# Patent

## Title
RNA-Seq transcriptome analysis of spirodela dormancy without reproduction and identification of molecular targets useful for improving biomass production for industrial applications

## Abstract

Compositions and methods are provided for altering carbon partitioning in biomass isolated from Duckweed.

## Background
This application claims priority to U.S. Provisional Application No. 61/912,328 filed Dec. 5, 2013, the entire disclosure being incorporated herein by reference.


FIELD OF THE INVENTION
This invention relates to the fields of plant molecular biology and recombinant manipulation of plant species in order to maximize production of biomass having desirable characteristics. More specifically, the invention provides valuable gene targets for manipulating carbon production in Duckweed based on results obtained from deep sequencing of the Duckweed genome.
BACKGROUND OF THE INVENTION
Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found within and at the end of the specification. The disclosure of each of these references is incorporated by reference.
Plants, unlike animals, do not have fur nor can they seek shelter to survive under food shortage and cold weather conditions. Consequently, they often become dormant to avoid adverse environments, such as poor nutrition, chilling temperature and drought. Dormancy is a complex state of plant development, in which the plant body exhibits little or no growth. Plants resume their growth once the conditions are favorable.
There are mainly two types of plant dormancy, e.g., forming seeds or buds. Seed dormancy has been observed for many plants species including our major crops [1-3]. Winter dormant buds are found for instance in woody plants, bulbs, rhizomes and tubers of herbaceous plants [4]. Studies on the molecular mechanisms of bud dormancy transitions in perennial woody plants have been conducted, including pear[5], oak[6], and poplar[7].
Spirodela polyrhiza, a floating aquatic monocot, develops a specific dormant organ called a turion during its life cycle, which alternates between periods of clonal propagation and dormancy. Its leaf, stem and bud are extremely compact forming a round-shaped frond, resembling a single leaf. Large numbers of Spirodela plants can be maintained in cell cultures under totally controlled medium and environmental conditions. They reproduce vegetatively through budding of fronds (growth phase) during spring and summer[8] and transition to turions (dormant phase), when there is a shortage of nutrients in the fall or when the temperature drops in the winter[9].
Noticeably, fronds perform photosynthesis and turions function as storage for starch and germinate in the following spring[10-13]. Turion cells exhibit dense intercellular space, thick cell wall and are also rich in anthocyanins[14]. Therefore, turionsprovide a unique system to study both bud and seed dormancy because they reproduce like buds without sexual hybridization but are functionally equivalent to seeds that could generate a progeny plant in the growing season. Previous studies have shown that addition of abscisic acid (ABA) into growth medium quickly leads to turion formation after 5 days of treatment in the laboratory[13, 15, 16]. Only 3 days after ABA treatment, the Spirodela primordium is irreversibly committed to turion development[15]. The ease of growth and its direct contact with water make Spirodela a model system to gain molecular insights into plant dormancy[17].
At the molecular level, some studies onturion development have already been performed. For example, the transcript level of D-myo-inositol-3-phosphate synthase is rapidly induced within 15 min of ABA application, an enzyme that plays a key role in the inositol metabolism of the cell wall[18, 19]. The expression of the key enzyme ADP-glucose pyrophosphorylase (APL) for starch production[13] is significantly changed during turion formation. Still, not much information is known about the global transcriptome profiling for turion formation in this model system.
SUMMARY OF THE INVENTION
In accordance with the present invention, a method for altering carbon partitioning from starch to lipids in biomass produced from Duckweed cultures is disclosed. In one embodiment the method comprises introducing an agent or gene variant, which modulates expression of a gene product identified in Table S1, wherein the agent or gene variant is effective to reduce starch production and increase lipid production in said culture relative to control untreated or unaltered cultures. The agent may be a nucleic acid, a small molecule, an antibody or a chemical compound. In another embodiment, the promoters of genes with high expression levels identified in Tables S1 can either be used to overexpress coding regions of key enzymes in lipid biosynthesis or RNA interference products against transcripts of starch biosynthesis as they have been identified in Tables S1 or S2.
In one aspect of the method, the agent or gene variant inhibits or increases expression of at least one gene product identified in Table S1, the inhibition or increase resulting in increased lipid production in biomass obtained from said Duckweed culture.
In a preferred aspect of the inventive method the agent modulates, (e.g., inhibits or increases) expression of at least one gene selected from the group consisting of AGPS1, AGPL3, GBSSI, APL1, ACCase4, GPAT1, and DGAT2. Alternatively, the agent modulates production of at least one gene in Table S2, which shifts carbon partitioning, in turn increasing lipid and or protein biosynthesis.
In yet another aspect of the invention, a Duckweed plant produced from any of the methods described above is provided. In a particularly preferred embodiment, the Duckweed plant is Spirodela polyrhiza. Also encompassed by the present invention are plant parts, progeny, seed or cells obtained from the duckweed plants described herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Comparison between frond and mature turion by TEM. A. A frond cell with a big vacuole and well-shaped chloroplasts but few and less starch granules, Bar=2 μm; B. A turion cell with thick cell wall and abundant starch granules, Bar=2 μm; Abbreviation: cell wall (CW), chloroplast (C), starch granule (S), vacuole (V) and nucleus (N).
FIG. 2. Biological variation for biological replicates from fronds and developing turions. Biological variation was represented by the square coefficient of variation of FPKM values for each gene (CV2).
FIG. 3. Comparison of APL gene expression from qRT-PCR vs. RNA-Seq. A. APL gene expression from qRT-PCR; B. APL gene expression from RNA-Seq data. Abbreviation: APL1-JN180634; APL2-JN180635; APL3-JN180636.
FIG. 4. Alignment of ABF domain from Spirodela. The amino acid sequences of bZIP protein sequence from Spirodela were aligned and the conserved regions were demonstrated here. The consensus amino acids were labeled from conserved regions and highlight as motif 1 and motif 2, the primary structure of bZIP domains (basic region and leucine zipper). All members contain these four domains except SpbZIP, which only has a basic region and a leucine zipper. SpABF1-Spipo4G0008600; SpABF2-Spipo6G0055300; SpABF3-Spipo15G0021000; SpABF4-Spipo4G0111500; SpABF5-Spipo7G0034500; SpABF6-Spipo3G0017700; SpABF7-Spipo13G0002500; SpbZIP-Spipo2G0055800. Motif 1 sequences are SEQ ID NOs: 1-9 from top to bottom, motif 2 sequences are SEQ ID NOs: 10-18 from top to bottom, bZIP domain sequences are SEQ ID NOs: 19-27.
FIG. 5. Alignment of the ERF domain from Arabidopsis and Spirodela. The bar and black arrows indicate β sheet motif, which interacts with the GCC box of target DNA. The cross-hatched box indicates the a helix. The consensus amino acids are underlined in ERF domain. The accession numbers are: AtERF1-BAA32418; AtERF2-BAA32419; AtERF3-BAA32420; AtERF4-BAA32421; AtERF5-BAA32422; SpERF1-Spipo0G0155100; SpERF2-Spipo3G0031800; SpERF3-Spipo20G0027700; SpERF4-Spipo11G0028200. Sequences are SEQ ID NOs: 28-37, from top to bottom.
FIG. 6. A model of development of Spirodela dormancy through the signal transduction in response to environmental stimuli. Phosphorylated proteins are labeled as pink circles with a P inside. Solid lines represent direct connections. The dotted line indicates indirect connection. Not all linkages and details of pathway are shown in this diagram in order to simplify the model. Abbreviations: ABA (abscisic acid), CPK (calcium-dependent protein kinase), MAPK (mitogen-activated protein kinase), ABF (ABA-responsive element binding factor), ERF (ethylene-responsive element binding factor), HSF (heat shock transcription factor), WRKY (WRKY transcription factors), ABRE (ABA-responsive element), ERE (ethylene-responsive element).



