Maize VIVIPAROUS1 Interacts with ABA INSENSITIVE5 to Regulate

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Agricultural and Environmental Chemistry

Maize VIVIPAROUS1 Interacts with ABA INSENSITIVE5 to Regulate GALACTINOL SYNTHASE2 Expression Controlling Seed Raffinose Accumulation Yumin Zhang, Qichao Sun, Chunxia Zhang, Guanglong Hao, Chunmei Wang, Lynnette M. A. Dirk, A. Bruce Downie, and Tianyong Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00322 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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Journal of Agricultural and Food Chemistry

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Maize VIVIPAROUS1 Interacts with ABA INSENSITIVE5 to

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Regulate GALACTINOL SYNTHASE2 Expression Controlling Seed

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Raffinose Accumulation

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Yumin Zhanga,b • Qichao Suna,b • Chunxia Zhang a,b •Guanglong Haoa,b • Chunmei

5

Wangc • Lynnette M.A. Dirk d •A. Bruce Downied • Tianyong Zhaoa,b*

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Running Title: VP1 and ABI5 Regulate Raffinose Biosynthesis

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aState

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Northwest A&F University, Yangling, Shaanxi, 712100, China.

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bThe

Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences,

Key Laboratory of Biology and Genetics Improvement of Maize in Arid Area of

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Northwest Region, Ministry of Agriculture, Northwest A&F University, Yangling,

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Shaanxi, 712100, China.

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c

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Northwest A&F University, Yangling, Shaanxi, China.

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dDepartment

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Environment, University of Kentucky, Lexington, KY 40546, USA.

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*Corresponding author

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Tianyong Zhao, Ph.D

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Professor

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Department of Biochemistry and Molecular Biology

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College of Life Sciences, Northwest A&F University

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Yangling, Shaanxi 712100, China

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Email: [email protected]

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Tel: 86-13324517154

The Biology Teaching and Research Core Facility, College of Life Sciences,

of Horticulture, Seed Biology, College of Agriculture, Food, and

1

ACS Paragon Plus Environment


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Abstract

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Raffinose, an oligosaccharide found in many seeds, plays an important role in seed

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vigor; however, the regulatory mechanism governing raffinose biosynthesis remains

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unclear. We report here that maize W22 wild type (WT) seeds, but not W22 viviparous1

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(zmvp1) mutant seeds, start accumulating galactinol and raffinose at 28 days after

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pollination (DAP). Transcriptome analysis of the zmvp1 embryo showed that the

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expression of GALACTINOL SYNTHASE2 (GOLS2) was down-regulated relative to

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WT. Further experiments showed that the expression of ZmGOLS2 was up-regulated

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by ZmABI5, but not by ZmVP1, and it was further increased by co-expression of

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ZmABI5 and ZmVP1 in maize protoplasts. ZmABI5 interacted with ZmVP1, while

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ZmABI5, but not ZmVP1, directly binds to the ZmGOLS2 promoter. Together, all the

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finding suggest that ZmVP1 interacts with ZmABI5 and regulates ZmGOLS2

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expression and raffinose accumulation in maize seeds.

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Key words: maize, seeds, viviparous, ABI5, GALACTINOL SYNTHASE, raffinose

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biosynthesis.

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Introduction

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Raffinose-family oligosaccharides (RFOs) accumulate during seed maturation1 and

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play important roles in seed vigor2. There are two commitment steps for raffinose

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biosynthesis. The first step is to use GALACTINOL SYNTHASE (GolS: EC 2.4.1.123)

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to catalyze galactose transfer from UDP-galactose to myo-inositol to form galactinol3.

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The second step is to synthesize raffinose by transfer of the galactosyl unit from

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galactinol to sucrose, which is catalyzed by RAFFINOSE SYNTHASE (RS, EC

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2.4.1.82)4. We have previously characterized two maize GALACTINOL SYNTHASE

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genes5 and the unique maize RAFFINOSE SYNTHASE2. Both ZmGOLS2 and ZmRAFS

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were expressed in the later stages of seed development2, 5. The seeds of Arabidopsis

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thaliana atgols1 mutant or AtGOLS1 RNAi lines, with decreased RFOs, completed

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germination faster than those of wild type6, suggesting that RFOs negatively influence

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the rapidity of completion of seed germination.

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Maize VIVIPAROUS1 (ZmVP1) encodes a transcription factor7 orthologous to the

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Arabidopsis thaliana ABA INSENSITIVE3 (AtABI3)8. ZmVP1 is a key regulator of

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seed maturation, its expression is induced by abiotic stress and ABA in mature embryos

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or leaves9. Seeds with null alleles at VP1 result in a loss of sensitivity to ABA, leading

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to vivipary (precocious germination), and loss of desiccation tolerance10. There are two

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independent zmvp1 mutant lines, vp1-R and vp1-Mc11. VP1 is not expressed in the vp1-R

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mutant kernels, and the zmvp1-R seeds accumulate only trace amounts of raffinose in

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the embryos11. The raffinose content in zmvp1-MC seeds is similar to that of the wild

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type, and the zmvp1-MC seeds acquire desiccation tolerance, suggesting that raffinose 3



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biosynthesis may occur in the absence of a fully functional vp1 gene product11.

