Comprehensive Transcriptome Analysis of Phytohormone

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Comprehensive Transcriptome Analysis of Phytohormone Biosynthesis and Signaling Genes in the Flowers of Chinese Chinquapin (Castanea henryi) Xiaoming Fan, Deyi Yuan, Xiaoming Tian, Zhoujun Zhu, Meilan Liu, and Heping Cao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03755 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Journal of Agricultural and Food Chemistry 1

Comprehensive Transcriptome Analysis of Phytohormone Biosynthesis and Signaling Genes in the Flowers of Chinese Chinquapin (Castanea henryi)

Xiaoming Fan1,2, Deyi Yuan*,1,2, Xiaoming Tian3, Zhoujun Zhu1,2, Meilan Liu1,2, Heping Cao*,4

1

Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees of Ministry of

Education, Central South University of Forestry and Technology, Changsha, Hunan, China 2

Key Laboratory of Non-Wood Forest Products of State Forestry Administration, Central South

University of Forestry and Technology, Changsha, Hunan, China 3

Hunan Forest Botanical Garden, Changsha, Hunan, China

4

U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research

Center, New Orleans, Louisiana, United States of America

Manuscript Correspondence: Heping Cao, PhD USDA-ARS-SRRC 1100 Robert E. Lee Blvd. New Orleans, LA 70124 Phone: 504-286-4351 E-mail: [email protected]

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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1

ABSTRACT

2

Chinese chinquapin (Castanea henryi) nut provides a rich source of starch and nutrients as food

3

and feed, but its yield is restricted by a low ratio of female to male flowers. Little is known about

4

the developmental programs underlying sex differentiation of the flowers. To investigate the

5

involvement of phytohormones during sex differentiation, we described the morphology of male

6

and female floral organs and the cytology of flower sex differentiation, analyzed endogenous

7

levels of indole-3-acetic acid (IAA), gibberellins (GAs), cytokinins (CKs) and abscisic acid

8

(ABA) in the flowers, investigated the effects of exogenous hormones on flower development,

9

and evaluated the expression profiles of genes related to biosyntheses and signaling pathways of

10

these four hormones using RNA-Seq combined with qPCR. Morphological results showed that

11

the flowers consisted of unisexual and bisexual catkins, and could be divided into four

12

developmental stages. HPLC results showed that CK accumulated much more in the female

13

flowers than that in the male flowers; GA and ABA showed the opposite results; while IAA did

14

not show a tendency. The effects of exogenous hormones on sex differentiation were consistent

15

with those of endogenous hormones. RNA-Seq combined with qPCR anlyses suggest that

16

several genes may play key roles in hormone biosynthesis and sex differentiation. This study

17

presents the first comprehensive report of phytohormone biosynthesis and signaling during sex

18

differentiation of C. henryi, which should provide a foundation for further mechanistic studies of

19

sex differentiation in Castanea Miller species and other non-model plants.

20

KEYWORDS

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Castanea Miller, flower development, hormone, RNA-seq, sex differentiation, transcriptome


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INTRODUCTION

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Castanea Miller (Fagaceae) is a monoecious plant genus with ecological and economic value.

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Castanea Miller species are cultured in many countries due to good taste and rich in nutrition of

25

the nuts. Chinese chinquapin (Castanea henryi) is widely distributed in the South of China.

26

However, the low ratio of female and male flowers (1/2000-1/3000) is one of the reasons for the

27

low seed yield of the species. In a previous study, we systematically investigated the micro- and

28

mega-sporogenesis in addition to male and female gametogenesis in C. henryi (Castanea Miller

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species).1 Our results have shown that the ovules primordia are immature at the time of

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pollination and require 6 weeks to become fully developed. During this 6-week period, 32% of

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ovules abort because of the inability to form an embryo sac. Approximately 16% of ovules abort

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because of abnormal development of embryo sacs. From 7 weeks to 8 weeks after pollination in

33

which double fertilization occurs, most mature ovules are also aborted because of cell

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degeneration in gametophytes. Only one ovule can develop into a ripe seed. Thus, the delayed

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fertilization in C. henryi may be necessary to increase the time for mate choice and selective

36

fertilization. A certain number of ovule abortions may be the result of delayed fertilization to

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maximize reproductive success. However, there is a lack of information on sex expression in

38

Chinese chinquapin.1 Therefore, it is important to explore the mechanism of floral sex

39

differentiation in the species; which may provide valuable information for increasing seed yield.

40

Reproductive biology has been studied in several species of Fagaceae family, such as

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cytological observation of floral biology and embryo development,2-4 pollen germination and

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tube growth,5 male-sterile,6 and delayed fertilization.7 In recent years, researchers have focused



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much of their attention on the studies of a novel natural bud mutant of Chinese chestnut

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(Castanea mollissima).8 Like Chinese chinquapin, Chinese chestnut has far more male flowers

45

than female flowers; which are limiting factors for nut yield. A naturally occurring mutation of

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male catkins was found on a single branch of a Chinese chestnut tree in the mountains near

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Beijing, China. The mutation at short catkin1 (sck1) resulted in the catkin length of sck1 mutant

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only 1/6 to 1/8 that of the wild-type male catkin on the same tree. The mutation was associated

49

with a greater number of female flowers and increased yield, but the information of sex

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expression in the species remains limited.

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Sex determination is under the control by both genetic factors and environmental

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conditions.9 Phytohormones have been known to modulate development throughout the plant life

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cycle.10 Gibberellins (GAs) are present in floral regulatory networks, essential for the

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development of reproductive organ, especially for floral determination and commitment.11,12

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Auxins play a major role in organ initiation and organogenesis; which not only determines

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whether flower primordia are formed, but also specifies floral organs.13 Cytokinins (CKs) are

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known to contribute to the control of flower sex type of Jatropha curcas14 and Luffa

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cylindrical,15 and have been used to induce female flower production for decades in horticultural

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practices.16,17 The phytohormone biosynthesis and signaling genes have been identified and

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reported to be involved in sex differentiation in several species.

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Although several phytohormones are known to have essential roles during gender

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differentiation, there is no inclusive study of their involvement in Castanea Miller species.

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Transcriptomics technology have been used in our laboratories in the studies of unigene-derived


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simple sequence repeat markers in developing seeds of tung tree (Vernicia fordii),18 fructose-1,6-

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bisphosphate aldolase gene family in the seeds of tea oil tree (Camellia oleifera),19 starch and

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sucrose metabolism in the seeds of Chinese chestnut,20 and glycolytic pathway genes in

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developing kernels of Eucommia ulmoides.21 In this study, we investigated several aspects of

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flowering and its regulation by plant hormones in Chinese chinquapin using the same

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technologies. Firstly, we carried out the morphological observation of sex differentiation and

70

flower development in C. henryi. Secondly, we analyzed the endogenous levels of four kinds of

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phytohormones, namely indole-3-acetic acid (IAA), GAs, CKs and abscisic acid (ABA) in the

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flower. Thirdly, we investigated the effects of the four exogenous phytohormones on the sex

73

differentiation. Fourth, transcriptome analysis was used to analyze cell type-specific expression

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profiles of the biosynthetic and signaling genes for these four phytohormones. Finally, we

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validated the expression of 18 unigenes encoding key enzymes in the phytohormone biosynthesis

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pathway by qPCR using RNAs from four stages of C. henryi flower. We expected that such

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systematic characterization will contribute to the understanding of genetic mechanisms of

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phytohormones involved in sex differentiation of C. henryi and other non-model plants, and

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eventually facilitating genetic engineering and breeding of C. henryi.

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MATERIALS AND METHODS

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Plant Materials and Sample Collection. Chinese chinquapin (Castanea henryi) ‘Huali 4’

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cultivar was obtained from Chengzhou, Hunan Province, China.1 Catkins/flowers were selected

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from 10 trees aged at 8 years. The samples were collected every ten days from April to May,



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which contained four time points according to preliminary research (Figure 1). As male and

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female flowers cannot be distinguished by the external morphology during the first time point,

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the entire flower was collected. The male and female flowers were collected at the other three

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time points as per the following protocol: only the first lowermost projection of the female

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flower and 6-20 projections of the male flower were collected from the mixed catkin (bisexual

90

catkin). Other tissues, except for floral organs, were removed thoroughly; at least 500

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branches/flowers were collected at each time point. In order to ensure the accuracy of the

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sampling, only the mixed catkins located on the second and higher bearing branches were

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collected (if a catkin was determined as a mixed catkin, then the nodes above could only give

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rise to mixed catkins).

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Light Microscope Observation of Female and Male Traits during C. henryi Sex

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Differentiation. The samples were prefixed in Carnoy’s solution (acetic acid:ethanol=1:3) for 5

97

h and subsequently maintained in 70% alcohol for 24 h. These were transferred to 70% ethanol

98

and stored at 4°C prior to sectioning. The sex differentiation process was observed using the

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conventional paraffin sectioning method, a modified version of the method described.22 Tissues

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were sectioned into 10-µm sections using a Leica RM2265 microtome (Leica Camera AG,

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Solms, Germany); these were stained with hematoxylin-eosin Y. Observations and

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photomicroscopy of the sections were conducted using a microscope (BX-51; Olympus, Tokyo

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Japan).

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Quantification of Phytohormones.

