Anda di halaman 1dari 26

Characterization and RNA-seq analysis

of underperformer, an activation-tagged
potato mutant

Sukhwinder S. Aulakh, Richard


E. Veilleux, Allan W. Dickerman,
Guozhu Tang & Barry S. Flinn

Plant Molecular Biology


An International Journal on Molecular
Biology, Molecular Genetics and
Biochemistry

ISSN 0167-4412
Volume 84
Number 6

Plant Mol Biol (2014) 84:635-658


DOI 10.1007/s11103-013-0159-4

1 23
Your article is protected by copyright and all
rights are held exclusively by Springer Science
+Business Media Dordrecht. This e-offprint
is for personal use only and shall not be self-
archived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com”.

1 23
Author's personal copy
Plant Mol Biol (2014) 84:635–658
DOI 10.1007/s11103-013-0159-4

Characterization and RNA-seq analysis of underperformer,


an activation-tagged potato mutant
Sukhwinder S. Aulakh · Richard E. Veilleux ·
Allan W. Dickerman · Guozhu Tang · Barry S. Flinn 

Received: 16 September 2013 / Accepted: 21 November 2013 / Published online: 4 December 2013
© Springer Science+Business Media Dordrecht 2013

Abstract  The potato cv. Bintje and a Bintje activation- using RNA-seq on leaves from 60 day-old plants revealed a
tagged mutant, underperformer (up) were compared. dataset of over 1,600 differentially expressed genes. Gene
Mutant up plants grown in vitro were dwarf, with abun- expression analyses suggested a variety of biological pro-
dant axillary shoot growth, greater tuber yield, altered tuber cesses and pathways were modified in the mutant, includ-
traits and early senescence compared to wild type. Under ing carbohydrate and lipid metabolism, cell division and
in vivo conditions, the dwarf and early senescence pheno- cell cycle activity, biotic and abiotic stress responses, and
types of the mutant remained, but the up plants exhibited proteolysis.
a lower tuber yield and fewer axillary shoots compared to
wild type. Southern blot analyses indicated a single T-DNA Keywords  Solanum tuberosum · Potato · BTB/POZ
insertion in the mutant, located on chromosome 10. Initial domain · Pleiotropic · Transcriptome · RNA-seq
PCR-based gene expression studies indicated transcrip-
tional activation/repression of several genes in the mutant
flanking the insertion. The gene immediately flanking the Introduction
right border of the T-DNA insertion, which encoded an
uncharacterized Broad complex, Tramtrac, Bric-a-brac; Advances in genomic technology have allowed the
also known as Pox virus and Zinc finger (BTB/POZ) sequencing and annotation of numerous plant genomes.
domain-containing protein (StBTB/POZ1) containing an Potato (Solanum tuberosum) is the 30th plant species and
Armadillo repeat region, was up-regulated in the mutant. first asterid for which a genome sequence has been pro-
Global gene expression comparisons between Bintje and up duced, providing significant new information on potato
gene models. The genome size of the doubled monoploid
potato (S. tuberosum Group Phureja DM 1-3 566 R44)
Electronic supplementary material  The online version of this selected for sequencing is 844 megabases (Mb), and the
article (doi:10.1007/s11103-013-0159-4) contains supplementary assembly and annotation of 86 % of this 844 Mb have pre-
material, which is available to authorized users. dicted 39,031 protein coding genes and a 62.2 % repeti-
tive DNA content (The Potato Genome Sequencing Con-
S. S. Aulakh · R. E. Veilleux · B. S. Flinn 
Department of Horticulture, Virginia Tech, Blacksburg, sortium 2011). Similarly, in the closely related tomato
VA 24061, USA genome, 900 Mb of sequence were generated from inbred
cultivar Heinz 1706; the assembly and annotation of
S. S. Aulakh · G. Tang · B. S. Flinn (*) 
760 Mb (84 %) of sequence predicted 34,727 protein cod-
The Institute for Sustainable and Renewable Resources (ISRR),
The Institute for Advanced Learning and Research (IALR), ing genes (The Tomato Genome Consortium, 2012). Sev-
Danville, VA 24540, USA eral other valuable genetic resources are also available for
e-mail: barry.flinn@ialr.org potato, including the transcriptome of the reference potato
genome (Massa et al. 2011), potato expressed sequence
A. W. Dickerman 
Virginia Bioinformatics Institute (VBI), Virginia Tech, tags (ESTs) from NCBI (249,761—dbEST release
Blacksburg, VA 24061, USA 130101) and microarray expression data (Kondrák et al.

13
Author's personal copy

636 Plant Mol Biol (2014) 84:635–658

2011). The potato genome reveals 2,642 genes specific to has yet to be demonstrated in potato, a potential model crop
this large angiosperm clade and provides a platform for for autopolyploid species.
genetic improvement of potato. Many traits of interest in Potato is the world’s third most important crop after
potato are quantitative in nature and access to the genome wheat and rice, and the most widely grown vegetable crop,
sequence will simplify their characterization and deploy- with more than half of its global harvest produced in devel-
ment in cultivars (The Potato Genome Sequencing Consor- oping countries (http://faostat.fao.org/). The cultivated
tium 2011). However, as for all plant genomes, scientists potato (Solanum tuberosum L.) belongs to a large genus
are faced with the enormous task of functional annota- that includes 160 tuber-bearing species, eight of which
tion of the various genes. Mutant populations are a valu- are cultivated. Most commercial potato cultivars are auto-
able resource for gene discovery, but few such resources tetraploid (2n = 4x = 48), having originated from diploid
are available for potato (Fischer et al. 2008) slowing the ancestors in the Andean Mountains of South America. The
process of gene discovery and annotation. Therefore, the development of resources and methods to elucidate the
challenge for the post-sequencing era is to create valuable function of potato genes remains an important goal for the
mutant resources in potato and to identify the biological realization of many of the potential benefits of the potato
functions of the predicted genes. Various approaches can genome sequence (The Potato Genome Sequencing Con-
be used to discover gene function, and the most direct is sortium 2011) and generation and characterization of acti-
to disrupt the gene(s) and analyze the consequences. How- vation tagged potato lines could provide one such resource.
ever, gene disruption approaches also have limitations. In a Another useful resource is knowledge of the transcrip-
tetraploid crop such as potato, disruption of a single allele tome. The main goal of whole transcriptome analysis is to
may not result in phenotypic changes, as the three unal- identify, characterize and catalogue all of the transcripts
tered alleles may compensate. expressed within a specific cell/tissue/organ, at a particu-
Activation tagging is used for generating dominant or lar stage, with the potential to determine the correct splic-
gain-of-function mutations, complementing conventional ing patterns and structure of genes, and to quantify the dif-
insertional mutagenesis and offering many advantages. In ferential expression of transcripts under physiological and
this method, T-DNA or a transposable element contain- pathological conditions (Costa et al. 2010). The arrival of
ing multimerized Cauliflower Mosaic Virus (CaMV) 35S Next Generation Sequencing (NGS) platforms [Genome
enhancers (Hayashi et al. 1992; Suzuki et al. 2001) is Sequence FLX (Roche), Genome Analyzer IIx and HiSeq
inserted into the genome. These enhancers can function in 2000 (Illumina), SOLiD 4 and 5500 Series Genetic Ana-
either orientation and at a considerable distance from cod- lyzer (Applied Biosystems)] over the last few years has
ing regions, and may cause transcriptional activation of allowed sequencing capabilities and associated gene
genes, resulting in dominant gain-of-function mutations. expression profiling capabilities to achieve a new level.
Such gene activation may produce novel phenotypes, and In this paper, we report on the characterization of the
can aid in determining gene functions, including those of potato activation tagged mutant AT615, which we termed
redundant members of a gene family or genes essential for underperformer (up), due to its general poor performance,
development and survival of an organism. However, the reduced organ size and early senescence compared to wild
activation tag may also integrate into a gene, disrupting type Bintje. We also report the results of RNA-seq analysis
gene function. Many genes are present in multiple copies to characterize gene expression changes on a global level
in the genome with partially redundant functions (Hiratsu in the leaves of the mutant compared to wild type Bintje.
et al. 2003), and this aspect is even more pronounced in This analysis resulted in a unique dataset of 1,632 genes
polyploid species like potato, making functional gene anno- that were differentially expressed between up and Bintje.
tation difficult. However, the activation tagging approach We attempted to present the broader picture of gene expres-
could prove useful, given that it activates the transcription sion changes, derive the biological meaning from this data
of flanking endogenous gene(s), and produces an obvious and relate this to the role of activation tagging in the mutant
phenotype. This tagging system has been used to identify phenotype.
and characterize a variety of genes in Arabidopsis (Huang
et al. 2001; Kakimoto 1996; Kardailsky et al. 1999; Weigel
et al. 2000), petunia (Zubko et al. 2002), tomato (Mathews Methods
et al. 2003), barley (Ayliffe et al. 2007) and poplar (Busov
et al. 2003; Harrison et al. 2007). The only prerequisite for Generation and screening of potato activation tagged
using activation tagging in any species is the need for at mutants
least one genotype to be efficiently transformed. Hence,
activation tagging has been growing in popularity in diverse The wild type Bintje and activation tagged AT 615 (up)
non-model plant species (Busov et al. 2011) but its utility mutant plantlets were received from BioAtlantech, having

