The cuticle of maize silks as a model system to define the
pathway of hydrocarbon biosynthesis
Wenmin Qin，Adarsh Jose, Sam Condon, Basil Nikolau and Marna Yandeau-Nelson
Department of Biophysics, Biochemistry and Molecular Biology, Iowa State University, Ames, IA, United States
Introduction
Hydrocarbon content in silks of NAM founders
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.
Rs-COOH
• 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.
Rm-COOH
Head-to-head
Condensation
RsCORm
Elongation
Rn-COOH
1
2
Rn-CHO
3
Decarboxylase
Rn-CH2OH
4
Decarbonylase
Dehydratase
RsCH(OH)Rm
CO2
Rs+m+1
CO
Rn
H2O
Rn
Odd-numbered hydrocarbon
Figure 3. Hydrocarbon content in 3-day emerged and encased maize silk
Each measurement is the average of 5 biological replicates.
Rn+1
Even-numbered hydrocarbon
Table 1. Range of relative amounts of individual hydrocarbon constituents of 3-day emerged maize silk across all inbreds tested
Figure 1
Constituent
Hydrocarbon accumulation across the length of
B73 silks
• Both emerged and encased silks from all inbreds tested contained odd numbered nonisoprenoid 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).
C29
-4
-3 -2
1
-1
2
3
C26 (internal standard)
4
C27 C29:2 and C29:1
C31
C25
C23
C27:2 and C27:1
Encased
Emerged
Figure 2C
Emerged
Figure 2D
C25:1
C25
C27:2
C27:1
C27
C27:1
ketone
C27
ketone
C29:2
C29:1
C29
C29:1
ketone
C31:1
C31
C33:1
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%
Identification of differentially expressed genes
between emerged and encased B73 maize silk
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.
C31:2 and C31:1
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.
Encased
C23
C27:2
ketone
Minimum
C33
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.
C23
ketone
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
Homologs of
Arabidopsis Homologs
Fatty Acid Genes2
No. of Genes
Expressed
Inside
28112
Only Out 2102
4
Outside 27352
>10
>5
<0.2
<0.1
Only In
Total
1
3
5
3
3
19
FC1
(Out/In)
No. of
Genes
144
475
483
94
1342
4640
Wax Synthase, P450, ELO,
KCS
LACS2
KCS1
Binding Protein -1, CER-1
CER-60, KCS
P450-like, CER-2, MS-2
1 – Fold Changes
2- Genes whose homologs in Arabidopsis are involved in fatty acid synthesis/ elongation/ enriched in the gene lists
3 - 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