Transcriptomics of maize silks: Understanding hydrocarbon
accumulation patterns on the cuticle of developing silks
Adarsh Jose1,2, Wenmin Qin1, Sam Condon1, Marna Yandeau-Nelson1 and Basil Nikolau1
1Department
of Biophysics, Biochemistry and Molecular Biology, Iowa State University, Ames, IA, United States
2Bioinformatics and Computational Biology, Iowa State University, Ames, IA, United States
1. Introduction
3. Bioinformatics Analysis Pipeline
The cuticle of maize silks, which functions as a water barrier and as a
protective layer against abiotic and biotic stresses, is largely comprised of
linear hydrocarbons (lengths from C23 to C33) and is the most abundant source
of hydrocarbons in maize. These surface hydrocarbons (SHCs) consist of
linear alkanes and alkenes and are thought to be produced via elongation of
fatty acids with subsequent conversion to hydrocarbons (Figure 1).
Rs-COOH
Illumina Paired End
Reference Genome
ZmB73_RefGen_v2.masked.fasta
QUALITY FILTER
(fastx toolkit)
[~15-20% reads filtered out]
RNA-Seq Data
[75 bp x ~15 million pairs x
2 Biological Replicates x
[Encased + Emerged Silks]
Rm-COOH
Head-to-head
Condensation
Elongation
RsCORm
Rn-COOH
Rn-CHO
1
2
3
RsCH(OH)Rm
Decarboxylase
CO2
Decarbonylase
4
Dehydratase
CO
Rn
Rs+m+1
Rn
Odd-numbered hydrocarbon
Genome
Annotations
Rn-CH2OH
[Interpro & GO
Annotations,
Arabidopsis
best hits]
H2O
Rn+1
We have previously demonstrated in the inbred B73 that SHCs accumulate
~5-fold more on silks that have emerged as compared to those still encased
within ear husks. Moreover, accumulation continues to increase between 3
and 6 days after silks first emerge from the husk leaves, suggesting that the
genes involved in the synthesis and accumulation of SHCs are actively
transcribed at 3-days post-emergence (Figure 2).
Although we have
established that SHC accumulation varies significantly across development
(Figure 2) and among genotypes, we do not understand the biochemical and
genetic mechanisms involved in SHC synthesis and accumulation.
To dissect the genetic and biochemical mechanism(s) involved in SHC
synthesis, we have performed transcriptome analyses (RNA-seq) on silks that
either hyper- or hypo-accumulate hydrocarbons (Figure 3).
READ COUNTs used to estimate gene
expression as FPKM*
(Cufflinks package)
•  SHC accumulation was examined along the lengths of emerged and
encased silks at 3- and 6-days post-silk emergence (Figure 2).
•  Total SHC content increased between the two time points, demonstrating
that the genes involved in SHC accumulation are actively transcribed in 3
days post-emergent silks (Figure 2A). Moreover, SHCs accumulate along
the length of the emerged silk at 3-days PSE allowing the entire lengths of
emerged silks to be used for transcriptome analysis (Figure 2B).
•  The transcriptomes of emerged and encased silks harvested 3 days postemergence were sequenced using the Illumina Genome Analyzer IIE. The
resulting ~48,000,000 high quality 75 bp paired-end reads were mapped to
the B73 genome.
•  ~25,000 genes from the maize Working Gene Set were identified to be
expressed in silks, ~2400 of which were differentially expressed between
emerged and encased silks (q-value < 0.05) [Table1].
•  The differentially expressed genes were mapped to MaizeCyc pathways
(Figure 4, Table 2) and subjected to GO term enrichment analyses (Table
2).
Emerged silks
2 3 4 Encased silks
-4
-2
-3
-1
B.
7 Total SHC (umol/g fresh wt)
6 Emerged
Encased
5 3 Days
4 6 Days
3 2 1 0 -­‐4 -­‐3 -­‐2 -­‐1 1 2 B73 silks (1 inch segments)
3 Extract expression of
Candidate Genes
DIFFERENTIAL EXPRESSED
GO ENRICHMENT
Gene Lists
(CuffDiff Package)
[topGO R package]
PATHWAY MAPPING
MaizeCyc Pathways
Map differentially expressed genes to curated pathways
from MaizeCyc Database, Cell Designer
Figure 3. Analysis pipeline. FPKM, Fragments per Kb gene length per million reads. Input
data, output data and software displayed in blue, red and green, respectively.
4. Differentially Expressed Genes
Fold Change
(Emerged/
Encased)
Working
Genes
[110,028]
Filtered
Genes
[39,656]
Transcription
Factors (Subset of
Working Genes)
[1390]
Candidate
Genes [78]
Differentially Expressed
Gene Products Involved in
Fatty Acid or Cuticle
Synthesis
Only
Emerged
152
42
4
0
--
FC > 10
104
41
1
3
Wax Synthase, Long-chain
Acyl-CoA Synthase
(LACS2), P450-like
2. Experimental Design and Methods
1 (Homologs of 89
hand-picked from
Arabidopsis
Thaliana)
Even-numbered hydrocarbon
Figure 1. Four possible mechanisms of hydrocarbon biosynthesis. The fatty acid headto-head condensation mechanism (1), the elongation-decarboxylation (2), and the fatty acid
elongation-decarbonylation (3) pathways produce odd-numbered alkanes and alkenes. The
final pathway (4) involves a primary alcohol intermediate and would result in even-numbered
hydrocarbons.
A.
