Encased Emerged Encased Emerged

C26 (internal standard)

C23

C25

C27

C27:2 and C27:1

C29

C29:2 and C29:1

C31

C31:2 and C31:1

C33

The cuticle of maize silks as a model system to define the pathway of hydrocarbon biosynthesis

Simple non-isoprenoid hydrocarbons (e.g., alkanes and alkenes) are major components in petroleum but are found only in discrete parts of the biosphere. Extant biological hydrocarbons are relatively abundant in the cuticle of plants and insects, and function to create a water barrier for these organisms. Although the biological production of hydrocarbons most likely occurs via the conversion of fatty acids to alkanes and alkenes, the biochemical mechanism of this conversion is not defined. Four metabolic mechanisms have been proposed for hydrocarbon biosynthesis (Figure 1).The cuticle of maize silk is largely comprised of linear hydrocarbons (lengths from C23 to C33) and is the most abundant source of hydrocarbons in maize. To understand the pathway of hydrocarbon biosynthesis, hydrocarbons were investigated in 26 diverse founder lines used in the Nested Association Mapping (NAM) population. In addition, a complementary RNA-seq approach is currently underway to identify differentially expressed genes within the transcriptomes of silks that have emerged, as compared to those still encased within the ear husks.

• For all inbreds tested, there was a ~6-fold range in total hydrocarbon accumulation in emerged silks. (Figure 3)• The relative amounts of hydrocarbon constituents varied among inbreds (Table 1). These results demonstrate that both the overall amount of hydrocarbons and the distribution of individual hydrocarbon constituents vary among inbreds.

Wenmin Qin ， Adarsh Jose, Sam Condon, Basil Nikolau and Marna Yandeau-NelsonDepartment of Biophysics, Biochemistry and Molecular Biology, Iowa State University, Ames, IA, United States

Hydrocarbon accumulation across the length of B73 silks

Introduction

Identification of differentially expressed genes between emerged and encased B73 maize silk

Hydrocarbon content in silks of NAM founders

• Both emerged and encased silks from all inbreds tested contained odd numbered non-isoprenoid hydrocarbons (C23 to C33) (Figure 2B).• The most prevalent constituents (>60% of total hydrocarbons) were saturated C27 and C29 alkanes, and C27:1 and C29:1 alkenes (Figure 2B). •Hydrocarbon content was measured across the length of B73 silks (Figure 2A). At 3-days post-emergence, hydrocarbon amount was higher in emerged as compared to encased silks (Figure 2C). Hydrocarbon content increased by 6-days post-emergence (Figure 2D). •This data indicates that hydrocarbons are actively synthesized in 3-days post-emergent B73 silk. Thus, mRNAs from 3-day B73 silks were used for Illumina sequencing to identify differentially expressed genes between emerged and encased silks (Figure 4).

Rn-CHO Rn-CH2OHRn-COOH

Rn Rn Rn+1

Decarboxylase Decarbonylase Dehydratase

CO H2O

Rm-COOH

Elongation

Rs-COOH

RsCORm

Head-to-head Condensation

RsCH(OH)Rm

CO2

Rs+m+1

Odd-numbered hydrocarbon Even-numbered hydrocarbon

11 22 33 44

Figure 1

Figure 3. Hydrocarbon content in 3-day emerged and encased maize silk Each measurement is the average of 5 biological replicates.

Table 1. Range of relative amounts of individual hydrocarbon constituents of 3-day emerged maize silk across all inbreds tested

Figure 2C

Constituent C23

C23 ketone C25:1 C25 C27:2 C27:1 C27

C27:2 ketone

C27:1

ketoneC27 ketone C29:2 C29:1 C29

C29:1 ketone C31:1 C31 C33:1

Minimum 0% 0% 0% 0.48% 0% 0.04% 14% 0% 0% 0% 0% 1.3% 15% 0% 0.43% 0.55% 0%

Maximum 2.8% 1.1% 3.6% 14% 5.0% 9.9% 39% 0.18% 0.89% 6.7% 9.9% 27% 54% 0.26% 7.5% 24% 0.28%

-1-3-4 -2321 4

Figure 2A. Maize silks from B73 were cut into 1 inch segments. Hydrocarbons were extracted with hexane and purified through silica gel from each lyophilized segment. Total hydrocarbons analyzed by GC spectrometry from each segment at both 3- and 6-days post-emergence are shown in Figure 2C.

Figure 2B. The data files were deconvoluted by NIST AMDIS software, and compared against an in-house compound library as well as the NIST compound library.

Figure 2D

Transcriptomes of the B73 post-emergent silks, which produce large amounts of hydrocarbons, and pre-emergent silks, which produce smaller amounts of hydrocarbons, have been sequenced using the Illumina Genome Analyzer IIE. Transcripts that are differentially expressed between pre- and post-emergent silks are potential candidate genes in the hydrocarbon biosynthetic pathway. The reads were mapped to B73 maize Working Gene Sets. We observed several genes, which have not previously been experimentally verified, indicating that they maybe uniquely expressed in the silk samples. Genes expressed at higher levels in post-emergent silks are enriched with functions associated with stress (e.g., arginine decarboxylase), environmental stimuli (e.g., light), defense responses (zeamatin precursor), and senescence etc.Genes expressed at higher levels in pre-emergent silks are enriched with functions associated with development, biosynthetic processes, increased metabolic activity (e.g., glycerol-phosphate acyl transferase), etc. 

Illumina Reads (2 Biological Replicates)

Source No. of Reads

Inside ~20,000,000

Outside ~16,000,000

Filter (Fastx) -> Map to Working Gene Set

(TopHat/Bowtie) -> Quantify (FPKM3)

Source No. of Genes Expressed

Inside 28112

Outside 27352

FC1 (Out/In)

No. of Genes

Homologs of Fatty Acid Genes2

Arabidopsis Homologs

Only Out 2102 4 Wax Synthase, P450, ELO, KCS

>10 144 1 LACS2

>5 475 3 KCS1

<0.2 483 5 Binding Protein -1, CER-1

<0.1 94 3 CER-60, KCS

Only In 1342 3 P450-like, CER-2, MS-2

Total 4640 19

1 – Fold Changes2- Genes whose homologs in Arabidopsis are involved in fatty acid synthesis/ elongation/ enriched in the gene lists3 - Fragments (Read-Pairs) /kb of Gene Length / Million Mapped Read Pairs

Figure 4: Bioinformatic results of transciptome of 3-day pre- and post-emergent B73 corn silk