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 637

been generated (Supplementary Materials and Methods) by recorded after 30 days. All in vitro experiments were done
the Canadian Potato Genome Project (Regan et al. 2006). in randomized complete block design with three replicates
and each experiment was repeated at least three times.
Determination of T‑DNA copy number by Southern blot Data were analyzed in JMP, version 9.0.0 (SAS Institute
analysis Inc., Cary, NC, USA) and tables and graphs were drawn in
Microsoft Excel (2010).
Genomic DNA of Bintje and up was digested with PciI
and PsiI restriction enzymes and separated on a 0.8 % Phenotyping under growth chamber and greenhouse
agarose gel. Digested DNA was transferred to positive conditions
charged Zeta probe nylon membrane (Bio-Rad; Hercules,
CA, USA) using downward capillary transfer and NaOH Phenotyping was done on plants grown in walk-in growth
buffer (Sambrook and Russell 2001). DNA was transferred chambers in the Department of Forest Resources and Envi-
to the membrane and rinsed in 2X SSC, then placed on a ronmental Conservation, Virginia Tech. Ten equal size (by
sheet of Whatman 3 MM filter paper and allowed to air dry weight) tubers of Bintje (average tuber weight 24 g) and up
for 30 min. The probe (640 bp) used for hybridization was (average tuber weight 22 g) were planted in 30 cm diam-
amplified from the 4X35S enhancer region of the pSKI074 eter pots filled with commercial Miracle-Gro potting mix
plasmid by PCR using primers (Supplementary Table S1) (Scotts Miracle-Gro, Marysville, OH, USA). Standard
and labeled using Amersham AlkPhos Direct Labeling plant growth practices were followed. Plants were grown
Reagents (GE Healthcare Life Sciences; Buckinghamshire, under 16 h photoperiod; 22 °C day/16 °C night and 350 μE
UK Catalog No. RPN3682) following the manufacturer’s light intensity and data were recorded 75 days after tuber
protocol. Overnight hybridization with the probe was done planting (45 days after plant emergence). Growing plants
in Alkphos Direct hybridization buffer (Amersham GE) at were supported by a plastic net and manually watered twice
55 °C. After hybridization, the blot was washed to remove per week or as needed. Ratings for potato tuber character-
excess probe and signal detected with CDP-Star (GE istics were based on the standardized rating codes detailed
Healthcare Life Sciences; Buckinghamshire, UK) chemilu- in Appendix 2 of the NC Potato Variety Trial Report (http://
minescent detection reagent. For final signal generation, the potatoes.ncsu.edu/pdf/NCPOTRPT05.pdf). The up mutant
blot was exposed to autoradiography film and the film was was also compared to Bintje under greenhouse conditions.
developed with Kodak GBX developer and fixer solution. Bintje and up plants were grown in the greenhouse of the
Institute for Sustainable and Renewable Resources (ISRR)
In vitro plant growth and tuberization at the Institute for Advanced Learning and Research
(IALR), Danville, VA, USA. Twenty tubers each of wild
For in vitro plant growth assays, Bintje and up plants were type and mutant (after equalizing the weight) were planted
grown from single node explants taken from 4 to 5 week directly into 30 cm diameter pots filled with commercial
old plantlets. Explants were placed on MS basal medium Miracle-Gro potting mix. Standard plant growth practices
with vitamins (Phytotech, Lenexa, KS, USA) supplemented were followed. The greenhouse was set to 24–30 °C. Data
with 3 % sucrose, pH 5.8 and solidified with 0.7 % agar. were recorded 75 days after planting (45 days after emer-
Plants were grown under 16 h photoperiod, 24 ± 1 °C and gence) and analyzed in JMP.
70–100  μE light intensity in a plant growth chamber and
data were recorded after 4 weeks. Identification of potato genomic regions flanking
For in vitro tuberization assays, stem sections, 3–5 cm the T‑DNA
long, with 4–5 nodes per section were prepared from 4 to
5 week old in vitro Bintje and up plantlets by removing We used the GenomeWalker universal kit (Clontech,
all leaves. These stem sections were grown in 40 ml liq- Mountain View; CA, USA) to identify and amplify the
uid propagation medium (MS basal medium + 148 mg l−1 genomic region flanking the T-DNA insertion. As the
NaH2PO4  + 0.4 mg l−1 thiamine HCl + 100 mg l−1 ino- pSKI074 vector has few unique sequences between the
sitol  + 3 % sucrose, pH 5.8) (Radouani 2003) at 16 h T-DNA right border and the tetramerized enhancer region,
photoperiod, 24 ± 1 °C and 70–100 μE for 3–4 weeks in we selected the left border region of pSKI074 for genome
250 ml conical flasks capped with ventilated plugs. After walking. Genomic DNA was extracted using Plant DNAzol
3–4 weeks, propagation medium was replaced with micro- Reagent (Life Technologies; Grand Island, NY, USA) fol-
tuberization medium (MS basal + 8 % sucrose, 2 × the lowing manufacturer’s instructions. Regions flanking the
amounts of NH4NO3, KH2PO4 and KNO3 and without T-DNA were amplified via GenomeWalker following the
NaH2PO4, pH 5.8) (Radouani 2003). Culture flasks were manufacturer’s instructions. Genomic DNA was digested
kept at 18–20 °C in the dark to induce tubers and data were with single restriction enzymes that leave blunt ends

13
Author's personal copy

638 Plant Mol Biol (2014) 84:635–658

(MscI, NaeI, ScaI and SspI) in separate tubes, followed the tubers (about 30 days after emergence) were pooled to
by ligation with GenomeWalker adapters to form Genom- form a composite sample for each genotype. Three repli-
eWalker “libraries.” Gene specific primers and nested gene cates of each composite sample were harvested, frozen in
specific primers were designed against the mannopine syn- liquid nitrogen and stored at −80 °C until needed. RNA
thase gene sequences and used with arbitrary primers 1 was extracted for RT-PCR analysis using the Concert Plant
(AP1) and 2 (AP2) to amplify the potato genomic region RNA Reagent (Invitrogen, Carlsbad, CA) following manu-
flanking the left border of the T-DNA (Supplementary facturer’s instructions. RNA was extracted for RNA-seq
Table S1). The primary PCR amplification was done with analysis using a combination of Trizol Reagent (Invitrogen,
outer adapter primer (AP1), gene specific primer (GSP1) Carlsbad, CA, USA) extraction and the RNA mini kit (Qia-
and GenomeWalker library DNA as templates which gen, Valencia, CA, USA) purification.
were followed by secondary PCR amplification using
nested adapter primer (AP2), nested gene specific primer Reverse transcriptase PCR analysis
(GSP2) (Supplementary Table S1) and diluted primary
PCR product as template. Secondary PCR amplification DNase treatment of RNA was done with the DNA-free
products were separated by electrophoresis and purified kit (Ambion; Foster City, CA, USA) following manu-
using a gel extraction kit (Qiagen; Valencia, CA, USA); facturer’s instructions. First strand cDNA was synthe-
cloned and sequenced using a CEQ 8800 genetic analysis sized using SuperScript III (Invitrogen; Carlsbad, CA,
system (Beckman Coulter, Brea, CA, USA). Recovered USA) using 1 μg of total RNA. After cDNA synthesis
genomic sequences flanking the left border of the T-DNA the final volume was adjusted to 100 μl of diluted cDNA
insertion sites were identified and used to perform Basic for each cDNA synthesis reaction. Reverse transcrip-
Local Alignment Search Tool (BLAST) searches in the tion polymerase chain reaction (RT-PCR) was performed
GenBank database (accessed on 4-5-2010) of the National using gene-specific primers (Supplementary Table S2).
Center for Biotechnology Information (NCBI), and in the Equal amounts of cDNA were used in each reaction and
potato genome version 4.0.3 (http://potatogenomics.plan Solanum tuberosum Elongation Factor 1-α (StEF1-α)
tbiology.msu.edu/blast.html) with default search param- gene expression was used as control. Gel images were
eters (expected threshold 10, maximum number of align- acquired using an Alpha Innotech gel doc system (San
ments 100, max number of descriptions 100, word length Leandro, CA, USA).
11, no filter and both strands). This allowed the T-DNA
insertion site to be located on Chromosome 10 (genomic RNA‑sequencing
coordinates; chr10:674572) of the potato genome.
Approximately 400 kb of potato sequence upstream RNA for RNA-seq was extracted from leaves of plants that
and downstream of the insertion site were analyzed for had been growing in the chambers for 60 days using a com-
the presence of annotated gene models using the potato bination of Trizol Reagent extraction and RNA mini kit
genome browser (http://potato.plantbiology.msu.edu/cgi- purification methods as described earlier. For DNase treat-
bin/gbrowse/potato). Sequence homology searches ment we used Turbo DNA free protocol following manu-
and analyses were performed using the NCBI BLAST facturer’s instruction with some modifications. Samples
server. Sequence alignments were carried out by the with an RNA Integrity Number (RIN) value >8 were sub-
CLUSTALW2 (Larkin et al. 2007) method and using jected to cDNA synthesis. The Illumina TruSeq RNA sam-
the EMBL server (http://www.ebi.ac.uk/Tools/msa/ ple preparation kit (low-throughput protocol) was used for
clustalw2/). Identification of conserved domains within the poly-A based mRNA enrichment and thermal mRNA frag-
predicted protein sequence of the identified tagged gene mentation, followed by cDNA synthesis using 4 μg of total
(StBTB/POZ domain-containing protein 1) was carried out RNA sample as starting material for each enrichment reac-
using InterProScan version 4.8 at the EMBL-EBI website tion. The fragmented mRNA samples were used for cDNA
(http://www.ebi.ac.uk/Tools/pfa/iprscan/). synthesis using reverse transcriptase (Super-Script II) and
random primers. The cDNA was further converted into
Preparation of RNA double stranded DNA using the reagent supplied in the kit
and resulting dsDNA used to prepare a library of template
Tuber samples from wild type and mutant plants were molecules suitable for high-throughput DNA sequencing
used for RNA extraction and subsequent RT-PCR, while using the TruSeq Illumina kit.
foliar samples were used for RT-PCR and RNA-seq anal- Sequencing (RNA-seq) was performed at the Research
yses. Ten lateral leaves, 5–6 cm long and 2–3 cm broad, Technology Support Facility (RTSF) of Michigan State
which were at the same developmental stage (not fully University using the Illumina Genome Analyzer (GAII)
mature) harvested from ten plants, 60 days after planting and HiSeq 2000 system, which uses a massively parallel

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 639

sequencing-by-synthesis four-dye approach to generate Visualization of gene expression data


billions of bases of high-quality DNA sequence per run.
A total of two biological replicates each consisting of two We used MapMan (version 3.5.1), which displays large data
technical replicates was sequenced. For one set of biologi- sets onto diagrams (Maps) that symbolically depict areas
cal replicates we used the GAII platform generating sin- of biological function. Maps are diagrams of pathways or
gle end 36 bp reads and for the second biological set we processes in which experimental data are displayed. Map-
used the HiSeq 2000 machine generating single end 50 bp Man organizes data into functional categories (BINs)
reads. which are themselves split into subcategories (subBINs).
Each Bin was given a corresponding numerical code (e.g.,
Bioinformatics and statistical analysis ‘photosynthesis’  = 1) which can be extended in a hierar-
chical manner (e.g., the subBINs ‘light reactions’ = 1.1;
Sequence reads were mapped to S. tuberosum Group ‘photorespiration’  = 1.2) and so on (Thimm et al. 2004).
Phureja DM1-3 516-R44 (DM) superscaffolds using The change of expression is displayed via false color code:
Tophat v2.0.4 (Trapnell et al. 2009), which made use of blue for increased and red for decreased expression. A set-
Bowtie. Reads were counted in regions of genes based on ting was selected that saturates at a value of 2 (22 = a four-
the PGSC_DM_v3_2.1.10_pseudomolecule annotation. fold change). Potato mapping file Stub_PGSC_DM_v3.4
Transcript abundance was expressed as raw read counts and was downloaded from the MapMan website (http://mapm
also in a normalized unit, reads per kilobase per million an.gabipd.org/web/guest/mapmanstore). The mapping file
reads (RPKM) as implemented in Cufflinks v2.0.2 (Trap- stores the association of genes, metabolites or proteins to
nell et al. 2010), which allow comparison both within and MapMan BINs to link experimental data with MapMan
among samples. Transcripts with RPKM value >0.001 and ontology. An experimental data file with potato peptide
95 % confidence interval were considered for further analy- IDs (PGSC0003DMP4000xxxxx) and relative expression
sis. Differentially expressed genes were flagged as signifi- levels (log2 fold values) was created and uploaded in the
cant based on multiple comparisons values, q. Only genes MapMan dataset folder. The experimental data file con-
with q values <0.05 were considered significant. Individual tained the measurements from the RNA-seq experiment in
datasets (GAII or HiSeq) as well as the combined dataset the log2 fold changes between a treatment (mutant up) and
(GA and HiSeq) were used for Cuffdiff analysis and the control (Bintje). MapMan not only displays a large data-
resulting output files were scanned for significant differ- set but also incorporates statistical analysis. For example
entially expressed genes. A stricter statistical analysis was deviation of response of items in a particular BIN from
performed using a differential expression analysis tool the response of all other items is calculated by Wilcoxon
based on negative binomial distribution (Anders and Huber test, which is similar to Student’s t test. MapMan also has
2010) and genes which had significantly different expres- a built-in multiple comparison test (Benjamini Hochberg)
sion level at α < 0.05 level were considered. The hierarchi- which calculates corrected P values (Usadel et al. 2009).
cal clustering of genes filtered using Cuffdiff analysis was
done using MapMan v3.5.1 software (Thimm et al. 2004).
Protein domain architecture analysis was done with Sim- Results
ple Modular Architecture Research Tool (SMART). To
carry out phylogenetic analysis of the BTB/POZ domain Generation and screening of potato activation tagged
containing protein family members from various plant spe- mutants
cies, we used the StBTB/POZ1 protein sequence (Genbank
No. AGT37251) to extract BTB/POZ1 orthologs from The activation tagging approach was used by the Cana-
GenBank and Phytozome v9.1 (www.phytozome.net). dian Potato Genome Project (Regan et al. 2006) to create
The deduced protein sequences from different plant spe- over 8,000 mutants of cv. Bintje using the activation tag-
cies were aligned using the Clustal W method in MegAlign ging vector pSKI074 (Weigel et al. 2000). The mutant line
(DNAStar Lasergene 11.0) and a rooted phylogenetic tree AT615, which we designated underperformer (up), was
was constructed. The length of each pair of branch repre- selected during an in vitro screen. After 1 month of in vitro
sents the distance between each sequence pair. The units at growth from single node explants, the up mutant was much
the bottom of the tree indicate the number of substitution shorter than wild type Bintje and several new axillary stems
events. Bootstrapping was used to test for consistency and were formed, leading to branching, whereas wild type
robustness of the tree created using ClustalW. The branch Bintje exhibited a single main stem (Supplementary Fig.
patterns with greater values are statistically more likely to S1). Transgenic plantlets were initially screened by poly-
occur and provide a measure of confidence in the optimum merase chain reaction (PCR) to confirm the presence of the
tree structure. T-DNA plasmid sequences in the plant (data not shown).