Approximately 100 mg (fresh weight) of each

sample was utilized for this analysis. According to our light microscope observation, the sex


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differentiation process of Chinese chinquapin flowers could be divided into four stages. The

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flower samples for hormone quantification included seven samples from all four stages of male

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and female flowers (refer “Results” section for details). The phytohormones were quantified

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using the autosampler of a HPLC-MSn system, utilizing no-waste mode as described.23

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Exogenous Hormone Application. A stock solution of GA3, IAA, 6-benzyladenine (BA) or

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ABA was prepared by dissolving 0.25 g of each in 50 ml of 95% ethanol except BA was

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dissolved in 1 N NaOH and bringing the final volume to 1 L with distilled water. Tween-20

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(0.05%) was used as a surfactant for dissolution of each working solution. The final

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concentration of each hormone was 0.25 g/L. Control treatment was sprayed with 5 mL of

115

distilled water containing equivalent amount of ethanol and Tween-20. The spraying was carried

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out thrice with the first application in mid-March before bud germination, and followed by 1-

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week intervals by using a hand sprayer. Forty branches from 15 plants were used for each

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treatment. The total number of bisexual catkins, staminate catkins, female flowers and male

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flowers of each fruiting branches were counted.

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RNA Isolation and cDNA Library Construction.

RNA was extracted from frozen

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bisexual catkins obtained 2 days after bisexual catkin formed (DAB, prior to or initial stages of

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sex differentiation), and from frozen female and male flowers of bisexual catkins collected at 12

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DAB (during sex differentiation) using a PureLink RNA Mini Kit (Omega BioTek, Norcross,

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GA, USA). The purified RNA was used for cDNA library construction and transcriptome

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analysis. Catkins/flowers at all four stages of development were subjected to qPCR analyses.

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RNA purification and cDNA library construction was performed as detailed.20



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Transcriptome Sequencing and Unigene Annotation. The cDNA sequencing was

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performed using the Illumina Solexa HiSeq 2000 sequencing system (Biomarker Technologies

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Co., China).21 Raw data were recorded and filtered to remove reads with low quality and

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contaminated adaptors. The remaining high-quality data were used to construct unigenes by

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Biomarker Technologies using an open software platform, the Trinity software platform, with

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default parameter settings (https://github.com/trinityrnaseq/trinityrnaseq/wiki) according to the

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published procedure.24 Unigene sequences were subjected to a basic local alignment search tool

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BLASTX (http://balst.ncbi.nlmm.nih.gov/Blast.cgi) search in the Nr database (National Center

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for Biotechnology Information (NCBI) non-redundant protein database), Swiss-Prot, and clusters

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of orthologous groups (COGs), for function annotation and classification. Subsequently, the

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InterProScan software database was used for Gene Ontology (GO) function annotation and

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classification. Lastly, the unigene sequences were annotated with the Kyoto encyclopedia for

139

genes and genomes (KEGG) data for studying the related metabolic pathways.

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Identification of Genes and Digital Analysis of Differentially Expressed Genes. In order

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to obtain information regarding the biosynthesis and signal transduction of four kinds of

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phytohormones (IAA, GA, CK and ABA), the transcripts involved in these pathways were

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initially filtered by KEGG maps. However, a significant number of known enzymes were not

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included in these pathways. Therefore, the corresponding genes, as well as genes related to floral

145

organ development, were also identified via a manual search. However, the annotations of the

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same transcript determined from the Nr, Swiss-Prot, and KEGG databases were not always

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consistent with each other. Some sequences with ambiguous annotations were confirmed online


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by comparisons with the Nr database, using BLASTX (http://balst.ncbi.nlmm.nih.gov/Blast.cgi),

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when the annotations were determined from less than two different databases.

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The expression levels of the unigenes from three samples, the bisexual catkins of stage 1

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(B0), the female flowers in bisexual catkins of stage 2 (F1) and the male flowers in bisexual

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catkins of stage 2 (M1) were compared and analyzed using the published method.25 This was

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done by comparing the reads sequence of sample sequencing with unigene library using Bowtie

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software.26 Query sequences were aligned using ClustalW version 1.81 (http://align. genome.jp/)

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followed by manual alignment.27 According to the alignment information, the expression level

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was estimated using RSEM.28 EBSeq software was used for differential expression analysis.29

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The false discovery rate (FDR) was used to determine the threshold P-value for multiple testing.

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The reads per kilobase per million mapped reads (RPKM) method was used to calculate unigene

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expression; that is, unigene is differentially expressed if RPKM values between two samples are

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≥ 2-fold difference. FDR < 0.001 and absolute value of the log2 RPKM ratio > 1 were used as

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the threshold to determine significant differences in gene expression.30

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qRT-PCR Analysis. Eighteen key genes, mostly coding for phytohormone biosynthesis,

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were identified from the unigene sequences for digital expression profile analysis. The

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expression of these 18 genes was analyzed by qPCR in seven flower samples from all four stages

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of male and female flowers. The unigene names, annotations, sequences of forward and reverse

166

primers, and properties of qPCR products for these key genes and GADPH gene of C. henryi

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serving as the reference gene were listed in Table 1. The qPCR primers were designed by the

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Primer Express software. qRT-PCR was performed in triplicate using the SYBR Green method



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and the Bio-Rad CFX system (Bio-Rad, Hercules, CA, USA) as detailed.31 The relative gene

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expression for qPCR data was calculated using GAPDH as loading control and B0 as sample

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control by the 2(-Delta Delta C(T)) Method.32 The significant differences among different

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samples were determined with Duncan’s multiple-range test, using IBM SPSS Statistics 20.0.

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The correlation between gene expression levels and endogenous hormones content was analyzed

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using the method of Pearson correlation coefficient with IBM SPSS Statistics 20.0.

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RESULTS

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Morphological Observation of Male and Female Floral Organs. The Chinese chinquapin

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flowers were grown on the catkin axis, consisting of two types of catkins: the unisexual

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staminate catkins (SC) located close to the bottom of the flower branches, and the bisexual

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catkins (BC) developed near the apex (Figure 1-I). Female flowers usually occurred singly at the

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base of the bisexual catkins. Male flowers were spirally inserted along the catkin axis in cluster

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of three. The differentiation and development of male flowers varied, depending on the

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arrangement of the flowers. In bisexual catkins, the female flowers and male flowers formed at

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the same time in early April. However, in unisexual staminate catkins, differentiation began

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earlier than April, and anthesis started late April. The onset of male catkin differentiation and

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development was earlier than that of the bisexual catkins; the development of female and male

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flowers on the staminate catkins was not synchronized. Therefore, this study only focused on the

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female and male flowers on the bisexual catkin. In order to ensure the accuracy of sampling, only

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the bisexual catkins located on the second and higher bearing branches were collected for


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analysis. Worth of mention is that the Chinese chinquapin flowers are naturally pollinated and

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this type of pollination has little correlation with the process of flower sex differentiation.

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Cytological Observation of Flower Sex Differentiation.

The sex differentiation

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process of Chinese chinquapin flowers could be divided into four stages (Figure 1-II, A-D).

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Stage 1 (B0) was within 2 days after bisexual catkin formed when sex differentiation is just

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beginning, and the stamen and carpel primordial begin to form (Figure 1-II, A). Stage 2 (F1, M1)

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was approximately 3-21 days after bisexual catkin formed when carpel and stamen primordia

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began to differentiate to 8-12 distinct carpels and 12 stamens (Figure 1-II, B). Stage 3 (F2, M2)

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was approximately 22-41 days after bisexual catkin formed when female flower bloomed (Figure

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1-II, C). Stage 4 (F3, M3) was approximately 42-52 days after bisexual catkin formed when male

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flower bloomed to male inflorescence segments withered and dropped (Figure 1-II, D). The

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flower samples were then divided into seven groups according to their development stages:

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sample 1 (B0), the bisexual catkins of stage 1; sample 2 (F1), the female flowers in bisexual

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catkins of stage 2; sample 3 (M1), the male flowers in bisexual catkins of stage 2; sample 4 (F2),

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the female flowers in bisexual catkins of stage 3; sample 5 (M2), the male flowers in bisexual

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catkins of stage 3; sample 6 (F3), the female flowers in bisexual catkins of stage 4; sample 7

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(M3), the male flowers in bisexual catkins of stage 4 (Figure 1-II, A-D).

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Sex differentiation occurred after the development of stamen and carpel primordia (Figure 1-

208

II). The female flowers experienced a period of bisexual flowers. When the carpel primordia

209

were developed, twelve stamen primordia were also developed along with the development of

210

the carpels (Figure 1-II, F). However, the elongation of the stamens occurred slower than that of



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the pistils. Upon blossoming, the female flower was enlarged and the stigma developed rapidly;

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the filament elongation was stopped and filament length was observed to be significantly shorter

213

than that of the stigma, whereas the anther developed normally (Figure 1-II, G). The anther’s

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maturation time in female flower is synchronized with its male flower, which was approximately

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20 days after blossoming of the female flower and in the period of megaspore development10

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(Figure 1-II, H). The differentiation period for female flower in a single catkin was short (22

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days). Unlike female flowers, during the development of male flowers, only stamen primordia

218

were developed (Figure 1-II, I-K). The flowering time of male flowers was 20 days after that of

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female flower, when anthers were dehiscent and distributed (Figure 1-II, L). The blossoming

220

period for male flower was 12 days. Hence, it was approximately 55 days for male flower in the

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bisexual catkin.

