that a seedling grows is thought to be a more

347

suitable indicator of flowering time than the day number to flowering35. Under normal

348

conditions (16 L/8 D), an3-1 and an3-4 mutant plants had increased leaf numbers

349

regardless of long or short days (Figure S4G;

350

normal conditions, an3-4 mutant plants produce leaves faster than the wild type.

351

Delayed flowering reveals the capacity of the mutation to offset the effect of sugars

352

on flowering timing; therefore, changing the sugar metabolism affects seed size5.

7,21).

These results suggested that under

16


Page 17 of 42


353

Thus, we investigated if the increased rosette leaf number in an3-4 lines can be

354

restored with high glucose (5%) or sucrose (6%) concentration. As speculated, the an3

355

mutant and wild type plants with high sugar had similar numbers of rosette leaves

356

(Figures 4G, L and S4G), indicating that abnormal an3 rosette leaf numbers depend

357

on sugar metabolism. This phenotype is opposite to gin1(glucose-insensitive1), a

358

mutant to glucose-insensitivity, which showed that flowering timing is not postponed

359

via glucose26. We observed that the yda-10 mutant showed early flowering and few

360

rosette leaves. However, similar to an3 mutants, the 6% sucrose restored the altered

361

rosette leaf number in yda-10 mutants (Figure S4G). Thus, abnormal rosette leaf

362

numbers in both an3 and yda mutant plants depend on sugar metabolism. The an3-4

363

abnormal rosette leaf number can be restored; however, the shape of an3-4 narrow

364

rosette leaves7 cannot be restored by 5% glucose (Figure 4G), indicating that the

365

shape of an3 leaf blades is not regulated by sugar. Interestingly, under high glucose

366

concentration, the an3-4 mutant revealed the cell elongation orientation in the root

367

meristem and elongation zone (Figures 5I and J). The an3 roots may be similar to

368

those of defective mutations in the biosynthesis of the cell wall (carbohydrate

369

metabolism), such as when cobra mutant affects the cell expansion orientation in the

370

roots and reduces the magnitude of crystalline cellulose of cell walls within the zone

371

of the root growth27, 28. Moreover, the phenotype of the yda roots resembles to the

372

carbon starvation caused via the decreased ability for the catabolism of sucrose in the

373

root cell, for example the extreme reduction in root growth (Figures 6A–C; 8) and the

374

loss of the root cap starch (Figures 5K and L) caused by abnormal cell elongation

17



Page 18 of 42

375

within the root elongation zone. The abnormal cell elongation in yda-1 roots may be

376

due to the lacking substrate in cell wall synthesis, but not a drawback in a specific

377

synthesis pathway of cell wall, which is similar to that in cinv1/cinv2 mutant that is

378

defective in cytosolic invertase29. Moreover, the obvious enhancement of root

379

elongation via exogenous glucose (Figures 6D and E) indicated that this phenotype is

380

partly due to carbon starvation. The outcomes of cell proliferation determine the final

381

seed size. Cell expansion involves the coordination of cell wall dynamics with

382

internal cellular activities, such as gene expression, during cell wall modification29.

383

Thus, seed size regulation in an3-4 and yda mutants might be due to embryonic cell

384

elongation secondary to cell wall modification.

385

Cotyledons are derived from embryos; therefore, we used cotyledons for studying

386

cell elongation in response to glucose. Cotyledons of the an3 mutants are large, but

387

those of yda mutants are smaller than those of control plants on MS medium with 1%

388

sucrose (Figures 4A and H). However, the abnormal cotyledon sizes in an3 and yda

389

mutants could be restored using MS medium supplemented with 5% glucose (Figures

390

4B and I). To confirm this, the anatomical structure of the cotyledons was observed.

391

The an3 and yda mutants had larger and smaller cotyledon cells, respectively, than the

392

control plants in solid MS medium supplemented with 1% sucrose. However, sizes of

393

5% glucose-supplementation cotyledon cells were not distinct from those of control

394

seedlings (Figures 4C, D, J and K). Similarly, the protein levels of AN3-3XGFP

395

declined (Figure 4E) and those of YDA-GFP increased in MS medium supplemented

396

with 5% glucose compared with 1% sucrose (Figure 4F). Therefore, by transpressing

18


Page 19 of 42


397

YDA, we proposed that AN3 regulates the sugar-mediated cotyledon cell elongation

398

and modulates cotyledon size. In summary, yda and an3 are sugar metabolism

399

mutants.