DETAILED DESCRIPTION OF THE INVENTION
Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing an as yet an untapped source for industrial applications.
We used next generation sequencing technology to examine the transcriptome of turion development triggered by exogenous ABA. A total of 208 genes showed more than a 4-fold increase compared with 154 down-regulated genes in developing turions. The analysis of up-regulated differential expressed genes in response to dormancy exposed an enriched interplay among various pathways: signal transduction, seed dehydration, carbohydrate and secondary metabolism, and senescence. On the other side, the genes responsible for rapid growth and biomass accumulation through DNA assembly, protein synthesis and carbon fixation are repressed. Noticeably, three members of late embryogenesis abundant protein family are exclusively expressed during turion formation. High expression level of key genes in starch synthesis are APS1, APL3 and GBSSI, which could artificially be reduced for re-directing carbon flow from photosynthesis to create a higher energy biomass.
The identification and functional annotation of differentially expressed genes opens a major step towards understanding the molecular network underlying vegetative frond dormancy. Moreover, genes have been identified that could be engineered in duckweeds for practical applications easing agricultural production of food crops.
In another aspect of the invention we have investigated the unusual mechanism of the regulation of organellar gene expression where the translation of organellar RNAs requires the replacement of a C with a U before translation, also referred to as RNA editing. Sequence specificity is achieved through a nuclear gene family that encodes PLS-type pentatricopeptide repeat proteins (PPRs). Within monocotelydonous plants, most genomes that have been sequenced belong to the order of Commelinids, but sequencing of species of the Alismatales, Spirodela (Spirodela polyrhiza), has providing us with a new reference for the evolution of PPR proteins, and means to modulate expression of the same to confer desirable phenotypes. We used deep sequencing of non-ribosomal total RNA to determine the number and conversion efficiency of editing sites of Spirodela organellar mRNA. There are 66 editing sites, of which 58 are in protein coding regions. Comparison to coconut, maize, and rice suggests that RNA editing originated from a common ancestor, but that the number of PPR genes and editing sites changed independently either by losses or gains of gene copies during evolution. Based on the expression of nuclear-encoded PPR genes and RNA editing efficiency in plastids, it appears that for  ̃24% of incomplete RNA editing is the result of lower level expression of individual PPR genes. Furthermore, 37 PPR genes are differentially expressed and 11 RNA editing sites show a significant change when growth is arrested at dormancy. Thus, it appears that RNA editing is regulated through the expression of gene copies in a developmental fashion.
Definitions
The term “duckweed system” or “duckweed culture” as used herein encompasses duckweed plant cultures, duckweed fronds or immature turion cultures, duckweed suspension cultures, and duckweed protoplast cell cultures.
The term “duckweed plant culture” as used herein refers to a culture comprising mostly fully differentiated duckweed plants.
A “differentiated cell,” as used herein, is a cell having at least one phenotypic characteristic (e.g., a distinctive cell morphology or the expression of a marker nucleic acid or protein) that distinguishes it from undifferentiated cells or from cells found in other tissue types. In some embodiments, the duckweed turion culture comprises duckweed turions as described elsewhere herein.
The term “duckweed suspension culture” as used herein refers to a culture comprising dispersed duckweed cells, for example dispersed duckweed callus cells. Generally, a duckweed suspension culture will comprise both single cells and unorganized cellular aggregates of varying sizes.
The term “duckweed protoplast cell culture” refers to a culture comprising duckweed cells where at least about 50%, 60%, 70%, 80% or 90% of the duckweed cells lack a cell wall. Methods for making protoplast cells from plant cells are described, for example, in Eriksson (1995) in Plant Protoplasts, Fowke et al., eds., CRC Press, herein incorporated by reference.
The term “biological function” as used herein refers to a biological activity or property of a nucleic acid molecule or polypeptide. For example, biological functions for nucleic acid molecules include modulating biological responses, coding for polypeptides, and modulating the expression of target nucleotide sequences. Examples of biological functions for polypeptides include modulating biological responses, conferring structural properties of interest, conferring biochemical activities of interest, and conferring regulatory activities of interest. Particular, non-limiting examples of modulatory and regulatory activities include the ability to bind a substrate of interest, the ability to bind a ligand of interest, the ability to catalyze a reaction of interest, the ability to modulate a response to a plant hormone, the ability to modulate a response to a plant growth regulator, the ability to modulate a response to environmental perturbation, the ability to modulate a response to physiological perturbation, the ability to modulate a response to one or more pathogens, and the ability to modulate a response to one or more toxins.
The term “duckweed” refers to members of the family Lemnaceae. This family currently is divided into four genera and 34 species of duckweed as follows: genus Spirodela (S. polyrrhiza, S. intermedia, S. punctata); genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa, Wa. microscopica, Wa. neglecta) and genus Wolfiella (Wl. caudata, Wl. denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda, Wl. rotunda, and Wl. neotropica). Any other genera or species of Lemnaceae, if they exist, are also aspects of the present invention. Lemna species can be classified using the taxonomic scheme described by Landolt (1986) Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae—A Monograph Study Geobatanischen Institut ETH, Stiftung Rubel, Zurich.
“Operably linked” as used herein in reference to nucleotide sequences refers to multiple nucleotide sequences that are placed in a functional relationship with each other. For example a promoter nucleotide sequence is operably linked to a second nucleotide sequence when it is positioned such that it can drive the transcription of the second nucleotide sequence.
“Polypeptide” refers to any monomeric or multimeric protein or peptide.
“Biologically active polypeptide” refers to a polypeptide that has the capability of performing one or more biological functions or a set of activities normally attributed to the polypeptide in a biological context. Those skilled in the art will appreciate that the term “biologically active” includes polypeptides in which the biological activity is altered as compared with the native protein (e.g., suppressed or enhanced), as long as the protein has sufficient activity to be of interest for use in industrial or chemical processes or as a therapeutic, vaccine, or diagnostics reagent. Biological activity can be determined by any method available in the art. For example, the biological activity of members of the interferon family of proteins can be determined by any of a number of methods including their interaction with interferon-specific antibodies, their ability to increase resistance to viral infection, or their ability to modulate the transcription of interferon-regulated gene targets.
“Nucleotide sequence of interest” as used herein refers to any nucleotide sequence encoding a polypeptide intended for expression in duckweed. For example, nucleotide sequences encoding therapeutic (e.g., for veterinary or medical uses) or immunogenic (e.g., for vaccination) polypeptides can be expressed using transformed duckweed according to the present invention.
The terms “expression” or “production” refer to the biosynthesis of a gene product, including the transcription, translation, and assembly of said gene product.
The term “genetically modified” as used herein refers to a plant cell or plant that is modified in its genetic information by the introduction of one or more foreign polynucleotides, and that the expression of the foreign polynucleotides leads to a phenotypic change in the plant. For example, a plant that is genetically modified to alter glycosylation is modified in its genetic information by the introduction of one or more foreign polynucleotides, where expression of the polynucleotide or polynucleotides leads to a change in glycosylation in the plant.
The transitional terms “comprising”, “consisting essentially of” and “consisting of”, when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim, an in the latter instance, impurities ordinarily associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or materials and those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
The following methods are provided to facilitate the practice of the present invention.
Sample Preparation
Spirodela polyrhiza 7498 was grown into half-strength Schenk and Hildebrandt basal salt mixture (Sigma, S6765) with 1% sucrose liquid medium under 6-hrs light, 8-hrs dark photoperiod. Plant tissues from four biological replicates for fronds without ABA treatment and developing turions with 3-day 10 μM ABA were collected and frozen in liquid nitrogen. 10 μg of total RNA was extracted for each sample by RNA-easy Qiagen kit with RLC buffer due to second metabolites. Ribosomal RNA was depleted with a kit from Epicenter (MRZPL116) in order to increase the coverage of other RNA classes. Vegetative fronds and turions with 14 days ABA treatment were fixed, embedded, and examined under transmission electron microscope as described[13, 20].
Library Construction and Sequence Quality Control
We started with  ̃300 ngrRNA-depleted total RNA, fragmented the RNA, performed reverse transcription and size-selected the cDNA, used Emulsion PCR to amplify the complex gene libraries and prevent formation of chimeric cDNA products. All steps followed the manufacturer's guide (SOLiDTM total RNA-Seq kit). To minimize potential experimental batch effect, eight samples were barcoded, pooled, and evenly distributed into three lanes. The single-end reads with the size of 75 bp were generated with our in-house SOLiD 5500 platform. The Exact Call Chemistry (ECC) module was utilized in the sequencing run, which is an optional kit that is used to further enhance sequencing accuracy by generating reference-free bases directly. After quality trimming with score of 20, reads with a minimum length of 40 bp were saved.
Read Mapping and Quantifying Gene Expression
The remaining reads were mapped to the reference genome Spirodela polyrhiza 7498 (GenBank Accession #ATDW01000000), which was recently sequenced, assembled, and annotated, by using TopHat 2[21] with Bowtie[22]. TopHat is a fast splice junction mapper for RNA-Seq reads. It aligns RNA-Seq reads to reference genomes using the ultra high-throughput short read aligner Bowtie, and then analyzes the mapping results to identify splice junctions between exons. Gene expression levels were normalized using fragments per kilobase of exon per million mapped reads (FPKM). Transcript abundance and deferential gene expression were calculated with Cufflinks[23]. DE genes were defined, as when their absolute value of log 2 fold change was higher than 2 and their P value was less than 0.01.
As a positive control for our measurements, we used independent data obtained in a separate study under the same induction conditions as in this study from the expression of ADP-glucose pyrophosphorylase genes with qRT-PCR [13]. As a negative control, we used northern blot data of the expression of the tur4 gene obtained in yet another study [24].
Functional Annotation and Cis-Element Predictions
For each DE gene, GO annotation was obtained with the program of blast2go, which uses a blast algorithm to assign GO terms to sequences based on similarity[25]. GO enrichment was performed in two groups of gene sets, respectively, one of highly expressed transcripts in turions, the other one of highly expressed transcripts in fronds based on the whole gene set of the Spirodela genome using GOseq, which adjusts the bias from gene lengths[26]. The cis-acting regulatory DNA elements were predicted by signal scan search from PLACE database[27]. PLACE is a database of motifs found in plant cis-acting regulatory DNA elements, all from previously published reports. We dissected 1-kb regions upstream of DE genes and scanned them for potential pairs of TFs and cis-elements.
The following example is provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way.
Example I
To further uncover the regulation of gene expression as the phase switches, we took advantage of RNA deep sequencing, and compared the transcriptome between fronds and developing turions. A more comprehensive understanding of the gene repertoire and its regulation during turion formation has great potential for industrial applications including the redirection of carbon flow into higher energy products.
Calibration and Selection of Tissue Samples
A comprehensive study for turion formation has been done using abscisic acid (ABA) induction[14, 15, 17, 28, 29]. Three days after ABA induction, the Spirodela primordium is committed to turion development, which cannot be reversed. All primary biosynthesis of protein, mRNA and DNA are shutdown resulting in the onset of the dormant state[28]. To calibrate our growing conditions with previous investigations, we used transmission electron microscopy (TEM) to investigate different developmental stages. We chose fronds and developing turions with 3 days after ABA treatment instead of 14 days because 14-day treatment is not a key transition state and RNA purification is greatly hampered by high content of starch, but mature turions with 14-days treatment provide a more complete structural image through TEM. Turion cells have thicker cell walls, multiple smaller vacuoles and distorted plastids filled with abundant starch granules, whereas frond cells differ with having well-shaped chloroplasts consistent with previous observations (FIG. 1). Therefore, growing conditions and turion induction appear to be reproducible.
Mapping RNA-Seq Reads
We used eight samples in total, with each condition having four biological replicates. To eliminate potentially technical variation from biological replicates, they were multiplexed, pooled, and sequenced with the SOLiD 5500 platform. A total of 15 ̃41 million quality reads per sample were generated after filtering raw reads (Table 1).
The high quality reads were mapped to chloroplast[30], mitochondria[31], and nuclear genomes[32], respectively. We could clearly divide sequence reads into these three classes. Surprisingly, there was an abundance of organelle-derived transcripts with 28 ̃39% of total reads. With this depth of data we could assemble sequences for complete plastid and mitochondrial transcriptomes. The high proportion of organelle reads stresses the important roles of their transcripts, provides us with their expression profiles and facilitates the phylogenetic analysis[33]. Based on the combined reads of nuclear and organelle RNAs, more than 89% of our RNA-Seq reads were mappable, attesting to the performance of the sequencing platform. It also suggests that part of previously unmapped reads in other studies remained undetected because of their organellar origin[5, 34-36]. We still found that 1 ̃9% of total reads were derived from ribosomal RNA, which is an indication that the protocol for the depletion of ribosomal RNA from samples was reasonably successful. Such efficiency is critical for mainly uncovering the desired transcriptome with complete coverage and in a cost-effective manner[37].
Among the total reads, 53-61% originated from nuclear DNA, lower than in other cases with about 80% of mappable sequences [34, 36]. The reason could be the method we used through ribosomal RNA removal rather than polyA selection. In case of polyA selection, organelle transcripts are automatically removed due to the lack of the polyA tail in organelle transcripts, whereas most of them were captured by our method of ribosomal RNA removal. Excluding the abundant organelle and rDNA reads, nuclear reads corresponded to 29 ̃72X coverage for all annotated genes (Table 1), demonstrating that the depth used in our study was sufficient to cover the Spirodela nuclear transcriptome.
Identification and Validation of Differentially Expressed Genes
Comparison of frond and developing turion samples provided us with 362 differentially expressed (DE) genes. A total of 117 had greater than 10-fold difference in mRNA levels and 208 genes were up-regulated and expressed at higher levels in developing turions than in fronds, whereas 154 genes were down-regulated, indicating lower expression in turions than in fronds (Table 2).
Previous studies had indicated that a small number of biological replicates might not be robust enough because it is impossible to know whether expression patterns are specific to individuals or are characteristic for the total population. Even for RNA deep sequencing, a sufficient number of biological replicates are still required to have confidence in the measurements[38-40]. Because two biological replicates usually are not sufficient to account for sample variability, we increased this number to four independent biological replicates. The coefficient of variation to the power of two (CV2), a normalized measure of cross-replicate variability that can be useful for evaluating the quality of RNA-Seq data, was calculated to exhibit the biological variation (FIG. 2). As expected, the data showed that the abundance of the genes varied between replicate RNA samples, especially for ones with lower FPKM values. However, with four biological replicates, which take variation within the target population into account and also counteract random technical variation[23, 41], we were very confident to assess gene expression levels with accuracy.