222

Measurement of Endogenous Levels of Phytohormones. The endogenous levels of

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GAs, IAA, CKs, and ABA in the flowers during C. henryi sex differentiation were measured

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(Table 2). The levels of total GA and total active GA in both female flowers (F1-3) and male

225

flowers (M1-3) during the sex determination process were lower than those in the mixed catkins

226

before sex differentiation (B0). This result indicates that a greater quantity of GAs is required

227

during the differentiation of floral primordia than that during the development of reproductive

228

organs. A high level of GA1, the 13-hydroxylated bioactive GA, but not GA4, the 13-non-

229

hydroxylated bioactive GA, was detected in the floral organs. The level of GA1 in female flowers

230

was lower than that in male flowers. In addition, consistently higher levels of 13-hydroxylated

231

GAs (GA1, GA20, and GA53) were detected in the floral organs compared with their 13-non-


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hydroxylated counterparts (GA4, GA9 and GA34). GA12 which was the first synthetic hormone

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showed the maximum value, and other GAs were undetectable (GA3, GA19 and GA44) in all of

234

the stages (Table 2). These results indicate that Chinese chinquapin predominantly uses GA1 in

235

the floral organs.

236

IAA content was relatively similar in different stages of female and male flowers (Table 2).

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However, IAA levels were significantly lower in flowers at stages 1-3 than those before sex

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differentiation (B0). Finally, IAA level was not significantly different between the male and

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female flowers at these development stages.

240

The level of trans-zeatin-type of CK (trans-zeatin, tZ) was higher in the female flowers than

241

that in the male flowers at stages 2 and 3 (F1 and F2 vs. M1 and M2) but similar levels in stage 3

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of both type of flowers (F3 vs. M3). The level of isopentenyl adenine-type of CK (N6-

243

isopentenyl adenine, iP) was 2.1 and 3.3 times higher in the female flowers at stages 3 and 4 (F2

244

and F3) than that in the male flowers (M1 and M2), respectively. The level of trans-zeatin-O-

245

glucoside (ZOG) was the highest in stage 1 (B0) undifferenciated flowers than those in male and

246

female flowers (M1-3 and F1-3) (Table 2). In addition, the levels of total active CK (tZ+iP) were

247

much higher in the female flowers than those in the male flowers and peaked at stage 3. Other

248

CKs

249

isopentenyladenine-7-glucoside (iP7G), N6-isopentenyl adenosine (iPR) except iP7G in F2 and

250

F3 flowers or undetectable including trans-zeatin-7-glucoside (Z7G), trans-zeatin-9-glucoside

251

(Z9G), trans-zeatinriboside (ZR), dihydrozeatinriboside (DHZR), and dihydrozeatin 9-glucoside

252

(DHZ9G) in all of the stages (Table 2). These results may indicate that the development of

were either very low including transzeatinriboside-O-glucoside (ZROG),


N6–


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female flowers requires more CKs than male flowers.

254

The levels of cis-abscisic acid (c-ABA) and trans-abscisic acid (t-ABA) in the female

255

flowers (F1-3) were much lower than those in the male flowers (M1-3) in their respecticve stages

256

(Table 2). While c-ABA levels were much higher in all but F1 stages than that in B0 stage, t-

257

ABA level was much higher in B0 than all but M3 stages.

258 259

Taken together, the above results suggest that GA, CK and ABA have important roles during C. henryi sex differentiation, whereas the involvement of IAA does not appear to be important.

260

Effects of Exogenous Application of Phytohormones on C. henryi Flower Development.

261

IAA treatment did not appeared to have a significant effect on the number of staminate catkins

262

and male flowers per fruiting branch compared to the control (Table 3, Figure 1-III, B vs. A).

263

GA3 treatment significantly increased the total number of staminate catkins and male but not

264

female flowers per fruiting branch compared to the control, which resulted in a 3.1 times more

265

staminate catkins than bisexual catkins, and the female:male ratio was only 1:4319 in fruiting

266

branches (Table 3, Figure 1-III, C vs. A). BA-treatment induced bisexual catkins and increased

267

more female flowers in each biseual catkin than the control branches (Figure 1-III, D vs. A). The

268

female:bisexual catkin ratio was increased from 1:1 in control branches to 1:3 in branches treated

269

with BA, resulted in a 3.3-fold increase (from 4.1 to 13.6) in female flowers per branch, and the

270

female:male ratio was reached at 1:738. In addtition to the normal male and female flowers

271

found in control inflorescence, bisexual flowers were induced in BA-treated inflorescences

272

(Figure 1-III, D). The phenomenon of the increase in the number of staminate catkin and male

273

flower was also occurred in ABA teatment; however, no female flower was induced in the


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bisexual catkin (Table3, Figure 1-III, E vs. A). In addition, ABA treatment resulted in the

275

staminate catkins being more prone to wilting and abscission, and part of shoot tips also wilting

276

(Figure 1-III, E vs. A). To summarize, the effects of exogenous hormones on sex differentaiton in

277

C. henryi were in consistent with those of endogenous hormones.

278

Expression Profile of GA Biosynthesis and Signaling Genes. To serve as an example for

279

the rest of the paper of how to calculate differential gene expression and compare relative gene

280

expression levels in the C. henryi flowers, we analyzed in details of the expression levels of

281

unigenes involved in GA biosynthesis, deactivation, and signaling in three flower samples, i. e.,

282

the bisexual catkins of stage 1 (B0), the female flowers in bisexual catkins of stage 2 (F1) and

283

the male flowers in bisexual catkins of stage 2 (M1). The false discovery rate (FDR) was used to

284

determine the threshold P-value for multiple testing. The reads per kilobase per million mapped

285

reads (RPKM) method was used to calculate unigene expression. FDR < 0.001 and absolute

286

value of the log2 RPKM ratio > 1 were used as the threshold to determine significant differences

287

in gene expression. Table 4 showed that CPS gene was down-regulated in the flowers with a B0

288

> F1 > M1 order of expression, which was corresponding to the decreases in color intensity in

289

the heat map of CPS (Figure 2-I). Similarly, KS was up-regulated in the flowers; whereas

290

GA20OX1 (c57580) but not GA20OX1 (C57926) was up-regulated in the flowers (Table 4,

291

Figure 2-I).

292

Synthesis of active GAs begins from trans-geranylgeranyl diphosphate (GGDP) (Figure 2-I).

293

During the initial steps of GA biosynthesis, four enzymes are involved in the conversion of

294

GGDP to GA12 or GA53, a C-13-hydroxylated derivative of GA12 (Figure 2-I, step 1). They are



Page 16 of 52 16

295

ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO),

296

and ent-kaurenoic acid oxidase (KAO). The second half of GA biosynthetic pathway starts from

297

GA12 or GA53 (Figure 2-I, step 2). The enzyme GA20ox catalyzes the conversion from

298

GA12/GA53 to bioactive GA precursors GA9/GA20. The conversion of GA9/GA20 to GA4/GA1 is

299

catalyzed by GA3ox. GA deactivation is subsequently triggered by GA2ox catalysis (Figure 2-I,

300

step 3). Upon synthesis of the GAs, they are recognized by the soluble receptor GID1 (Figure 2-

301

I). GID1 ligated with GA binds to the DELLA protein. DELLA is a negative regulatory factor.

302

The degradation of DELLA in the GA-GID1-DELLA complex is triggered by SCFGID2, leading

303

to the expression of GA-responsive genes.33 GAMYB is as a positive trans-acting factor

304

downstream of the DELLA.34

305

In our studies, CPS, KS, KO, KAO and GA20ox were encoded by single genes but there were

306

three paralogs of genes for GA2ox (GA2ox1; GA2ox2; GA2ox8) and two paralogs of genes for

307

GA3ox (GA3ox1; GA3ox4) (Figure 2-I). The numbers of paralogs of genes were based on

308

sequence comparisons with other published plant genomes. Unigenes coding for the later steps of

309

GA biosynthesis (GA20ox, GA3ox and GA2ox) accounted for 77.7% of the total RPKM for GA

310

biosynthesis (Figure 2-II, A). The higher expression level of the later steps of GA synthesis genes

311

(GA20OX and GA3OX) than those initial steps of GA biosynthesis (CPS, KS, KO and KAO) may

312

indicate that the expression of genes in the later steps of GA biosynthesis is more important for

313

flower development in C. henryi. The expression levels of CPS, KS, KAO, and KO were

314

significantly higher in the female chinquapin flowers compared to the male flowers, while higher

315

expression levels of GA20ox and GA3ox were found in the male flowers (Figure 2-II, A). The


Page 17 of 52


316

expression of the deactivating genes GA2ox was higher in female flower than that in male

317

flower, whereas the expression of GA20ox and GA3ox in female and male flowers showed the

318

opposite pattern (Figure 2-II, A). GA20ox1 was the only homolog for GA20ox family, and

319

GA3ox1 and GA2ox1 were the main genes expressed in GA3ox and GA2ox families, respectively

320

(Figure 2-II, B and C). Taken together, these results suggest that GA20ox1, GA3ox1 and GA2ox1

321

are the major enzymes responsible for the accumulation of GA1 during C. henryi sex

322

differentiation.

323

In our study, the homologs of GA signaling factors GID1, GID2, DELLA, SPY and GAMYB

324

were single copy gene. GID2, SPY and GAMYB each contained only one unigene in the

325

transcriptomes (Figure 2-I). The general tendency of expression of GID1, GID2, DELLA, and

326

SPY was similar among the three tissues but GAMYB was preferentially expressed at M1 (Figure

327

2-II, D). These results suggest that GA signaling may preferentially occur in male flower of C.

328

henryi.