400

The ratio of sucrose to hexose shows a positive association with embryo cell

401

elongation rather than cell division in an3-4 and yda-10 mutants

402

Sucrose cleavage is catalysed by the hydrolysis of sucrose invertase and produces

403

fructose and glucose (hexoses)30. For testing the content of these metabolites, we

404

performed high-performance liquid chromatography analysis using an3-4 and Col-0

405

or yda-1/+ and Ler developing seeds as materials. Glucose and fructose

406

concentrations in an3-4 developing seeds declined at 5, 8 and 11 days after pollination

407

(DAP) compared with corresponding developing seeds in the wild type (Figure 5A).

408

By contrast, although the sucrose concentration was lower in 5-DAP an3-4

409

developing seeds than in corresponding wild-type seeds, the concentrations were

410

higher 8 and 11 DAP (Figure 5B). As a result, although the ratios of sucrose to hexose

411

were not significantly different in 5 DAP developing seeds of an3-4 and wild-type

412

plants, these ratios were seven and five times higher in 8 and 11 DAP developing

413

seeds, respectively, of an3-4 plants relative to those of the Col-0 (Figure 5C). On the

414

other hand, the glucose and fructose concentrations declined at 5, 8 and 11 DAP in

415

yda-1/+ compared with corresponding Ler developing seeds (Figure 5E). However,

416

the sucrose concentration was not significantly different between 5 and 8 DAP

417

developing seeds in yda-1+ and Ler plants; therefore, sucrose concentration was

418

higher in 11 DAP yda-1/+ seeds (Figure 5F). Consequently, the ratio of sucrose to

19



Page 20 of 42

419

hexose levels was higher in 5, 8 and 11 DAP developing seeds of yda-1/+ compared

420

with Ler plants (Figure 5G). In general, a low ratio of sucrose to hexose is closely

421

related to cell division during early seed development; by contrast, a high ratio of

422

sucrose to hexose is closely relative to cell elongation during late seed development5,

423

31, 32.

424

developing seeds indicates that both AN3 and YDA regulate seed size by modulating

425

cell elongation but not cell division during the late phase of seed development. The

426

finding agrees with that of cytological experiments (Figures 1 and 3), i.e., an3 large

427

embryos are caused by embryo cell elongation, whereas yda small embryos are

428

caused by the suppression of embryo cell elongation during the late phase of seed

429

development. Moreover, the above data indicated that AN3 and YDA control soluble

430

sugar accumulation during seed development.

Therefore, a high ratio of sucrose to hexose in both an3-4 and yda-1/+

431

The changes in sucrose and glucose concentrations are regarded as results of

432

altered invertase activity17; therefore, we speculated that invertase activity may be

433

changed in an3 and yda mutant plants. In Arabidopsis29, rice33 and legumes34, neutral

434

invertase is essential to normal plant growth and development. For analyzing if

435

neutral invertase activity is changed in an3 and yda mutants, the neutral invertase

436

protein levels were assayed using enzyme-linked immunosorbent assay in developing

437

seeds of the mutant and control plants. Neutral invertase activity in an3-4 mutants was

438

enhanced by 28% compared with that in Col-0, whereas that in yda-1/+ lines was

439

decreased by 22% relative to that in Ler (Figure 5D). These are consistent with the

440

finding that the phenotype of the yda roots resembles to carbon starvation caused by

20


Page 21 of 42


441

the decreased ability for sucrose catabolism in the root cell (Figures 6A–C;

18).

To

442

confirm this, we determined the cell wall invertase activity. We observed a high level

443

of nitroblue tetrazolium precipitate in the developing seeds in an3-4 mutant plants,

444

but observed none in the corresponding yda-1/+ seeds (Figure 5H). The precipitate

445

specifically indicated that the cell wall invertase had higher activity in the an3

446

mutants than in the yda developing seeds. Therefore, high and low endogenous

447

sucrose accumulations in the an3 and yda mutants are caused by low and high neutral

448

invertase activities, respectively (Figures 4 and 5).

449

AN3 and YDA act maternally to affect seed size

450

For determining the genetic control of seed mass, we need know if AN3 and YDA act

451

maternally or zygotically when regulating seed size. Reciprocal cross experiments

452

between yda and Ler or an3-4 and Col-0 plants were performed. When Col-0 or an3-4

453

and yda or Ler pollens were used as donors, and an3-4 or yda mutant plants as

454

acceptors, the influence on seed size was not altered upon changing donors (Figure

455

S4H). Similarly, when Col-0 or an3-4 and yda or Ler pollens were used as the donors

456

and Col-0 or Ler pollens plants as acceptors, the influence on seed size was not

457

altered with the alteration of the donor (Figure S4H). In summary, these results

458

indicated that an3-4 and yda mutants have maternal influence on mutants and affect

459

seed size.