As another quality control, we could rely on our measurements of the 3 transcripts of ADP-glucose pyrophosphorylases (APLs) for starch synthesis[13], which were done with qRT-PCR, and compared with the RNA-Seq data. Indeed, the correlation co-efficient of 0.992 indicated that the two independent measurements were consistent and showed similar patterns: APL1 (GenBank Accession #JN180634) was highly expressed in fronds and APL3 (GenBank Accession #JN180636) showed the most abundance in developing turions. However, APL2 (GenBank Accession #JN180635) was not identified as DE gene due to only 1.5 times of difference at the time point of 0 and 3rd day by the threshold value of 4 (FIG. 3). A fourth gene, tur4, provided us with a negative control from an independent study [24]. The tur4 gene has the Gene ID Spipo7G0013500 in the sequenced genome of Spirodela. Expression of this gene during turion formation was studied with Northern blot analysis. Although the tur4 gene responded to ABA treatment within hours, it appeared to return to nearly normal levels of expression thereafter. Northern blot analysis showed no induction at day 3 after ABA treatment, whereas we could still detect a 2-fold increase in tur4 expression with RNA-Seq, indicating that our method is more sensitive than Northern blot analysis. However, given both the APLs and tur4 results, we selected a cut-off for DE genes at 4-fold expressional change.
Response to ABA Stimulus
The plant hormone abscisic acid (ABA) plays a major role as a signal in seed development and plant dormancy[42, 43] and regulates many important aspects, such as the synthesis of seed storage proteins, starch and lipids[44, 45]. In Spirodela, exogenous ABA effectively triggers entry into the dormant state (turions) from growth phase (fronds)[15]. We found 25 up-regulated DE genes in response to ABA stimulus or regulation based on their GO annotation (Table 3 and S1). The pathway of ABA signal transduction and response seemed to be interwoven with enzyme metabolism (kinase, synthase, and phosphatase) and other signaling pathways (transporter, ethylene). Northern blot analysis shows that ABA rapidly up-regulates tur4 transcriptional level that encodes a peroxidase, which could stimulate turion formation and growth inhibition[24].
Growth Inhibition
Dormancy is generally defined by the lack of visible growth. The shoot apices cease active growth in perennial plants when a state of dormancy is reached. The seed dormancy is observed in seeds with a quiescent phase preventing germination. The same phenomenon was investigated for Spirodela in the presence and absence of growth. When we looked at DE genes associated with Spirodela growth by RNA-Seqdata, we found genes of histone H3 (Spipo9G0039400, Spipo0G0046100 and Spipo13G0007500) and H4 (Spipo28G0019000), ribosomal protein (Spipo1G0126300), expansins (Spipo22G0026300), aquaporins (Spipo11G0033800, Spipo17G0045100), ribulose-1,5-bisphosphate carboxylase oxygenases (RuBisCO) (Spipo19G0027700, Spipo23G0013400) for carbon fixation were down-regulated in turions (Table 4). In eukaryotic cells, DNA replication requires the synthesis of histone proteins to package newly replicated DNA into nucleosomes. Expansins are a key endogenous regulator of plant cell enlargement[46]. Aquaporins support cell growth and especially contributes to cell expansion and cell division. The gene that is highly expressed in fronds (69 times higher than in turions) is aquaporin (Spipo11G0033800) (Table 4). Over-expression of aquaporin stimulates cell growth in tobacco[47] or in Arabidopsis[48]. These results further confirm our knowledge that fronds are mainly responsible for rapid growth through actively DNA assembly, protein synthesis and carbon fixation, leading to a quick biomass increase, in comparison to the turions, where these processes are greatly decreased. Previous studies also suggested this mechanism of the turion formation by measuring DNA, RNA and protein content, which showed that DNA, protein and RNA biosynthesis were largely inhibited, resulting in the decrease of cell division, expansion and differentiation[28].
Late Embryogenesis Abundant Protein (LEA) Genes are a Valuable Marker for Dormancy
On the other hand, we found some specific mRNAs were increased in developing turions, for example LEAs. Although there were five members of LEA genes (Spipo14G0001200, Spipo5G0015500, Spipo0G0166800, Spipo1G0033500, Spipo26G0007700) with increased expression in turions, the LEA gene (Spipo0G0166800) was the most up-regulated DE gene, two other LEA genes (Spipo5G0015500 and Spipo14G0001200) were exclusively expressed in developing turions (Table 5). Indeed, the promoter of these LEA genes would be ideal to ensure expression of other coding regions exclusively in turions through transgenic approaches. Additionally, LEA was found to protect other proteins against desiccation, cold, and high salinity[49] and especially accumulates when plant seeds desiccate[50]. Given their high induction, they provide valuable markers for dormancy in general. In response to dehydration, endogenous ABA levels increased dramatically followed by induction of LEA[51]. As expected, when Spirodela fronds are destined to dormant turions triggered by ABA, desiccation is an indispensable step, in which LEA proteins play pivotal roles to preserve the cellular structures and nutrients in turions.
Genes Involved in Carbon Partitioning
Starch is the major carbon reserve in plant storage organs, and ABA has a signaling role by inducing starch biosynthetic gene expression and co-ordinate carbohydrate partitioning[52]. In our study, four genes (Spipo12G0062400, Spipo18G0038500, Spipo16G0027000 and Spipo27G0011300) (Table S1) participating in starch biosynthesis were significantly enhanced in developing turions. The qRT-PCR experiment confirmed the key enzyme of large-subunit ADP-glucose pyrophosphorylase 3 (APL3) for starch biosynthesis was highly expressed in turion development[13]. The RNA-Seq study for Landoltia punctata also revealed gene expression involved in starch biosynthesis was up-regulated under nutrient starvation[53]. Another way to accumulate starch content is to redirect carbon flow to starch biosynthesis. We found seven genes participate in the degradation of lipids by alpha- (Spipo0G0156600, Spipo0G0180000, Spipo0G0156500, Spipo5G0040500) or beta-oxidation (Spipo0G0179100, Spipo3G0031300, Spipo1G0110400), which probably allocate carbon to starch rather than fatty acids to achieve denser turions that sink to the bottom of streams during seasons (Table S1). Previously, it has been shown that the carbon flow into seeds can be rebalanced between different macromolecules with different energy content [54]. Reallocation of carbon is critical for the improvement of oil production in novel crops in the future. In oilseed species, numerous biotechnological approaches have been carried out that were aimed to maximize the flow of carbon into oil by over-expression of enzymes of the TAG assembling network[55]. Although one might argue that turions would no longer be able to sink in water when filled with lipids, in those applications biomass would be accumulated under constant temperature.
Another way to investigate the balance of carbon partitioning can be derived from the average FPKM value (Fragments Per Kilobase of transcript per Million mapped reads) of all the key genes encoding both pathways. The genes encoding for lipid production were expressed relatively low with FPKM of 28 and 22 in fronds and turions, respectively. Therefore, the level of lipids remains low throughout development (Table S2). Given the high level of starch in turions, genes in lipid production are not induced, whereas the ones for starch biosynthesis are during turion formation, providing us with a correlation between metabolic products and the regulation of the corresponding pathways. Given this correlation, we hypothesize that we could redirect carbon flow into lipids by blocking key genes of such as AGPS1, AGPL3, GBSSI and ACCase4, GPAT1, DGAT2, and over-express transcripts of the lipid pathway (Table S2) together with turion-specific promoters, like LEAs (Spipo14G0001200, Spipo5G0015500, Spipo0G0166800)(Table 5).
Turion-Specific Pathways
We found that the transcriptome also closely links the turion phenotypic variation with thick cell wall and abundant secondary metabolites like pigment. The expressions of eight members of the UDP-glycosyltransferase superfamily (Spipo2G0010600, Spipo2G0043800, Spipo16G0044000, Spipo2G0039000, Spipo14G0034300, Spipo2G0124000, Spipo5G0014300, Spipo2G0077900) and two of the cellulose synthases (Spipo28G0017100, Spipo7G0044000) involved in cell wall biosynthesis were increased (Table S1). Three dihydro flavonol reductases (Spipo7G0010700, Spipo10G0000200, Spipo14G0054900) and one flavonoid 3′,5′-hydroxylase (Spipo0G0155000) involved in the anthocyanin pathway were up-regulated (Table S1). In addition, we found the average FPKM value for all key enzymes of lignin biosynthesis were 23 in fronds but 41 in turions, which may explain the rigidity of cell wall in turion cells to defend water pressure at the bottom of waters (Table S2).
To gain a broad overview into the biological functions for DE genes, we next performed an analysis of gene ontology (GO) enrichment (Methods). We found a total of 24 enriched pathways (p<0.01) in developing turions, whereas no enriched GO was found in fronds under the null hypothesis of the entire gene set of Spirodela (Young et al., 2010). The clustered DE genes were mainly related to response to ABA, fatty acid oxidation, and ion transportation. The GO functions of leaf senescence and cell wall modification were also highlighted (Table 6).
Transcriptional Regulation of Differentially Expressed Genes
Transcription factors (TFs) are crucial components of regulatory systems, which initiate vital changes in gene expression. Thus, we examined TF gene models and found nine TFs were significantly changed including two ABA-responsive element binding factors (bZIP, Spipo4G0008600 and Spipo2G0055800), four Ethylene-responsive element binding factors (ERFs, Spipo0G0155100, Spipo3G0031800, Spipo20G0027700 and Spipo11G0028200), two heat shock TFs (HSFs, Spipo8G0037600 and Spipo9G0002000), and one WRKY TF (Spipo8G0045500) (Table 5 and S1).
ABA-Responsive Element Binding Factor
The bZIP transcription factors regulate plant development through a basic region and a leucine zipper dimerization motif that binds to DNA[56, 57]. In the complete sequence of Spirodela genome[32], an exhaustive search of the bZIP superfamily was performed and 41 members identified. Among them, seven genes belong to the ABA-responsive element binding factors (ABFs), i.e., the bZIP superfamily group A due to their structural features with conserved regions C1-C2, basic regions, and leucine zippers (FIG. 4)[56, 58]. This group is thought to play a central role in controlling ABA-responsive gene expression in seeds and vegetative tissues via binding to ABA-responsive-elements (ABREs). For example, ABI5, one member of ABFs, induces LEA expression by binding to its promoters during seed maturation[58]. Here, all seven genes showed differentially increased expression levels, whereas only SpABF1 (Spipo4G0008600) was defined as a DE gene due to a significant change (Table 5). Noticeably, SpbZIP (Spipo2G0055800), another bZIP transcription factor, was significantly decreased in developing turions (Table 5). It shared leucine residues in the basic domain but missing other 2 conserved regions, corresponding to bZIP group I in Arabidopsis. Studies of group I genes from several species indicate that they might play a role in vascular development[56]. SpbZIP might positively regulate xylem and phloem development, too. Because both structure and function of turions are equivalent to seeds, less vascular tissue is needed in turions compared to fronds and the expression of SpbZIP is decreased accordingly. Thus, we conclude that a specific subset of bZIP transcription factors are involved in turion formation.
Other TFs Involved in ABA-Mediated Gene Expression
In addition to ABF TFs, other TFs were also identified to be involved in turion development. Ethylene-responsive element binding factors (ERFs) are transcription factors that are specific to plants. A highly conserved DNA binding domain, known as the ERF domain interacting directly with the GCC box in the ethylene-responsive-element (ERE), is the unique feature of this protein family[59] (FIG. 5). ERFs also play a role in a variety of developmental processes such as flower, seed development[60], and fruit ripening[61]. We identified 57 ERF genes in the Spirodela genome, where SpERF1 (Spipo0G0155100), SpERF2 (Spipo3G0031800), and SpERF3 (Spipo20G0027700) were significantly up-regulated and SpERF4 (Spipo11G0028200) down-regulated in response to turion development (Table 5). It had been reported that AtERF1, AtERF2, ATERF5 functioned as activators of GCC box-dependent transcription in Arabidopsis leaves, but AtERF3 and AtERF4 acted as repressors[57, 59]. It also was shown that ERF2 and ERF4 enhanced the transcription of a reporter gene in tobacco protoplasts[62]. The three highly up-regulated ERFs in Spirodela turions should therefore play an important role in turion development.
Heat shock transcription factors (HSFs) are transcriptional activators of heat shock genes. An increasing number of studies indicated that some HSFs appeared during the maturation stage of the seed, when cell division ceased and seeds adapted to desiccation and long-term survival[63]. Here, the increased expression of two HSFs (Spipo9G0002000 and Spipo8G0037600) (Table 5) might also indicate an important function for turion desiccation and survival during long periods of winter.
WRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy, and seed germination[64]. In vivo and in vitro promoter-binding studies showed that WRKY TFs could either activate or repress the expression of downstream ABFs through W-box sequences present in their promoters[65]. However, whether the Spirodela WRKY TF (Spipo8G0045500) (Table 5) is a repressor or activator needs to be further investigated.
Together, the significant changes in the expressions of ABFs, ERFs, HSF and WRKY TF reflected their obligatory regulation during turion development. Their involvement in the transition from fronds to turions and their control of spatial and temporal expression of target genes provides us also with new tools to create specialized traits through tailoring of chimeric genes.
Cis-Element
Control of gene expression is achieved through the binding of transcription factors to specific cis-elements in promoter regions of target genes[66]. To predict potential pairs of TFs and cis-elements, we scanned a 1-kb region upstream of DE genes with the PLACE database[27]. We found 30 up-regulated DE genes containing the cis-element of ABA-responsive element (ABRE: YACGTGGC) and 119 with ethylene-responsive element (ERE: GCCGCC) (Table S1). These target genes of ABFs and ERFs are associated with seed dehydration (like late embryogenesis abundant proteins), regulatory transcription factor, protein kinases and phosphatases (like CPK, MAPK), carbohydrate and secondary metabolism (like cellulose synthase and stachyose synthase), and senescence-associated proteins (like Glutathione-S-transferase).
Discussion
ABA is essential for seed maturation and also enforces a period of seed dormancy so that the seeds do not germinate prematurely during unseasonably conditions. The same behavior is seen in dormant Spirodela turions that are induced by low temperature, limited nutrition, or exogenous ABA[67]. The external stimuli rapidly induce both Ca2+ influx and endogenous ABA synthesis[68]. In maturing seed, ABA-regulated genes include those required for the synthesis of storage reserves and the acquisition of desiccation tolerance. Ca2+ can act as secondary messenger to activate the expression of cascade components of calcium-dependent protein kinase (CPK) and mitogen-activated protein kinase (MAPK). The structure of CPK shows there are four Ca2+-binding EF hand domains allowing the protein to function as a Ca2+ sensor. In addition to Ca2+, reversible phosphorylation also regulates kinase activity[69]. A number of studies have demonstrated that MAPKs in Arabidopsis are associated with hormone biosynthesis and signaling including ethylene and ABA[43]. Both of CPK and MAPK could phosphorylate a wide range of target proteins, including other kinases and/or transcription factors[44, 57], in particular SpERF of Spipo0G0155100, Spipo3G0031800 and Spipo20G0027700, SpABF of Spipo4G0008600 and Spipo2G0055800, SpHSF of Spipo8G0037600 and Spipo9G0002000, and SpWRKY of Spipo8G0045500 (Table 5). The activation of TFs ultimately regulates their target genes to cease cell division but begin to accumulate secondary metabolites. As shown in flowering seeds, aspects of reserve accumulation and late embryogenesis abundant (LEA) gene expression are controlled largely by the coordinated action of transcription factors[44]. Taken together, we generated a model summarizing the signal transduction leading to Spirodela dormancy based on integration of our result and previous knowledge (FIG. 6).
CONCLUSIONS
Many studies have been concerned with seed development in plants. Seeds are the product of sexual reproduction and the segregation of Mendelian traits. They also represent a dormant state in the life cycle of the plant and they compartmentalize nutrients for growth in the absence of photosynthesis. Agriculture could not exist without these properties of plants. Here, we studied a plant that propagates by clonal division and can undergo dormancy without forming seeds. The aquatic plant Spirodela could not survive on water surface without human intervention, when the water freezes. It simply switches to dormancy and accumulates starch that allows it to sink to the bottom of the water to escape the ice. Besides low temperature, however, the same switch can be achieved with the hormone ABA that has been shown to perform the same change for seed maturation. Using such an induction with Spirodela, we can study genes that regulate dormancy. Here, we isolated total RNA exclude ribosomal RNA before and at the onset of dormancy, sequenced them with next-generation technology, and identified the transcripts by mapping them back to the genome sequence. The detailed analysis of the transcriptional landscape of differentially expressed genes provides the first comprehensive view at the dormancy of aquatic plants. On the other hand, research studies have been initiated with the goal of developing duckweed species as an alternative to algae for oil production with the fact of fast growth and quick biomass accumulation[70]. The expression data for lipid and starch biosynthesis together with the turion-specific transcriptional genes from our RNA-Seq data would be the ideal targets to develop duckweeds into oil crops.