329

Expression Profile of IAA Biosynthesis and Signaling Genes. IAA is the predominant

330

endogenous auxin in plants.35 It has been proposed that there are two IAA biosynthetic pathways:

331

tryptophan (Trp)-dependent and Trp-independent pathways (Figure 3-I). The Trp-dependent

332

pathway is likely to be dominant, which includes anthranilate synthase (composed of the two

333

subunits, ASA and ASB), YUCCA protein, tryotophan aminotransferase-1 (TAA1), cytochrome

334

P450 proteins (CYP79B2/CYP79B3), nitrilase (NIT) and IAAId oxidase 1 (AAO1).27 After IAA

335

is synthesized, it is perceived by the F-box-containing auxin receptor TIR1 and its homologs

336

AFBs36 (Figure 3-I). The downstream of IAA signaling pathway includes IAA/AUX proteins,



Page 18 of 52 18

337

auxin response factors (ARFs) and gene targets.37 The pattern of auxin signaling pathway is

338

closely related to auxin concentration.38

339

Our studies showed that C. henryi has single gene for ASA (ASA1), ASB (ASB1) and TAA1

340

(TAA1), two homologs of NIT genes (NIT1, NIT4), and six homologs of YUCCA genes

341

(YUCCA2, 4, 6, 8, 9, and 10) (Figure 3-I). There are one gene for TIR and AFB, 12 homologs for

342

IAA (IAA1, 4, 7, 9, 11, 13, 16, 17, 20, 26, 27, 29) and 12 homologs for ARF (ARF1-ARF9,

343

ARF17-ARF19) (Figure 3-II). The expression of the five IAA biosynthesis genes was not

344

consistent. ASA1 and ASB1 genes were preferentially expressed at B0, YUCCA at F1, but NIT at

345

M1 (Figure 3-II, A). Six members of the YUCCA gene family including two differentially

346

expressed genes (c57743 coding for YUCCA2; c65674 coding for YUCCA4) also did not show

347

synchronized expression patterns (Figure 3-II, B; Figure 3-I). The expression of IAA/AUX, ARF

348

and most members of the two gene families showed the synchronism (Figure 3-II, C-E), which

349

occurred predominantly in female flower of the three samples. In addition, four differentially

350

expressed genes (c48111 coding for IAA1; c50648 coding for IAA13; c23595 coding for IAA16;

351

c37989 coding for IAA17) did not show synchronized expression (Figure 3-I). These results may

352

be related to IAA level, which was not significantly different between the male and female

353

flowers at these development stages (Figure 3-II).

354

Expression Profile of CK Biosynthesis and Signaling Genes.

Figure 4-I shows the

355

synthesis of biologically active CKs involving the following steps: 1) the transfer of the prenyl

356

group of dimethylallyl diphosphate (DMAPP) to the N6 position of adenine nucleotides (ATP,

357

ADP or AMP), which is catalyzed by adenosine phosphate-isopentenyltransferase (IPT); 2)


Page 19 of 52


358

hydroxylationone of the methyl groups on the prenyl side chain by CYP735A; and 3) the

359

removal of ribose 5’-monophosphate by LONELY GUY (LOG).39,40 The deactivation of CKs is

360

accomplished by cytokinin oxidase/dehydrogenase (CKX) (Figure 4-I).41 In the CK signaling

361

pathway, histidine kinase (HK) functions as a CK receptor, and the phosphor-relay signaling is

362

transferring the phosphate group to the down-stream target histidine phosphor-transfer protein

363

(HP), and then to A- or B-type response regulators42 (A-type RRs or B-type RRs, respectively)

364

(Figure 4-II).

365

We found 5 homologs of LOG, 6 homologs of CKX, but only one homolog of IPT in C.

366

henryi (Figure 4-I). However, we did not find any unigene coding for CYP735A. Since CK

367

accumulated to high levels in the female flower (Table 2), we expected coordinated expression of

368

CK synthetic genes. Unigenes coding for LOG were the highest expressed genes which

369

accounted for 74% of the total RPKM from the 3 families of unigenes (Figure 4-II, A). The

370

expression of unigenes coding for LOG1, LOG3 and LOG5 was higher than the other two

371

homologs of LOG family (Figure 4-II, B). Interestingly, most members of CKX gene family were

372

preferentially expressed in male flower, and the expression levels of unigenes coding CKX was

373

higher in M1 (male flower) than those in F1 (female flower) (Figure 4-II, C). Among the 20

374

unigenes coding for LOG, CKX, and IPT, the expression levels of LOG3 and LOG7 were

375

increased by 2-fold in F1 flowers (female) compared to those in M1 flowers (male), while

376

expression levels of CKX3 and CKX5 were decreased by 2-fold in F1 flowers compared to those

377

in M1 flowers (Figure 4-II, C).

378

C. henryi has five paralogs of genes for HK (HK1-HK5), two for HP (HP1, HP4), three for



Page 20 of 52 20

379

A-type RRs (RR2, RR11 and RR12) and four for B-type RRs (RR9, RR10, RR15 and RR17)

380

(Figure 4-I). Our transcriptome profiling demonstrated that unigenes coding for HK were the

381

highest expressed genes with 230, 243, and 205 RPKM in B0, F1 and M1 samples, respectively;

382

which accounted for 52.4% of the total RPKM (Figure 4-II, D). Most of the CK signaling gene

383

families were preferentially expressed in F1 flower (Figure 4-II, D). The overall expression

384

pattern of signaling factors was similar within each gene family, excepted for HK3 and HP1

385

(Figure 4-II, E, F), which was also preferentially expressed in female flower (Figure 4-II, G, H).

386

However, there were some ambiguous results (Figure 4-II). The expression pattern of c53377

387

(HP4-1) showed different from those of c53377 (HP4-2) and c65153 (HP4-3), which were

388

expressed predominantly in M1. Although the expression patterns of ARR2, ARR11 and ARR12

389

(A-type RR) were essentially the same as those of the CK synthesis and signaling genes (Figure

390

4-II, G), A-type RRs are suggested to be negative regulators of CK signaling.24 However, high

391

level expression of CK synthesis and CK signaling genes and low level expression of CKX in F1

392

suggests that CK biosynthesis and signaling functions actively in female flower of C. henryi.

393

Expression Profile of ABA Biosynthesis and Signaling Genes. ABA is synthesized from

394

zeaxanthin (β−carotene compound) by a series of enzymes including zeaxanthin epoxidase

395

(ZEP), neoxanthin synthase (NSY; ABA4), 9-cis-epoxycarotenoid dioxygenase (NCED),43,44 a

396

short-chain dehydrogenase/reductase (encoded by ABA2) and aldehyde oxidase (encoded by

397

AAO3)45,46 (Figure 5-I). The deactivation of ABA is accomplished by a subfamily of cytochrome

398

P450 monooxygenase CYP707A (ABA8OX)47 (Figure 5-I). ABA signaling pathway requires the

399

participation of a series of enzymes including phospholipase D alpha, protein phosphatase 2C


Page 21 of 52


400

(PP2C), SAPK and TRAB-related bZIP proteins, but only PP2C protein is a negative regulator of

401

ABA signaling.48,49 (Figure 5-I).

402

In our analysis, ZEP, NCED1, ABA2 and AAO3 were encoded by single genes, whereas

403

ABA8OX was encoded by four genes (ABA8OX1-4) in C. henryi (Figure 5-I). ZEP expressed the

404

highest levels among all of the family genes but without dominant expression among flower

405

samples, which was the same expression pattern as those of ABA2 and AAO3 (Figure 5-II, A).

406

The expression of NCED1 and ABA8OX genes was observed predominantly in M1 (Figure 5-II,

407

A). Three ABA8OX family genes had relatively high levels of expression in M1, while only

408

ABA8OX4 was preferentially expressed in B0 (Figure 5-II, B). There were 4 unigenes (c66762

409

coding for ZEP; c63125 coding for NCED1; c58472 coding for ABA8OX1; c58078 coding for

410

ABA8OX2) out of 18 unigenes coding for ABA synthesis that were differentially expressed

411

significantly. Interestingly, the expression levels of these 4 unigenes were much higher in M1

412

than those in B0 and F1 (Figure 5-I). Therefore, these results indicate that expression of genes

413

involved in ABA biosynthesis in male flower is prior to female flower, although the function of

414

ABA8OX gene family is deactivation of ABA. The gene activity of ABA synthesis pathway was

415

stronger than that of activation pathway.

416

The C. henryi transcriptome contains 47 homologs for PP2C, 4 homologs for SAPK

417

(SAPK1-3, SAPK10), 3 homologs for bZIP, and single gene for PLD and VP1 (Figure 5-I). The

418

expression levels of these 5 family genes were almost the same in the three samples, and PP2C

419

had the highest expression level among the 5 family genes (Figure 5-II, C). We did not perform a

420

detailed analysis of PP2C gene expression because of the enormous number of unigenes coding



Page 22 of 52 22

421

for PP2C. It is worth mentioning that only 3 of the 66 unigenes coding PP2C were differentially

422

expressed, and the expression of three unigenes (c68435 cording for PP2C 38, c57524 cording

423

for PP2C 44 and c55114 cording for PP2C 51) occurred predominantly in F1 (Figure 5-I).

424

Quantitative Analysis of 18 Selected Genes Involved in Phytohormone Biosynthesis.