460 461

DISCUSSION

462

In a previous study19, although the authors did not mention the seed size of an3 21



Page 22 of 42

463

mutants, the cotyledon area of the an3-4 lines was bigger relative to that of the Col-0

464

(see Figures 5A and S3A therein). These data suggest that our observation might be

465

correct. Moreover, it has been currently reported that AN3-MINI3 gene cascade

466

regulates seed mass41. In this report, we only use an3-4 lines, but not an3-1 lines,

467

which is not sufficient in evidence. For better proving AN3 function, we analyzed

468

an3-1 and an3-4 mutants in seed mass and embryo mass (Figure 1 and Figure S1).

469

The gene cascade of AN3–YDA has crucial biology functions in regulating seed

470

size

471

In this work, YDA mutation significantly decreases seed size in the an3 mutant, and

472

AN3 is downstream of YDA. The above data suggested a negative relationship

473

between YDA and AN3 when regulating seed size. More findings of the impact of

474

AN3 associated with the promoter of YDA in vivo (Figure 2), on endogenous sugar

475

levels (Figure 5), and on invertase activity (Figure 5), on sugar sensing (Figure 4)

476

conﬁrmed the negative regulatory effects of AN3 on YDA.

477

In summary, the gene cascade of AN3–YDA has crucial biology functions in

478

regulating seed size.

479

yda and an3 are mutants of sugar metabolisms

480

It is well known that the embryo mass is determined via embryo cell size and number.

481

In terms of cytological data (Figures 1 and 3), we concluded that the enhanced

482

embryo mass of an3-4 lines was as a result of the embryo cell elongation, and the

483

decreased embryo size of yda-10 was because of the suppressed embryo cell

484

elongation. Further analysis revealed that the alternation of cell elongation in the

22


Page 23 of 42


485

embryos of an3-4 and yda-10 was triggered by sugar metabolism and/or signalling.

486

This finding is based on the comparison of glucose response phenotypes in cotyledons,

487

this stability of YDA and AN3 proteins in the concentration of high glucose,

488

restoration of the defects of delayed flowering in an3-4 mutant by glucose, the

489

orientation of cell elongation in root meristem, the elongation zone in an3-4 mutant

490

and the loss of the root cap starch in yda-1 mutant.

491

Microarray analysis22 showed that a lot of sugar-related genes result to twice

492

higher transcripts in an3 mutants relative to Col-0 seedlings. Alterations were

493

analyzed in the transcripts of several genes, especially in an3-4, including those in

494

carbohydrate metabolism (e.g., sugar transport: At1g34580; carbohydrate metabolism:

495

At1g69830), cell wall metabolism (e.g., pectinesterase: At4g02330; At3g10720) and

496

response to fructose, glucose and sucrose (At1g74670)22. In a previous study22, genes

497

involved in secondary metabolism, amino acid metabolism, lipid metabolism and

498

stress had twice lower expression levels in an3-4 mutants; however, genes involved in

499

major carbohydrate metabolism and transport had twice higher expression levels.

500

These results suggested that AN3 participates in sugar metabolism, i.e., the changed

501

transcripts in an3 mutants mostly affect metabolism rather than developmental

502

modulation.

503

In a previous report8, microarray observation of yda lines revealed that fourteen

504

of 8000 genes had alternations in transcripts of two times; they covered those in

505

sucrose response (AT5G13930), sugar metabolism (AT4G15760, AT2G43570 and

506

AT5G57550), cell wall synthesis (At2g45220) and sugar-mediated signal pathways

23



Page 24 of 42

507

(AT3G27660). Moreover, over 1/2 are close correlation with sugar signalling and/or

508

metabolism. In another yda microarrays25, differentiated epidermis of the cell walls

509

were significantly effected, such as, over 11% up-regulated genes in yda lines were

510

participated in the differentiation of cell wall. The findings further suggested that YDA

511

is participated in sugar signalling and/or metabolism. Furthermore, the genes that

512

encode glucokinases, including at1g12080 and at2g16790, were remarkably altered in

513

yda plants. In summary, our findings further revealed that yda and an3 are mutants of

514

sugar metabolism.