TABLE 1







Summary of sequence read alignments to three genome


references.
















Reads #
Map







Qualified
map nuclear
nuclear
Nuclear
Map
Map
Map


Sample
total reads
genome
genome
coverage
chloroplast
mitochondria
rDNA

















fronds 1
24,356,014
12,795,916
53%
42
35%
1%
4%


fronds 2
41,310,111
22,039,845
53%
72
37%
3%
4%


fronds 3
28,333,911
16,444,539
58%
54
29%
2%
6%


fronds 4
28,188,669
16,282,775
58%
53
30%
2%
9%


turions 1
26,484,522
15,431,023
58%
50
28%
2%
1%


turions 2
28,466,211
16,123,639
57%
53
34%
2%
2%


turions 3
25,754,050
15,697,393
61%
51
26%
2%
3%


turions 4
14,996,833
8,824,987
59%
29
29%
2%
1%
















TABLE 2







Fold change in differentially expressed genes between in fronds and


developing turions at FDR <0.01.









Fold change














4.0-5.0
5.1-10
10.1-15
15.1-20
>20
Sum





Genes expressed
37
73
12
10
22
154


lower in turions








than fronds








Genes expressed
38
97
25
15
33
208


higher in turions








than fronds
















TABLE 3







FPKM for Up-regulated DE genes in response to ABA stimulus.












Fold
Frond
Turion



Gene ID
change
FPKM
FPKM
Annotation














Spipo6G0001100
146
0.3
45.3
Peripheral-type benzodiazepine receptor


Spipo5G0029200
57
0.8
48.1
Major facilitator superfamiiy protein


Spipo19G0014500
43
0.5
22.4
Galactinol synthase


Spipo26G0007700
17
8.4
140.0
Late embryogenesis abundant protein LEA


Spipo8G0058900
16
1.2
19.4
Flowering locus T/Terminal flower 1-like protein


Spipo4G0016300
15
15.2
235.2
Annexin


Spipo18G0029800
15
28.7
420.3
O-acetyltransferase-like


Spipo3G0078900
14
0.4
6.2
Stachyose synthase, putative


Spipo0G0155100
13
1.7
21.5
Ethylene-responsive transcription factor 1


Spipo0G0130700
9
1.6
15.5
C4-dicarboxylate transporter


Spipo7G0041900
7
1.4
9.8
ABC transporter G family member


Spipo3G0031800
7
10.3
74.2
Ethylene-responsive transcription factor 2


Spipo8G0062500
6
7.2
44.3
Receptor-like protein kinase


Spipo14G0026800
5
4.0
18.3
Eukaryotic aspartyl protease family protein


Spipo12G0003900
4
37.9
162.9
myb domain protein 73


Spipo5G0040500
8
23.1
189.7
Alpha-dioxygenase


Spipo0G0156500
6
15.5
97.9
Alpha-dioxygenase


Spipo0G0180000
6
98.0
561.3
Alpha-dioxygenase


Spipo0G0156600
5
19.5
104.4
Prostaglandin G/H synthase


Spipo8G0046200
67
0.2
15.6
Protein phosphatase 2c, putative


Spipo3G0013100
38
0.5
20.2
NAC domain-containing protein 67


Spipo23G0012800
32
1.1
34.2
Protein phosphatase 2C


Spipo21G0022300
10
6.1
59.1
Protein phosphatase 2c, putative


Spipo1G0021700
6
3.2
18.9
Protein phosphatase 2c, putative


Spipo6G0056800
4
11.5
46.5
NAC domain-containing protein 67
















TABLE 4







FPKM for Down-regulated DE genes associated with Spirodela growth.












Fold
Frond
Turion



Gene ID
change
FPKM
FPKM
Annotation














Spipo11G0033800
69
33.6
0.5
Aquaporin


Spipo17G0045100
5
86.3
17.8
Aquaporin


Spipo22G0025300
5
186.4
40.4
Expansin


Spipo9G0039400
7
68.2
9.4
Histone H3


Spipo0G0046100
7
112.4
16.4
Histone H3


Spipo13G0007500
6
159.5
27.5
Histone H3


Spipo28G0019000
5
77.9
14.8
Histone H4


Spipo3G0024800
14
1018.4
71.2
Pre-rRNA-processing






protein PNO1


Spipo1G0126300
5
1371.4
293.1
60S ribosomal protein






L10-like protein


Spipo19G0027700
29
6951.7
241.3
Ribulose bisphosphate






carboxylase small






chain


Spipo23G0013400
5
476.1
93.1
Ribulose-1 5-






bisphosphate






carboxylase/oxygenase






activase
















TABLE 5







FPKM for Turion-specific genes and DE transcriptional factors.













Fold
Frond
Turion




Gene ID
change
FPKM
FPKM
Directionality
Annotation















Spipo14G0001200
NA
0.0
31.0
up-regulated
Late embryogenesis abundant protein LEA


Spipo5G0015500
NA
0.0
45.8
up-regulated
Late embryogenesis abundant protein LEA


Spipo0G0166800
170
1.4
235.2
up-regulated
Late embryogenesis abundant protein LEA


Spipo1G0033500
34
3.4
114.8
up-regulated
Late embryogenesis abundant protein LEA


Spipo26G0007700
17
8.4
140.0
up-regulated
Late embryogenesis abundant protein LEA


Spipo4G0008600
5
6.1
33.1
up-regulated
bZIP transcription factor A


Spipo8G0037600
11
1.1
11.6
up-regulated
Heat shock transcription factor A2


Spipo9G0002000
5
14.2
67.8
up-regulated
Heat shock transcription factor A2


Spipo0G0155100
13
1.7
21.5
up-regulated
Ethylene-responsive transcription factor 1


Spipo3G0031800
7
10.3
74.2
up-regulated
Ethylene-responsive transcription factor 2


Spipo20G0027700
5
10.6
53.1
up-regulated
Ethylene-responsive transcription factor 3


Spipo11G0028200
7
32.7
4.4
down-regulated
Ethylene-responsive transcription factor 4


Spipo8G0045500
7
11.3
1.7
down-regulated
WRKY transcription factor, putative


Spipo2G0055800
4
17.1
4.0
down-regulated
bZIP transcription factor I
















TABLE 6







Functional GO enrichment in developing turions.










Enriched GO ID
description






GO: 0001561
fatty acid alpha-oxidation



GO: 0033539
fatty acid beta-oxidation using acyl-CoA




dehydrogenase



GO: 0010167
response to nitrate



GO: 0015706
nitrate transport



GO: 0055114
oxidation-reduction process



GO: 0009830
cell wall modification involved in abscission



GO: 0009651
response to salt stress



GO: 0010106
cellular response to iron ion starvation



GO: 0010150
leaf senescence



GO: 0009737
response to abscisic acid stimulus



GO: 0006826
iron ion transport



GO: 0001676
long-chain fatty acid metabolic process



GO: 0001666
response to hypoxia



GO: 0046487
glyoxylate metabolic process



GO: 0071732
cellular response to nitric oxide



GO: 0010286
heat acclimation



GO: 0071446
cellular response to salicylic acid stimulus



GO: 0072329
monocarboxylic acid catabolic process



GO: 0019579
aldaric acid catabolic process



GO: 0009751
response to salicylic acid stimulus



GO: 0042542
response to hydrogen peroxide



GO: 0046686
response to cadmium ion



GO: 0009788
negative regulation of abscisic acid mediated




signaling pathway



GO: 0009414
response to water deprivation









Abbreviations
ABA: abscisic acid; FPKM: Fragments Per Kilobase of transcript per Million mapped reads; DE gene: differentially expressed genes; LEA: late embryogenesis abundant protein; ABF: ABA-responsive element binding factors; ERF: Ethylene-responsive element binding factors; CPK: calcium-dependent protein kinase; MAPK: mitogen-activated protein kinase; CCR: Cinnamoyl-CoA reductase; CAD: cinnamyl-alcohol dehydrogenase; APS: ADP-glucose pyrophosphorylase small subunit; APL: ADP-glucose pyrophosphorylase large subunit; SS: starch synthase; GBSS: Granule-bound starch synthase; BE: Starch branching enzyme; DBE: Starchdebranching enzyme; ACCase: Acetyl-CoA carboxylase; GPAT: Glycerol-3-phosphate acyltransferase; AGPAT: Acylglycerophosphateacyltransferase; DGAT: Diacylglycerolacyltransferase.
