425

We used qPCR to evaluate the expression profiles of 7 genes involved in GA biosynthesis, 7

426

genes involved in CK biosynthesis, and 4 genes involved in ABA biosynthesis, using primers

427

designed on the basis of the respective unigene sequences (Table 1). qPCR showed that CPS

428

mRNA levels were the highest in B0 stage and were significantly decreased in later stages of

429

flower development, especially much more reduced in male flower than female flower collected

430

at stages 2 and 3 (Figure 6A). The same trend appeared in the expression of c57580 (GA20OX1),

431

which was the highest in B0 stage and significantly decreased in later stages of flower

432

development, but there was no difference in the expression levels between male and female

433

flowers at all stages (Figure 6C). The expression levels of 69504 (KS) (Figure 6B), c53790

434

(GA2OX1) (Figure 6F), c62258 (LOG3) (Figure 6I) and c50806 (LOG5) (Figure 6J) in female

435

flower were significantly higher than those in male flower at three stages. By contrast, the

436

expression levels of c57926 (GA20OX1) (Figure 6D), c49197 (GA3OX1) (Figure 6E), c50625

437

(CKX1) (Figure 6L), c62597 (CKX3) (Figure 6M) and c63125 (NCED1) (Figure 6P) in male

438

flower were superior to female flower at all stage. The expression levels of c56312 (GA2OX2)

439

and c51765 (LOG1) were the lowest in B0 stage and were significantly increased in later stages

440

of flower development. The gene c56312 (GA2OX2) had the highest expression levels in male

441

flower at stage 3 (Figure 6G); whereas gene c51765 (LOG1) showed the highest levels in male


Page 23 of 52


442

flower at stage 4 (Figure 6H). The expression levels of c45740 (LOG7) of female flower

443

increased in the early stage and then decreased, while in male flower appeared opposite trend and

444

lower than B0 (Figure 6K). The expression levels of c64368 (CKX5) were significantly increased

445

in male flower at stages 2 and 3 but decreased at stage 4, while its expression level was

446

significantly decreased in female flower to the lowest at stage 2 and then increased (Figure 6N).

447

The expression level of c66762 (ZEP) showed a growth trend in female and male flower at stage

448

2 to 4 (Figure 6O). The expression patterns of c58078 (ABA8OX2) and c58472 (ABA8OX1) in

449

flower stage were similar in which the lowest expression levels appeared in stage 3 (Figure 6R

450

and Q).

451

Correlation between Gene Expression and Endogenous Hormone Concentration. In

452

order to explore the consequence of gene expression in hormone accumulation, we carried out

453

the correlation analysis between gene expression levels and phytohormone levels by SPSS

454

software (Table 4). Expression levels of six genes among the selected 18 genes were highly

455

correlated with the amount of active hormones with correlation coefficients close to or greater

456

than 0.8 (bold faced in Table 4). These six genes were GA20OX1 (c57926) and GA3OX1

457

(c49197) for GA biosynthesis, LOG3 (c62258), LOG5 (c50806) and CKX1 (c50625) for CK

458

biosynthesis, and NCED1 (c63125) for ABA biosynthesis. It is worth of pointing out that the

459

conclusion derived from this correlation study needs to be confirmed by other experimental

460

approaches such as proteomics and metabolomics.

461 462

DISCUSSION



Page 24 of 52 24

463

The Chinese Castanea henryi nut is used for food and feed, but its yield is low due to low ratio

464

of female to male flowers. In the previous study, we systematically investigated the micro- and

465

mega-sporogenesis in addition to male and female gametogenesis in C. henryi.1 The ovaries are

466

not yet mature (primordial) at the time of pollination and require an additional 6 weeks to

467

become fully developed. Flowering time of male flowers in male inflorescence is consistent with

468

female flowers; however the flowering time of male inflorescence on bisexual inflorescence was

469

20 days later than that of female flowers. In other words, male inflorescence is the main pollen

470

source for female flowers during the pollination process. Therefore, in the current study, we

471

selected male flowers on male inflorescence and female flowers collected between 3 days after

472

flowering (pollination period) to 7 weeks after flowering. We investigated the involvement of

473

phytohormones during C. henryi sex differentiation. We first described the morphology and

474

cytology of male and female floral organs. We then measured the endogenous levels of four plant

475

hormones (IAA, GAs, CKs and ABA) in the flowers and investigated the effects of exogenous

476

hormones on flower development. Finally, we evaluated the expression profiles of genes related

477

to these phytohormone biosyntheses and signaling pathways using RNA-Seq and qPCR. Our

478

results showed that C. henryi flowers consisted of unisexual and bisexual catkins which could be

479

divided into four developmental stages, that CK accumulated much more in the female flower

480

than that in the male flower and the effects of exogenous hormones on sex differentaiton in C.

481

henryi were consistent with those of endogenous hormones, and that several gnes may play key

482

roles in phytohormone biosynthesis and sex differentiation of C. henryi. The relationships

483

between plant hormone and sex differentiation are described in details as below.


Page 25 of 52


484

Relationship between GA and Sex Differentiation of C. henryi. GA synthesis continues

485

throughout all stages of flower bud development50 and plays a key role in flower development,

486

especially in the regulation of stamen development.11,51 It has been shown that higher levels of

487

active GA are expressed in male flowers compared to female flowers, suggesting that the high

488

levels of GA facilitate differentiation in male flowers.52 The expression of early GA synthesis

489

genes at a low level in catkins or flower implies that GA may not be actively synthesized in floral

490

organ. However, high level of GA12, the product of early GA biosynthetic pathway, suggests that

491

it may play an active role in the floral organ. The possible reason for the incongruous observation

492

is that GA12 could be transported into the floral organ from the other parts of the plant, which

493

allow them to be converted to bioactive GAs in the floral organs. The high level of GA1

494

accumulation in the male flower and high expression level of GA biosynthetic genes with the

495

synchronized expression of GA signaling genes is suggestive that GA1 might be used

496

immediately for flowering during C. henryi sex differentiation.

497

Genes that encode the enzymes for the initial steps of GA biosynthesis were expressed

498

during the early synthesis of active GAs. We observed a significantly higher expression of all

499

related catalytic enzymes in the female flowers compared to the male flowers. On the other hand,

500

during the later stages of GA biosynthesis, GA20ox and GA3ox expression was significantly

501

lower in female flowers than male flowers.

502

Our qPCR profiles indicated that GA20ox1 and GA3ox1 are dominantly involved in

503

bioactive GA synthesis in the floral organ. The GA20 oxidase family in Arabidopsis thaliana is

504

comprised of five members: AtGA20ox1, AtGA20ox2, AtGA20ox3, AtGA20ox4, and



Page 26 of 52 26

505

AtGA20ox5.53 GA20ox1 and GA20ox2 play important roles in the regulation of flower

506

development. GA20ox1 is mainly expressed in the stem and catkins and is crucial for filament

507

elongation growth and anther fertility.54 GA20ox3-5 isoforms do not have significant regulatory

508

effect on the development of flowers.55 GA3ox family has four members; GA3ox1 (among these)

509

is mainly expressed in the filaments and receptacles.56 During the developmental stages, the

510

expression levels of GA20ox1 and GA3ox1 in male chinquapin flowers were significantly higher

511

than those in female flowers, which is in accordance with the observation that the filament

512

elongation rate of male flowers is much faster than that of female flowers. In other words,

513

GA20ox1 and GA3ox1 mediated-regulation of filament growth might contribute to the regulatory

514

role of GAs in sex determination in chinquapin flowers.

515

Relationship between IAA and Sex Differentiation of C. henryi. Auxin is necessary for

516

the formation of flowers and floral organ patterning, which not only determines whether flower

517

primordial are formed, but also plays an essential role in specifying floral organs and

518

determining the pattern formation within a floral organ.13 Although IAA synthesis and signaling

519

in the flower of C. henryi was not clearly demonstrated because of the asynchronism expression

520

of related genes, the high content of IAA in female and male flowers indicated that IAA is

521

essential for pistil and stamen primordium induction, filament elongation,57 anthers dehiscing58

522

and ovary development events, but have little effect on sex differentiation of C. henryi. Our

523

exogenous IAA application further supported this view. The inflorescence meristems of C. henryi

524

treated with IAA shown a larger floral organ (not show), but no marked difference between male

525

and female flowers ratio in compared with the control.


Page 27 of 52


526

Relationship between CK and Sex Differentiation of C. henryi. CK is considered a

527

“female hormone” because it was shown to play a significant role in the development of female

528

flowers in a number of species.59,60 BA treatment induces bisexual flowers of Jatropha curcas,

529

which are not found in control inflorescences, and substantially increases the ratio OF female-to-

530

male flowers.61 Our data are consistent with these observations. High accumulation of tZ+iP and

531

preferential expression of CK synthetic genes in female flowers support the idea that CK

532

synthesized in female flower is important for pistil primordium induction. The high level of

533

tZ+iP accumulation in the female flower at stage 3 and exogenous CK treatment inducing

534

bisexual flowers suggest that CK is crucial for carpel development.62

535

LOG proteins are produced at specific locations and time points, especially at active growth

536

regions during plant development.63 In C. henryi, four LOG genes (LOG1, LOG3, LOG5 and

537

LOG7) were predicted, and the fluorescence quantitative data and correlation analysis indicated

538

that LOG3 and LOG5 were dominantly involved in bioactive CK synthesis in flower of C. henryi

539

during sex expression, however, the action mechanism of these two LOG members in C. henryi

540

were not clear at the present. CKX enzyme is thought to play an essential role in fine-tuning

541

cytokinin levels for control of plant growth and development.64 The predominant expression of

542

CKX genes and low-level of active CKs (tZ+iP) in male flower suggests that CKs are deactivated

543

during male flower differentiation compared with female flower. The expression of CKX1 was

544

negatively correlated with CK content, indicating that CKX1 among of CKX family is the major

545

gene that affects sex differentiation in C. henryi, which support the conclusion that the main

546

catalytic substrates of CKX1 include tZ and iP. We recognize that more direct evidence using



Page 28 of 52 28

547

other experimental approaches at the protein and metabolite levels should be used to confirm this

548

conclusion derived from correlation studies.