515

The different sucrose to hexose ratios caused by altered cell wall invertase

516

provides a different signalling for cell proliferation

517

Overall, a low sucrose to hexose ratio is closely related to cell division in the early

518

phase stage of seed development; by contrast, a high ratio is correlated with cell

519

elongation in the late phase stage of seed development5,

520

immature fava bean embryos cultured in high hexose concentrations underwent cell

521

division; by contrast, embryos cultured in high sucrose concentrations performed cell

522

elongation31, 32. Thus, a low sucrose to hexose ratio generates a signal of cell division,

523

whereas a high ratio produces a signal of cell elongation. In the early stage of seed

524

development (5 DAP) (Figure 5), a low sucrose to hexose ration was present and

525

generated a cell division signal. In the middle and later stages of seed development (8

526

and 11 DAP) (Figure 5), a high sucrose to hexose ratio was present and generated a

527

signal for cell elongation.

528

30, 31.

In a related study,

Acid invertase can supply carbohydrates into the sink, and it is an important

24


Page 25 of 42


529

transition regulator of source–sink by keeping the gradient of sucrose concentration17.

530

Therefore, cell wall invertase was measured using a staining technique for

531

quantitative and vacuolar invertase, which are potentially important in determining

532

sucrose to hexose ratios (Figure 5H). Thus, these data indicated sound grounds for

533

conclusions on why sucrose to hexose ratios may have differed between the two

534

genotypes (cell division and elongation).

535 536

In summary, AN3 and YDA regulate seed size via sugar-mediated embryonic cell elongation caused by altered cell wall invertase (Figure 7).

537

Seed mass of higher plants is central to a lot of situations of evolutionary fitness.

538

Seed size constitutes an important agricultural trait. In agricultural crop, large seed

539

size not only indicates yield enhancement, but also suggests the enhancement of other

540

merits, for example, the seed biotinylated protein36; seed protein and oil5, seed

541

defense37; blueberry seed oils38; sugar beet seed39.

542 543

■ AUTHOR INFORMATION

544

L.-S.M. designed experiments. L.-S.M, M.-K.X, D.L and M.-M Z performed the

545

experiments. L.-S.M., M.-K.X, D.L and M-M Z completed statistical analysis of data.

546

L.-S.M and J-H.J wrote, edited and revised this manuscript.

547 548

ACKNOWLEDGMENTS

549

This study was supported by grants from the Agricultural High Technology Research

550

of Xuzhou City (KC16NG063). The Doctoral Scientific Research Founding of

25



551

Page 26 of 42

Jiangsu Normal University.

552 553

Supporting Information description

554

Figure S1.an3-1 has increased seed size than does S96.

555

Figure S2. the gel analysis of protein.

556

Figure S3. Expression analysis of AN3 and YDA.

557

Figure S4. an3-4 and yda-10 restore leaf number in high concentration of 6% sucrose.

558

an3-4 and yda-10 act maternally for regulating seed size.

559 560

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6. Zhou, Y.; Zhang. X.J.; Kang, XJ.; Zhao, X.Y.; Zhang, X.S.; Ni, M. SHORT HYPOCOTYL UNDER BLUE1 Associates with MINISEED3 and HAIKU2 Promoters in Vivo to Regulate Arabidopsis Seed Development. Plant Cell. 2009, 21, 106-117. 7. Horiguchi, G.; Kim, G.T.; Tsukaya, H. The transcription factor AtGRF5 and the transcription coactivator AN3 regulate cell proliferation in leaf primordia of Arabidopsis thaliana. Plant J. 2005, 43, 68-78. 8. Lukowitz, W.; Roeder, A.; Parmenter, D.; Somerville, C. A MAPKK kinase gene regulates extraembryonic cell fate in Arabidopsis. Cell. 2004, 116, 109-119. 9. Kang, C.Y.; Lian, H.L.; Wang, F.F.; Huang, J.R.; Yang, H.Q. Cryptochromes, phytochromes, and COP1 regulate light-controlled stomatal development in Arabidopsis. Plant Cell. 2009, 21, 26242641. 10. Meinke, D.; Sweeney, C.; Muralla, R. Integrating the genetic and physical maps of Arabidopsis thaliana: identiﬁcation of mapped alleles of cloned essential (EMB) genes. PLoS ONE. 2009, 4, e7386 11. Kawade, K.; Horiguchi, G.; Tsukaya, H. Non-cell-autonomously coordinated organ size regulation in leaf development. Development. 2010,137, 4221–4227. 12.Meng, L.S. Transcription Coactivator Arabidopsis ANGUSTIFOLIA3 Modulates Anthocyanin Accumulation and Light-Induced Root Elongation through Transrepression of Constitutive Photomorphogenic1.Plant Cell Environ.2015, 38, 838-851. 13. Meng, L.S.; Yao, S.Q. Transcription co-activator Arabidopsis ANGUSTIFOLIA3 (AN3) regulates water-use efficiency and drought tolerance by modulating stomatal density and improving root architecture by the transrepression of YODA (YDA). Plant Biotechnol. J. 2015, 13, 893-902.