TABLE S1






Fold
Frond
Turion
Direction-







Gene ID
change
FPKM
FPKM
ality
Annotation
ABRE
ERE
GO
Function
























Spipo14G0001200
NA
0.0
31.0
up-regulated
Late embryogenesis abundant
NO
YES









protein LEA






Spipo5G0015500
NA
0.0
45.8
up-regulated
Late embryogenesis abundant
YES
YES









protein LEA






Spipo0G0166800
170
1.4
235.2
up-regulated
Late embryogenesis abundant
NO
NO









protein LEA






Spipo6G0001100
146
0.3
45.3
up-regulated
Peripheral-type benzodiazepine
NO
YES
GO: 0009737
response to







receptor



abscisic acid











stimulus


Spipo10G0013800
99
0.1
9.5
up-regulated
70 kDa heat shock protein
NO
YES




Spipo21G0043200
97
0.7
66.3
up-regulated
Protein of unknown function,
NO
YES









DUF599






Spipo25G0008400
95
0.2
18.0
up-regulated
myb domain protein 73
NO
NO




Spipo2G0010600
84
0.2
19.7
up-regulated
UDP-Glycosyltransferase
NO
NO









superfamily






Spipo2G0035100
72
0.3
18.2
up-regulated
Unknown protein
NO
YES




Spipo1G0017700
71
0.5
34.3
up-regulated
(R)-limonene synthase, putative
NO
NO




Spipo8G0046200
67
0.2
15.6
up-regulated
Protein phosphatase 2c, putative
YES
YES
GO: 0009788
negative











regulation of











abscisic acid











mediated











signaling











pathway


Spipo13G0026700
58
4.9
285.5
up-regulated
Sulfite oxidase
NO
NO




Spipo5G0029200
57
0.8
48.1
up-regulated
Major facilitator superfamily protein
NO
NO
GO: 0009737
response to











abscisic acid











stimulus


Spipo2G0043800
54
6.4
340.8
up-regulated
UDP-Glycosyltransferase superfamily
NO
YES




Spipo16G0044000
52
0.2
8.8
up-regulated
UDP-Glycosyltransferase superfamily
NO
YES




Spipo11G0050900
49
20.2
986.4
up-regulated
Kunitz trypsin inhibitor
NO
YES




Spipo19G0014500
43
0.5
22.4
up-regulated
Galactinol synthase
NO
NO
GO: 0009737
response to











abscisic acid











stimulus


Spipo4G0003000
42
0.5
22.8
up-regulated
Glutamate decarboxylase, putative
NO
YES




Spipo25G0001000
42
1.0
42.4
up-regulated
Exostosin family protein
NO
YES




Spipo14G0016400
41
0.8
33.7
up-regulated
Lachrymatory-factor synthase
NO
NO




Spipo3G0013100
38
0.5
20.2
up-regulated
NAC domain-containing protein 67
NO
NO
GO: 0009788
negative











regulation of











abscisic acid











mediated











signaling











pathway


Spipo2G0039000
38
0.4
13.6
up-regulated
UDP-Glycosyltransferase superfamily
NO
NO









protein






Spipo1G0033500
34
3.4
114.8
up-regulated
Late embryogenesis abundant
NO
YES









protein LEA






Spipo15G0020300
34
1.5
52.2
up-regulated
Cytochrome P450
NO
NO




Spipo12G0048100
33
0.0
1.6
up-regulated
Pentatricopeptide repeat-containing
NO
YES









protein, putative






Spipo10G0053200
33
0.1
3.6
up-regulated
Sucrose phosphate synthase
NO
NO




Spipo23G0012800
32
1.1
34.2
up-regulated
Protein phosphatase 2C
NO
YES
GO: 0009788
negative











regulation of











abscisic acid











mediated











signaling











pathway


Spipo30G0012600
31
2.8
89.1
up-regulated
Unknown protein
NO
YES




Spipo9G0021500
29
0.7
19.2
up-regulated
Nucleotide-sugar transporter family
YES
NO









protein






Spipo12G0044900
29
0.4
11.1
up-regulated
CBL-interacting protein kinase 9
YES
YES




Spipo0G0148600
27
23.1
635.9
up-regulated
Unknown protein
NO
NO




Spipo15G0047700
27
0.1
1.6
up-regulated
Cytochrome P450
NO
YES




Spipo0G0175800
2.1
1.4
28.2
up-regulated
EC metallothionein-like protein
NO
YES




Spipo0G0138500
19
10.6
206.5
up-regulated
Unknown protein
YES
YES




Spipo4G0033500
19
0.1
1.0
up-regulated
Pentatricopeptide repeat-containing
NO
NO









protein, putative






Spipo2G0012200
19
0.4
7.8
up-regulated
Glutamate dehydrogenase
NO
YES




Spipo4G0112100
19
1.6
31.2
up-reglated
9-cis-epoxycarotenoid dioxygenase 1
NO
NO




Spipo1G0101000
18
0.1
1.6
up-regulated
Pentatricopeptide repeat-containing
NO
YES









protein






Spipo21G0039100
18
12.7
232.4
up-regulated
Unknown protein
NO
NO




Spipo26G0007700
17
8.4
140.0
up-regulated
Late embryogenesis abundant
YES
YES
GO: 0009737
response to







protein LEA



abscisic











acid











stimulus


Spipo10G0023800
17
0.8
13.8
up-regulated
Class 1 heat shock protein
NO
NO




Spipo14G0034300
16
3.9
62.6
up-regulated
UDP glycosyltransferase
YES
NO




Spipo9G0006300
16
2.1
32.6
up-regulated
Phosphoserine aminotransferase
NO
NO




Spipo8G0058900
16
1.2
19.4
up-regulated
Flowering locus T/Terminal flower
NO
YES
GO: 0009737
response to







1-like protein



abscisic acid











stimulus


Spipo1G0068300
15
8.6
133.2
up-regulated
Heavy metal transport/detoxification
YES
YES









superfamily protein






Spipo4G0016300
15
15.2
235.2
up-regulated
Annexin
NO
NO
GO: 0009737
response to











abscisic acid











stimulus


Spipo2G0064500
15
3.6
56.0
up-regulated
Cytochrome P450
NO
YES




Spipo6G0047600
15
0.7
10.1
up-regulated
Beta glucosidase like protein
NO
NO




Spipo28G0015900
15
0.1
1.6
up-regulated
Trehalose-6-phosphate synthase
NO
YES




Spipo18G0029800
15
28.7
420.3
up-regulated
O-acetyltransferase-like
NO
NO
GO: 0009737
response to











abscisic acid











stimulus


Spipo1G0076000
14
0.6
8.6
up-regulated
Putative lysine decarboxylase family
NO
NO









protein






Spipo8G0061900
14
4.6
64.7
up-regulated
LOB domain-containing protein,
YES
YES









putative






Spipo11G0038800
14
0.9
13.4
up-regulated
Arabidopsis protein of unknown
NO
YES









function (DUF241)






Spipo3G0078900
14
0.4
6.2
up-regulated
Stachyose synthase, putative
YES
NO
GO: 0009737
response to











abscisic acid











stimulus


Spipo18G0020700
14
10.6
143.8
up-regulated
Formate dehydrogenase
NO
NO




Spipo4G0080100
13
0.2
2.3
up-regulated
Cytochrome P450, putative
NO
YES




Spipo0G0155100
13
1.7
21.5
up-regulated
Ethylene-responsive transcription
YES
YES
GO: 0009737
response to







factor 1



abscisic











acid











stimulus


Spipo28G0001000
13
3.3
42.3
up-regulated
Phosphofructokinase, putative
NO
YES




Spipo15G0037300
13
3.8
48.6
up-regulated
Mitogen-activated protein kinase
NO
YES









kinase 1 (MAPK)






Spipo2G0067900
12
0.3
4.0
up-regulated
Heat shock 70 kDa protein 1
NO
YES




Spipo26G0000300
12
6.6
79.8
up-regulated
unknown protein
NO
YES




Spipo2G0124000
12
0.6
7.7
up-regulated
UDP-Glycosyltransferase superfamily
NO
NO









protein






Spipo4G0079600
12
30.7
364.1
up-regulated
12-oxophytodienoate reductase
NO
YES




Spipo2G0096800
12
13.9
160.7
up-regulated
unknown protein
NO
YES




Spipo1G0074600
11
22.6
257.1
up-regulated
Unknown protein
NO
YES




Spipo7G0045000
11
12.2
137.5
up-regulated
Sucrose phosphate synthase
NO
YES




Spipo8G0037600
11
1.1
11.6
up-regulated
heat shock transcription factor A2
NO
YES




Spipo21G0039300
11
3.2
35.0
up-regulated
Unknown protein
NO
NO




Spipo5G0014300
11
3.4
36.1
up-regulated
UDP-Glycosyltransferase superfamily
NO
YES









protein






Spipo28G0003500
11
30.5
322.2
up-regulated
Gibberellin-regulated protein
NO
YES




Spipo1G0058300
10
0.1
1.0
up-regulated
Pentatricopeptide repeat-containing
NO
YES









protein






Spipo3G0032200
10
10.4
105.2
up-regulated
Epoxide hydrolase 1
NO
YES




Spipo0G0116100
10
6.2
62.5
up-regulated
Beta-glucosidase, putative
NO
YES




Spipo4G0092300
10
45.1
447.5
up-regulated
Alcohol dehydrogenase
NO
YES




Spipo21G0022300
10
6.1
59.1
up-regulated
Protein phosphatase 2c, putative
NO
YES
GO: 0009788
negative











regulation of











abscisic acid











mediated











signaling











pathway


Spipo19G0036000
10
1.2
11.2
up-regulated
Aspartic proteinase, putative
NO
NO




Spipo19G0016500
10
2.8
27.3
up-regulated
Isocitrate dehydrogenase [NADP]
NO
NO




Spipo0G0130700
9
1.6
15.5
up-regulated
C4-dicarboxylate transporter/malic
NO
NO
GO: 0009737
response to







acid transport protein



abscisic acid











stimulus


Spipo7G0066600
9
164.7
1539.8
up-regulated
Glyceraldehyde-3-phosphate
YES
YES









dehydrogenase






Spipo22G0037200
9
18.8
173.7
up-regulated
Amino acid selective channel protein
NO
NO




Spipo6G0047400
9
5.3
47.4
up-regulated
Universal stress protein
NO
NO




Spipo25G0008100
9
1.5
13.5
up-regulated
Alpha-mannosidase-like protein
NO
YES




Spipo5G0050700
9
0.6
5.3
up-regulated
Sugar phosphate exchanger 2
NO
NO




Spipo24G0000900
9
0.3
2.9
up-regulated
Kinase, putative
NO
NO




Spipo2G0047500
8
17.8
148.0
up-regulated
SAUR-like auxin-responsive protein
YES
YES









family






Spipo11G0001500
8
22.3
185.3
up-regulated
Abscisic acid 8′-hydroxylase 3
NO
NO




Spipo5G0064900
8
10.7
88.2
up-regulated
Tetratricopeptide repeat (TPR)-like
NO
YES









superfamily protein






Spipo5G0040500
8
23.1
189.7
up-regulated
Alpha-dioxygenase
NO
NO
GO: 0009737;
response to










GO: 0001561
abscisic acid











stimulus;











fatty











acid alpha-











oxidation


Spipo27G0011300
8
13.8
112.1
up-regulated
Beta-glucosidase A
NO
NO
GO: 0019252
starch











biosynthetic











process


Spipo1G0069600
8
32.9
266.4
up-regulated
26S proteasome regulatory subunit
NO
YES









S3, putative






Spipo11G0031500
8
3.9
31.8
up-regulated
Cytochrome P450, putative
NO
NO




Spipo2G0040400
8
0.8
6.1
up-regulated
Cytochrome P450
YES
YES




Spipo11G0039200
8
5.8
45.4
up-regulated
Alpha-amylase, putative
NO
YES




Spipo3G0041300
8
0.2
1.3
up-regulated
Pentatricopeptide repeat-containing
NO
YES









protein






Spipo14G0010200
8
1.8
13.9
up-regulated
Protein CHUP1
NO
YES




Spipo11G0060400
8
1.5
11.7
up-regulated
Folate/biopterin transporter family
NO
YES









protein






Spipo5G0023500
8
6.1
47.4
up-regulated
Protein of unknown function
NO
NO









(DUF1262)






Spipo1G0032700
8
11.8
91.9
up-regulated
2-succinylbenzoate-CoA ligase
NO
NO




Spipo16G0027000
8
4.4
33.4
up-regulated
Glutathione-S-transferase (GST)
NO
YES
GO: 0019252
starch











biosynthetic











process


Spipo0G0176700
8
0.6
4.9
up-regulated
Ovate family protein 1
NO
YES




Spipo18G0038500
8
36.0
291.5
up-regulated
Glucose-1-phosphate
NO
NO
GO: 0019252
starch







adenylyltransferase



biosynthetic











process


Spipo11G0025200
7
2.1
15.6
up-regulated
methionine gamma-lyase
NO
YES




Spipo13G0053800
7
6.9
51.6
up-regulated
Glyceraldehyde 3-phosphate
YES
NO









dehydrogenase






Spipo5G0077000
7
0.4
2.7
up-regulated
Pentatricopeptide repeat-containing
NO
NO









protein






Spipo23G0007500
7
1.5
10.9
up-regulated
O-fucosyltransferase family protein
NO
NO