549

Relationship between ABA and Sex Differentiation of C. henryi. There are currently only

550

a few reports concerning the role of ABA in floral development or sex differentiation. More

551

recent studies have shown that ABA regulates reproductive development such as early anther

552

development,65 sporogenesis66 and pollen fertility.67 In the present study, male flower contained

553

much higher levels of both free and bound ABA than female flower at all development stages.

554

The possible reason for this phenomenon is that the anther of male flower is much larger than

555

that of female flower, therefore requires more ABA participation in male flower development.

556

The sustained increases of bound ABA from stage 3 to stage 4 may imply that ABA synthesized

557

during stamen development is not used immediately, but is stored for later pollen maturity and

558

organ abscission events. The observation of a high level of ABA accumulation at stage 4 when

559

anthers develop to mature and begin to decline in flower of C. henryi supports the idea that ABA

560

is involved in the regulation of the anther development.

561

NCED is considered to catalyze the rate-limiting step in ABA biosynthesis.68 Our results

562

showed that expression of NCED1 was detected in the male flower to some extent consistent

563

with high ABA level, and that the expression level of NCED1 was positively correlated with

564

ABA content in C. henryi. The high level expression of NCED1 in the male flower may imply

565

that NCED1 is involved in the regulation of the development of stamen in C. henryi.

566

In summary, our current study provides the first comprehensive report of phytohormone

567

biosynthesis and signaling during sex differentiation of C. henryi. The results described in this


Page 29 of 52


568

report should provide the foundation for studying sex differentiation in Castanea Miller species

569

and other non-model plants in the future.

570 571

572

Corresponding Authors

573

*(D.Y.) E-mail: [email protected].

574

*(H.C.) E-mail: [email protected]. Phone: (504)286-4351. Fax: (504) 286- 4367.

AUTHOUR INFORMATION

575 576

Funding

577

This work was supported by Chinese National Science and Technology Pillar Program

578

(2013BAD14B04), The Education Department of Hunan Province (17B280), and USDA-ARS

579

Quality and Utilization of Agricultural Products Research Program 306 through CRIS 6054-

580

41000-103-00-D. Mention of trade names or commercial products in this publication is solely for

581

the purpose of providing specific information and does not imply recommendation or

582

endorsement by the U.S. Department of Agriculture.

583 584

Notes

585

The authors declare no competing financial interest. The RAW data are accessible at NCBI under

586

bioproject

587

(SRR4434437); biosample for female flower (SAMN05913064): F1 (SRR4434436); biosample

588

for male flower (SAMN05913065): M1 (SRR4434438).

(PRJNA348647),

biosample

for

bisexual

catkin


(SAMN05913063):

B0


Page 30 of 52 30

589 590

591

ABA, abscisic acid; c-ABA, cis-abscisic acid; t-ABA, trans-abscisic acid; ABA8OX, abscisic

592

acid 8’-hydroxylase; CPS, copalyl diphosphate synthase; CK, cytokinin; CKX, Cytokinin

593

dehydrogenase; DHZ9G, dihydrozeatin 9-glucoside;

594

gibberellins; GA20OX1, gibberellin 20 oxidase 1; GA3OX1, gibberellin 3 oxidase 1; GA2OX1,

595

gibberellin 2-oxidase 1; GA2OX2, gibberellin 2-oxidase 2; iP, isopentenyl adenine; iP7G,

596

isopentenyladenine-7-glucoside; iPR, isopentenyl adenosine; KS, ent-kaurene synthase; LOG,

597

Cytokinin riboside 5’-monophosphate phosphoribohydrolase; NCED1, 9-cis-epoxycarotenoid

598

dioxygenase; tZ, trans-zeatin; ZEP, Zeaxanthin epoxidase; Z7G, trans-zeatin-7-glucoside; Z9G,

599

trans-zeatin-9-glucoside; ZOG, trans-zeatin-O-glucoside; ZR, trans-zeatinriboside; ZROG,

600

transzeatinriboside-O-glucoside.

ABBREVIATIONS USED

DHZR, dihydrozeatinriboside; GA,


Page 31 of 52


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Developmental Biology - Biotechnological Perspectives: Volume 2, Pua, E. C.; Davey, M. R., Eds.; Springer Berlin Heidelberg: Berlin, Heidelberg, 2010; pp 237-254. (60) Werner, T.; Schmulling, T. Cytokinin action in plant development. Curr. Opin. Plant Biol. 2009, 12 (5), 527-538. (61) Fu, Q.; Niu, L.; Zhang, Q.; Pan, B. Z.; He, H.; Xu, Z. F. Benzyladenine treatment promotes floral feminization and fruiting in a promising oilseed crop Plukenetia volubilis. Ind. Crops Prod. 2014, 59 (Supplement C), 295-298. (62) Tarkowski, P.; Tarkowska, D.; Novak, O.; Mihaljevic, S.; Magnus, V.; Strnad, M.; Salopek-Sondi, B. Cytokinins in the perianth, carpels, and developing fruit of Helleborus niger L. J. Exp. Bot. 2006, 57 (10), 2237-


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Figure Legends

Figure 1. Morphology, cytology and exogenous hormone effects on flower development and sex expression of C. henryi. I. Morphological observation of inflorescence in C. henryi (longitudinal section). BC, bisexual catkin; SC, staminate catkin; Red circle represents the female flower (longitudinal section). II. Cytological observation of the differentiation in male and female flowers. A-D: Bisexual catkins. (A) 2 days, (B) 12 days, and (C) 22 days after the appearance of mixed catkins, female flowers began to bloom; (D) 42 days after the appearance of the mixed catkins, male flowers began to bloom. E-H: Female flower differentiation and development. (E) Early differentiation stage, (F) developmental stage, (G) blossom stage, (H) 10 days after blossom. I-L: Male flower differentiation during development. (I) early differentiation stage, (J) developmental stage-Phase I, (K) developmental stage-Phase II, and (L) blossoming stage. The red circle represents the female flower. The black circle and white circle show the sampling points of the female and male flowers respectively. A, anther; G, gynoecium; GP, gynoecial primordia; S, stamen; SP, stamen primordial. Scale bars: E, F, I-K = 200 µm；G, H = 500 µm；L = 50 µm。 III. Effects of exogenous hormone on flower development and sex expression of C. henryi. . (A) Inflorescence from control plants. (B) Inflorescence from IAA-treated plants. (C) Inflorescence from GA3-treated plants. (D) Inflorescence from BA-treated plants. (E) Inflorescence from ABA-treated plants. The red circle represents the female flower. BC, bisexual catkin.

Figure 2. Representative gene expression profiles of the GA biosynthesis, deactivation and


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signaling pathways in the flower of C. henryi. I. Representative gene expression profiles of the GA biosynthesis, deactivation and signaling pathways in the flower of C. henryi. The color intensity in the heat map of each unigene represents the relative expression levels of that unigene among the three tissue samples compared. The rectangles marked with blue frames represent highly expressed genes in the corresponding tissues. The scale of color intensity shown in the lower right panel represents the fold of differential gene expression. CPS (c48715); KS (c69504); KO (c57819); KAO (c88172); GA20OX1-1 (c57580); GA20OX1-2 (c57926); GA3OX1 (c49197); GA3OX4 (c28384); GA2OX1 (c53790); GA2OX2 (c56312); GA2OX8 (c87683); GID1 (c54766); GID2 (c62628); SPY (c68972); DELLA-1 (c23705); DELLA-2 (c59953); DELLA-3 (c65360); DELLA-4 (c67334); DELLA-5 (c67354); GAMYB (c68228). Red and green colors indicate higher and lower expression, respectively. The color scale (representing the average log 2 Z-value normalized by R software) is shown at the bottom. B0, bisexual catkin; F1, female flower at stage 2; M1, male flower at stage 2. II. Expression profiles of the GA biosynthesis and signaling pathway unigenes in the flower of C. henryi. The x-axis represents the proteins coded for by the unigenes followed by the number of unigenes discovered in the flower transcriptomes. The y-axis represents total RNA levels expressed as the sum of RPKM values from all of the unigenes identified in the same gene or gene family. B0, bisexual catkin; F1, female flower at stage 2; M1, male flower at stage 2.

Figure 3. Representative gene expression profiles of the IAA biosynthesis, deactivation and signaling pathways in the flower of C. henryi. I. Representative gene expression profiles of the IAA biosynthesis, deactivation and signaling pathways in the flower of C. henryi. The color intensity in the heat map of each unigene



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represents the relative expression levels of that unigene among the three tissue samples compared. The rectangles marked with blue frames represent highly expressed genes in the corresponding tissues. The scale of color intensity shown in the lower right panel represents the fold of differential gene expression. ASA1 (c65431); ASB1 (c60746); YUCCA2 (c57743); YUCCA4 (c65674); YUCCA6 (c65926); YUCCA8 (c88419); YUCCA9 (c66966); YUCCA10 (c63669); TAA1 (c10184); NIT1 (c57649); NIT4 (c64500); TIR (c57921); AFB2 (c63322); IAA1 (c48111); IAA13 (c50648); IAA16 (c23595); IAA17 (c37989); ARF1 (c68868); ARF7 (c63554); ARF9 (c65310); ARF17 (c64535). II. Expression profiles of the IAA biosynthesis and signaling pathway unigenes in the flower of C. henryi. The x-axis represents the proteins coded for by the unigenes followed by the number of unigenes discovered in the flower transcriptomes. The y-axis represents total RNA levels expressed as the sum of RPKM values from all of the unigenes identified in the same gene or gene family. B0, bisexual catkin; F1, female flower at stage 2; M1, male flower at stage 2.