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14. Meng, L.S.; Wang, Y.B.; Yao, S.Q.; Liu, A. Arabidopsis AINTEGUMENTA mediates salt tolerance by trans-repressing SCABP8. J Cell Sci. 2015, 128, 2919-2927. 15. Meng, L.S.; Wang, Z.B.; Yao, S.Q.; Liu, A.The ARF2–ANT–COR15A gene cascade regulates ABA signaling-mediated resistance of large seeds to drought in Arabidopsis. J Cell Sci. 2015, 128, 39223932. 16. Kovtun, Y. Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. USA. 2000, 97, 2940-2945. 17. Lou, Y.; Gou, J.Y.; Xue, H.W. PIP5K9, an Arabidopsis phosphatidylinositol monophosphate kinase, interacts with a cytosolic invertase to negatively regulate sugar-mediated root growth. Plant Cell. 2007, 19, 163-181. 18. Kuhn, C.; Barker, L.; Burkle, L.; Frommer, W.B. Update on sucrose transport in higher plants. J Exp Bot.1999, 50, 935-953. 19. Ferjani, A.; Horiguchi, G.; Yano, S.; Tsukaya, H. Analysis of leaf development in fugu mutants

of Arabidopsis reveals three compensation modes that modulate cell expansion in determinate organs. Plant Physiol. 2007, 144, 988-999. 20. Li, Y.; Zheng, L.; Corke, F.; Smith, C.; Bevan, M.W. Control of final seed and organ size by

the DA1 gene family in Arabidopsis thaliana. Gene Dev. 2008, 22, 1331–1336. 21. Kim, J.H.; Kende, H. A transcriptional coactivator, AtGIF1, is involved in regulating leaf growth and morphology in Arabidopsis. Proc. Natl. Acad. Sci. USA. 2004, 101, 13374-13379. 22. Horiguchi, G.; Nakayama, H.; Ishikawa, N.; Kubo, M.; Demura, T. ANGUSTIFOLIA3 Plays Roles in Adaxial/Abaxial Patterning and Growth in Leaf Morphogenesis. Plant and Cell Physiol 2011, 52, 112-124.

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Kim, M.C.; Jaeger, K.E.; Busch, W.; Schmid, M.; Weigel, D. Integration of spatial

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Plant Sci. 1997, 2, 169-174. 33. Jia, L.; Zhang, B.; Mao, C.; Li, J.; Wu, Y. OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (Oryza sativa L.). Planta.2008, 228, 51-59. 34. Welham, T;

Pike, J.; Horst, I.; Flemetakis, E.; Katinakis, P. A cytosolic invertase is required for

normal growth and cell development in the model legume, Lotus japonicus. J Exp Bot. 2009, 60, 3353-3365. 35. Mcnellis, T.W.; Vonarnim, A.G.; Araki, T.; Komeda, Y.; Misera, S.; Deng, X.W. Genetic and Molecular Analysis of an Allelic Series of Cop1 Mutants Suggests Functional Roles for the Multiple Protein Domains. Plant Cell. 1994, 6, 487-500. 36. Riascos, J.J.; Weissinger, S.M.; Weissinger, A.K.; Kulis, M.; Burks, A.W.; Laurent Pons. The seed biotinylated protein of soybean (glycine max): a boiling-resistant new allergen (gly m 7) with the capacity to induce IgE mediated allergic responses. J. Agric. Food Chem. 2016, 64, 3890−3900. 37. Mora, C.A.; Halter, G.J; Adler, C.; Hund, A.; Anders, H.; Yu, K.; Stark, W.J. Application of the prunus spp. Cyanide seed defense system onto wheat: reduced insect feeding and field growth tests. J. Agric. Food Chem. 2016, 64, 3501−3507. 38. Li, Q.; Wang, J.; Shahidi, F. Chemical characteristics of cold-pressed blackberry, black raspberry, and blueberry seed oils and the role of the minor components in their oxidative stability. J. Agric. Food Chem. 2016, 64, 5410−5416. 39. Wettstein, F.E.; Kasteel, R.; Garcia Delgado, M.F.; Hanke, I.; Huntscha, S.; Balmer, M.E.; Poiger, T.; Bucheli, T.D. Leaching of the neonicotinoids thiamethoxam and imidacloprid from sugar beet seed dressings to subsurface tile drains. J. Agric. Food Chem. 2016, 64, 6407−6415. 40. Fiehn, O.; Kopka, J.; Dormann, P.; Altmann, T.; Trethewey, R.N.; Willmitzer, L. Metabolite