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Similar to the maize zmvp1 mutant, the RFOs (raffinose and stachyose) accumulate

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in the Arabidopsis thaliana desiccation-tolerant wild-type and atabi3-1 mutant seeds

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while the RFOs are not detectable in the more penetrant atabi3-5 mutant lines12. During

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seed maturation, VP1 activated the anthocyanin pathway and inhibited the germination-

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specific alpha-amylase genes in aleurone cells in the developing seed13. In Arabidopsis

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thaliana, ABI3 directly controls the seed chlorophyll breakdown process through

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regulation of STAY GREEN2 (SGR2) expression during seed maturation14. ABA

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INSENSITIVE5 (ABI5) is a basic leucine zipper (bZIP) transcription factor which

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plays a key role in the regulation of seed maturation and seed germination15. Medicago

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truncatula Mtabi5 seed accumulate fewer RFOs than that of the wild type control

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during seed maturation15. ABI5 acts downstream of ABI3 and activates late

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embryogenesis programs16. ABI5 binds to the ABRE motif or G-box in the promoter

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of some ABA responsive genes, such as AtEm1 and AtEm6, and regulates gene

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expression17. Despite the extensive studies on the regulatory network of ABI3 and ABI5

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controlling events during late embryogenesis whether the control of RFO production is

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directly influenced by these transcription factors is unknown. However, alterations in

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the abundance of raffinose in maize zmvp1 allelic series; in the Arabidopsis thaliana

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atabi3 allelic series; in atabi5- or Mtabi5-mutant seed leaves little doubt that both the

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biochemistry of production and/or genetic mechanism of control of RFO production is

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linked, directly or indirectly, to this transcription factor cascade in multiple species.

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In this study, we have demonstrated that the two important transcription factors 4


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ZmVP1 and ZmABI5 in the ABA signaling transduction pathway, interact with each

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other and synergistically regulate ZmGOLS2 expression and raffinose accumulation in

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maize seed. These findings provide new insights into the molecular mechanism of

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raffinose biosynthesis.

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Materials and methods

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Plant material

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Maize (Zea mays L.) vp1-R mutant seeds (#326B) in the W22 background, were

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obtained from the Maize Genetics Cooperation Stock Center and were maintained in

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Yangling (34°17'47.9"N 108°04'28.2"E). The zmvp1-R homozygous seeds were

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obtained by self-pollination of the heterozygous stocks. The WT or heterozygous

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kernels are purple, and the homozygous zmvp1-R mutant seeds are yellow. The WT,

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zmvp1-R embryos and endosperms were separated from 20, 22, 24, 26, 28, 30, 32, 34

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and 36 days after pollination (DAP) kernels, then frozen in liquid nitrogen and stored

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at -75 °Cuntil use.

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Soluble Carbohydrate Extraction and HPLC–ELSD Analysis of Sugars

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Soluble sugar extraction followed a published protocol2. Isolated plant tissues (0.5

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g for embryos, 1.5 g for endosperms) were ground into powder in liquid nitrogen. Four

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aliquots of 1 mL of 80 % (v/v) ethanol were sequentially added, homogenized to a

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slurry, and transferred to a 50 mL Falcon tube. Another 3 mL of 80 % ethanol was used

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to wash the mortar and added to the Falcon tube. The Waters Xbridge amide column

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was washed using Acetonitrile: H2O (65:35) as a mobile phase at 1.0 mL/min for 5



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separation of soluble carbohydrate components. An evaporative light-scattering

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detector (ELSD, Waters 2424) was applied to monitor the sugar signal.

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RNA Seq

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Maize embryos separated from 26 DAP seeds of W22 or zmvp1-R were sent to

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BGI-Shenzhen,

China

for

total

RNA

extraction

and

RNA-seq

analysis

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(https://www.bgi.com/us/sequencing-services/rna-sequencing-solutions/

110

transcriptome-sequencing/ ). RNA library preparation and sequencing were performed

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with BGISEQ-500 Platform. The data set was analyzed according to the BGI

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bioinformatics protocols for RNA-seq.

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Identification of ZmABI5

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The seed-specific expressed bZIP transcription factors were selected according to

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the data in the published database18-19. The ZmGOLS2 expression and that of the

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selected bZIP genes was examined by Pearson correlation analysis using a two-tailed

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method (IBM SPSS Statistics 20). The expression of 9 bZIP genes were positively

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correlated with ZmGOLS2 expression. The protein sequences of the 9 selected bZIPs

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and another 9 known bZIP transcription factors were aligned using CLUSTAL W20 and

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then input into MEGA721. The neighbor-joining (NJ) method was used to construct a

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phylogenetic tree22 with the following sequences as given by their accession numbers:

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GRMZM2G175280 (Zea mays, NP_001147562.1); GRMZM2G060216 (Zea mays,

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NP_001104893.1);

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GRMZM2G030877

(Zea

6


mays,

XP_008677589.1);

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NP_001105272.2);

GRMZM2G055413

(Zea

mays,

NP_001148839.1);

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NP_001131340.1);

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(Oryza sativa, BAA 83740.1); MtABI5 (Medicago truncatula, XP_003625810.1);

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AtABF2 (Arabidopsis thaliana, NP_001185157.1); AtABF1 (Arabidopsis thaliana,

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NP_001189934.1); AtbZIP67 (Arabidopsis thaliana, NP_566870.1); AtbZIP12

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(Arabidopsis thaliana, NP_565948.1); HvABI5 (Hordeum vulgare, AAO06115.1); and

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OsABI5 (Oryza sativa, XP_015628684.1).