Figure 4. Representative gene expression profiles of the CK biosynthesis, deactivation and signaling pathways in the flower of C. henryi. I. Representative gene expression profiles of the CK biosynthesis, deactivation and signaling pathways in the flower of C. henryi. The color intensity in the heat map of each unigene represents the relative expression levels of that unigene among the three tissue samples compared. The rectangles marked with blue frames represent highly expressed genes in the corresponding tissues. The scale of color intensity shown in the lower right panel represents the fold of differential gene expression. IPT (c28464); LOG1 (c51765); LOG3 (c62258); LOG5 (c50806); LOG7 (c45740); LOG8 (c45313); CKX1 (c50625); CKX3 (c62597); CKX4 (c76701); CKX5 (c64368); CKX6 (c77409); CKX7 (c60857); HK1 (c71550); HK2 (c68781); HK3


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(c69166); HK4 (c66356); HK5 (c21471); HP1 (c51031); HP4-1 (c49962); HP4-2 (c53377); HP4-3 (c65153). ARR2 (c33930); ARR11 (c65887); ARR12 (c62315); ARR9 (c69850); ARR10 (c43409); ARR15 (c38743); ARR17 (c69009). II. Expression profiles of the CK biosynthesis and signaling pathway unigenes in the flower of C. henryi. The x-axis represents the proteins coded for by the unigenes followed by the number of unigenes discovered in the flower transcriptomes. The y-axis represents total RNA levels expressed as the sum of RPKM values from all of the unigenes identified in the same gene or gene family. B0, bisexual catkin; F1, female flower at stage 2; M1, male flower at stage 2.

Figure 5. Representative gene expression profiles of the ABA biosynthesis, deactivation and signaling pathways in the flower of C. henryi. I. Representative gene expression profiles of the ABA biosynthesis, deactivation and signaling pathways in the flower of C. henryi. The color intensity in the heat map of each unigene represents the relative expression levels of that unigene among the three tissue samples compared. The rectangles marked with blue frames represent highly expressed genes in the corresponding tissues. The scale of color intensity shown in the lower right panel represents the fold of differential gene expression. ZEP (c66762); NCED (c63125); ABA2 (c65304); AAO3 (c67867); ABA8OX1 (c58472); ABA8OX2 (c58078); ABA8OX3 (c86031); ABA8OX4 (c49840); PLD (c53711); PP2C1 (c65222); PP2C38 (c68435); PP2C44 (c57524); PP2C51 (c55114); SAPK1 (c21512); SAPK2 (c51758); SAPK3 (c45816); SAPK10 (c60550); VP1 (c65271); bZIP9 (c64010); Bzip60 (c64010); bZIP63 (c64010). II. Expression profiles of the ABA biosynthesis and signaling pathway unigenes in the flower of C. henryi. The x-axis represents the proteins coded for by the unigenes followed by the number of unigenes discovered in the flower transcriptomes. The y-axis represents total RNA levels



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expressed as the sum of RPKM values from all of the unigenes identified in the same gene or gene family. B0, bisexual catkin; F1, female flower at stage 2; M1, male flower at stage 2.

Figure 6. Expression profiles of GA, CK and ABA biosynthesis genes in C.henryi flowers. qPCR was used to quantify the mRNA levels using the total RNAs from four flower developmental stages in Huali No. 4 of C. henryi. Different letters show a significant difference in the mRNA level (P < 0.05). (A) c48715 (CPS); (B) c69504 (KS); (C) c57580 (GA20OX1); (D) c57926 (GA20OX1); (E) c49197 (GA3OX1); (F) c53790 (GA2OX1); (G) c56312 (GA2OX2); (H) c51765 (LOG1); (I) c62258 (LOG3); (J) c50806 (LOG5); (K) c45740 (LOG7); (L) c50625 (CKX1); (M) c62597 (CKX3); (N) c64368 (CKX5); (O) c66762 (ZEP); (P) c63125 (NCED1); (Q) c58472

(ABA8OX1);

(R)

c58078


(ABA8OX2).

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Table 1. Primers Used for qPCR Analysis No. unigene

annotation

sequence (5’−3′)

A.

c48715 a

copalyl diphosphate synthase (CPS)

B.

c69504

ent-kaurene synthase (KS)

C.

c57580

gibberellin 20 oxidase 1 (GA20OX1)

D.

c57926


E.

c49197


F.

c53790

gibberellin 2-oxidase 1 (GA2OX1)

G.

c56312

gibberellin 2-oxidase 2 (GA2OX2)

H.

c51765

I.

c62258

J.

c50806

K.

c45740

L.

c50625

Cytokinin dehydrogenase 1 (CKX1)

M. c62597


N.

c64368


O.

c66762

Zeaxanthin epoxidase (ZEP)

P.

c63125

9-cis-epoxycarotenoid dioxygenase (NCED1)

Q.

c58472

Abscisic acid 8’-hydroxylase 1 (ABA8OX1)

R.

c58078

Abscisic acid 8’-hydroxylase 2 (ABA8OX2)

Cytokinin riboside 5’-monophosphate phosphoribohydrolase 1 (LOG1) Cytokinin riboside 5’-monophosphate phosphoribohydrolase 3 (LOG3) Cytokinin riboside 5’-monophosphate phosphoribohydrolase 5 (LOG5) Cytokinin riboside 5’-monophosphate phosphoribohydrolase 7 (LOG7)

reference gene a

GADPH

ATTGGAGGCTATTGTGACGCATGG TCTCCTTGCTGCCTATCACCTTCA GGCACTTCACCGCACAATCTGT ACGGACTTGTCTCTCAACCACTCT GGCTCAACTACTATCCACCATGTCA ATTGATCGCCATTCCTCGTCTACA GGGACTGGAAGGAGGTGTTTGA TGAACTCGGGCGGGTACTGA ACCTTGCTCACAATTCTCCACCAA TCACCTATGTTGACCACCAGTGC CCATGCCCAGACTTTCAGACCTTG CCAGATGCGTTGTTGGACCTCAG AGTCCTCAGGCTCAATCACTATCCA AGGGTCAGAATGCTCTCCAAATCC GGAGTGGAAGAGGGTTTCATAGAGG CTTTGTCCCAGTGCTACCTTGTCAT TCAAGATTCCGACGTGTCTGTGTT CATCAAGCCAATACTTCCTCCTCCA TCCCTAGCGTTTGGAGCAGAGAC GCCTGTGGGATTGCTTAATGTCGAT GCCAACCGCACGTCGCATTA AGTCTATCCACTCCTTCCCAAACCA TTGACCTCTGCAATGCCATCTTCC GCTTGCCACTCTTCCTGCGTATT ATGCCACACTTGAACATGGACTTG CTGATCTGAGGACCATAACGGAAGG GGTCCTCAGATTAGCAATGTCT ACCAAGAACAGCATGGAACAG GCAGCTTTAGAAGATGGTGTTG CCTCTCATGTTGCTCCTTGTC GCCGTATCAAGTCCGAATCAA TGAATGACATCGTAGCTGAGTG CCAGTGTTCTTCAGGCAGTCA GTTATTGGCATCTTCTTGGTGTCT AACCAGCCATTAAGTTGGAGTC CACATCAGCCACTGCTTCTC GGTGGTGCTAAGAAGGTTGT GGTGGTGCTAAGAAGGTTGT

annealing temp (°C)

amlicon (bp)

53.9

108

53.6

144

54.2

142

55.5

126

57

116

54.1

104

53.3

112

53.5

128

55

144

54.6

129

54.8

126

54.4

149

54.9

124

51.2

108

50.9

114

52.8

150

53.1

112

51.9

120

54.8

214

The “c” and the number in the “unigene” column represent plant species “Castanea henryi” and the identification number of the unigene in the

transcriptomes.