13
Author's personal copy

640 Plant Mol Biol (2014) 84:635–658

Determination of T‑DNA copy number by Southern blot (Fig.  3). The number of stolons also increased for Bintje
analysis but not up.

To determine the T-DNA copy number in the up mutant, Phenotyping under growth chamber and greenhouse
we performed Southern blot analysis with genomic DNA, conditions
using two restriction enzymes (PciI and PsiI) that cut
within the T-DNA, in the region just left of the tetramer- Additional phenotype information was obtained for up
ized enhancer sequences. Using a probe that corresponded plants when grown ex vitro in soil, in addition to the traits
primarily to the enhancer region of pSKI074, we observed observed under in vitro conditions. Mutant plants were
a strong, single hybridizing band in the mutant for both modified in various above-ground and tuber characteristics
restriction enzymes, indicating the presence of a single (Table 1; Figs. 4, 5, 6), had smaller sized leaves (Figs. 4a,
T-DNA insertion (Supplementary Fig. S2). b, 5), produced long and fingerlike tubers (Figs. 4c, d, 5),
had small floral buds which did not open and dropped pre-
In vitro plant growth and tuberization maturely (Fig. 4e–g), and exhibited early senescence of the

We multiplied Bintje and up plants from single node


explants and recorded data after 4 weeks of in vitro growth.
Mutant up plants exhibited a smaller mean stem length and
a larger mean number of axillary branches, which were
significantly different from the Bintje control. No signifi-
cant differences were found in number of roots per plant
(Fig. 1). In vitro tuberization experiments were conducted
to assess tuberization ability of the up mutant. All recorded
parameters (number of tubers/explant, number of stolons/
explant and tuber weight/explant) were significantly greater
for up (Fig. 2a) compared to wild type Bintje. We also
observed differences in tuber shape and size between Bintje
and up plants. Bintje tubers were round and more uniform
compared to the longer tubers of up (Fig. 2b). As cytokinin
is known to impact tuberization, kinetin effects on tuberiza-
tion were assessed with both Bintje and up, to see if any
variation in responsiveness existed in the mutant. In vitro
tuberization medium supplemented with 10 μM kinetin
improved the number and weight of tubers for both Bintje
and up, but the increase was somewhat greater for Bintje

Fig. 2  In vitro tuberization response of wild type Bintje and up


mutant plants. a Stem explants 3–4 cm long with 4–5 nodes were
cultured in liquid propagation medium for 3–4 weeks under 16 h
photoperiod and then transferred to high sucrose liquid tuberization
medium and transferred to the dark, and data recorded after an addi-
Fig. 1  In vitro plant growth of wild type Bintje and underper- tional 30 days. The values are mean ± SE. Asterisks statistical signifi-
former (up) mutant plants. Single node explants from 4 to 5 week- cance of means in the same parameter estimated using Tukey’s HSD
old potato plantlets of Bintje and the up mutant were placed on MS test (*P < 0.05–0.005; **P < 0.005–0.0005; ***P < 0.0005). b Pho-
medium with vitamins, and data recorded after 30 days. The values tographic representation of the response of the Bintje and up mutant
are mean ± SE. Asterisks statistical significance of means in the 30 days after plantlets were placed on liquid tuberization medium
same parameter estimated using Tukey’s HSD test (*P < 0.05–0.005; (upper panel), and the harvested tubers of both (lower panel), show-
**P < 0.005–0.0005, ***P < 0.0005) ing the different shape and size of the mutant tubers. Bar 2 cm

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 641

misshapen, cracked and rotted tubers) and a lower specific


gravity (Table 1).

Identification of potato genomic regions flanking the


T‑DNA

Using GenomeWalker technology (Clontech), we posi-


tioned the T-DNA insertion on chromosome 10:674572
(Supplementary Fig. S3a). Further PCR and sequence
analysis of the left border T-DNA/plant DNA junction
site revealed that a vector backbone of 250 bp (7,802 to
8,053 sequence of pSKI074) outside of the T-DNA was
also found in the up mutant (data not shown). In a simi-
Fig. 3  The effect of kinetin on in vitro tuberization response of wild lar analysis of the right border T-DNA/plant DNA junc-
type Bintje and up mutant plants. Stem explants 3–4 cm long with tion site in the up mutant, we found that 22 bp of the
4–5 nodes were cultured in liquid propagation medium for 3–4 weeks T-DNA right border (4,273 to 4,294 sequence of pSKI074)
under 16 h photoperiod and then transferred to high sucrose liquid
tuberization medium without (control) or with 10 μm kinetin, trans-
and the same number of base pairs of the potato genome
ferred to the dark, and data recorded after an additional 30 days. The (chr10:674550..674571) were lost during T-DNA integra-
values are mean ± SE. Asterisks statistical significance of means in tion (data not shown). Using the potato genome browser
the same parameter estimated using Tukey’s HSD test (*P < 0.05– (http://potato.plantbiology.msu.edu/cgi-bin/gbrowse/
0.005; **P < 0.005–0.0005; ***P < 0.0005)
potato), we identified several gene model predictions on
either side of the T-DNA insertion. The first predicted
Table 1  Various phenotypic measurements for ex vitro-grown Bintje potato gene open reading frame flanking both right and left
and up plants borders of the T-DNA occurred approx. 5.2 and 3 kb from
Description Mean values + SE P value the T-DNA, respectively (Supplementary Fig. S3a).
Bintje up
Transcript analysis of potato genes flanking the T‑DNA
Plant height (cm) 72.9 ± 1.3 41.8 ± 1.4 <0.0001*
Chlorophyll content index 46.1 ± 1.7 40.8 ± 1.5 0.0229* We used reverse transcriptase PCR for transcript analysis
Tuber shape 4.3 ± 0.4 8.6 ± 0.2 <0.0001* of potato genes flanking the T-DNA with primers designed
Tuber color 6.6 ± 0.2 6.8 ± 0.2 0.6186 against the reading frame of each gene model prediction
No. of eyes/tuber 13.8 ± 0.9 35 ± 2.2 <0.0001* (Supplementary Table S2), and with cDNA made from
Tuber eye depth 8 ± 0.2 5.1 ± 0.2 <0.0001* leaf RNA. We analyzed approximately 400 kb regions
Tuber skin texture 7 ± 0.3 7.3 ± 0.3 0.5057 both upstream and downstream of the insertion site, and
Specific gravity 1.11 ± 0.006 1.08 ± 0.004 0.0205* observed up- and down-regulation of various potato genes
flanking both borders (Fig. 7). The most significant change
in gene expression in the mutant was a strong up-regula-
leaves (Fig. 4h, i). When freshly harvested tubers of Bintje tion of the first two predicted genes (conserved genes of
were replanted in soil without any treatment to break tuber unknown function), which started 5.2 and 11.5 kb from the
dormancy, they exhibited normal dormancy, with no spout- right border (Fig. 7a). Another gene whose expression was
ing observed within 40 days (Fig. 4j). In contrast, over the up-regulated in the mutant was a predicted SAM-dependent
same time period, 60 % of up tubers sprouted (Fig. 4j), methyltransferase, located 41 kb downstream of the right
indicating reduced tuber dormancy in the mutant. The border. In addition several genes within the 400 kb region
plant and tuber data (Table 1; Figs. 4, 5), indicated that of the right border were down-regulated (F-box family pro-
the up mutant exhibited significantly reduced plant size, tein, GA2-oxidase 1, P21-rho-binding domain-containing
smaller leaves and tubers and fewer tubers. Biomass and protein, adenosine deaminase, Dicer homolog, DNA-3
yield traits (aboveground fresh weight, total fresh weight, methyladenine glycosylase) in the mutant. In the region
total dry biomass weight, total tuber weight per plant) analyzed, one gene off of the left border of the T-DNA, a
were also significantly lower in the mutant compared to conserved gene of unknown function, was observed to be
wild type Bintje (Fig. 6). Other tuber qualitative traits highly up-regulated in the up mutant, whereas a Protein
(tuber shape, tuber color, tuber eye depth) also differed phosphatase 2C and another conserved gene of unknown
between the two genotypes (Table 1). The mutant had function were down-regulated in the mutant (Fig. 7b). This
more tubers that exhibited external defects (such as green, transcript analysis of the flanking genes on both sides of the

13
Author's personal copy

642 Plant Mol Biol (2014) 84:635–658

Fig. 4  Phenotyping of Bintje and up mutant plants under growth of senescence observed 35 days after planting with Bintje plants,
chamber and greenhouse conditions. a Bintje and b up plants growing while up plants (i) exhibited distinct senescence. j Tuber dormancy
in a walk-in-chamber, 80 days after planting. c Bintje and d up tubers phenotypes for Bintje (left) and up (right) 30 days after tubers were
after harvest. e Bintje and f up inflorescences. g A close up of repre- planted without cold storage. Note the reduced tuber dormancy and
sentative flower buds from Bintje (left) and up (right) plants. h Lack sprouting of the mutant tubers

Fig. 5  Growth characteris-
tics of Bintje and up plants
grown in a walk-in-growth
chamber. Plants were grown
from tubers directly planted
in soil after breaking the tuber
dormancy (cold storage for
45 days). Data were recorded
75 days after planting (DAP).
Tuber data were recorded at
final harvest (130 DAP). The
values are mean ± SE. The
asterisks indicate statistical
significance of means in the
same parameter estimated using
Tukey’s HSD test (*P < 0.05–
0.005; **P < 0.005–0.0005;
***P < 0.0005). L length, B
breadth