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Characterization of zmvp1-R mutant plants

GRMZM2G168079 (Zea mays, AQK 99305.1); OsTRAB1

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Genomic DNA was isolated from maize embryos using the CTAB method23. PCR

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was performed to characterize the genotype of the plants using ZmVP1 gene-specific

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primers (F1 and R1, F2 and R2; as listed in Supplemental Table 1). The primer F2a and

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R2 were used for RT-PCR analysis of ZmVP1 gene expression (Primers were listed in

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Supplemental Table 1).

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RNA extraction and real time RT‑PCR

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Total RNA was extracted from embryos following a published protocol24. TRIzol

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Reagent (Takara, Japan) was used for extraction of RNA from protoplasts. The reverse

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transcription (RT) products were diluted fifty-fold with DEPC-treated water. Different

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primers (as listed in Supplemental Table 1) were used for real time RT-PCR analysis

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of gene expression, for ZmGOLS2, primers F3 and R3 were used; for ZmABI5, primers

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F4 and R4 were used; for ZmGAPDH, primers F5 and R5 were used. The expression of 7



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tested genes was normalized to the ZmGAPDH gene expression. The experiments were

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repeated at least three times with independent biological samples.

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Analysis of ZmGOLS2 promoter

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To further investigate whether ZmVP1 was able to directly bind to the promoter of

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the ZmGOLS2 gene and up-regulate its transcription, the 1496 bp ZmGOLS2 promoter

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sequence was analyzed for all putative cis-acting elements using the databases,

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PlantCARE

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CARE.html), PLACE (http://www.dna.affrc.go.jp/PLACE/)25 and Neural Network

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Promoter Prediction (http://www.fruitfly.org/seq_tools/promoter.html).

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Vector construction

156 157

(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/search_

For all manipulations involving PCR, the primer sequences are listed in Supplemental Table 1.

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For construction of vectors used for maize protoplasts transformation, the coding

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sequence of ZmVP1 or ZmABI5 was amplified by PCR from cDNA synthesized from

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RNA that was isolated from mazie B73 embryos (DAP 20). The PCR products were

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purified and inserted into pGL3-basic vector between MluI and XbaI as described26.

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The restriction enzyme sequences were designed in the primers to facilitate cloning (for

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ZmVP1, primers F6 and R6; for ZmABI5, primers F7 and R7).

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For construction of vectors used for characterization of the ZmGolS2 promoter

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activity, overlapping PCR was performed to generate the single mutation or double

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mutation of the G-box in the ZmGOLS2 promoter in a dual luciferase expression vector, 8


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in which the Rluc expression was controlled by a 1093 bp ZmGOLS2 promoter (named

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V6)27. For mutation of the ABRE motif at -647 bp, the 5’ part of ZmGOLS2 promoter

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was amplified by PCR from V6 using primers F8 and R10, the 3’ part of ZmGOLS2

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promoter was amplified by PCR from V6 using primers F10 and R9. The mutated

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ZmGOLS2 promoter (M1) was obtained by PCR using a mixture of the 5’ and 3’

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amplicons as templates with primers F8 and R9. For mutation of the G-box element at

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-237 bp, the 5’ part of the ZmGOLS2 promoter was amplified by PCR from V6 using

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primers F8 and R8, and the 3’ part of the ZmGOLS2 promoter was amplified by PCR

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from V6 using primers F9 and R9. The mutated ZmGOLS2 promoter (M2) was obtained

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by PCR using the two PCR amplicons as templates with primers F8 and R9. For

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mutation of both the ABRE motif and the G-box elements at -647 bp and -237 bp, the

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5’ part of the ZmGOLS2 promoter was amplified by PCR from M1 using primers F8

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and R8 and the 3’ part of the ZmGOLS2 promoter was amplified by PCR from M1 using

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primers F9 and R9. The mutated ZmGOLS2 promoter (M3) was obtained by PCR using

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the two PCR amplicons as templates with primers F8 and R9.

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For expression of ZmVP1 protein used for antibody preparation, the truncated

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coding sequence of ZmVP1 with deletion of its acid A domain was amplified by PCR

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from the cloned vector (used for maize protoplast transformation) using primer F11 and

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R11. For construction of bacterial expression vectors of ZmABI5, the coding sequence

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of ZmABI5 was amplified by PCR from the cloned vector (used for maize protoplast

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transformation) using primer F12 and R12. For both ZmVP1 and ZmABI5, the PCR

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products were purified and inserted into a pET-28a vector using the EcoRI and HindIII 9



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sites which results in a fusion protein with hexahistidyl tag on both N-terminal and C-

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terminal. An additional bacterial expression vector of ZmVP1 was also constructed for

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purification of the protein for EMSA experiments. The DNA fragment of ZmVP1

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encoding the B2 and B3 domain was amplified by PCR from previously cloned vectors

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using primer F13 and R13. The PCR products were purified and inserted into the

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pGEX-4T-1 vector between EcoRI and XhoI.