Page 42 of 52 42

Table 2. Phytohormone Content in Male and Female Flowers during C. henryi Sex Differentiation

GA1 GA3 GA4 GA9 GA12 GA19 GA20 GA34 GA44 GA53 IAA tZ Z7G Z9G ZOG ZR ZROG DHZR DHZ9G iP iP7G iPR c-ABA

B0 a 459.57 ± 37.44 a b ND 30.35 ± 5.18 a 86.45 ± 6.23 a 682.67 ± 59.43 a ND 169.96 ± 13.98 a 19.24 ± 1.20 a ND 236.72 ± 21.78 a 3927.56 ± 378.40 a 8.84 ± 1.04 b ND ND 5.07 ± 0.62 a ND 0.34 ± 0.02 a ND ND 2.50 ± 0.10 d 0.18 ± 0.04 c 0.56 ± 0.05 b 173.61 ± 28.66 d

F1 178.22 ± 22.55 d ND 1.15 ± 0.19 d 48.32 ± 2.97 c 235.27 ± 30.28 c ND 83.26 ± 10.24 c 5.98 ± 0.43 c ND 57.20 ± 8.92 d 1994.07 ± 193.26 c 12.10 ± 1.30 a ND ND 1.96 ± 0.73 cd ND 0.23 ± 0.02 b ND ND 2.51 ± 0.15 d 0.11 ± 0.01 c 0.34 ± 0.08 d 165.05 ± 12.84 d

F2 216.75 ± 38.45 cd ND 0.34 ± 0.05 d 30.98 ± 3.54 d 262.80 ± 19.81 c ND 76.78 ± 6.32 c 12.79 ± 0.93 b ND 107.39 ± 10.89 c 2312.03 ± 423.14 bc 10.93 ± 1.93 a ND ND 3.51 ± 0.25 b ND 0.27 ± 0.02 b ND ND 9.69 ± 1.73 b 2.69 ± 0.53 b 0.21 ± 0.12 d 575.47 ± 97.75 c

flower stage F3 288.75 ± 44.39 bc ND 1.42 ± 0.22 d 28.10 ± 2.75 d 364.44 ± 28.38 b ND 119.26 ± 7.92 b 15.72 ± 0.24 b ND 100.79 ± 14.32c 2912.78 ± 578.98 b 4.58 ± 0.08 c ND ND 3.65 ± 0.51 b ND 0.22 ± 0.04 b ND ND 14.03 ± 1.83 a 3.82 ± 0.71 a 0.32 ± 0.09 d 401.75 ± 65.10 cd

M1 334.51 ± 40.04 b ND 3.68 ± 0.60 c 62.35 ± 3.51 b 381.25 ± 40.91 b ND 124.44 ± 8.27 b 13.66 ± 2.41 b ND 118.97 ± 20.15 c 2570.53 ± 478.09 bc 5.78 ± 0.70 c ND ND 1.25 ± 0.49 d ND 0.12 ± 0.06 c ND ND 3.05 ± 0.45 cd 0.12 ± 0.02 c 0.65 ± 0.05 a 524.98 ± 56.85 c

M2 332.94 ± 56.78 b ND 6.91 ± 0.75 b 51.34 ± 3.86 c 338.04 ± 25.73 b ND 118.75 ± 12.71 b 20.55 ± 1.89 a ND 114.40 ± 16.44 c 2056.66 ± 25.20 c 5.12 ± 0.98 c ND ND 1.86 ± 0.12 cd ND 0.12 ± 0.03 c ND ND 4.56 ± 0.10 c 0.14 ± 0.02 c 0.43 ± 0.01 c 1508.20 ± 277.42 a

M3 306.72 ± 5.62 bc ND 5.80 ± 0.49 b 50.23 ± 4.06 c 215.16 ± 19.23 c ND 70.54 ± 5.32 c 19.68 ± 1.43 a ND 161.32 ± 10.23 b 2575.67 ± 451.81 bc 6.48 ± 0.79 c ND ND 2.14 ± 0.51 c ND 0.15 ± 0.02 c ND ND 4.21 ± 0.76 cd 0.27 ± 0.01 c 0.54 ± 0.01 b 1007.86 ± 188.00 b

t-ABA

7054.97 ± 947.98 b

2422.37 ± 195.12 e

3049.62 ± 339.04 de

6211.38 ± 1152.03 bc

4622.40 ± 740.74 cd

3357.69 ± 659.12 de

9826.48 ± 1808.43 a

hormone types

GA c

IAA

CK

ABA

a

Flower stage: B0, the bisexual catkins of stage 1; F1, F2, and F3, the female flowers in bisexual catkins of stage 2, 3, and 4, respectively; M1, M2, M3, the male flowers in bisexual catkins of stages 2, 3, and 4, respectively. b Endogenous phytohormone levels were measured in triplicate and the mean values ± SD (ng.g-1) are shown in each sample. Different letters in each row indicate significantly differences between samples at 5% level. ND, not detected. c Hormone abbreviations: ABA, abscisic acid; c-ABA, cis-abscisic acid; t-ABA, trans-abscisic acid; CK, cytokinin; DHZR, dihydrozeatinriboside; DHZ9G, dihydrozeatin 9-glucoside; GA, gibberellins; IAA, indole-3-acetic acid; iP, N6 -isopentenyl adenine; iP7G, N6–isopentenyladenine-7-glucoside; iPR, N6 isopentenyl adenosine; tZ, trans-zeatin; Z7G, trans-zeatin-7-glucoside; Z9G, trans-zeatin-9-glucoside; ZOG, trans-zeatin-O-glucoside; ZR, trans-zeatinriboside; ZROG, transzeatinriboside-O-glucoside .


Page 43 of 52


Table 3. Effects of Exogenous Hormone Treatment on Flower Number and Sex Ratio in C. henryi a exogenous

bisexual catkin

staminate catkin

treatment

female flower

male flower

/a bearing branch

B:S c ratio

F:B ratio

F:M ratio

Control

3.14 ± 0.21 b b

6.68 ± 1.09 b

4.09 ± 0.36 b

8089.24 ± 890.89 b

1:2.1

1.3:1

1:1977.8

IAA

3.09 ± 0.18 b

6.65 ± 0.87 b

3.82 ± 0.71 b

9001.93 ± 963.33 b

1:2.2

1.2:1

1:2356.5

GA3

3.2 ± 0.31 b

9.92 ± 1.23 a

3.61 ± 0.50 b

15593.12 ± 2559.96 a

1:3.1

1.1:1

1:4319.4

6-BA

4.14 ± 0.32 a

6.16 ± 1.07 b

13.62 ± 1.24 a

10053.60 ± 1423.54 b

1:1.5

3.3:1

1:738.1

ABA

0

8.03 ± 1.10 ab

0c

13320.33 ± 2067.12 ab

0

-

0

a

The control treatment represents that plants were sprayed with 5 mL of distilled water containing equivalent amount of ethanol and Tween-20 to those

in the four hormone treatments. The spraying was carried out thrice with the first application in mid-March before bud germination, and followed by 1-week intervals by using a hand sprayer. b

Different letters in each column indicate significantly differences between samples at 5% level.

c

Abbreviations: B, bisexual catkin; F, female flower; M, male flower; S, staminate catkin.



Page 44 of 52 44

Table 4. Identification of Differentially Expressed Genes in GA Biosynthesis Pathway Annotation (abbreviation)

unigene

B0 vs. F1a

B0 vs. M1

FDR

Log2FC

regulated

FDR 1.44E-

M1 vs. F1

Log2FC

regulated

FDR

Log2FC

regulated

-4.20367

down

0.00249

2.665297

up

copalyldiphosphate synthase (CPS)

c48715

0.001523

-1.53672

down

ent-kaurenesynthase (KS)

c69504

0.000114

1.020016

up

-b

-

-

-

-

-


c57580

1.55E-07

-1.25625

down

-

-

-

-

-

-


c57926

1.25E-10

-3.73607

down

-

-

-

7.03E-09

3.501989

down


c49197

-

-

-

1.29059

up

0

-1.92453

down

c53790

-

-

-

-

-

-

1.05E-10

1.189288

up

c56312

-

-

-

-

-

-

0.001446

-2.27289

down

Gibberellin receptor GID1

c54766

-

-

-

1.356782

up

-

-

-

DELLA protein RGL1

c65360

-

-

-

-

-

0.002457

-1.24789

down

Transcription factor GAMYB

c68228

-

-

-

1.826271

up

1.21E-08

-1.3147

down

Gibberellin 2-beta-dioxygenase 1 (GA2OX1) Gibberellin 2-beta-dioxygenase 2 (GA2OX2)

15

3.07E07

8.13E06 1.39E11

a

B0, bisexual catkin; F1, female flower at stage 2; M1, male flower at stage 2. B0 and F1 before “vs.” represents the reference items, and F1 and M1 after “vs.” represents the comparisons. b ”-“ represents no significant difference in gene expression levels between two samples.


Page 45 of 52


Table 5. Correlation between Gene Expression Levels and Endogenous Hormones Content in C. henryi Flowers ID a

unigene

annotation

GA1b

ID

unigene

annotation

tZ+iP

ID

unigene

annotation

c-ABA

A

c48715

CPS

0.501

H

c51765

LOG1

-0.164

O

c66762

ZEP

0.744

B

c69504

KS

-0.145

I

c62258

LOG3

0.910**

P

c63125

NCED1

0.928**

C

c57580

GA20OX1

0.413

J

c50806

LOG5

0.786*

Q

c58472

ABA8OX1

-0.652

D

c57926

GA20OX1

0.782*

K

c45740

LOG7

0.493

R

c58078

ABA8OX2

-0.635

E

c49197

GA3OX1

0.775*

L

c50625

CKX1

-0.785*

F

c53790

GA2OX1

-0.449

M

c62597

CKX3

-0. 518

G

c56312

GA2OX2

-0.358

N

c64368

CKX5

-0.416

a

Bolded ID, unigene, and annotations represent those genes showing correlation between mRNA levels and endogenous hormone levels.

b

The top nominations at the forth columns (GA1, tZ+iP, c-ABA) mean that these hormone species were the most active hormones with high content in

C. henryi flowers. They were used to analyze the correlation between gene expression and endogenous hormone concentration. “*”and “**” after the correlation coefficients represent the correlation between their mRNA levels and endogenous hormone levels significantly different at P < 0.05 and P < 0.01, respectively.



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III


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TOC Graphic

Chinese Chinquapin Flower Transcriptome and Phytohormone Biosynthesis and Signaling Genes


Comprehensive Transcriptome Analysis of Phytohormone

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