reads is their indels and mismatches which can
be corrected via deeper PacBio sequencing of the
same read, alignment with the reference genome,
and alignment with short-read sequencing, all of
which can be combined (An et al. 2018a). With
these technologies, it is now possible to accurately characterize and measure the transcriptome
of virtually any species with a reference genome
or through de novo assembly.
When looking at the RNA-seq studies of the
duckweed family, we see that most of the gene
expression analyses have been done in the
recently sequenced Lemna minor, or in Landoltia
punctata and Lemna aequinoctialis with de novo
transcriptomes assembled from small reads (An
et al. 2018b). These species have genome sizes
ranging from 379 to 650 Mb and de novo transcriptomes of 74,797 and 72,105 unique contigs,
while Lemna minor has 22,382 annotated genes,
and Spirodela has 19,623 and 18,507 in strains
7498 and 9509, respectively, suggesting a wide
variety of possible mRNAs from the roughly
20,000 genes found in Lemnaceae genomes (Tao
et al. 2013; Wang et al. 2014a, 2016; Van Hoeck
et al. 2015; Michael et al. 2017; Yu et al. 2017).
In addition to the transcriptomic analysis of
ABA-induced turion formation, there have been
a couple of other RNA-seq experiments within
Spirodela polyrhiza that measure aspects other
than gene expression to understand the full
complexity of these plant transcriptomes. A reinvestigation of turion development RNA-seq
identified the chloroplast genes that undergo
mRNA editing and how this relates to the rest of
the monocots. Another study was the sequencing
of the degraded RNAs in Spirodela within eight
conditions as a measure of miRNA induced
cleavage, which could also show a rough measure of expression. These three studies provide
researchers a chance to witness mRNA expression, editing, and degradation.
One unique aspect of the duckweed lifecycle
is the formation of turions. Their role as an
asexual organ of perennation makes them analogous to both seeds, which sexually give rise to
an entire organism, and tubers or buds, which
asexually survive winter and other unfavorable
conditions. In 2014, an RNA-seq study was
performed on Spirodela during the development
of turions, making it the first genome wide-study
of gene expression in a plant tuber, which was
followed by a study of potatoes in 2015 (Wang
et al. 2014b). It was previously discovered that
3 days of exposure to 10 lM abscisic acid,
ABA, induced irreversible turion development
and an increase of two enzymes involved in
starch and cell wall production. This study,
therefore, compared four biological replicates of
Spirodela fronds with and without 3 days of
exposure to ABA by sequencing 15–41 million
75 bp reads of ribosomal, rRNA, depleted total
RNA on a SOLiD 5500 instrument. They were
able to map reads to the nuclear, mitochondrial,
and chloroplast genomes, with 28–39% of reads
deriving from the organelles. Reads were aligned
with bowtie and tophat, normalized and compared in cufflinks, and annotated for GO term
enrichment through blast2go and GOseq. The
results showed 154 genes down-regulated during
turion development, meaning that they were
minimally four-fold less abundant, with roughly
half being 0.2–0.1 the expression compared to
control. These results had a false discovery rate,
FDR, less than 0.01 thanks to the eight biological
replicates. The 154 down-regulated genes were
largely involved in carbon fixation, protein synthesis, DNA replication, and growth in general
since turions no longer grow. For the 208
upregulated genes, the GO term enrichment
showed that many of these upregulated genes
functioned in starch and anthocyanin production,
hormone response and signal transduction, cell
wall synthesis, and seed dehydration. There were
13 genes in cell wall and anthocyanin production
that were specific to turion induction. Similar to a
desiccating seed of a terrestrial plant developing
turions upregulated five and expressed two previously silent genes of the late embryogenesis
abundant protein family. These LEA family
proteins protect other proteins and confer resistance to dehydration, salinity, and cold stress.