Spipo5G0014900
7
1.0
7.1
up-regulated
Cytochrome P450, putative
YES
YES




Spipo29G0009400
7
3.4
24.9
up-regulated
Dof zinc finger protein
NO
NO




Spipo7G0041900
7
1.4
9.8
up-regulated
ABC transporter G family member
NO
YES
GO: 0009737
response to











abscisic acid











stimulus


Spipo3G0031800
7
10.3
74.2
up-regulated
Ethylene-responsive transcription
NO
NO
GO: 0009737
response to







factor 2



abscisic











acid











stimulus


Spipo21G0009000
7
4.7
33.4
up-regulated
Auxin responsive protein
YES
YES




Spipo0G0124700
7
2.0
14.1
up-regulated
G-type lectin S-receptor-like
NO
YES









serine/threonine-protein kinase






Spipo19G0037600
7
21.2
144.8
up-regulated
Hydroxyacylglutathione hydrolase
NO
YES




Spipo3G0073100
7
14.3
96.6
up-regulated
Peroxiredoxin
NO
YES




Spipo22G0042300
7
24.8
166.4
up-regulated
Soul heme-binding family protein
NO
YES




Spipo10G0025700
7
1.1
7.6
up-regulated
Histidine decarboxylase
NO
NO




Spipo7G0061700
7
140.8
936.4
up-regulated
Cysteine-rich secretory protein
NO
YES




Spipo18G0031500
7
2.8
18.8
up-regulated
LOB domain-containing protein 41
NO
YES




Spipo8G0027700
7
1.4
8.9
up-regulated
MATE efflux family protein
YES
NO




Spipo28G0017100
7
1.6
10.6
up-regulated
Cellulose-synthase-like C5
NO
YES




Spipo0G0155000
6
1.5
9.8
up-regulated
Cytochrome P450 flavonoid 3′,5′-
NO
NO









hydroxylase






Spipo7G0010700
6
10.1
64.8
up-regulated
Dihydroflavonol 4-reductase
NO
YES




Spipo10G0000200
6
26.2
167.8
up-regulated
Dihydroflavonol 4-reductase
NO
NO




Spipo15G0031400
6
3.3
21.1
up-regulated
Major facilitator superfamily protein
NO
YES




Spipo0G0156500
6
15.5
97.9
up-regulated
Alpha-dioxygenase
NO
NO
GO: 0009737;
response to










GO: 0001561
abscisic acid











stimulus;











fatty











acid alpha-











oxidation


Spipo1G0110400
6
5.8
36.8
up-regulated
Electron transfer flavoprotein-
NO
NO
GO: 0033539
fatty acid







ubiquinone oxidoreductase



beta-











oxidation


Spipo6G0053900
6
6.5
39.9
up-regulated
Major facilitator superfamily
NO
NO









antiporter, putative, expressed






Spipo8G0062500
6
7.2
44.3
up-regulated
Receptor-like protein kinase
NO
YES
GO: 0009737
response to











abscisic acid











stimulus


Spipo14G0009000
6
1.7
10.6
up-regulated
Major facilitator superfamily protein
NO
NO




Spipo15G0028800
6
2.5
15.1
up-regulated
Cysteine-rich receptor-like protein
NO
YES









kinase






Spipo28G0011200
6
2.3
13.5
up-regulated
Chaperone clpb, putative
NO
NO




Spipo5G0051100
6
7.8
46.5
up-regulated
Cytochrome P450, putative
NO
YES




Spipo11G0020200
6
116.8
695.6
up-regulated
Kunitz trypsin inhibitor 4
NO
NO




Spipo1G0021700
6
3.2
18.9
up-regulated
Protein phosphatase 2c, putative
NO
YES
GO: 0009788
negative











regulation of











abscisic acid











mediated











signaling











pathway


Spipo7G0059600
6
28.6
168.7
up-regulated
AP-1 complex subunit gamma-2,
NO
NO









putative






Spipo3G0031300
6
16.3
95.9
up-regulated
Acyl-CoA dehydrogenase
NO
YES
GO: 0033539
fatty acid











beta-











oxidation


Spipo26G0019100
6
3.1
17.7
up-regulated
Integral membrane protein TmpA
NO
YES




Spipo8G0065700
6
1.2
6.9
up-regulated
Core-2/I-branching beta-1,6-N-
NO
NO









acetylglucosaminyltransferase family











protein






Spipo17G0009300
6
1.5
8.7
up-regulated
CBS domain-containing protein,
YES
YES









putative, expressed






Spipo10G0038500
6
2.1
12.1
up-regulated
Phosphatidylinositol kinase family-
NO
YES









like protein






Spipo0G0180000
6
98.0
561.3
up-regulated
Alpha-dioxygenase
NO
NO
GO: 0009737;
response to










GO: 0001561
abscisic acid











stimulus;











fatty











acid alpha-











oxidation


Spipo24G0001200
6
5.6
31.8
up-regulated
Adiponectin receptor protein
NO
NO




Spipo10G0030200
6
11.2
63.7
up-regulated
Chaperone DnaJ-domain superfamily
YES
YES









protein






Spipo8G0040300
6
1.9
10.8
up-regulated
3-hydroxyisobutyrate dehydrogenase
NO
NO




Spipo23G0017700
6
1.6
9.0
up-regulated
GTPaseobg
NO
NO




Spipo0G0148500
6
34.3
192.2
up-regulated
Unknown protein
NO
YES




Spipo16G0046000
5
17.7
96.7
up-regulated
Beta-amylase
YES
YES




Spipo3G0073400
5
5.0
27.4
up-regulated
tolB protein-related
NO
NO




Spipo4G0008600
5
6.1
33.1
up-regulated
bZIP transcription factor A
NO
NO




Spipo3G0064800
5
14.2
76.7
up-regulated
Beta-1,3-glucanase
NO
NO




Spipo0G0156600
5
19.5
104.4
up-regulated
Prostaglandin G/H synthase
NO
NO
GO: 0009737;
response to










GO: 0001561
abscisic acid











stimulus;











fatty











acid alpha-











oxidation


Spipo11G0043800
5
10.3
54.6
up-regulated
Calcium-binding EF hand family
NO
YES









protein






Spipo23G0006300
5
7.3
38.4
up-regulated
RNA-binding protein
YES
YES




Spipo12G0062400
5
2.8
14.6
up-regulated
Glycogen debranching enzyme
NO
YES
GO: 0019252
starch











biosynthetic











process


Spipo18G0028400
5
0.8
3.9
up-regulated
NHL domain-containing protein
NO
YES




Spipo7G0060200
5
8.5
44.3
up-regulated
Unknown protein
NO
YES




Spipo10G0017900
5
5.8
30.4
up-regulated
ABC transporter G family member
NO
YES




Spipo2G0099700
5
1.4
7.5
up-regulated
unknown protein
NO
NO




Spipo11G0031600
5
2.9
15.1
up-regulated
Cytochrome P450
NO
NO




Spipo16G0030600
5
4.1
21.5
up-regulated
Glutamine synthetase
NO
NO




Spipo2G0061100
5
3.4
17.5
up-regulated
Prolyl 4-hydroxylase alpha subunit,
NO
YES









putative






Spipo20G0017000
5
4.5
22.9
up-regulated
Isoflavonereductase-like protein
NO
NO




Spipo4G0013100
5
55.2
282.1
up-regulated
Inositol-3-phosphate synthase
NO
YES




Spipo4G0033200
5
18.5
94.4
up-regulated
MtN19-like protein
NO
YES




Spipo14G0054900
5
2.0
9.9
up-regulated
Dihydroflavonol 4-reductase
NO
YES




Spipo2G0071700
5
0.4
1.9
up-regulated
Pentatricopeptide repeat-containing
NO
YES









protein, putative






Spipo0G0030300
5
47.3
238.4
up-regulated
Aldehyde dehydrogenase
YES
YES




Spipo3G0002000
5
1.6
7.8
up-regulated
Xanthine/uracil permease family
YES
YES









protein






Spipo20G0027700
5
10.6
53.1
up-regulated
Ethylene-responsive transcription
NO
NO









factor 3






Spipo7G0013600
5
8.2
40.9
up-regulated
2-oxoglutarate (2OG) and Fe(II)-
NO
NO









dependent oxygenase superfamily






Spipo0G0013700
5
2.4
12.0
up-regulated
Unknown protein
YES
YES




Spipo2G0018300
5
9.6
47.7
up-regulated
Early light-induced protein
NO
NO




Spipo7G0048800
5
16.7
82.8
up-regulated
NAC domain protein
NO
YES




Spipo8G0040000
5
1.2
5.5
up-regulated
2-hydroxy-3-oxopropionate reductase
NO
YES




Spipo9G0002000
5
14.2
67.8
up-regulated
Heat shock transcription factor A-2
NO
NO




Spipo9G0033600
5
123.3
585.4
up-regulated
Fructose-bisphosphatealdolase
NO
YES




Spipo23G0001600
5
3.2
15.2
up-regulated
Kinase, pfkB family protein,
NO
YES









expressed






Spipo7G0044000
5
4.4
20.5
up-regulated
Cellulose synthase-like B3
YES
YES




Spipo6G0053800
5
3.7
17.3
up-regulated
Major facilitator superfamily
NO
NO









antiporter, putative, expressed






Spipo2G0077900
5
6.1
28.4
up-regulated
UDP-Glycosyltransferase superfamily
NO
NO




Spipo14G0026800
5
4.0
18.3
up-regulated
Eukaryotic aspartyl protease family
NO
YES
GO: 0009737
response to







protein



abscisic acid











stimulus


Spipo0G0179100
5
11.5
53.2
up-regulated
Electron transfer flavoprotein-
NO
YES
GO: 0033539
fatty acid







ubiquinone oxidoreductase



beta-











oxidation


Spipo1G0003700
5
7.6
35.1
up-regulated
Calcium-dependent protein kinase
YES
NO









(CPK)






Spipo15G0048400
5
7.7
34.9
up-regulated
Cathepsin L-like cysteine proteinase
NO
YES




Spipo0G0154800
5
4.4
19.7
up-regulated
Xanthine dehydrogenase/oxidase
NO
YES




Spipo2G0033500
4
7.7
34.6
up-regulated
Lysine-
NO
NO









ketoglutaratereductase/saccharopine











dehydrogenase






Spipo4G0038800
4
3.9
17.3
up-regulated
GDSL esterase/lipase
NO
NO




Spipo13G0039800
4
16.2
70.6
up-regulated
Cytochrome P450
NO
NO




Spipo3G0073500
4
2.9
12.4
up-regulated
tolB protein-related
NO
NO




Spipo7G0066500
4
1.6
6.9
up-regulated
Multidrug resistance protein ABC
YES
YES









transporter family






Spipo24G0015900
4
2.8
12.2
up-regulated
Unknown protein
NO
NO




Spipo9G0002200
4
105.3
452.4
up-regulated
Thioredoxin
NO
YES




Spipo12G0003900
4
37.9
162.9
up-regulated
Myb domain protein 73
NO
YES
GO: 0009737
response to











abscisic acid











stimulus


Spipo2G0096700
4
55.9
238.4
up-regulated
Peroxidase 73, putative
NO
YES




Spipo26G0017500
4
5.7
24.1
up-regulated
Exostosin family protein
NO
YES




Spipo7G0028500
4
23.1
97.9
up-regulated
Potassium transporter 11
NO
NO




Spipo5G0012100
4
17.9
75.8
up-regulated
CCR4-NOT transcription complex
NO
YES









subunit 7






Spipo19G0030800
4
6.0
25.1
up-regulated
Inositol-pentakisphosphate 2-kinase,
NO
NO









putative






Spipo20G0012400
4
1.5
6.2
up-regulated
RING/U-box superfamily protein
NO
YES




Spipo23G0021500
4
5.4
22.8
up-regulated
SEC14 cytosolic factor family
NO
YES









protein/phosphoglyceride transfer











family protein






Spipo22G0012700
4
31.1
130.2
up-regulated
Rubber elongation factor protein
NO
YES









(REF)