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 643

(Fig.  9). MapMan displays large datasets of metabolic


pathways or other processes as pictorial diagrams that sym-
bolically depict areas of biological function. The classifica-
tion was based on hierarchical functional categories (Bins,
subBins, individual enzymes). The major MapMan path-
ways were classified to represent metabolism, cell function,
regulation, cellular response, stress secondary metabolism,
large enzyme families and RNA protein synthesis. Table 2
summarizes each of these pathways, their various numbers
of up- and down-regulated genes, and the highest up-regu-
lated and down-regulated gene in each pathway classifica-
tion for the up mutant relative to wild type Bintje.
Fig. 6  Yield traits of Bintje and up plants grown in walk-in-growth In the following sections, we will describe the major
chambers. Plants were grown from tubers directly planted in soil after biological pathways/processes, those most relevant to
breaking the tuber dormancy (cold storage for 45 days). Biomass
data were recorded, 75 days after planting (DAP). Tuber data were
our RNA-seq dataset and into which our differentially
recorded at final harvesting (130 DAP). The values are mean ± SE. expressed genes were clustered. The detailed Bin ids, Bin
The asterisks indicate statistical significance of means in the same description, peptide ids, gene annotation and log2 fold
parameter estimated using Tukey’s HSD test (*P < 0.05–0.005; change values are given in Supplementary Table S3. The
**P < 0.005–0.0005; ***P < 0.0005)
information linking gene ids with peptide ids is given in
Supplementary Table S4. We use the terms up- and down-
T-DNA revealed that insertion of the T-DNA had resulted regulation in gene expression levels of mutant genes rela-
in modification of the expression of many genes in the up tive to wild type Bintje gene expression. The majority of
mutant, not only the first (or few) genes immediately adja- the genes identified were down-regulated in the up mutant.
cent to the right border and associated tetramerized enhanc-
ers. In order to more fully characterize gene expression Metabolism overview
changes, we used RNA sequencing (RNA-seq) of leaf tran-
scripts to obtain a more comprehensive global gene expres- Many genes were identified which were modified in their
sion profile of the mutant. expression patterns in the mutant and were associated with
metabolism (Fig. 9a, Supplementary Table S3). These
RNA‑sequencing genes were primarily associated with photosynthesis [Bin:
1], major carbohydrate metabolism [Bin: 2], minor carbo-
We generated >207 million RNA-seq reads in total, of which hydrate metabolism [Bin: 3], cell wall synthesis, composi-
183 million (88 %) were mapped to the potato genome tion, modification and degradation [Bin: 10], lipid metabo-
assembly PGSC_DM_v3_2.1.10_pseudomolecules.zip lism [Bin: 11], amino acid metabolism [Bin: 13], secondary
using TopHat v2.0.4. Our combined dataset flagged 1,632 metabolism [Bin: 16] and nucleotide metabolism [Bin: 23].
genes as showing significantly different expression lev- The majority of the identified photosynthesis genes related
els between the two genotypes. Of these differentially to photosystem subunit proteins, ATP synthase activity,
expressed genes, 661 (40.5 %) were up-regulated and electron flow (e.g., similar to PIF1 and NDF4) and the Cal-
971 genes (59.5 %) were down-regulated in the mutant. vin cycle (e.g., similar to glyceraldehyde 3-phosphate dehy-
Although the T-DNA insertion in the mutant was previ- drogenase subunits and fructose-1,6,-bisphosphatase) were
ously identified as occurring on chromosome 10, the dis- up-regulated in the mutant. In addition, most of the identi-
tribution of up- and down-regulated genes followed a ran- fied major carbohydrate metabolism genes (e.g., similar to
dom pattern across all 12 potato chromosomes (Fig. 8). SUS4, SPF1F and AMY1) and minor carbohydrate metab-
The greatest numbers of up- (n = 110) and down-regulated olism genes (e.g., similar to DIN10, GOLS1 and TPS11)
(n  = 133) genes were found on chromosomes 9 and 2, were also up-regulated in the mutant. Regarding cell wall
respectively. metabolism, a majority of the identified genes associated
with cell wall degradation (e.g., similar to pectate lyases,
Visualization of gene expression data BURP domain-containing proteins) and cell wall modi-
fication (e.g., similar to XTH27, EXP8 and EXPL1) were
We used MapMan software to localize the 1,632 differen- also up-regulated, while a mix of genes encoding pectin
tially-expressed genes to various categories and subcatego- esterase family proteins were up- and down-regulated. In
ries based on GO annotation, and the genes were shown contrast, a majority of the identified genes associated with
as different elements for major plant pathways/processes lipid metabolism and relating to fatty acid synthesis and

13
Author's personal copy

644 Plant Mol Biol (2014) 84:635–658

Fig. 7  Reverse transcriptase PCR analysis of various potato genes of the arrow indicates the orientation of the predicted open reading
flanking the right border (a) and left border (b) of the activation tag frame, and the distance of each predicted gene from the T-DNA inser-
T-DNA insertion of the up mutant. RNA was extracted from leaves of tion is indicated in kb. Information on primers used for RT-PCR is
60 day old plants grown in a walk-in-growth chamber. The direction given in Supplementary Table S2

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 645

[Bin: 35.2]. Amongst the up-regulated genes, the high-


est-fold increases in expression levels (7.3 and 6.8
log2 fold) were observed for a gene involved in regu-
lation of transcription [PGSC0003DMG400031871;
PGSC0003DMP400055149] and for a homeobox tran-
scription factor family gene [PGSC0003DMG400014020]
encoding peptide PGSC0003DMP400024694 [Bins: 27.3.9
and 27.3.22, respectively]. Amongst the down-regulated
genes in the mutant, the gene which showed the great-
est level of down-regulation (−5.3 log2 fold) was found
to be a glutaredoxin gene [PGSC0003DMG400002360;
PGSC0003DMP400004213] [Bin: 21.4].

Fig. 8  Chromosomal distribution of genes showing modified leaf Genes assigned to cell organization, cell division and cell
expression patterns in the up mutant compared to wild type Bintje. cycle, DNA/RNA synthesis/processing/repair
The 1,632 significantly differentially expressed genes identified using
RNA-seq were grouped according to their location on the potato
chromosomes. Leaf RNA from 60 day old plants was used for RNA- A closer look at these various functional classifications
seq. Significance cutoff was based on q < 0.05 values (Fig.  9b, Supplementary Table S3) revealed the majority
of identified genes associated with cell organization [Bin:
31.1], cell division [Bin: 31.2] and cell cycle [Bin: 31.3]
elongation (e.g., similar to KAS1, KCS5 and CER2) and were down-regulated in their expression (e.g., similar
lipid degradation (e.g., similar to triacylglyerol lipases) to various kinesins, the mitotic spindle checkpoint gene
were down-regulated in the mutant, although some up- MAD2 and the regulator of chromatin condensation gene
regulation of lysophospholipase and fatty acid desaturease RCC1). Similarly, numerous identified genes associated
(FAD2) gene expression was noted in the mutant. with DNA synthesis [Bin: 28.1], and various genes asso-
Several genes associated with amino acid metabolism ciated with RNA processing [Bin: 27.1], RNA synthesis
were identified, and of those associated with amino acid [Bin: 27.2], and DNA repair [Bin: 28.2] were down-regu-
synthesis, many were down-regulated in the mutant (e.g., lated, including those similar to various histone proteins,
similar to tryptophan synthase, tryptophan-associated C2 DNA-directed DNA polymerases and transducer family
domain-containing protein, DHS1 and ASN1). Similarly, proteins.
several identified genes associated with amino acid deg-
radation (e.g., similar to fumarylacetoacetase and proline Genes assigned to stress
oxidase) were also down-regulated. In the area of second-
ary metabolism, the majority of the identified genes in the Several genes associated with biotic and abiotic stress
mutant associated with terpenes, flavonoids, phenylpro- (Bin: 20) were modified in their expression patterns in the
panoids and phenolics were down-regulated (e.g., simi- mutant, with very distinct patterns associated with biotic
lar to APR3, MVA1, HMG1, TPS1, 4CL, CAD8, CAD9, or abiotic stress-associated genes (Fig. 9b, Supplementary
TT4). Finally, a variety of genes associated with nucleo- Table S3). The majority of the identified genes associated
tide metabolism was also modified in their expression pro- with biotic stress (e.g., similar to various pathogenesis-
files in the mutant, with almost all (e.g., similar to CTP related PR proteins, thaumatin and osmotin-like proteins,
synthase, thymidine kinase, deoxyuridine 5′-triphosphate HIN1 and JAZ1) was down-regulated in the mutant, while
nucleotidohydrolase and the ribonucleotide reductase the majority of those genes associated with abiotic stress
RNR1) being down-regulated. was up-regulated (e.g., similar to those coding for germin-
like proteins, allergen family proteins, heat shock proteins,
Cell function overview SAG20 and COR413-PM2).

In addition to metabolism, differentially expressed genes Genes assigned to regulation of transcription


and their affiliation with cell function processes were
identified (Fig. 9b, Supplementary Table S3), with vari- One of the largest groups of genes identified represented
ous processes (e.g., cell division and cell cycle, DNA those associated with the regulation of transcription [Bin:
synthesis, regulation of transcription) specified. Approxi- 27.3]. A variety of the identified transcription factor classes
mately one-third of the genes could not be assigned to was primarily down-regulated in gene expression in the
any functional category, and so were grouped together mutant (Fig. 9b, Supplementary Table S3), with several

13
Author's personal copy

646 Plant Mol Biol (2014) 84:635–658

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 647

◂Fig. 9  MapMan overview display of differentially-expressed genes proteins (e.g., similar to SPL9, SPL8, SPL14) and numer-
assigned to a metabolism and b cell function. Changes in leaf tran- ous other genes (e.g., similar to a gene encoding a tropomy-
script levels at 60 DAP were measured from leaves harvested from
activation tagged up mutant and wild type Bintje plants. Transcript osin-related protein, the gene for protodermal factor PDF1,
abundance was expressed in reads per kb of exon model per million the gene OFP8 for an ovate-family protein, and a gene for
mapped reads (RPKM) and converted to relative (up/Bintje) log2 a dem-related protein). A few identified genes were up-reg-
fold values. Relative log2 fold values were visualized using MapMan ulated in the mutant, such as the flowering locus gene FT,
software. The change in expression levels were displayed via false
color code: blue for increased and red for decreased expression in those encoding various nodulin family proteins (e.g., genes
the mutant. Each square represents one gene (peptide). A color set- similar to MtN21 and MtN3), and those encoding tetraspa-
ting was selected in which it saturates at a value of 2 (22  = a four- nins (e.g., similar to the genes TET7 and TET8).
fold change). Genes with significantly (q < 0.05) different expres-
sion levels were displayed. Number near each cluster corresponds to
MapMan BIN ids. Grey dots were genes not present in our dataset. Genes assigned to hormones
Detailed information for each data point including BIN ids, BIN
description, peptide ids, gene annotation and log2 fold values are Genes assigned to various hormone-associated functions
given in Supplementary Table S3 (Bin: 17) were modified in their expression profiles, with
various up- and down-regulation patterns in the mutant
AP2/EREBP members (e.g., genes similar to cytokinin (Fig.  9b, Supplementary Table S3). Many auxin-induced/
response factor CRF4, AINTEGUMENTA and AINTEG- regulated/response/activated and transduction genes were
UMENTA-LIKE), GATA transcription factor family mem- primarily up-regulated (e.g., similar to auxin response
bers, YABBY family members (e.g., genes similar to YAB1 family genes, PIN5, DFL1 and SAUR12) in the mutant,
and YAB3), GRAS family members (e.g., genes similar to although a small proportion was down-regulated (e.g., sim-
SCR), C2H2 zinc finger family members, Squamosa pro- ilar to the indole 3-acetic acid-amido synthetase GH3.1). In
moter binding protein family members (e.g., genes similar addition, a few cytokinin synthesis/degradation-associated
to SPL8 and SPL9), WRKY family members (e.g., genes genes (e.g., similar to the cytokinin oxidase/dehydrogenase
similar to WRKY21 and WRKY70), TCP family members gene CKX1 and the isopentenyl transferase 3 gene IPT3)
(e.g., genes similar to TCP4 and TCP12), general tran- were also up-regulated in the mutant. Genes associated
scription factor family members (e.g., genes similar to with ethylene synthesis/degradation/signal transduction
growth regulating factors GRF1, GRF2 and GRF3), chro- were both up-regulated (e.g., similar to 2OG-Fe(II) oxy-
matin remodeling factors (e.g., gene similar to DDM1), genase, 2-oxoglutarate-dependent dioxygenase genes) and
DNA methyltransferases (e.g., genes similar to CMT3 down-regulated (e.g., similar to senescence-related SRG1
and MET1) and SET-domain family members (e.g., genes and ethylene response factor ERF 1 and ERF15 genes), as
similar to SUVH5 and SUVH6) showing this pattern. Other were gibberellin synthesis/degradation/induced/regulated
identified transcription factor classes displayed a combina- genes (e.g., similar to the ENT-Kaurenoic acid hydroxylase
tion of up- or down-regulation in the mutant, such as bHLH gene KAO2, a GA-responsive protein gene, a GASA1 gene,
family members (e.g., genes similar to UNE10 and SPCH, and GA3- and GA20-oxidases). Some jasmonate metabo-
respectively), DOF zinc finger family members (e.g., genes lism and signal transduction-associated genes were modi-
similar to CDF3 and OBP1, respectively), Homeobox fam- fied in the mutant (e.g., up-regulation of an allene oxide
ily members (e.g., genes similar to HDG8 and BLH11, synthase gene, and down-regulation of JAZ1), as were
respectively), MYB family members (e.g., genes similar to some genes associated with salicylic acid synthesis/degra-
MYB111 and MYB106, respectively), bZIP family members dation (e.g., up-regulation of genes similar to an SAM car-
(e.g., genes similar to TGA1 and bZIP44, respectively) and boxymethyltransferase gene, and down-regulation of IAA
MADS family members (e.g., genes similar to AGL8 and carboxymethyltransferase gene IAMT1).
SVP, respectively). The only transcription regulation fam-
ily member class where the modified genes were all up-reg- Genes assigned to regulation
ulated were those of the identified AUX/IAA family (e.g.,
genes similar to IAA29 and SHY2). When considering genes assigned to a regulation func-
tion (Bin: 30), a majority of genes involved in signal-
Genes assigned to development ing was down-regulated in the mutant (Fig. 9b, Sup-
plementary Table S3), including those for various
In the development function category (Bin: 33), many of leucine-rich repeat receptor kinases (e.g., genes similar
the identified genes in the mutant were down-regulated to FLS2 and IMK2), DUF26 receptor kinases (e.g., genes
(Fig. 9b, Supplementary Table S3), including various stor- similar to WAK2, LYM1 and ARK3), calcium signaling
age protein genes (e.g., similar to patatin-like proteins molecules (e.g., genes similar to those for various calm-
PLP1, PLP6, PLP9), genes for squamosa promoter-binding odulin-binding proteins, like IQD6, IQD18 and NPGR1)