This transcriptomic study was properly timed to
observe not only the structural changes of turion
development, but the signaling pathway. They
noticed upregulation of seven ABA-responsive,
three ethylene-responsive, and two heat shock
134
P. Fourounjian
be corrected via deeper PacBio sequencing of the
same read, alignment with the reference genome,
and alignment with short-read sequencing, all of
which can be combined (An et al. 2018a). With
these technologies, it is now possible to accurately characterize and measure the transcriptome
of virtually any species with a reference genome
or through de novo assembly.
When looking at the RNA-seq studies of the
duckweed family, we see that most of the gene
expression analyses have been done in the
recently sequenced Lemna minor, or in Landoltia
punctata and Lemna aequinoctialis with de novo
transcriptomes assembled from small reads (An
et al. 2018b). These species have genome sizes
ranging from 379 to 650 Mb and de novo transcriptomes of 74,797 and 72,105 unique contigs,
while Lemna minor has 22,382 annotated genes,
and Spirodela has 19,623 and 18,507 in strains
7498 and 9509, respectively, suggesting a wide
variety of possible mRNAs from the roughly
20,000 genes found in Lemnaceae genomes (Tao
et al. 2013; Wang et al. 2014a, 2016; Van Hoeck
et al. 2015; Michael et al. 2017; Yu et al. 2017).
In addition to the transcriptomic analysis of
ABA-induced turion formation, there have been
a couple of other RNA-seq experiments within
Spirodela polyrhiza that measure aspects other
than gene expression to understand the full
complexity of these plant transcriptomes. A reinvestigation of turion development RNA-seq
identified the chloroplast genes that undergo
mRNA editing and how this relates to the rest of
the monocots. Another study was the sequencing
of the degraded RNAs in Spirodela within eight
conditions as a measure of miRNA induced
cleavage, which could also show a rough measure of expression. These three studies provide
researchers a chance to witness mRNA expression, editing, and degradation.
One unique aspect of the duckweed lifecycle
is the formation of turions. Their role as an
asexual organ of perennation makes them analogous to both seeds, which sexually give rise to
an entire organism, and tubers or buds, which
asexually survive winter and other unfavorable
conditions. In 2014, an RNA-seq study was
performed on Spirodela during the development
of turions, making it the first genome wide-study
of gene expression in a plant tuber, which was
followed by a study of potatoes in 2015 (Wang
et al. 2014b). It was previously discovered that
3 days of exposure to 10 lM abscisic acid,
ABA, induced irreversible turion development
and an increase of two enzymes involved in
starch and cell wall production. This study,
therefore, compared four biological replicates of
Spirodela fronds with and without 3 days of
exposure to ABA by sequencing 15–41 million
75 bp reads of ribosomal, rRNA, depleted total
RNA on a SOLiD 5500 instrument. They were
able to map reads to the nuclear, mitochondrial,
and chloroplast genomes, with 28–39% of reads
deriving from the organelles. Reads were aligned
with bowtie and tophat, normalized and compared in cufflinks, and annotated for GO term
enrichment through blast2go and GOseq. The
results showed 154 genes down-regulated during
turion development, meaning that they were
minimally four-fold less abundant, with roughly
half being 0.2–0.1 the expression compared to
control. These results had a false discovery rate,
FDR, less than 0.01 thanks to the eight biological
replicates. The 154 down-regulated genes were
largely involved in carbon fixation, protein synthesis, DNA replication, and growth in general
since turions no longer grow. For the 208
upregulated genes, the GO term enrichment
showed that many of these upregulated genes
functioned in starch and anthocyanin production,
hormone response and signal transduction, cell
wall synthesis, and seed dehydration. There were
13 genes in cell wall and anthocyanin production
that were specific to turion induction. Similar to a
desiccating seed of a terrestrial plant developing
turions upregulated five and expressed two previously silent genes of the late embryogenesis
abundant protein family. These LEA family
proteins protect other proteins and confer resistance to dehydration, salinity, and cold stress.
This transcriptomic study was properly timed to
observe not only the structural changes of turion
development, but the signaling pathway. They
noticed upregulation of seven ABA-responsive,
three ethylene-responsive, and two heat shock
134
P. Fourounjian