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For construction of vectors used for yeast two-hybrid assay, the coding region of

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ZmVP1 was cloned by PCR from previously described vector using primer F14 and

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R14. The amplicons were purified and inserted into pGADT7 vector between EcoRI

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and SacI, which is in frame with the GAL4 DNA-activation domain. The coding region

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of ZmABI5 was cloned by PCR from a previously described vector using primer F15

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and R15. The PCR products were purified and inserted into the pGBKT7 vector

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between EcoRI and SalI, which is in frame with the GAL4 DNA-binding domain.

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For construction of vectors used for bimolecular fluorescence complementation

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(BiFC ) assay, the coding region of ZmVP1 was amplified by PCR from previously

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cloned vector using primer F16 and R16. The PCR products were purified and inserted

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into PTF-486 NYFP between SpeI and XhoI. The coding region of ZmABI5 was

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amplified by PCR from previously cloned vectors using primer F17 and R17. The PCR

207

products were purified and inserted into PTF-486 CYFP between SpeI and XhoI.

208

For construction of vectors used in the sub-cellular localization experiment, the

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coding sequence of ZmVP1 or ZmABI5 without the stop codon was amplified using 10


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PCR from a previously cloned vector (for ZmVP1, primer F18 and R18 were used; for

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ZmABI5, primer F19 and R19 were used). The PCR products were purified and inserted

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into pGL3-YFP vector between NheI and MluI28.

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Antibody preparation, protein extraction and western blot

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ZmVP1- or ZmABI5-His6 fusion proteins were expressed in BL21(DE3) E. coli

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strain. The ZmVP1- or ZmABI5-His6 fusion proteins were purified over Nickel

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columns and were used to immunize the rabbit following a published protocol27.

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Western Blot analysis of ZmGOLS2 and ZmABI5 protein expression in maize embryos

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at 20-, 26-, 32- and 36-DAP were performed following a published protocol27.

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Maize protoplast preparation and transformation

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The protoplast preparation and culture followed a published protocol26.

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Preparation of GST‑ZmVP1, His-ZmABI5 fusion protein and electrophoretic

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mobility shift assay (EMSA)

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The bacterial expression vector pGEX-4T-1-ZmVP1 was transformed into the E.

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coli strain BL21 (DE3), and the pET-28a-ZmABI5 was transformed into the E. coli

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(Rosetta gami2, DE3; EMD Millipore) cells. Bacterial cultures were grown to OD600

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nm = 0.6 at 37°C, then supplemented with IPTG (the final concentration was 0.42 mM),

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and further cultured at 25°C for 12 hr. GST-ZmVP1 protein or GST-tag alone (used as

228

a negative control) was purified using a Glutathione Sepharose 4B column (GE

229

Healthcare) according to the manufacturer’s protocol. His-ZmABI5 protein was

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purified using a GenScript High Affinity Ni-Charged Resin (L00250) according to the 11



231

manufacturer’s protocol. For gel mobility shift assays, the doublestranded DNA

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containing the identified G-box and the mutated G-box were prepared by annealing two

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complementary oligonucleotides (G-box-F and G-box-R; mG-box-F and mG-box-R;

234

(Supplemental Table 1)) following a published protocol26. The annealed, double-

235

stranded DNA was used directly for DNA EMSA. The binding reaction of DNA and

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purified protein was conducted using an EMSA kit (Invitrogen, Carlsbad, CA, USA).

237

Mixtures of 10 ng of DNA and different amounts of purified protein were incubated in

238

a binding buffer (25 mM HEPES, 40 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA,

239

and 8% glycerol, pH 8.0) for 30 min at room temperature and then applied to a 6% non-

240

denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide). Gel electrophoresis

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and staining followed the kit protocol. The gel image was captured using ChemiDoc

242

MP (Bio-Rad Inc., Hercules, CA, USA).

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Yeast two-hybrid assay

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Yeast two-hybrid assay was performed following a published protocol28. The

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vectors were co-transformed into yeast strain AH109 through the lithium acetate

246

method29. After 3 days culture, the transformed single colonies were picked up from

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synthetic drop-out (SD)-Leu-Trp (LT) solid medium

248

medium. The exponentially grown yeast cells were centrifuged at 5000 ×g at room

249

temperature and adjusted to OD600 = 0.6 with sterilized double-distilled water. The yeast

250

cells were then diluted 10×, 100× and 1000× with distilled water. Two microliters of

251

the above yeast cells were spotted onto SD-LT and SD-LT-His-Ade (LTHA) medium

252

and grown at 30°C for 2 d (SD-LT plates) or for 5 d (SD-LTHA plates, without or with

and cultured in SD-LT liquid

12


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30 or 60 mM 3-amino-1,2,4-triazole [AT]) before being photographed.

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Subcellular localization and BiFC assay

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For subcellular localization of ZmVP1 and ZmABI5 analysis, different vectors

256

and nuclear localization signal (NLS)-mCherry vector were co-transformed into maize

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protoplasts. For the BiFC assay, vector DNA (10 μg of each) were co-transformed into

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maize protoplasts. After culture for 12 h, YFP and NLS-mCherry signals were

259

monitored and differential interference contrast (DIC) imaging conducted using a

260

scanning confocal microscope (Andor Revolution WD, UK).

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Statistical analysis

262

Statistical analysis was performed using SPSS 20.0. Significance (P < 0.05) was

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assessed by using the Student’s t test or by Duncan's multiple range test.

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Results

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Raffinose is not detectable in zmvp1-R mutant seed.