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profiling for plant functional genomics. Nat. Biotechnol. 2000. 18: 1157–1161. 41. Meng, L.S.; Wang, Y.B.; Loake, G.J.; Jiang, J.H. Seed Embryo Development Is Regulated via an AN3-MINI3 Gene Cascade.Front. Plant Sci. 2016, 7, 1645.

Figure Legend Fig 1. AN3 Negatively Regulates Seed Size.

561

(A). Representative mature dry seeds of an3-4 (a), CS4002 (b), WT (Col-0) (c), and 35S-AN3+an3-4

562

(d), respectively. Bar = 0.5mm for (a) to (d).

563

(B). (a) and (b). Representative epidermal cell layer of cotyledon embryos from WT and an3-4 seeds,

564

respectively. Bars = 100um for (a) to (b). (c) and (d). Representative the panes in (a) and (b) are

565

amplified, respectively. Bars = 10um for (c) to (d).

566

(C), (D) and (E). Bar graph exhibiting the difference in average seed weight/100 seeds (C), seed length

567

(D) and seed width (E) between WT, an3-4,an3-4+35S-AN3 and CS4002 seeds. Error bars represent

568

SD [n=3 in (C); n=30 in (D); n=30 in (E)]. Heteroscedastic t test analysis showed significant

569

differences (**P < 0.01). These experiments were repeated at least three times (bio-replicates) with

570

similar results.

571

(F). Representative epidermal cell layer derived from hypocotyls in WT (a) and an3-4 mutant (b). Bars

572

= 10um for (a) to (b).

573

(G). Bargraph exhibiting the difference in cotyledon embryo areas between WT and an3-4 seeds. (H).

574

Bargraph exhibiting the difference in the cell area of cotyledon embryos between WT and an3-4 seeds.

575

(I). Bargraph exhibiting the difference in average cell numbers from three columns in the central region

31



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576

of the hypocotyl plus the embryonic root in WT and an3-4 seedlings. Error bars represent SD [n=14 in

577

(G); n=40 in (H); n=40 in (I)]. Heteroscedastic t test analysis showed significant differences (**P
20). Representative images of

591

GUS staining are shown. and screening method about seedlings is described in METHODS.Bar= 1.0

592

cm for (a) to (b).

593

(D). Schematic diagram of the YDA loci and a few amplicons with initiating from ATG of YDA.

594

(E). Bargraph exhibiting the difference interaction between AN3 and YDA promoter. ChIP was

595

performed to analysize the in vivo interaction between AN3 with the YDA promoter. Input was

596

chromatin before immuno precipitation. Anti-GFP antibody was used for precipitating chromatin

597

associated with 35S-AN3-3XGFP. HA was used as a negative control for the specificity of

32


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598

immunoprecipitation. The YDA promoter region associated with AN3 was amplified by Q-RT-PCR

599

using YDA promoter-specific primers for distinct regions. In (A) and (B), Col-0 is set as 1.0.

600

Quantifications were normalized to the expression of UBQ5. Error bars represent SD (n=3).

601

Heteroscedastic t test analysis showed significant differences (***P < 0.001; **P < 0.01). These

602

experiments were repeated at least three times (bio-replicates) with similar results.

603

Fig 3. YDA Mutation Suppresses Large Seed Size of an3-4 mutant.

604

(A). Representative mature dry seeds of an3-4 (a), WT (b), yda-1(c), yda-10 (d) and an3-4 yda-10 (e),

605

respectively.Bar = 0.5 mm for (a) to (e).

606

(B). (a) and (b): Representative embryos derived from WT(Col-0) (a) and yda-10 (b) seeds. (c) and (d):

607

Representative the panes in (a) and (b) are amplified, respectively. Bars = 100μm for (a) to (b). Bars =

608

10 μm for (c) to (d).