Candidate
Genes
MAP reads to Reference Genome
[ ZmB73_5a_WGS.gff,ZmB73_5b_FGS.gff]
(Tophat+Bowtie)
4 Figure 2. SHC content varies
along length of silks. (A)
Emerged silks were cut at the
point of emergence and
sectioned into 1-inch segments
(1 through 4).
Husks were
removed from the cob and
encased silks were cut into 1inch segments (-1 through -4).
(B) SHCs were extracted in
hexanes and analyzed via GCMS. The total SHC content
increases between 3 days and
6 days post emergence and
accumulates along the length
of the silk.
2 < FC < 10
579
469
20
8
Keto-acyl-CoA Synthases
(KCS3, KCS12, KCS19,
CER60)
0.5 < FC < 2
206
179
0
1
CER1
0.1 < FC < 0.5
973
829
38
15
Acetyl-CoA Carboxylase,
ATP Citrate Lyase A and B,
Enoyl CoA Reductase
FC < 0.1
223
150
5
4
Fatty Acid Reductase (MS2),
KCS20, LACS2
Only Encased
192
70
8
0
--
Total
2429
1779
76
31
Table 1. Differentially Expressed Genes (q-value <0.05). The candidate gene list consists
of maize homologs of 89 genes in Arabidopsis known to be associated with lipid
biosynthesis and fatty acid elongation (see below).
5. Genetic mechanisms of SHC synthesis
•  SHCs are thought to be generated via conversion of very long chain fatty acid
(VLCFA) precursors to SHCs. We have identified a total of 78 maize homologs of
89 genes in Arabidopsis thaliana known to be involved in lipid biosynthesis and
fatty acid elongation, which are processes needed to support the biosynthesis of
the precursors of hydrocarbons. Of these, 59 genes were active in silks and 31
were differentially expressed between encased and emerged silks (Table 1).
•  The genetic pathways responsible for both de novo and VLCFA synthesis are active
in both encased and emerged silks. Several genes in VLCFA synthesis are
upregulated in emerged silks (e.g. several ketoacyl-CoA synthases, which initiates
the 1st reaction in fatty acid biosynthesis) (Figure 4).
•  Several genes that are specifically involved in the synthesis of the cuticle are
expressed more highly in emerged silks, including Glossy1, a fatty acid acyl-ACP
reductase (MS2), a Cer1 homolog, and a putative wax synthase homolog.
Figure 4. Very long chain fatty acid elongation pathway (modified from
MaizeCyc) expressed in maize silks. For both emerged and encased silks,
at least one gene is expressed at each step in VLCFA elongation.
6. Genetic Pathways Expressed in Silks
•  This transcriptome dataset provides global information about the
expression of genetic pathways in encased and emerged silks and
addresses the environmental impact of silk emergence from the
protective husks.
•  Metabolism- and development-related activities are enriched in encased
silks, whereas stress- and defense-related activities are enriched in
emerged silks, which are exposed to increased abiotic and biotic
stresses.
Table 2. Enrichment Patterns for Highly Expressed Genes
Encased Silk
Emerged Silk
GO Terms
•  Anatomical and structural development
and cell growth
Processes: Syntheses, Assembly and
Organization of Chromosome, Chromatin,
Nucleotides, Cell wall, Cellulose
Components: Organelles (non-membrane
bound), Chromatin, Nucleosome
Functions: Auxin stimulus response
•  Stress response
Processes: Response to Defense, Heat, Biotic
Stress, Fungus, Temperature and Light
Components: Intracellular Organelles,
Mitochondrial Membrane, Plastids and Stroma
Functions: Cellular Lipid Metabolism,
Phosphatase Activity and Hydrolase Activity
Pathways (Maize-Cyc)
•  Development – Syntheses and metabolism of •  Metabolism and Degradation of Sugar,
amino acids and nucleotides.
Cellulose and Glucose
•  Exposure to light - photorespiration, aerobic
respiration, Calvin-Benson cycle.
•  Biosynthesis of fatty acids, amino acids,
Coenzyme-A, Vitamins, NAD and heme.
•  Metabolism - TCA cycle, glyoxylate •  Water-loss prevention (Suberin)
•  Response to abiotic stress (Jasmonic acid)
•  Defense – GDP- Mannose biosyntheses.
•  Senescence-related (Glutathionine)
Transcription Factors
•  Gibberellin signaling (WRKY69, involved in
cell elongation)
•  Morphogenesis and development (Rolled
leaf1).
•  Pathogen defense (WRKY53).
•  Homoiothermy, response to freezing
•
•
•
•
Glycolysis regulation
Heat response regulator (WRKY51)
Ethylene-responsive (B6T5Z6)
Sensitivity to bacteria (MADS-X)
(GRMZM2G065896).
•  Control, development and organ identity
(B6TDB4, MADS-box )
7. Complementary Approaches
•  This work supports the hypotheses that fatty-acids are precursors of
SHCs. We are currently developing bioinformatics methods to combine
sequence similarity and gene co-expression to identify novel genes
involved in the conversion of fatty-acids to SHCs and its regulation in
maize.
•  Quantitative genetic approach using the high-resolution intermated
B73xMo17 populations to localize the chromosomal positions associated
with SHC accumulation. These RNA-Seq transcriptome analyses will
complement the results obtained in the quantitative genetics studies and
will allow for the narrowing of candidate gene pools.
•  Similar transcriptome analyses are being conducted in three additional
biological systems that can hyper- or hypo-accumulate HCs under
different growth conditions or stages of development. We are currently
developing bioinformatic approaches for comparing and contrasting the
genetic mechanisms involved in SHC production among the four
systems.