13

648

13
Table 2  Summary of RNA-seq gene expression changes in foliar tissues of the up mutant relative to the Bintje control as identified using MapMan
Major MapMan No. of major No. of differentially No. of genes No. of genes Highest up-regulated gene Highest down-regulated gene
pathways MapMan BINs expressed genes up-regulated down-regulated [PGSC0003DMP4000..; log2- [PGSC0003DMP4000..; log2-
in mutant in mutant fold change] fold change]

Metabolism 58 196 94 102 Tetrapyrolle synthesis gene Secondary


glutamyl-tRNA reductase metabolism.phenylpropanoids
[55149; 3.9] [47121; −4.8]
Cell function 22 1,441 560 881 Regulation of transcription Not assigned.unknown
[55149; 7.3] [28930; −6.5]
Regulation 22 452 178 274 RNA.regulation of transcription. Redox.glutaredoxins
unclassified [04,213; −5.3]
[55149; 7.3]
Cellular response 14 163 66 97 Development.unspecified Redox.glutaredoxins
[40404; 5.2] [04213; −5.3]
Stress 24 322 145 177 Stress.abiotic.unspecified Redox.glutaredoxins
[2994; 5.1] [04213; −5.3]
Secondary metabolism 11 44 10 34 Secondary metabolism.simple Secondary
phenols metabolism.phenylpropanoids
[35339; 3.7] [47121; −4.8]
Large enzyme families 12 110 38 72 Misc.GDSL-motif lipase Misc.nitrilases, *nitrile lyases,
Author's personal copy

[38664; 2.7] berberine bridge enzymes,


reticuline oxidases, troponine
reductases
[44963; −5.3]
RNA protein synthesis 8 141 67 74 Protein.degradation.ubiquitin. Protein.degradation.ubiquitin.
E3.SCF.FBOX E3.SCF.FBOX
[35351; 3] [47928; −4.6]
Plant Mol Biol (2014) 84:635–658
Author's personal copy
Plant Mol Biol (2014) 84:635–658 649

and G-protein-associated signaling (e.g., genes similar to family protein member. A few genes were down-regulated
ROPGEF1, ROPGEF2, ROPGEF5 and RABA1D). How- in the mutant, and included additional glutaredoxin family
ever, some signaling-associated genes were up-regulated protein members, a dehydroascorbate reductase gene (e.g.,
(e.g., genes similar to the lectin protein kinase family, the similar to DHAR1), a protein disulfide isomerase gene
histidine phosphotransfer kinase AHP3 and calcium-bind- (e.g., similar to PDI6) and a thioredoxin-disulfide reductase
ing protein PBP1). gene (e.g., similar to NTRC).
A few genes associated with metal handling/binding,
Genes assigned to protein synthesis, amino acid activation, chelation and storage (Bin: 15) were modified in their
protein modification and protein degradation expression profiles in the mutant (Fig. 9b, Supplementary
Table S3), with a nicotianamine synthase gene (e.g., simi-
Many of the genes associated with protein synthesis (Bin: lar to NAS3) strongly up-regulated, and genes encoding
29.2) and modification (Bin: 29.4) that were modified in a metal ion binding protein and a heavy metal-associated
expression in the mutant were down-regulated (Fig. 9b, domain containing protein down-regulated.
Supplementary Table S3), including those for various ribo-
somal structural proteins (e.g., genes encoding 40S ribo- Genes assigned to transport
somal protein S15A and the 60S ribosomal proteins L26
and L38), post-translational protein modifiers (e.g., similar The majority of transport-associated genes (Bin: 34) altered
to the histidine kinase gene AUR1, the cyclin-dependent in their expression profiles was up-regulated in the mutant
kinase genes CDBK1 and CDBK2, genes for numerous (Fig.  9b, Supplementary Table S3). Many of these genes
protein kinase family proteins). However, some genes were encoded transporters for a variety of compounds, includ-
up-regulated in the mutant (e.g., similar to genes encod- ing sugars, amino acids, ammonium, calcium, sulphate,
ing protein phosphatase 2C, genes encoding octicosapepti phosphate, potassium, nucleotides, peptides/oligopeptides,
de/Phox/Bem1p (PB1) domain-containing proteins, and the unspecified cations and anions (e.g., genes similar to car-
protein kinase gene WAG1). bohydrate transporter MSS1, amino acid transporter AAP3,
In contrast to protein synthesis and modification, many ammonium transporter AMT1, sulfate transporter AST91,
genes associated with protein degradation (Bin: 29.5) were phosphate transporter ATPT2, purine permease PUP1, cop-
up-regulated in the mutant (Fig. 9b, Supplementary Table per transporter COPT1, peptide transporter PEPT3, potas-
S3), including genes encoding numerous subtilases and sium transporter KT2, calcium exchanger CAX7). However,
other serine proteases, cysteine proteases, aspartate pro- some transporter classes exhibited a greater proportion of
teases and a metalloprotease (e.g., genes similar to XSP1, down-regulation of gene expression in the mutant, such
SCPL48 and XCP1). In addition, various ubiquitination as genes for members of the ABC transporters/multidrug
pathway proteins were altered in their expression profiles resistance systems, members of the MATE efflux protein
(up- or down-regulated) in the mutant, including vari- family and members of the SEC14 cytosolic factor protein
ous ubiquitin conjugating enzymes (e.g., genes similar to family.
UBC9, UBC19, UBC36 and UBC37). Various members
of the E3 ubiquitin ligase classes were also modified, with Genes assigned to large enzyme families
approximately two-thirds of the SCF E3 ligases modi-
fied in their expression being down-regulated (e.g., simi- Many of the genes encoding large enzyme families (Bin:
lar to genes ASK4, VB1, SLY2), one-half of the RING E3 26) altered in their expression profiles were down-regulated
ligases down-regulated (e.g., similar to genes VIM1, PUB8, in the mutant (Fig. 9b, Supplementary Table S3). These
BRCA1), and our tagged gene, which classified as an appar- genes represented enzyme families such as UDP gluco-
ent member of the Cullin BTB/POZ E3 ligases, being syl and glucoronyl transferases (e.g., similar to GT72B1),
up-regulated. beta 1,3 glucan hydrolases (e.g., similar to BGL2),
nitrilases/nitrile lyases/berberine bridge enzymes/reticuline
Genes assigned to redox and metal handling oxidases/troponine reductases (e.g., similar to MES10),
cytochrome P450 s (e.g., similar to CYP71B34), peroxi-
The majority of the genes altered in their expression pro- dases (e.g., similar to PER12), dynamins (e.g., similar to
files associated with redox (Bin: 21) was up-regulated in ADL4) and GDSL-motif lipases. In contrast to the above,
the mutant (Fig. 9b, Supplementary Table S3), and included relatively few large enzyme families displayed an up-
thioredoxin members (e.g., similar to genes ACHT4 and regulation of their identified genes, such as invertase/pec-
ATHM4), genes for various glutaredoxin family protein tin methylesterase inhibitor family proteins (e.g., similar
members, and genes encoding a superoxide dismutase to C/VIF1) and glutathione S transferases (e.g., similar to
(e.g., similar to gene FSD2), and a SOUL heme-binding ATGSTU25).

13
Author's personal copy

650 Plant Mol Biol (2014) 84:635–658

Tuber gene expression Fig. 11  Analysis of the identified StBTB/POZ domain-containing ▸