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The maize viviparous1 (vp1) mutant was characterized by PCR (Supplemental Fig.

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1A). A 2 kb or a 7 kb DNA fragment was amplified from the wild type or zmvp1-R

268

plants, respectively, using primers which span the insertion site (Supplemental Fig. 1A

269

and B; sequence detail in Supplemental Table 1), while a 2 kb DNA fragment was

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amplified from both WT and zmvp1-R plants using primers beyond the Mu element-

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insertion site (Supplemental Fig. 1A and B). PCR and sequencing of the resultant

272

amplicons revealed a typical 5-bp duplication flanking the Mu element (Supplemental

273

Fig. 1A). The transcription of ZmVP1 was detected in WT plants, but not in zmvp1-R 13



274

plants, as determined by RT-PCR using F2a and R2 primers (Supplemental Fig. 1C).

275

Consistent with the mRNA expression, ZmVP1 protein accumulation was detected in

276

WT plants, but not in zmvp1-R plants using western blot hybridization (Supplemental

277

Fig. 1D).

278

The zmvp1-R seeds started to complete germination on the cob at 26 DAP and all

279

kernels had completed germination by 36 DAP while WT seed did not complete

280

germination precociously, even at the end of maturation (55 DAP; Fig. 1A). The

281

glucose and sucrose contents in the zmvp1-R embryo were significantly greater than in

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the WT embryo between 20- and 36-DAP as determined by HPLC (Fig. 1B-C,

283

Supplemental Fig. 2). Myo-inositol accumulation in the zmvp1-R embryo was greater

284

than that of WT at 20- and 22-DAP, and was less than that of WT at 26-, 32-, 34- and

285

36-DAP (Fig. 1D). The raffinose and galactinol amount in WT embryos started to

286

accumulate at 28 DAP and reached to its peak at 32- or 34-DAP and then declined,

287

while they were not detected at any point in the zmvp1-R embryos (Fig. 1E-F). The

288

glucose content in the endosperm of zmvp1-R seeds was higher than that of WT at 34-

289

and 36-DAP (Supplemental Fig. 3A). The sucrose in the endosperm of zmvp1-R seeds

290

was less than that of WT at 32- and 34-DAP, but was higher than that of WT at 36 DAP

291

(Supplemental Fig. 3B). Myo-inositol, galactinol and raffinose content of the

292

endosperm were all below the detection limit in both WT and the zmvp1-R mutant

293

(Supplemental Fig. 4).

294

ZmGOLS2 is down-regulated in the zmvp1-R embryo

295

GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE are two enzymes 14


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296

that are responsible for raffinose biosynthesis (Fig. 2A). To investigate the molecular

297

mechanism underlying the absence of raffinose in the zmvp1-R embryo, the

298

transcriptome was compared between WT and zmvp1-R developing embryos at 26 DAP,

299

a time point prior to raffinose accumulation but when GALACTINOL SYNTHASE2 and

300

RAFFINOSE SYNTHASE transcript and ZmGOLS2 protein is present in considerable

301

abundance (Fig. 2; Supplemental Fig. 5; Supplemental Table 3). The expression of both

302

ZmGOLS2 and ZmRAFS was significantly decreased in the zmvp1-R embryo as

303

compared with that of WT (Supplemental Table 3). The expression of ZmGOLS2 or

304

ZmRAFS in the zmvp1-R embryo was significantly less than that of WT at 20-, 26-, 32-

305

and 36-DAP as determined by quantitative RT-PCR (Fig. 2B; Supplemental Fig. 5).

306

The ZmGOLS2 protein accumulation in the zmvp1-R embryo at 26-, 32- or 36-DAP

307

was less than that of WT as determined by western blot analysis (Fig. 2C).

308

The predicted cis-elements in the 5’ regulatory region of ZmGOLS2 are depicted

309

in the sequence provided (Supplemental Fig. 6). There was no RY motif in the

310

ZmGOLS2 promoter (Supplemental Fig. 6), suggesting that ZmVP1 may not directly

311

bind to the ZmGOLS2 promoter as this is the known element to which ZmVP1 binds30.

312

ABI5 binds to the G-box or ABRE motif in the promoter of its target genes17. There are

313

3 G-boxes and 2 ABRE elements in the promoter of ZmGOLS2 (Supplemental Fig. 6),

314

suggesting that ABI5 may directly regulate ZmGOLS2 expression.

315

ZmVP1 and ZmABI5 synergistically up-regulate the expression of ZmGOLS2.

316

GFP, ZmVP1 or ZmABI5 expression vectors were constructed (Fig. 3A). The

317

ZmABI5 or ZmVP1/ZmABI5 expression vectors were co-transformed with a variety of 15



318

dual luciferase expression vectors into maize protoplasts (Fig. 3A). In these

319

experiments, the Rluc expression in the dual luciferase expression vector was controlled

320

by the ZmGOLS2 promoter (V6), or by a mutated ZmGOLS2 promoter in which one

321

ABRE motif (M1) or one G-box (M2), or both ABRE and G-box (M3) were mutated

322

(Fig. 3A). Rluc expression, regardless of ZmGOLS2 promoter, was not changed by

323

simultaneous ZmVP1 expression (Fig. 3B). However, Rluc expression, when controlled

324

by the V6 or the M1 fragment, was significantly increased by simultaneous ZmABI5

325

over-expression and this was further and dramatically increased by the co-expression

326

of ZmVP1/ZmABI5, as compared with the GFP expression (Fig. 3B). Rluc expression

327

controlled by the M2 or M3 fragment, was not changed by simultaneous expression of

328

ZmVP1, ZmABI5 or ZmVP1/ZmABI5 as compared with the GFP expression (Fig. 3B).