609

(C). Bargraph exhibiting the difference in the cotyledon embryo area between WT(Col-0) and yda-10

610

seeds. (D). Bargraph exhibiting the difference in the cell area of cotyledon embryo between WT(Col-0)

611

and yda-10 seeds. (E). Bargraph exhibiting the difference in average seed weight/100 seeds between

612

WT, yda-1/+ and yda-10 seeds. (F). Bargraph exhibiting the difference in average seed area between

613

WT, yda-10, an3-4 and an3-4 yda-10 seeds. Error bars represent SD [n=20 in (C), n= 87 in (D), n=3 in

614

(E), n=33 in (F)]. Heteroscedastic t test analysis showed significant differences (**P < 0.01). These

615

experiments were repeated at least three times (bio-replicates) with similar results. Data in (C), (D),

616

(E) and (F) are means ± SD from at least 10 independently propagated WT and mutant lines.

617

Fig 4. The Phenotype Defects in Cotyledons of an3 and yda Mutant can Be Restored by High Glucose

618

Concentration.

619

(A). Representative cotyledons of 6-day-old Ler, yda-1, yda-2, an3-1, an3-4 and Col-0 seedlings grown

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620

on solid MS medium with 1% sucrose. Bar= 2.0 mm.

621

(B). Representative cotyledons of 10-day-old Ler, yda-1, yda-2, an3-1, an3-4 and Col-0 seedlings

622

grown on solid MS medium with 5% glucose. Bar= 2.0 mm.

623

(C). Representative cotyledon cells of 6 or 10-day-old an3-4 and Col-0 seedlings grown on solid MS

624

medium with 1% sucrose or 5% glucose, respectively. Bar= 20 μm.

625

(D). Representative cotyledon cells of 6 or 10-day-old yda-1 and Ler seedlings grown on solid MS

626

medium with 1% sucrose or 5% glucose, respectively. Bar= 20 μm.

627

(E).Representative 5% glucose treatment accelerates AN3 protein degradation. The 10-day-old

628

35S:AN3:3X:GFP seedlings grown on solid MS medium with 1% sucrose and 5% glucose. Materials

629

were from at least 10 independently propagated lines.

630

(F).Representative 5% glucose treatment

631

PHB:EMB71/YDA:GFP seedlings grown on solid MS medium with 1% sucrose and 5% glucose.

632

Materials were from at least 10 independently propagated lines.

633

(G). Representative seedlings of an3-4 and Col-0 grown on solid MS medium with 5% glucose for 20

634

days. Bar=2.0 cm.

635

(H). Bargraph exhibiting the difference in the cotyledon area between Ler, yda-1, yda-2, an3-1, an3-4

636

and Col-0 seedlings grown on solid MS medium with 1% sucrose. (I). Bargraph exhibiting the

637

difference in the cotyledon area between Ler, yda-1, yda-2, an3-1, an3-4 and Col-0 seedlings grown on

638

solid MS medium with 5% glucose. (J). Bargraph exhibiting the difference in the cotyledon cell size

639

between an3-4 and Col-0 seedlings grown on solid MS medium with 1% sucrose and 5% glucose,

640

respectively. (K). Bargraph exhibiting the difference in the cotyledon cell size between yda-1 and Ler

641

seedlings grown on solid MS medium with 1% sucrose and 5% glucose, respectively. (L).Bargraph

stabilizes EMB71/YDA protein. The 10-day-old

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642

exhibiting the difference in the rosette leaf number between Wild-type (Col-0) and an3-4 seedlings

643

grown on solid MS medium with 1% sucrose and 5% glucose for 3 weeks under long light (16h

644

light/8h dark). Error bars represent SD [n=23 in (H), n= 23 in (I), n=87 in (J), n=84 in (K), n= 14 in

645

(L)]. Heteroscedastic t test analysis showed significant differences (**P < 0.01; *P < 0.05). These

646

experiments were repeated at least two times (bio-replicates) with similar results.

647

Fig 5. Sugar Metabolite Analysis.

648

(A). Bar graph exhibiting the difference in the concentrations of hexose (glucose and fructose)

649

between an3-4 and Col-0 seeds. (B). Bar graph exhibiting the difference in the sucrose

650

concentrations between an3-4 and Col-0 seeds. (C). Bar graph exhibiting the difference in the

651

ratio of the sucrose/hexose between an3-4 and Col-0 seeds. (D). Bar graph exhibiting the

652

difference in the neutral invertase activity between developing siliques of an3-4 and Col-0 or Ler

653

and yoda-1 plants. (E). Bar graph exhibiting the difference in the concentrations of hexose

654

(glucose and fructose) between Ler and yoda-1 seeds. (F). Bar graph exhibiting the difference in

655

the sucrose concentrations between Ler and yoda-1 seeds. (G). Bar graph exhibiting the difference

656

in the ratio of the sucrose/hexose between Ler and yoda-1 seeds. (H). (a) Representative nitroblue

657

tetrazolium (NBT) precipitation in the developing seeds of an3-4 wild-type and yda-1/+ plants

658

grown under long light (16L/8D) conditions on MS medium supplemented with 1% sucrose. (b).