protein 1 (StBTB/POZ1) sequence. a Nucleotide sequence encoding
the predicted open reading frame (ORF). b Amino acid translation of
While the primary focus of our gene expression analyses the predicted ORF, showing the location of the C-terminal BTB/POZ
was the above-ground tissues, our observed tuber pheno- domain (red highlight) and the N-terminal ARM repeat domain
types of elongated tuber shape and early sprouting were (black highlight). c InterProScan results showing the presence of the
similar to those reported by Morris et al. (2006), who BTB/POZ and ARM repeat domains
modified tuber isoprenoid biosynthesis via the plastid MEP
(2-C-methyl-d-erythritol) pathway. As Morris et al. (2006) region was found to be 2.3-fold (log2 value) greater in the
characterized DXS (1-deoxy-d-xylulose 5-phosphate syn- mutant, providing secondary verification of our PCR results.
thase), PSY (phytoene synthase) and PDS (phytoene desat- The potato gene models in the PGSC database showed
urase) transcript levels in their study, we carried out a simi- little physical separation between the two genes mod-
lar assessment of these transcripts for both wild type Bintje els (Supplementary Fig. S3a, b), approximately 100 bp.
and up tubers (Fig. 10). Using RT-PCR, we noted increased Based on these observations, and the similar level of
levels of DXS and PSY transcripts in tubers of the mutant, expression noted for these two genes in the up mutant, we
which was similar to that reported by Morris et al. (2006) hypothesized that the PGSC potato gene model was incor-
in their transgenics exhibiting elongated and early sprout- rect. Recent models generated by the Tomato Genome
ing tuber phenotypes. Annotation Group (ITAG) also suggest that the potato
(Sotub10g006050.1.1), tomato (Solyc10g005600.2.1) and
A BTB/POZ domain‑containing protein tobacco (NbS00001319g0217.1) gene model predictions
comprise a single gene containing these exons (Supplemen-
Our data showed that the first two potato genes flank- tary Fig. S3b). Interestingly, our RNA-seq transcript reads
ing the right border were significantly up-regulated in were similar to the predicted exon reads, except for the
the up mutant. In the potato genome browser, these two reads which aligned with the second intron of potato gene
genes (PGSC0003DMG401011239 and PGSC0003DMG (PGSC0003DMG401011239; chr10:665860..666280).
402011239) are annotated as two separate conserved genes The RNA-seq tracks in the PGSC database showed simi-
of unknown function (Supplementary Fig. S3a, b). RT- lar reads in the corresponding region (Supplementary Fig.
PCR results indicated that both genes were up-regulated S3b). A further database search using the 421 bp long
(Fig.  7a), and RNA-seq reads corresponding to this gene sequence covering these reads (chr10:665860..666280) as
region (Supplementary Fig. S3c) covered two annotated query revealed that this sequence has a secondary, perfect
potato gene models PGSC0003DMG402011239 and hit (3.9e−88; 100 % identity) in a region of chromosome 12
PGSC0003DMG401011239 (PGSC0003DMP400019889). that is not annotated for PGSC loci but houses ITAG gene
The raw reads from the Illumina GAII platform indicated models for tomato and potato (ITAG-Sotub12g020170.1.1;
similar, greater read counts for these two predicted genes chr12:35542420..35542540). One interpretation is that
for the mutant underperformer compared to Bintje (Sup- these RNA-seq reads are misaligned with our conserved
plementary Fig. S3c), illustrating the up-regulation of tran- gene of unknown function on chromosome 10 and actu-
script level in the mutant, and the low level of expression ally represent expression of the ITAG gene model on chro-
in Bintje. The corresponding gene expression value for this mosome 12. The PGSC RNA-seq data also indicated that
the gene expression pattern is similar across many differ-
ent potato tissues (Supplementary Fig. S3b). Furthermore,
while our RNA-seq reads across the predicted exons are
more abundant in the up mutant when compared with
wild type Bintje, our RNA-seq reads for the second intron
region show little difference in abundance between Bintje
and up (Supplementary Fig. S3b), providing further sup-
port for the idea that these reads are misaligned with this
gene model.
To confirm that the two predicted PGSC gene mod-
Fig. 10  Reverse transciptase PCR analysis of various potato isopre- els actually did represent a single gene, we used prim-
noid biosynthesis pathway genes in tubers of wild type Bintje and the ers designed against the start and stop codons based on
up mutant. RNA was extracted from freshly harvested mature potato the ITAG gene model Sotub10g006050.1.1, and carried
tubers. Primer information used in RT-PCR is given in Supplemen-
out RT-PCR using leaf cDNA from the mutant. If the
tary Table S2. EF-1α potato elongation factor 1-α (control), DXS
1-deoxy-d-xylulose 5-phosphate synthase, PSY phytoene synthase, ITAG gene models were correct, an amplification prod-
PDS phytoene desaturase uct of approximately 3 kb would result. Our PCR results

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 651

13
Author's personal copy

652 Plant Mol Biol (2014) 84:635–658

generated a 3 kb fragment (Supplementary Fig. S4), indi- eyes. These suggested that the single insertion resulted
cating that the two predicted genes of the PGSC potato in numerous pleiotropic effects, causing a range of phe-
model actually comprised a single gene. To obtain the notypes. Our in vivo and in vitro growth data supported
complete transcript sequence, we used 5′ and 3′ RACE each other, with the exception of the number and weight
to identify a full-length transcript, representing an open of tubers and number of axillary branches. In vitro plants
reading frame of 3,051 bp (Fig. 11a; Genbank Acces- of the up mutant produced more axillary branches; how-
sion No. KC979135), encoding a predicted protein of ever we observed fewer axillary branches in the mutant up
1,017 amino acids (Fig. 11b). A BLAST analysis of the plants during in vivo plant growth compared to wild type
predicted protein yielded hits to several hypothetical/ Bintje This trait may explain, to some extent, the greater
predicted plant orthologs (castor bean, soybean, cucum- number of tubers in up, and the subsequently greater tuber
ber, grape, tomato), representing a BTB/POZ domain- weight per mutant under in vitro conditions. Under green-
containing protein (Supplementary Fig. S5). As such, our house and growth chamber experiments, up mutant plants
potato gene (StBTB/POZ domain-containing protein 1) exhibited earlier senescence, resulting in less time for
yielded a predicted translation product with a BTB/POZ tuber formation and bulking, possibly causing lower tuber
(Broad complex, Tramtrac, Bric-a-brac; also known as number and overall reduction in yield. Observed differ-
Pox virus and Zinc finger) conserved domain near the ences in mutant phenotypes between in vitro and ex vitro
C-terminus of the predicted protein, as well as an Arma- growth conditions could be due to environmental factors
dillo (ARM) repeat domain towards the N-terminus (e.g., gaseous environment, in vitro sucrose supply, tem-
(Fig. 11b, c). perature and light) to which the mutant may be sensitive,
During this study, our focus on endogenous expres- which are less controlled in soil grown plants compared to
sion of StBTB/POZ1 was on foliar tissues, and our results in vitro plants.
indicated a relatively low level of endogenous expression In this study, reverse transcriptase PCR results showed
in wild type potato, compared to the up mutant. Given the both up- and down-regulation of genes on both sides of the
novelty of this gene, to further assess normal expression of T-DNA insertion. In almost all cases of activation tagging
StBTB/POZ1 in potato, an analysis of PGSC RNA-seq data using the CaMV 35S enhancer, the gene immediately adja-
was carried out, and indicated that this gene is expressed cent to the right border has been up-regulated (Weigel et al.
in many tissues/organs under various conditions (Supple- 2000; Ahn et al. 2007; Wan et al. 2009), leading eventually
mentary Fig. S3b; data not shown). Using leaf expression to the observed phenotype(s). In our study, the first two pre-
as the baseline, low levels of StBTB/POZ1 expression was dicted genes adjacent to the right border were strongly up-
also noted for stem, flowers and young/mature tubers. In regulated in the up mutant. On closer analysis, it became
contrast, greater expression was noted for roots, stolons, apparent that the potato gene model was incorrect, and that
sprouting tubers and fruit. RNA-seq data also indicated that these two predicted open reading frames actually repre-
StBTB/POZ1 expression was induced under certain stress sented a single open reading frame. This was much more
conditions. While wounding, heat shock and mannitol treat- in line with the gene model predictions generated by ITAG.
ments were non-inductive, salt stress induced StBTB/POZ1 Hence, the up-regulated gene adjacent to the right border
expression, and Phytophthora infestans treatment sig- identified here, located on chromosome 10 and predicted
nificantly enhanced abundance (data not shown). These to be a BTB/POZ domain-containing protein, is believed to
RNA-seq profiles can be accessed through the Spud DB be the actual tagged gene ultimately leading to the down-
Genome Browser (http://potato.plantbiology.msu.edu/cgi- stream phenotypes.
bin/gbrowse/potato/), viewing Landmark or Region In addition to the above gene, the PCR-based gene
chr10:659000..670000. expression analysis of the predicted genes extending up
to 400 kb from the left and right border of the insertion
revealed that several other genes were modified in their
Discussion expression profiles, not only the first gene adjacent to the
right border/enhancers. Generally, most activation tag
The activation tagged mutant underperformer (up) was studies have assessed gene expression in close proximity
initially identified by its dwarf and axillary branching to the insertion (Weigel et al. 2000; Imaizumi et al. 2005;
phenotype under in vitro growth conditions. Further in Wan et al. 2009) and have not considered chromosomal
vitro and in vivo studies revealed additional mutant phe- locations further upstream or downstream of the inser-
notypes, including loss of flowering (characterized by tion. However, gene expression profiling of the Arabidop-
small floral buds and premature floral bud abscission), sis activation tagged mutant K3571 revealed 1,273 genes
early senescence, reduced tuber dormancy and changes in with greater than twofold RNA abundance compared to
tuber size, tuber shape, tuber number and number of tuber control. Among 244 genes in a 984 kb region off of the

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 653

right border, 201 genes were up-regulated and six were Gene expression, pathway modifications, and up mutant
down-regulated (Ahn et al. 2007). Several explanations for characteristics
the activation of multiple genes were suggested, such as:
(1) the scanning action of an activator bound to the 35S The mutant phenotypes noted here represented those occur-
enhancer, which scans the genome and activates the genes ring above-ground (dwarf stature, slow growth, prema-
in its path until it meets with an insulator (Bell and Felsen- ture floral bud abscission, premature leaf senescence) and
feld 1999), (2) chromatin changes and re-arrangements below-ground (elongated tuber shape, reduced tuber dor-
may occur due to the T-DNA insertion, reducing gene mancy and early sprouting, other tuber quality traits). Our
repression, and (3) the T-DNA insertion serves to differ- foliar gene expression profiling results indicated numer-
entially express a nearby gene, such as a transcription fac- ous gene expression perturbations for different pathways.
tor, which has a subsequent impact on downstream genes. Various genes in the photosynthesis pathway, hormone
At present, it is unknown how many of these explanations metabolism, abiotic and biotic stress pathways, starch and
may have contributed to the observed up gene expression sucrose metabolism, minor carbohydrate metabolism, cell
changes. wall degradation proteins, proteolysis pathway, and redox
reaction pathway and transport were up-regulated in the
Visualization and assessment of gene expression data mutant. Furthermore, there appeared to be a shift in car-
bon metabolism, with increased gene expression towards
A more comprehensive comparison of global gene expres- carbohydrate metabolism, and reduced gene expression
sion between the Bintje control and the up mutant using towards lipid metabolism in the mutant. In contrast, numer-
RNA-seq revealed that more than 1,600 genes, represent- ous genes involved in DNA synthesis, cell division and cell
ing loci across all 12 chromosomes, were differentially cycle, cell organization, development, transcription factors,
expressed in foliar tissues of the mutant. Using Map- signaling, protein modification and secondary metabolism
Man software (Thimm et al. 2004), we viewed the data were down-regulated in the mutant. At this stage, it is dif-
at different levels of resolution, from the global level, the ficult to assess the contribution of each modified gene to
level of discrete processes, or down to the single gene the observed phenotypes of the mutant, although there are
level. The overview of expression levels indicated that the many changes in the mutant as a result of our activation tag
total number of genes down-regulated was greater than insertion. As cell division and cell expansion are key con-
the total number of genes up-regulated in the up mutant. tributors to overall plant size and form, the strong down-
These differentially expressed genes sorted into numer- regulation observed for DNA synthesis, cell cycle and cell
ous categories, representing different metabolic and func- division genes in the up mutant are expected to be key
tional pathways, and indicated that multiple perturbations contributors to the phenotype. We can also identify vari-
of normal plant gene expression and functional/metabolic ous genes modified in their expression, and shown in other
pathways were taking place within the mutant. This is studies to be associated with similar phenotypes. For exam-
not surprising, and can be expected to vary depending on ple, several Aintegumenta/Aintegumenta-like genes were
where the tagged locus fits into various pathways within down-regulated in the up mutant. Given the importance of
the plant. For example, an activation-tagged Arabidop- these genes in regulating cell division and associated flo-
sis line overexpressing a MYB transcription factor gene ral organ and shoot development (Krizek 2009; Mizukami
(PAP1) was found to over-express 38 genes (Tohge et al. and Fischer 2000), their reduced expression in the mutant
2005). In contrast, ectopic expression in an activation- could contribute to the smaller stature and reduced floral
tagged Arabidopsis line of the ORE7/ESC gene, an AT- bud development observed. In addition, our results indi-
hook DNA binding protein, resulted in 1,096 genes show- cated the up-regulation of DFL1 in the up mutant, whose
ing at least a two-fold change in gene expression in the over-expression has previously been described as causing
mutant compared to wild type (Lim et al. 2007). Hence, inhibition of shoot elongation, and subsequent dwarfness
our large number of observed gene expression changes in Arabidopsis (Nakazawa et al. 2001). We also noted that
can be viewed as reasonable. In addition, while many several members of the Growth Regulating Factor (GRF)
genes were altered in their expression profiles in foliar gene family were down-regulated in the mutant, and these
samples of our mutant, previous studies have indicated transcription factors play a role in the regulation of GA-
tissue specific modification of gene expression in activa- induced internode/stem elongation (van der Knaap et al.
tion-tagged mutants (Weigel et al. 2000). Therefore, while 2000). Similarly, plant β-1,3-glucanases, involved in path-
we were able to catalog the gene expression changes in ogen defense as well as a wide range of normal develop-
leaf tissues using RNA-seq, it is probable that a different mental processes like cell division and cell wall remodeling
set of differentially-expressed genes may occur in other (Doxey et al. 2007) were also down-regulated in the up
tissues. mutant.