329

These data show that the G-box in the ZmGOLS2 promoter is functional in regulation

330

of its expression in response to ZmABI5. Furthermore, endogenous ZmGOLS2

331

expression and protein abundance, while not enhanced by ZmVP1 expression, was

332

significantly increased in maize protoplasts expressing ZmABI5 and this was further

333

augmented by the co-expression of ZmVP1/ZmABI5 as determined by real time RT-

334

PCR and western blot analysis, as compared with the GFP expression (Fig. 3 C-E).

335

DNA-EMSA results showed that purified ZmABI5 protein was able to bind to the G-

336

box elements of the ZmGOLS2 promoter in vitro, while the purified ZmVP1 protein, or

337

the GST tag failed to bind to the G-box element of the ZmGOLS2 promoter (Fig. 3F).

338

ZmVP1 interacts with ZmABI5

339

There are three G-box motifs in the ZmGOLS2 promoter (Supplemental Fig. 6). It 16


Page 16 of 34

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340

has been reported that bZIP transcription factors are capable of binding G-box elements

341

due to the core ABRE motif they contain31 and regulate their target genes’ expression.

342

There are 125 bZIP genes predicted in the maize genome18. By searching the published

343

maize embryo and endosperm transcriptome19, we found the expression of 9 bZIP genes

344

that were positively correlated with the expression of ZmGOLS2 in maize embryos

345

(Supplemental Table 4). Phylogenetic analysis of these 9 maize bZIP transcription

346

factors and the known ABI5s from other species showed that GRMZM2G168079 is

347

most closely related to OsABI5 and AtABI5 (Fig. 4A). This GRMZM2G168079

348

protein was named ZmABI5. The mRNA and protein expression of ZmABI5 is

349

significantly decreased in the zmvp1-R embryo as determined by RT-PCR and western

350

blot analysis (Fig. 4B-C).

351

To validate the expected nuclear sub-cellular localization of the two transcription

352

factors, ZmVP1-YFP and ZmABI5-YFP fusion protein expression vectors were

353

constructed and separately transformed into maize protoplasts (Supplemental Fig. 7).

354

The YFP signal was monitored by confocal microscopy to determine protein subcellular

355

residency. The signal of both ZmVP1-YFP and ZmABI5-YFP was detected in the

356

nucleus as anticipated (Supplemental Fig. 7).

357

bZIP transcription factors are also known to interact with ABI3, an Arabidopsis

358

thaliana homolog of VP132. Yeast two-hybrid assay showed that ZmVP1 and ZmABI5

359

interacted with each other (Fig. 5A). The interaction of ZmVP1 and ZmABI5 was

360

further confirmed using BiFC in maize protoplasts (Fig. 5B). The YFP signal was not

361

detected in the protoplasts transformed with control constructs but was detected in 17



362 363

protoplasts transformed with ZmVP1-YN and ZmABI5-YC (Fig. 5B). Taken together, our data showed that ZmVP1 interacts with ZmABI5 and

364

regulates ZmGOLS2 expression and raffinose biosynthesis in maize seeds (Fig. 6).

365

Discussion

366

The regulation of raffinose biosynthesis

367

GOLS and RAFS are two key enzymes for raffinose biosynthesis (Fig. 2). The

368

genes encoding GOLS are induced by environmental stresses in plants. Over-

369

expression of GOLS genes isolated from different species increased the amounts of both

370

galactinol and raffinose and improved drought tolerance in transgenic plants27, 33. These

371

results suggest that GALACTINOL SYNTHASE may be a bottleneck for raffinose

372

accumulation. The consistent benefits on plant abiotic stress tolerance of GOLS

373

hyperproduction through transgenic means warrants an examination of how

374

endogenous GOLS is regulated in order to embark on more nuanced manipulations of

375

GOLS transcription, ensuing galactinol and raffinose production, and the level of

376

protection this affords crop productivity in stressful conditions. A better understanding

377

of the various elements acting to boost transcription of GOLS will aid interpretation of

378

intraspecific, natural variability in galactinol and raffinose production upon the

379

induction of stress. To this end, we have found that ZmGOLS2 is induced by

380

dehydration or heat stress5, 26, 34. In addition, we identified that ZmDREB2A regulates

381

the expression of ZmGOLS2 by binding to the DRE elements of the ZmGOLS2

382

promoter in maize leaf protoplasts27, suggesting that dehydration stress or ZmDREB2A

383

up-regulates raffinose biosynthesis in plant leaves. A WRKY transcription factor has 18


Page 18 of 34

Page 19 of 34


384

also been characterized to bind to the W-box motif of the Boea hygrometrica

385

GALACTINOL SYNTHASE promoter and regulate its expression35. ZmVP1 is

386

predominantly expressed in maize seed9. ZmVP1 positively regulates ZmGOLS2

387

expression and does so dependent upon, and synergistically with, ZmABI5 (Fig. 3).