Spipo18G0007900
4
70.2
289.4
up-regulated
Aspartate aminotransferase
NO
YES




Spipo0G0085700
4
14.1
57.9
up-regulated
Unknown protein
NO
NO




Spipo16G0033600
4
2.5
10.2
up-regulated
Alpha-galactosidase 1
YES
NO




Spipo3G0022400
4
11.3
46.0
up-regulated
Glutathione-S-transferase (GST)
NO
YES




Spipo6G0056800
4
11.5
46.5
up-regulated
NAC domain-containing protein 67
YES
YES
GO: 0009788
negative











regulation of











abscisic acid











mediated











signaling











pathway


Spipo3G0033300
4
8.9
35.9
up-regulated
Protein of unknown function,
NO
NO









DUF538






Spipo5G0073900
4
9.2
37.0
up-regulated
Alternative oxidase
NO
YES




Spipo12G0023200
4
0.5
1.9
up-regulated
Disease resistance protein
NO
NO




Spipo11G0033800
69
33.6
0.5
down-
Aquaporin
NO
NO








regulated







Spipo17G0047900
66
####
1921.3
down-
Chitobiosyldiphosphodolichol beta-
YES
YES








regulated
mannosyltransferase






Spipo1G0071900
54
####
1538.8
down-
Chitobiosyldiphosphodolichol beta-
NO
YES








regulated
mannosyltransferase






Spipo13G0047300
50
608.3
12.1
down-
Ycf68 protein
NO
NO








regulated







Spipo12G0026200
50
8.6
0.2
down-
Eukaryotic aspartyl protease family
YES
YES








regulated
protein






Spipo1G0120500
45
72.3
1.6
down-
Lectin
NO
YES








regulated







Spipo0G0129700
38
103.0
2.7
down-
Bifunctional inhibitor/lipid-transfer
NO
YES








regulated
protein/seed storage 2S albumin











superfamily






Spipo1G0120400
37
27.1
0.7
down-
Lectin
NO
NO








regulated







Spipo26G0006100
36
27.7
0.8
down-
BURP domain-containing protein
NO
NO








regulated







Spipo1G0108500
35
44.8
1.3
down-
Unknown protein
NO
NO








regulated







Spipo14G0020800
30
498.4
16.6
down-
Arabinogalactan peptide 20-like
YES
YES








regulated







Spipo19G0027700
29
####
241.3
down-
Ribulosebisphosphate carboxylase
NO
NO








regulated
small chain






Spipo21G0033600
27
####
487.3
down-
Oxidative stress 3
NO
NO








regulated







Spipo1G0121700
26
1530.8
59.8
down-
Bifunctional inhibitor/lipid-transfer
NO
NO








regulated
protein/seed storage 2S albumin











superfamily






Spipo23G0012000
25
6197.7
243.6
down-
Unknown protein
NO
YES








regulated







Spipo0G0022100
24
51.0
2.1
down-
Protein of unknown function,
NO
NO








regulated
DUF538






Spipo7G0031300
24
184.7
7.7
down-
Unknown protein
NO
NO








regulated







Spipo6G0029000
22
15.6
0.7
down-
O-acyltransferase WSD1
YES
NO








regulated







Spipo0G0067700
22
309.9
14.0
down-
Unknown protein
NO
YES








regulated







Spipo2G0110300
22
28.5
1.3
down-
Cysteine-rich secretory protein
NO
NO








regulated







Spipo1G0078600
21
19.1
0.9
down-
Peroxidase
NO
NO








regulated







Spipo12G0010100
20
7.7
0.4
down-
Nucleobaseascorbate transporter
NO
NO








regulated







Spipo18G0014800
20
95.2
4.9
down-
FASCICLIN-like arabinogalactan 6
NO
YES








regulated







Spipo6G0002600
19
503.1
26.1
down-
Unknown protein
NO
NO








regulated







Spipo3G0074100
19
11.9
0.6
down-
fucosyltransferase 1
NO
YES








regulated







Spipo5G0036600
19
1.7
0.1
down-
Receptor kinase
NO
YES








regulated







Spipo10G0007200
19
7698.9
414.6
down-
Unknown protein
NO
NO








regulated







Spipo11G0017100
18
19.8
1.1
down-
Blue copper protein
NO
YES








regulated







Spipo8G0067000
17
11.2
0.7
down-
Unknown protein
YES
YES








regulated







Spipo14G0059300
17
323.8
19.5
down-
Bifunctional inhibitor/lipid-transfer
NO
NO








regulated
protein/seed storage 2S albumin-like











protein






Spipo1G0007400
16
258.6
16.6
down-
Unknown protein
NO
NO








regulated







Spipo0G0159700
15
108.2
7.2
down-
BURP domain-containing protein
NO
NO








regulated







Spipo3G0024800
14
1018.4
71.2
down-
Pre-rRNA-processing protein PNO1
NO
NO








regulated







Spipo9G0045500
14
30.0
2.2
down-
Carboxyvinyl-
NO
NO








regulated
carboxyphosphonate-











phosphorylmutase,











putative, expressed






Spipo9G0031000
13
5.1
0.4
down-
Kinase, putative
NO
YES








regulated







Spipo5G0017800
13
1348.5
105.8
down-
Unknown protein
NO
NO








regulated







Spipo8G0062200
12
11.6
0.9
down-
Protein kinase family protein
NO
YES








regulated







Spipo0G0025800
12
5.8
0.5
down-
Leucine-rich repeat protein kinase-
NO
NO








regulated
like protein






Spipo26G0006600
12
68.2
5.9
down-
BURP domain-containing protein
YES
NO








regulated







Spipo20G0016500
11
64.9
5.8
down-
Plant cadmium resistance 2
NO
YES








regulated







Spipo7G0011900
11
33.1
3.1
down-
Early nodulin 20, putative
NO
NO








regulated







Spipo20G0037200
11
####
1037.5
down-
Unknown protein
NO
YES








regulated







Spipo30G0013100
10
3.4
0.3
down-
Protein kinase family protein
NO
YES








regulated







Spipo8G0009900
10
15.2
1.5
down-
Peroxidase 39
NO
NO








regulated







Spipo0G0134100
10
4.7
0.5
down-
Protein kinase family protein
NO
NO








regulated







Spipo2G0109900
10
141.9
14.4
down-
CAP (Cysteine-rich secretory proteins
NO
NO








regulated
and Pathogenesis-related 1 protein)






Spipo0G0190200
10
486.5
49.4
down-
Purple acid phosphatase 1
NO
NO








regulated







Spipo10G0029700
10
2.4
0.2
down-
Disease resistance gene homolog 9N
NO
NO








regulated







Spipo30G0015100
9
7.6
0.8
down-
9-cis-epoxycarotenoid dioxygenase 1
NO
YES








regulated







Spipo28G0025100
9
21.4
2.3
down-
Cation transport regulator-like protein
YES
YES








regulated







Spipo2G0091100
9
7.1
0.8
down-
Unknown protein
NO
YES








regulated







Spipo2G0100400
9
223.5
25.3
down-
Nucleotide-sugar transporter family
YES
YES








regulated
protein






Spipo2G0110400
9
218.2
24.7
down-
Cysteine-rich secretory protein
NO
YES








regulated







Spipo5G0027100
9
18.7
2.1
down-
Myb domain protein 73
YES
YES








regulated







Spipo1G0079200
9
14.9
1.7
down-
Peroxidase
NO
NO








regulated







Spipo14G0014700
9
####
1293.9
down-
Unknown protein
NO
YES








regulated







Spipo4G0110100
9
58.4
6.8
down-
Fructose-1,6-bisphosphatase class 1
YES
YES








regulated







Spipo19G0016700
8
1666.6
196.3
down-
Unknown protein
NO
YES








regulated







Spipo11G0063600
8
168.4
20.3
down-
Unknown protein
NO
YES








regulated







Spipo9G0030500
8
3.8
0.5
down-
Kinase, putative
NO
YES








regulated







Spipo14G0033900
8
5094.7
626.7
down-
Carbonic anhydrase
NO
YES








regulated







Spipo1G0023600
8
6.2
0.8
down-
HXXXD-type acyl-transferase family
NO
NO








regulated
protein






Spipo1G0018600
8
52.1
6.6
down-
RING finger and CHY zinc finger
NO
NO








regulated
domain-containing protein 1






Spipo3G0031100
8
47.7
6.1
down-
Pectinesterase
NO
NO








regulated







Spipo7G0045100
8
855.2
109.7
down-
Unknown protein
NO
NO








regulated







Spipo13G0020200
8
433.6
56.5
down-
Phosphoglycolate phosphatase
YES
YES








regulated







Spipo0G0001400
8
4.2
0.6
down-
Cationic amino acid transporter
NO
YES








regulated







Spipo20G0013200
8
117.3
15.6
down-
Acid phosphatase, putative, expressed
NO
YES








regulated







Spipo3G0049000
7
127.9
18.6
down-
Glucose-1-phosphate
NO
NO








regulated
adenylyltransferase






Spipo11G0028200
7
32.7
4.4
down-
Ethylene-responsive transcription
NO
YES








regulated
factor 4






Spipo8G0035200
7
71.3
9.6
down-
Putative nuclease HARBI1-like
NO
YES








regulated







Spipo0G0152100
7
15.2
2.1
down-
Beta-glucosidase-like glycosyl
NO
NO








regulated
hydrolase






Spipo3G0031200
7
5.9
0.8
down-
Endo-1,4-beta-glucanase
NO
NO








regulated







Spipo0G0000400
7
108.8
15.0
down-
Short-chain dehydrogenase/
YES
YES








regulated
reductase 2






Spipo9G0039400
7
68.2
9.4
down-
Histone H3
NO
NO








regulated







Spipo9G0045200
7
18.8
2.6
down-
Carboxyvinyl-
NO
YES








regulated
carboxyphosphonate-











phosphorylmutase






Spipo1G0125300
7
186.7
26.8
down-
Lectin
NO
NO








regulated







Spipo0G0046100
7
112.4
16.4
down-
Histone H3
YES
YES








regulated







Spipo4G0069000
7
23.5
3.5
down-
Tyrosine-rich hydroxyproline-rich
NO
NO








regulated
glycoprotein






Spipo10G0030500
7
7.2
1.1
down-
Subtilisin-like serine protease
NO
NO








regulated







Spipo8G0045500
7
11.3
1.7
down-
WRKY transcription factor, putative
NO
NO








regulated







Spipo18G0023200
7
13.3
2.0
down-
Naphthoate synthase, putative
NO
YES








regulated







Spipo0G0068600
7
168.0
25.6
down-
Fanconi anemia group I protein
NO
NO








regulated







Spipo32G0009000
7
83.6
12.8
down-
G-type lectin S-receptor-like
NO
YES








regulated
serine/threonine-protein kinase






Spipo4G0061000
6
10.4
1.6
down-
GDSL esterase/lipase
NO
NO








regulated







Spipo7G0024900
6
2151.8
336.5
down-
Auxin-repressed protein
NO
NO








regulated







Spipo14G0048100
6
229.3
36.3
down-
Aminomethyltransferase
NO
NO








regulated







Spipo14G0022500
6
4.7
0.7
down-
Disease resistance protein
NO
NO








regulated
(CC-NBS-LRR)