13
Author's personal copy

654 Plant Mol Biol (2014) 84:635–658

Given the significant role of hormones in controlling mutant displayed up- and down-regulation of various Myb
plant development, it was not surprising that various hor- transcription factors, up-regulation of numerous proteases
mone-associated pathways appeared modified in the up (serine, cysteine, aspartate, metallo), various ubiquitin-con-
mutant. For example, auxins are closely connected with jugating enzymes and E3 ligases, several pectate lyases and
numerous plant growth and development processes (e.g., polygalacturonases, allene oxide synthase, down-regulation
lateral root differentiation and apical dominance), and they of several GDSL-motif lipases, osmotin-like proteins. The
are also known to induce the expression of several genes modification of the same types of pathways and genes in
like DFL1 and SAURs (Nakazawa et al. 2001). In addition, the up mutant would be expected to contribute to the earlier
the overexpression of the IAMT1 gene, which encodes an abscission and senescence characteristics.
indole-3-acetic acid (IAA) carboxyl methyltransferase that In consideration of the tuber phenotypes observed for
converts IAA to methyl-IAA ester (MeIAA), affects auxin- the up mutant, several of the pathways noted as being mod-
regulated processes (Qin et al. 2005). In the present study, ified, based on RNA-seq data, could have direct implica-
DFL1 (discussed above) and SAUR were up-regulated, tions for the observed phenotype. Carbohydrate metabo-
whereas IAMT1 was down-regulated in the up mutant, sug- lism perturbations can have distinct effects on potato tubers
gesting modifications in auxin response potential. (Frommer and Sonnewald 1995; Morris et al. 2006). For
The phytohormones auxin and ethylene, and gradients example, transgene-mediated reduction of plastid ATP/
associated with these also play a role in abscission and ADP-transporter activity and consequent reduction in tuber
senescence (Basu et al. 2013; Taylor and Whitelaw 2001), starch content results in an elongated tuber phenotype
traits which were noted to occur prematurely in the up (Tjaden et al. 1998). Our RNA-seq results indicated that
mutant. Auxin also inhibits transcription of various genes gene expression in the up mutant was modified for various
associated with both processes (Noh and Amasino 1999; genes associated with major (starch, sucrose) and minor
Tucker et al. 2002) and the auxin response factors, ARF1 carbohydrate metabolism, as well as photosynthesis, and
and ARF2, have been shown to regulate senescence and this may have contributed to the overall tuber phenotype.
floral organ abscission in Arabidopsis (Ellis et al. 2005). Several phytohormones are known to contribute to the
In addition, these traits are also induced by reduced avail- control of tuber dormancy and sprouting (Suttle 2004).
ability of carbon assimilate (Marcelis et al. 2004). As men- ABA is needed for dormancy maintenance, and declines
tioned above, RNA-seq data indicated that carbohydrate during dormancy progression, while sensitivity to cytokinin
metabolism and auxin response potential appeared modi- increases as dormancy progresses, and cytokinin increase
fied in the mutant, and may contribute to the early floral is associated with dormancy release and sprouting. In con-
bud abscission and leaf senescence observed. trast, GA is a negative regulator of tuberization (Bachem
Various expression profiling studies have characterized et al. 2001; Xu et al. 1998). The knockdown of a steroid
the genes associated with pedicel/floral organ abscission dehydrogenase transcript (GANGLY) in potato resulted in
and senescence (Breeze et al. 2011; Cai and Lashbrook a modification of active GA levels, and also elongated tuber
2008; Nakano et al. 2013). During these processes, expres- and sprouting phenotypes (Bachem et al. 2001), similar to
sion of genes associated with different functional path- the observations noted for the up mutant. We noted that
ways are modified. These include genes associated with expression of various GA-regulated, GA-responsive and
transcriptional regulation (e.g., Myb transcription factors, GA homeostasis genes were modified in the up mutant and
ERF ethylene response factors, ARF auxin response fac- could impact overall tuber phenotype.
tors), receptor-like kinases (e.g., FLS2, wall associated A study by Morris et al. (2006) modified tuber iso-
kinase), cell wall degrading, modifying and modeling pro- prenoid biosynthesis via the plastid MEP (2-C-methyl-
teins (e.g., cellulose synthase, expansin, invertase/pectin d-erythritol) pathway, which resulted in significantly elon-
methylesterase inhibitor, pectate lyase, polygalacturonase, gated tubers, as well as early sprouting. These phenotypes
XTH endotransglucosylase/hydrolase), hormone-associ- closely resembled phenotypes observed with the up mutant.
ated regulators for biosynthesis, action and transport (e.g., In addition, when we assessed DXS (1-deoxy-d-xylulose
allene oxide synthase, auxin efflux carrier, indole-3-acetic 5-phosphate synthase), PSY (phytoene synthase) and PDS
acid-amido synthetase, gibberellin oxidase), lipid metabo- (phytoene desaturase) transcript levels for both wild type
lism (e.g., GDSL-motif lipase/hydrolase family), stress Bintje and up tubers, we noted increased levels of DXS and
response (e.g., osmotin, pathogenesis-related protein) and PSY transcripts in the mutant, and no difference for PDS
proteolytic degradation (e.g., subtilase, ubiquitin conjugat- transcript levels, results which paralleled those of Morris
ing enzyme). RNA-seq analysis of the up mutant in this et al. (2006), suggesting that similar mechanisms are asso-
present study revealed that many of the same pathways ciated with the up tuber characteristics. The MEP pathway
and gene types were modified in the mutant. As examples provides intermediates for the phytohormones cytokinin,
of the types of genes listed above, foliar tissues of the up gibberellic acid (GA) and abscisic acid (ABA), and Morris

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 655

et al. (2006) noted enhanced levels of cytokinin, trans-zea- The process by which StBTB/POZ1 could lead to
tin riboside in tubers of their transgenic potatoes. While we the complex phenotype manifested by the up mutant is
did not quantify cytokinin levels in up tubers, we did note unknown. However, in the CUL3-BTB multi-subunit E3
that cytokinin metabolism gene expression (cytokinin oxi- ligase, the BTB/POZ domain interacted with CUL3 (Diet-
dase/dehydrogenase, isopentenyl transferase 3) was modi- erle et al. 2005), and the ARM repeat domain would be
fied in the mutant. All of the above results further suggest expected to interact with other proteins, providing sub-
that some modulation of plant phytohormone signaling strate specificity for the target protein to be ubiquitinated.
and response was associated with the various above- and The two previously mentioned Arabidopsis proteins
below-ground up mutant phenotypes. encompassing the ARM and BTB/POZ domains (Dieterle
We have described several potential genetic contribu- et al. 2005) encoded ARIA (Kim et al. 2004) and ABAP1
tions to the up tuber phenotype above. van Eck et al. (1994) (Masuda et al. 2008). ARIA represents a component of the
described multiple alleles that control tube shape, with the ABA signaling pathway, while ABAP1 is involved in DNA
round (Ro) locus, the major QTL for shape, mapping to replication and cell proliferation. In both cases, the ARM
chromosome 10, the same chromosome on which our activa- repeats interacted with transcription factors; ARIA with the
tion tag is located. It remains to be determined if the tag has ABA response element binding factor ABF2 (Kim et al.
directly impacted the Ro locus, or if the long tuber pheno- 2004), and ABAP1 with several members of the NAC, AP2
type of up is due to pleiotropic effects of the activation tag. and TCP transcription factor families (Masuda et al. 2008).
Hence, StBTB/POZ1 action may involve the modulation of
The candidate tagged gene: a BTB/POZ domain‑ and ARM various transcription factors, with the potential to alter the
repeat domain‑containing protein expression of many genes and pathways in the up mutant.
Overexpression of ABAP1 limited DNA replication during
Based on its location off of the right border of the T-DNA mitosis, and decreased cell proliferation, which could be
and adjacent to the CaMV 35S enhancers, as well as the considered somewhat similar to the reduced levels of cell
clearly up-regulated gene expression in foliar tissues of the cycle-associated and cell division-associated gene expres-
up mutant, the candidate activation tagged gene is believed sion noted for the StBTB/POZ1-overexpressing up mutant.
to encode a predicted protein with a C-terminal BTB/POZ Several BTB/POZ domain-containing proteins are interac-
domain, as well as an N-terminal Armadillo (ARM) repeat tion partners with the Cullin component of the E3 ubiqui-
domain, which we named StBTB/POZ domain contain- tin ligase complex (Thomann et al. 2005) and several plant
ing protein 1 (StBTB/POZ1). BLAST analyses indicated proteins, including those associated with plant hormone
close similarity to several hypothetical/predicted proteins, signaling (Santner and Estelle 2010) are targeted for deg-
indicating that StBTB/POZ1 represents a novel, yet-to-be radation via the E3 ubiquitin ligase complex. In addition,
characterized protein. The BTB/POZ domain-containing the strong up-regulation of various protease classes in the
proteins play an important role, possibly affecting global up mutant suggest that proteolysis plays a significant role
repression of transcription factors (Hur et al. 2001; Liu in the mutant, observations which may help to explain
et al. 2011), plant defense (Boyle et al. 2009; Qu et al. the molecular basis of the multiple phenotypic changes
2010), protein degradation (Yuasa et al. 2008) and cell observed, as well as the apparent broad gene expression
death (Sadanandom et al. 2008). The BTB/POZ and ARM and metabolic pathway perturbations in the up mutant.
repeat domains are both known as protein–protein inter- This paper has characterized an activation tagged potato
action domains (Perez-Torrado et al. 2006; Tewari et al. mutant with several interesting phenotypic traits, at the
2010), suggesting that the protein product of StBTB/POZ1 developmental, growth form and global gene expression
may be modulating its effects through key protein–protein levels. Based on all of our information, we have put forward
associations. Two Arabidopsis proteins exhibiting a simi- a candidate gene (StBTB/POZ1) believed to be responsible
larly-organized N-terminal ARM repeat domain and C-ter- for this. To confirm this hypothesis, transgenic experiments
minal BTB/POZ domain, although with little sequence taking two different approaches are in progress. In one
similarity (8e−05) to StBTB/POZ1, were previously iden- approach, the StBTB/POZ1 ORF has been cloned behind
tified as Class V BTB domain-containing proteins. These a standard 35S CaMV promoter and introduced into wild
were shown to interact via their BTB domain with Cullin type Bintje, to see if over-expression of StBTB/POZ1 reca-
3A (Dieterle et al. 2005), thus flagging these proteins as pitulates the up mutant phenotype. In a second approach,
components of the multi-subunit CUL3-BTB E3 ubiquitin an RNAi construct targeting StBTB/POZ1 will be intro-
ligases. These results suggest that StBTB/POZ1 is also a duced into the up mutant, to see if this will cause a rever-
member of the CUL3-BTB E3 ubiquitin ligases, an impli- sion of the mutant back to a wild type form. The results of
cation which is supported by our MapMan analyses that these experiments will confirm if StBTB/POZ1 is truly the
placed StBTB/POZ1 within this pathway. tagged gene, responsible for the up mutant phenotype.