388

These data suggest that ZmGOLS2 is regulated by different transcription factors under

389

different spatio-temporal conditions. It now appears as though ZmVP1 and ZmABI5

390

are key regulators for raffinose biosynthesis in the maize embryo.

391

Multiple transcription factors, such as bZIPs, B3s, MYBs, and DOFs, function in

392

the zygotic embryo during seed maturation36. The bZIP53 protein specifically binds to

393

the G-box element in the promoter of ALBUMIN 2S2 gene and the formation of the

394

heterodimer of bZIP53 with bZIP10 or bZIP25, significantly enhances the dimer’s

395

DNA binding activity and synergistically increases its target gene expression over that

396

of bZIP53 alone36. The bZIP53 does not directly interact with ABI3, but the ternary

397

complex formation between the bZIP heterodimers and ABI3 increases target gene

398

expression still further36. Our results showed that ZmABI5 directly interacted with

399

ZmVP1 (Fig. 5) and co-expression of ZmABI5 and ZmVP1 in maize protoplasts

400

synergistically enhanced ZmGOLS2 expression (Fig. 3). Our data cannot exclude the

401

possibility that another bZIP transcription factor, up-regulated by ZmVP1, may

402

participate to form a heterodimer with ZmABI5 and that this is the cause of the observed

403

ZmVP1 stimulation of ZmABI5 transcriptional control of ZmGOLS2. If there was a

404

heterodimer of ZmABI5 and another bZIP factor, it was not required for the interaction

405

of ZmABI5 and ZmVP1 in yeast where the existence of an endogenous bZIP 19



406

transcription factor capable of mediating the ZmABI5-ZmVP1 interaction is

407

improbable. The more parsimonious explanation of our results is that ZmABI5 binds

408

the G-box most proximal to the ZmGOLS2 transcriptional start site and that ZmVP1

409

binds ZmABI5 to boost the transcriptional stimulation of ZmGOLS2.

410

RAFFINOSE SYNTHASE (ZmRAFS) is the key enzyme for raffinose

411

biosynthesis. However, to date, the transcriptional regulation of ZmRAFS has not been

412

characterized. Though ZmRAFS mRNA expression was significantly decreased in the

413

zmvp1-R embryo (Supplemental Fig. 5), using the same strategy used to characterize

414

the transcriptional regulation of ZmGOLS2, we found that the ZmRAFS gene was not

415

regulated by ZmVP1, ZmABI5, or their dimer in maize protoplasts (Supplemental Fig.

416

8), suggesting that the regulation of ZmRAFS deviates considerably from that of

417

ZmGOLS2.

418

The dependence of raffinose biosynthesis on the ABA signaling pathway

419

The regulation of raffinose biosynthesis appears to be independent of the abscisic

420

acid (ABA) signaling pathway, at least during dehydration stress in Arabidopsis

421

thaliana37. ZmVP1, like its Arabidopsis thaliana orthologue, ABI3, is responsible for

422

the plant cell to react to ABA9 and ABI3 in Arabidopsis thaliana, regulates the

423

expression of ABI516. Similarly, the expression of ZmABI5 was down-regulated in the

424

zmvp1-R mutant (Fig. 4B, C). VP1/ABI3 controls target gene expression by binding to

425

the RY/Sph element through its B3 domain while ZmABI5 does so by binding to G-

426

box (ABRE) elements. A synopsis of our results follows: in the absence of a functional

427

VP1, neither galactinol nor raffinose accumulate in the maize embryo (Fig. 1), 20


Page 20 of 34

Page 21 of 34


428

ZmGOLS2, ZmRAFS, and ZmABI5 transcripts are all repressed below WT in embryos

429

(Fig. 2B; Fig. 4B, C; Supplemental Fig. 5), and ZmGOLS2 and ZmABI5 protein

430

amounts are also much below WT (Fig. 2C, 4C); yet there are no RY/Sph motifs in the

431

ZmGOLS2 promoter to which VP1 could bind while there are three G-box motifs

432

(Supplemental Fig. 6); overexpression of ZmVP1 in maize protoplasts failed to up-

433

regulate the expression of reporter or endogenous ZmGOLS2 or of endogenous

434

ZmABI5 protein (Fig. 3); using the Arabidopsis thaliana ABI3/ABI5 interaction

435

paradigm we identified the maize bZIP most similar to ABI5 and named this putative

436

ortholog ZmABI5 (Fig. 4A); ZmABI5 transcript and protein abundance is severely

437

reduced in the zmvp1 embryo (Fig. 4B, C); ZmVP1 interacts with ZmABI5 in both yeast

438

and in maize protoplasts subjected to BiFC assays (Fig. 5); ZmABI5 overexpression in

439

maize protoplasts results in up-regulation of ZmGOLS2; the hyper-production of

440

ZmVP1, when ZmABI5 is also present in superabundance, synergistically increased

441

ZmGOLS2 expression and protein amounts above that of ZmABI5 alone (Fig. 3). Taken

442

together, these data suggest that ZmVP1 not only regulates the expression of ZmABI5,

443

at least in developing embryos, but also interacted with ZmABI5 to enhance its

444

transcriptional activation of at least ZmGOLS2. There is no difference in AtGOLS1 and

445

AtGOLS2 expression or RFO accumulation between the ABA deficient mutant atnced3

446

and WT under dehydration stress38, suggesting that galactinol biosynthesis, at least