Spipo4G0004200
6
47.5
7.6
down-
Acid phosphatase, putative, expressed
NO
NO








regulated







Spipo29G0003800
6
24.4
3.9
down-
Heavy metal transport/detoxification
NO
YES








regulated
superfamily protein






Spipo20G0028500
6
15.7
2.6
down-
Sulfate transporter
NO
NO








regulated







Spipo9G0012700
6
246.1
40.5
down-
Adenylosuccinatesynthetase
NO
NO








regulated







Spipo3G0061200
6
2685.1
442.0
down-
Chlorophyll a/b-binding protein
YES
NO








regulated







Spipo3G0041200
6
65.9
11.0
down-
CASP-like protein
YES
YES








regulated







Spipo0G0035400
6
33.9
5.7
down-
Pectinesterase
NO
NO








regulated







Spipo2G0072200
6
38.3
6.4
down-
Protein of unknown function,
NO
NO








regulated
DUF642






Spipo3G0023000
6
14.9
2.6
down-
Cyclin B1
NO
YES








regulated







Spipo3G0026500
6
11.4
2.0
down-
Triacylglycerol lipase, putative
NO
YES








regulated







Spipo2G0093200
6
44.1
7.6
down-
Unknown protein
NO
NO








regulated







Spipo13G0007500
6
159.5
27.5
down-
Histone H3
YES
YES








regulated







Spipo12G0058200
6
2360.0
407.0
down-
Xyloglucanendotransglucosylase/
NO
NO








regulated
hydrolase






Spipo5G0067200
6
14.6
2.6
down-
Cellulose synthase-like protein
NO
YES








regulated







Spipo20G0004100
6
91.6
16.1
down-
Nucleoside diphosphate kinase
NO
NO








regulated







Spipo0G0015500
6
8.4
1.5
down-
Urea active transporter-like protein
NO
NO








regulated







Spipo29G0004700
6
10.5
1.9
down-
Cyclin B1
NO
YES








regulated







Spipo2G0022100
6
1774.3
315.0
down-
Dynein light chain 1 cytoplasmic-like
NO
YES








regulated
protein






Spipo8G0020200
6
651.1
117.2
down-
Thylakoid membrane phosphoprotein
NO
NO








regulated
14 kDa






Spipo3G0020200
5
21.1
3.8
down-
Alpha-L-arabinofuranosidase
YES
YES








regulated







Spipo1G0117200
5
117.9
21.5
down-
Laccase 11
NO
NO








regulated







Spipo8G0039700
5
22.8
4.3
down-
Glucan endo-1,3-beta-glucosidase,
NO
YES








regulated
putative






Spipo28G0019000
5
77.9
14.8
down-
Histone H4
NO
YES








regulated







Spipo2G0093000
5
50.4
9.6
down-
Unknown protein
NO
YES








regulated







Spipo28G0012500
5
328.4
64.0
down-
Chaperone protein dnaJ
YES
YES








regulated







Spipo0G0114100
5
259.2
50.5
down-
Protodermal factor 1.3
NO
YES








regulated







Spipo23G0013400
5
476.1
93.1
down-
Ribulose-1 5-bisphosphate
NO
YES








regulated
carboxylase/oxygenaseactivase






Spipo12G0016000
5
10.2
2.0
down-
O-fucosyltransferase family protein
NO
YES








regulated







Spipo26G0012100
5
894.9
176.9
down-
Arabinogalactan protein 20
NO
YES








regulated







Spipo31G0007300
5
3.4
0.7
down-
Leucine-rich repeat receptor-like
NO
NO








regulated
protein kinase family protein






Spipo8G0005400
5
14.9
3.0
down-
Glutamate receptor
NO
NO








regulated







Spipo1G0080800
5
2505.2
507.5
down-
Unknown protein
YES
NO








regulated







Spipo3G0012100
5
5.4
1.1
down-
Protein kinase-like protein
NO
NO








regulated







Spipo20G0028600
5
9.6
2.0
down-
Sulfate transporter, putative
NO
NO








regulated







Spipo17G0045100
5
86.3
17.8
down-
Aquaporin
NO
YES








regulated







Spipo2G0059000
5
5.8
1.2
down-
Major facilitator superfamily protein
NO
YES








regulated







Spipo9G0038700
5
53.8
11.2
down-
U-box domain-containing protein
NO
NO








regulated







Spipo1G0126300
5
####
293.1
down-
60S ribosomal protein L10-like
NO
NO








regulated
protein






Spipo3G0024100
5
24.6
5.3
down-
Nicotianamine synthase, putative
NO
NO








regulated







Spipo16G0011700
5
25.7
5.5
down-
L-lactate dehydrogenase
NO
NO








regulated







Spipo6G0071400
5
125.5
27.1
down-
Pectinacetylesterase family protein
NO
YES








regulated







Spipo22G0026300
5
186.4
40.4
down-
Expansin
NO
YES








regulated







Spipo2G0039900
5
15.3
3.3
down-
Amine oxidase, putative
NO
YES








regulated







Spipo2G0092600
5
66.7
14.6
down-
Early nodulin-like protein 17
YES
YES








regulated







Spipo0G0183100
5
14.4
3.2
down-
Heparanase-like protein 2
NO
YES








regulated







Spipo15G0035800
5
429.5
94.0
down-
Glutamine synthetase
NO
YES








regulated







Spipo17G0007500
5
101.0
22.2
down-
Methyltransferase type 11
NO
YES








regulated







Spipo22G0045000
5
13.9
3.1
down-
Mitochondrial carrier family
NO
YES








regulated







Spipo13G0011300
4
3034.8
676.8
down-
Ferredoxin I
NO
NO








regulated







Spipo9G0063000
4
13.1
2.9
down-
Beta-galactosidase
NO
YES








regulated







Spipo3G0083800
4
297.7
66.7
down-
Bifunctional inhibitor/lipid-transfer
NO
NO








regulated
protein/seed storage 2S albumin











superfamily






Spipo29G0021000
4
61.1
14.2
down-
PsbP-like protein 2
NO
YES








regulated







Spipo2G0055800
4
17.1
4.0
down-
bZIP transcription factor I
NO
YES








regulated







Spipo3G0101700
4
12.6
3.0
down-
Receptor kinase
NO
YES








regulated







Spipo29G0004100
4
288.3
68.2
down-
unknown protein
NO
YES








regulated







Spipo11G0012500
4
104.1
24.7
down-
Antiholin-like protein LrgB
NO
NO








regulated







Spipo11G0008200
4
1110.6
266.8
down-
Acyl-CoA-binding protein
NO
YES








regulated







Spipo9G0043500
4
42.3
10.2
down-
Phosphatidylinositol transfer protein
YES
YES








regulated
SFH5






Spipo23G0035900
4
68.8
16.6
down-
Omega-3 fatty acid desaturase
NO
YES








regulated







Spipo27G0016000
4
50.1
12.1
down-
Ubiquitin-conjugating enzyme E2 C,
NO
NO








regulated
putative






Spipo7G0026400
4
32.6
7.9
down-
DNA-3-methyladenine glycosylase
NO
YES








regulated







Spipo20G0006200
4
7376.0
1797.6
down-
Acyl-CoA dehydrogenase
NO
NO








regulated







Spipo9G0025800
4
15.7
3.9
down-
D-arabinono-1,4-lactone oxidase-like
NO
YES








regulated
protein






Spipo0G0009000
4
40.9
10.2
down-
Fructose-bisphosphatealdolase
NO
YES








regulated







Spipo4G0036100
4
49.7
12.3
down-
Pectatelyase
NO
NO








regulated







Spipo11G0009400
4
38.9
9.7
down-
Mitochondrial carrier protein
NO
NO








regulated






















TABLE S2








FPKM
FPKM
Average
Average





in
in
FPKM in
FPKM in


Pathway
Gene ID
Enzyme
frond
turion
frond
turion





















Lignin
Spipo0G0185100
CCR1
32.6
23.5
23
41



Spipo11G0026200
CCR2
3.4
10.2





Spipo6G0037000
CCR3
45.3
35.5





Spipo28G0002300
CCR4
1.0
0.9





Spipo23G0040600
CCR5
6.7
16.7





Spipo11G0026400
CCR6
5.3
10.8





Spipo10G0016700
CCR7
0.3
0.6





Spipo10G0000200
CCR8
26.2
167.8





Spipo8G0071400
CCR9
20.0
24.2





Spipo5G0064600
CCR10
16.7
18.6





Spipo7G0010700
CCR11
10.1
64.8





Spipo0G0172200
CCR12
215.3
306.7





Spipo7G0010800
CCR13
0.4
1.4





Spipo14G0054900
CCR14
2.0
9.9





Spipo12G0004300
CAD1
18.1
32.5





Spipo17G0012300
CAD2
10.8
7.8





Spipo1G0069500
CAD3
2.6
0.7





Spipo2G0124600
CAD4
3.9
2.3




Starch
Spipo28G0001400
APS1
264.1
242.5
70
86



Spipo3G0049000
APL1
127.9
18.6





Spipo6G0024200
APL2
23.1
34.2





Spipo18G0038500
APL3
36.0
291.5





Spipo26G0026900
SSI
21.7
28.6





Spipo0G0050800
SSII
3.4
1.6





Spipo14G0048800
SSIII
33.4
24.0





Spipo14G0042000
SSIV
45.7
15.6





Spipo1G0057900
GBSSI
327.8
333.2





Spipo1G0057400
BEI
19.6
10.3





Spipo0G0008100
BEII
40.8
72.0





Spipo12G0062400
ISA1
2.8
14.6





Spipo3G0051400
ISA2
8.1
12.9





Spipo20G0022100
ISA3
25.1
98.0




Lipid
Spipo0G0127900
ACCase1
24.0
17.4
28
22



Spipo10G0023400
ACCase2
6.3
4.6





Spipo12G0034900
ACCase3
4.5
2.5





Spipo12G0063600
ACCase4
85.6
44.3





Spipo15G0009000
ACCase5
22.4
21.4





Spipo4G0043600
ACCase6
20.9
11.0





Spipo4G0047600
ACCase7
73.7
56.7





Spipo30G0006700
GPAT1
127.7
52.2





Spipo7G0013300
GPAT2
21.0
20.4





Spipo3G0111400
AGPAT1
1.4
0.3





Spipo4G0068200
AGPAT2
17.6
18.2





Spipo6G0030100
AGPAT3
15.8
13.5





Spipo7G0018900
AGPAT4
4.3
3.3





Spipo7G0051900
AGPAT5
1.6
0.9





Spipo21G0027500
DGAT1
6.0
5.7





Spipo28G0006400
DGAT2
70.9
117.6





Spipo1G0066600
DGAT3
0.9
1.5





Spipo20G0011900
DGAT4
11.2
8.2





Spipo3G0079500
DGAT5
14.6
23.1









The data presented in Table S2 reveal suitable targets for altering carbon partitioning in duckweed. For example, reducing expression of GPAT1 in fronds should increase protein content in duckweed cells. In another approach, introduction of a LEA promoter operably linked to an agent (e.g., an siRNA) to APL1 should increase the lipid content of in the resulting duckweed culture. In yet another approach, one could simultaneously target two or more genes in duckweed which are differentially expressed to alter carbon partitioning in the resulting cells. Methods for introducing transgenes into Duckweed are described in Canto-Pastor, A., Mollá, Morales, A., Ernst, E., Dahl, W., Zhai, J., Yan, Y., Meyers, B. C., Shanklin, J., Martienssen, R. (2014), Efficient transformation and artificial miRNA gene silencing in Lemna minor. Plant Biology. doi: 10.1111/plb.12215. Sequence information is available on the world wide web at Waksman.rutgers.edu/spirodela/genome.
REFERENCES


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While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

## Claims

1. A method for altering carbon partitioning from starch to lipids in biomass produced from Duckweed cultures, comprising introducing a transgene comprising a LEA (late embryogenesis abundant protein) promoter operably linked to at least one sequence encoding an RNA interference product targeting a gene selected from APL1, APL2, and APL3, and/or a transgene comprising an LEA promoter operably linked to at least one coding region for a gene selected from the group consisting of ACCase1, ACCase2, ACCase3, ACCase4, ACCase5, ACCase6, and ACCase7, wherein expression of said transgene or transgenes is effective to reduce starch production and increase lipid production in said culture relative to control untreated cultures.

2. The method of claim 1, wherein introduction of said transgene results in increased lipid production in biomass obtained from said Duckweed culture.

3. The method of claim 2, wherein said transgene is an RNAi and inhibits expression of at least one gene selected from the group consisting of APL1, APL2, and APL3.

4. A Duckweed plant produced from the method of claim 1, wherein the Duckweed plant comprises a transgene comprising a LEA promoter operably linked to at least one sequence encoding an RNA interference product targeting a gene selected from APL1, APL2, and APL3, and, or a transgene comprising a LEA promoter operably linked to at least one coding region for a gene selected from the group consisting of ACCase1, ACCase2, ACCase3, ACCase4, ACCase5, ACCase6, and ACCase7.

5. The Duckweed plant of claim 4, which is Spirodela polyrhiza.

6. A plant part, progeny, seed or cell obtained from the plant of claim 5.

7. A method for altering carbon partitioning from starch to lipids in biomass produced from duckweed cultures comprising introducing a transgene comprising a LEA promoter operably linked to an siRNA targeting the APL3 gene and culturing said duckweed under conditions promoting turion formation, said transgene causing a decrease in APL3 expression, thereby increasing lipid content of said cultures relative to control untreated cultures.

8. A transgenic duckweed as claimed in claim 7.

9. The duckweed plant of claim 8, which is Spirodela polyrhiza.

10. A plant part, progeny, seed or cell obtained from the plant of claim 9.