659

The intensity of cell wall invertase was quantified through using Adobe Photoshop CS (Adobe

660

Systems Inc.; San Jose, CA, USA) software, as has been described by Meng et al (2015b). Wild-

661

type is set as 1.0. Error bars represent SD [n=3 in (A), (B), (C), (D), (E), (F) and (G); n= 12 in (H)].

662

Heteroscedastic t test analysis showed significant differences (**P < 0.01). These experiments were

663

repeated at least two times (bio-replicates) with similar results. Data in (A), (B), (D), (E) and (F)

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664

are means ± SD from at least 10 independently propagated WT and mutant lines. yoda-1 in (E), (F)

665

and (G) indicates yoda-1/+ (for heterozygous lines).

666

(I) and (J). Meristem (I) and elongation (J) zones in the primary roots of 15-day-old wild-type and

667

an3-4 plants on solid MS medium with 5% glucose. Seedlings were from the same plate.

668

Magnifications are the same. Arrows indicate cell elongation orientation. Bar= 100μm for (a) to

669

(b).

670

(K) and (L). The tip of

671

solid MS medium with 1% sucrose. Seedlings were from the same plate. Magnifications are the

672

same. Bar= 50μm for (K) to (L).

673

Fig 6. The yda plants were involved in sugar metabolism and/or signaling.

674

(A). The primary roots in 6- and 12-day-old yda and Ler plants on MS medium supplemented with 1%

675

sucrose. Bar = 1.0 cm.

676

(B). The primary root elongation in the Ler and yda plants grown on MS medium supplemented

677

with 1% sucrose. The length of the primary roots was measured at the indicated time points. The

678

values are the mean ± SD of three independent experiments. (C). Lateral roots in the yda and Ler

679

plants grown on MS medium supplemented with 1% sucrose. The number of lateral roots was

680

counted at the indicated time points. Error bars represent SD [n=20 in (B) and (C)]. These

681

experiments were repeated at least two times (bio-replicates) with similar results.

682

(D).The yda roots were not sensitive to 5% glucose. The plants are photographed at 12 days after

683

sowing. Bar = 0.5 cm.

684

(E).The analysis of the primary root length of yda and Ler plants in (D). Standard deviations (bars)

685

were estimated from the results of three independent experiments (n > 30; ***P < 0.001).

primary roots

of 8-day-old

yoda-1 (K) and Ler (L) seedlings

on

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686

Fig 7. Soluble sugar accumulation effects on the AN3-YDA gene cascade.

687

Soluble sugar accumulation can effect on AN3 expression, in turn, AN3 is associated to YDA

688

promoter for regulating its expression, which effects on the sucrose levels. On the other hand, the

689

alternation of sucrose levels leads to soluble sugar accumulation. Moreover, drought stress can induce

690

the change of AN3 expression13. By the AN3-YDA gene cascade, the change of AN3 expression effects

691

on sucrose levels, which results in soluble sugar accumulation.

692 693

Graphic for manuscript:

694

Soluble sugar accumulation effects on the AN3-YDA gene cascade.

695

Soluble sugar accumulation can effect on AN3 expression, in turn, AN3 is associated to YDA

696

promoter for regulating its expression, which effects on the sucrose levels. On the other hand, the

697

alternation of sucrose levels leads to soluble sugar accumulation. Moreover, drought stress can induce

698

the change of AN3 expression13. By the AN3-YDA gene cascade, the change of AN3 expression effects

699

on sucrose levels, which results in soluble sugar accumulation.

700 701 702 703 704

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705 706

707 708 709 38


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710 711

712 713 714 39



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717 718 719 40


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721 722

723

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of Agricultural Food Chemistry Title: Soluble Sugar Accumulation Can InfluenceJournal Seed Size viaand AN3–YDA Gene Cascade

Graphic for manuscript:

Soluble Sugar Accumulation

drought stress

AN3 YDA

Sucrose levels


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Soluble Sugar Accumulation Can Influence Seed Size via AN3âYDA

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