447

from these two AtGOLS family members, was regulated by ABA-independent

448

pathways. Conflicting with this finding, the ABA-deficient and ABA-insensitive

449

aba1:abi3-1 double mutant seeds are viable but with reduced desiccation tolerance; 21



450

supplementation of ABA to the aba1:abi3-1 double mutant seed increased RFO

451

biosynthesis and promoted desiccation tolerance39. Raffinose biosynthesis was

452

stimulated by abiotic stress, such as drought-, salt-, or heat-stress, in plant vegetative

453

tissues. This type of regulation is mediated by some transcription factors, such as

454

DREB2A, and can be ABA-independent27. ABA promotes seed maturation and inhibits

455

completion of seed germination in many plant species40. During maize seed maturation,

456

the accumulation of raffinose is regulated by VP1 (Fig. 1), suggesting that the presence

457

of ABA stimulates the biosynthesis of oligosaccharides while suppressing precocious

458

germination.

459

The soluble oligosaccharides in seed and their possible functions

460

The absolute and relative abundance of different soluble oligosaccharides in seeds

461

determines the seed physiological status41. The conversion of the small, soluble

462

carbohydrate pool from predominately reducing monosaccharides to predominately

463

non-reducing sugar oligomers during orthodox seed maturation reduces the absolute

464

number of these dissolved solutes, protects against Maillard reactions by tying up

465

reducing ends in inter-monosaccharide covalent bonds, while creating oligosaccharides

466

capable of forming a glassy state conducive to the intracellular milieu withstanding

467

desiccation42. As well, the relative abundance of sucrose to RFO appears to influence

468

seed vigor2. The zmvp1-R mutant seed exhibits increased glucose and sucrose and

469

decreased galactinol and raffinose, compared with that of WT, and showed a viviparous

470

phenotype (Fig. 1B-D). This phenomenon was also observed in Arabidopsis thaliana

471

abi3-5 mutant seeds43. Raffinose accumulates in the late stages of seed development 22


Page 22 of 34

Page 23 of 34


472

and is hydrolyzed during seed germination2, 44. The maize zmvp1-R mutant seed and the

473

Arabidopsis thaliana mutant lines (abi3-5, lec1-1 and fus3-3)45 with reduced raffinose,

474

exhibited a desiccation-intolerant phenotype (a drastically curtailed longevity),

475

supporting the previous findings that the ratio of raffinose to sucrose determines seed

476

longevity in maize seeds2. RFOs are considered to be anti-nutritional46-47 while, on the

477

contrary, RFOs are an important energy resource for beneficial microflora in the small

478

intestine48. Understanding the regulatory mechanism for raffinose biosynthesis will aid

479

interpretation of intraspecific, natural variability in galactinol and raffinose production

480

upon the induction of stress, providing plant breeders with a more sophisticated model

481

of whole-genome alterations directed at using this biochemical pathway to combat

482

productivity loss due to plant stress. At a more fundamental level, an understanding of

483

the precise physical function of RFO-mediated protection of stress susceptible cellular

484

constituents will serve to pinpoint which entities in the cell are most susceptible to stress

485

while demonstrating one means by which plants protect these entities from stress as a

486

paradigm on which to focus synthetic biological research.

487

Supporting Information

488

The information about ZmVP1 mutant characterization, ZmRAFS expression and

489

regulation, Analysis of ZmGOLS2 promoter, HPLC analysis of endosperm sugar profile,

490

subcellular localization of ZmVP1 and ZmABI5, the RNAseq data, the primer list and

491

the abbreviation list were provided as supplemental data. The Supporting Information

492

is available free of charge on the ACS Publications website.

493

Author Information 23



494

Corresponding Author Tianyong Zhao; Email: [email protected]; ORCID ID:

495

0000-0002-1278-2842; Co-authors: Lynnette M.A. Dirk ORCID ID: 0000-0002-7564-

496

5095; A. Bruce Downie ORCID ID: 0000-0001-6680-0551.

497

Funding

498

This research was funded by the National Key Research and Development Plan

499

(2018YFD0100901) and the NSFC (31671776) to TZ. For LMAD and ABD, this work

500

was supported by the USDA National Institute of Food and Agriculture, Hatch project

501

1002299.

502

Acknowledgements

503

The HPLC experiments were conducted in The Biology Teaching and Research Core

504

Facility at College of Life Sciences, Northwest A&F University.

505

Notes

506

The authors declare no conflicts of interest. All the abbreviations are listed in

507

Supplemental Table 2.

508

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509 510 511

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Figure Captions

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Figure 1. Raffinose accumulates in wild type W22 maize seed but was not detectable

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in the zmvp1-R mutant seed during seed maturation.

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(A) Zmvp1 mutant seed germinated on the cob during seed development stage and its

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morphology can be distinguished from wild type W22. DAP: days after pollination.

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Bar = 5 mm. (B-F) Comparison of different sugar contents in embryos between WT

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and the zmvp1-R mutant. B: Glucose; C: Sucrose; D: myo-inositol; E: Galactinol; F:

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Raffinose. (µmol•g-1 DW: micromolar per gram dry weight). Values are means ± SE

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(n=3). **p

Maize VIVIPAROUS1 Interacts with ABA INSENSITIVE5 to Regulate

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