13
Author's personal copy

656 Plant Mol Biol (2014) 84:635–658

Acknowledgments  This work represents a portion of S. Aul- Molecular and functional characterization of Arabidopsis Cullin
akh’s doctoral dissertation. We want to thank Jared Carter (Depart- 3A. Plant J 41:386–399
ment of Horticulture, Virginia Tech) for help with growth chamber Doxey AC, Yaish MW, Moffatt BA, Griffith M, McConkey BJ (2007)
experiments, Robin Buell and Brieanne Vaillancourt, (Department of Functional divergence in the Arabidopsis beta-1,3-glucanase gene
Plant Biology, Michigan State University) for assistance with RNA- family inferred by phylogenetic reconstruction of expression
sequencing, and Kavita Aulakh for help with image preparation. This states. Mol Biol Evol 24:1045–1055
work was funded through Special Grants (2003-38891-02112, 2008- Ellis CM, Nagpal P, Young JC, Hagen G, Guilfoyle TJ, Reed JW
38891-19353 and 2009-38891-20092) from the United States Depart- (2005) AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE
ment of Agriculture, Hatch Project VA-135853, and operating funds FACTOR2 regulate senescence and floral organ abscission in
from the Commonwealth of Virginia to the Institute for Advanced Arabidopsis thaliana. Development 132:4563–4574
Learning and Research. Fischer L, Lipavska H, Hausman JF, Opatrny Z (2008) Morphologi-
cal and molecular characterization of a spontaneously tuberizing
potato mutant: an insight into the regulatory mechanisms of tuber
induction. BMC Plant Biol 8:117
References Frommer WB, Sonnewald U (1995) Molecular analysis of carbon par-
titioning in solanaceous species. J Exp Bot 46:587–607
Ahn JH, Kim J, Yoo SJ, Yoo SY, Roh H, Choi JH, Choi MS, Chung Harrison EJ, Bush M, Plett JM, McPhee DP, Vitez R, O’Malley B,
KS, Han EJ, Hong SM, Jung SH, Kang HJ, Kim BK, Kim MD, Sharma V, Bosnich W, Séguin A, MacKay J, Regan S (2007)
Kim YK, Kim YH, Lee H, Park SH, Yang JH, Yang JW, Yoo Diverse developmental mutants revealed in an activation-tagged
DH, Yoo SK, Lee JS (2007) Isolation of 151 mutants that have population of poplar. Can J Bot 85:1071–1081
developmental defects from T-DNA tagging. Plant Cell Physiol Hayashi H, Czaja I, Lubenow H, Schell J, Walden R (1992) Activation
48:169–178 of a plant gene by T-DNA tagging: auxin-independent growth in
Anders S, Huber W (2010) Differential expression analysis for vitro. Science 258:1350–1353
sequence count data. Genome Biol 11:R106 Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant
Ayliffe MA, Pallotta M, Langridge P, Pryor AJ (2007) A barley activa- repression of target genes by chimeric repressors that include
tion tagging system. Plant Mol Biol 64:329–347 the EAR motif, a repression domain, in Arabidopsis. Plant J
Bachem CW, Horvath B, Trindade L, Claassens M, Davelaar E, Jordi 34:733–739
W, Visser RG (2001) A potato tuber-expressed mRNA with Huang SS, Cerny RE, Bhat DS, Brown SM (2001) Cloning of an
homology to steroid dehydrogenases affects gibberellin levels Arabidopsis patatin-like gene, STURDY, by activation T-DNA
and plant development. Plant J 25:595–604 tagging. Plant Physiol 125:573–584
Basu MM, González-Carranza ZH, Azam-Ali S, Tang S, Shahid AA, Hur MW, Lee DK, Kwon HS (2001) Molecular mechanism of tran-
Roberts JA (2013) The manipulation of auxin in the abscission scription repression by HDAC and BTB/POZ-domain transcrip-
zone cells of Arabidopsis flowers reveals that indoleacetic acid tion factors. FASEB J 15:A517
signaling is a prerequisite for organ shedding. Plant Physiol Imaizumi R, Sato S, Kameya N, Nakamura I, Nakamura Y, Tabata S,
162:96–106 Ayabe S, Aoki T (2005) Activation tagging approach in a model
Bell AC, Felsenfeld G (1999) Stopped at the border: boundaries and legume, Lotus japonicus. J Plant Res 118:391–399
insulators. Curr Opin Genet Dev 9:191–198 Kakimoto T (1996) CKI1, a histidine kinase homolog implicated in
Boyle P, Le Su E, Rochon A, Shearer HL, Murmu J, Chu JY, Fobert cytokinin signal transduction. Science 274:982–985
PR, Després C (2009) The BTB/POZ domain of the Arabidop- Kardailsky I, Shukla VK, Ahn JH, Dagenais N, Christensen SK,
sis disease resistance protein NPR1 interacts with the repression Nguyen JT, Chory J, Harrison MJ, Weigel D (1999) Activation
domain of TGA2 to negate its function. Plant Cell 21:3700–3713 tagging of the floral inducer FT. Science 286:1962–1965
Breeze E, Harrison E, McHattie S, Hughes L, Hickman R, Hill C, Kim S, Choi HI, Ryu HJ, Park JH, Kim MD, Kim SY (2004) ARIA,
Kiddle S, Kim YS, Penfold CA, Jenkins D, Zhang C, Morris an Arabidopsis arm repeat protein interacting with a transcrip-
K, Jenner C, Jackson S, Thomas B, Tabrett A, Legaie R, Moore tional regulator of abscisic acid-responsive gene expression,
JD, Wild DL, Ott S, Rand D, Beynon J, Denby K, Mead A, is a novel abscisic acid signaling component. Plant Physiol
Buchanan-Wollaston V (2011) High-resolution temporal profil- 136:3639–3648
ing of transcripts during Arabidopsis leaf senescence reveals Kondrák M, Marincs F, Kalapos B, Juhász Z, Bánfalvi Z (2011)
a distinct chronology of processes and regulation. Plant Cell Transcriptome analysis of potato leaves expressing the tre-
23:873–894 halose-6-phosphate synthase 1 gene of yeast. PLoS ONE
Busov VB, Meilan R, Pearce DW, Ma C, Rood SB, Strauss SH (2003) 6:e23466
Activation tagging of a dominant gibberellin catabolism gene Krizek B (2009) AINTEGUMENTA and AINTEGUMENTA-LIKE6
(GA 2-oxidase) from poplar that regulates tree stature. Plant act redundantly to regulate Arabidopsis floral growth and pattern-
Physiol 132:1283–1291 ing. Plant Physiol 150:1916–1929
Busov V, Yordanov Y, Gou J, Meilan R, Ma C, Regan S, Strauss S Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan
(2011) Activation tagging is an effective gene tagging system in PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R,
Populus. Tree Genet Genom 7:91–101 Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and
Cai S, Lashbrook CC (2008) Stamen abscission zone transcrip- Clustal X version 2.0. Bioinformatics 23:2947–2948
tome profiling reveals new candidates for abscission control: Lim PO, Kim Y, Breeze E, Koo JC, Woo HR, Ryu JS, Park DH, Bey-
enhanced retention of floral organs in transgenic plants overex- non J, Tabrett A, Buchanan-Wollaston V, Nam HG (2007) Over-
pressing Arabidopsis ZINC FINGER PROTEIN2. Plant Physiol expression of a chromatin architecture-controlling AT-hook pro-
146:1305–1321 tein extends leaf longevity and increases the post-harvest storage
Costa V, Angelini C, De Feis I, Ciccodicola A (2010) Uncovering the life of plants. Plant J 52:1140–1153
complexity of transcriptomes with RNA-Seq. J Biomed Biotech- Liu Q, Yao F, Wang M, Zhou B, Cheng H, Wang W, Jin L, Lin Q,
nol 2010:853916 Wang JC (2011) Novel human BTB/POZ domain-containing zinc
Dieterle M, Thomann A, Renou JP, Parmentier Y, Cognat V, Lem- finger protein ZBTB1 inhibits transcriptional activities of CRE.
onnier G, Müller R, Shen WH, Kretsch T, Genschik P (2005) Mol Cell Biochem 357:405–414

13
Author's personal copy
Plant Mol Biol (2014) 84:635–658 657

Marcelis LF, Heuvelink E, Hofman-Eijer LR, Bakker JD, Xue LB Santner A, Estelle M (2010) The ubiquitin-proteasome system regu-
(2004) Flower and fruit abortion in sweet pepper in relation to lates plant hormone signaling. Plant J 61:1029–1040
source and sink strength. J Exp Bot 55:2261–2268 Suttle J (2004) Physiological regulation of potato tuber dormancy. Am
Massa AN, Childs KL, Lin H, Bryan GJ, Giuliano G, Buell CR J Potato Res 81:253–262
(2011) The transcriptome of the reference potato genome Sola- Suzuki Y, Uemura S, Saito Y, Murofushi N, Schmitz G, Theres K,
num tuberosum Group Phureja clone DM1-3 516R44. PLoS ONE Yamaguchi I (2001) A novel transposon tagging element for
6:e26801 obtaining gain-of-function mutants based on a self-stabilizing Ac
Masuda HP, Cabral LM, De Veylder L, Tanurdzic M, de Almeida derivative. Plant Mol Biol 45:123–131
Engler J, Geelen D, Inzé D, Martienssen RA, Ferreira PC, Taylor JE, Whitelaw CA (2001) Signals in abscission. New Phytol
Hemerly AS (2008) ABAP1 is a novel plant Armadillo BTB 151:323–340
protein involved in DNA replication and transcription. EMBO J Tewari R, Bailes E, Bunting KA, Coates JC (2010) Armadillo-repeat
27:2746–2756 protein functions: questions for little creatures. Trends Cell Biol
Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, 20:470–481
Matheis N, Schuster DK, Menasco DJ, Wagoner W, Lightner J, The Potato Genome Sequencing Consortium (2011) Genome
Wagner DR (2003) Activation tagging in tomato identifies a tran- sequence and analysis of the tuber crop potato. Nature
scriptional regulator of anthocyanin biosynthesis, modification, 475:189–194
and transport. Plant Cell 15:1689–1703 The Tomato Genome Consortium (2012) The tomato genome
Mizukami Y, Fischer RL (2000) Plant organ size control: AINTEGU- sequence provides insights into fleshy fruit evolution. Nature
MENTA regulates growth and cell numbers during organogen- 485:635–641
esis. Proc Natl Acad Sci USA 97:942–947 Thimm O, Bläsing O, Gibon Y, Nagel A, Meyer S, Krüger P, Selbig
Morris WL, Ducreux LJ, Hedden P, Millam S, Taylor MA (2006) J, Müller LA, Rhee SY, Stitt M (2004) MAPMAN: a user-driven
Overexpression of a bacterial 1-deoxy-D-xylulose 5-phosphate tool to display genomics data sets onto diagrams of metabolic
synthase gene in potato tubers perturbs the isoprenoid metabolic pathways and other biological processes. Plant J 37:914–939
network: implications for the control of the tuber life cycle. J Exp Thomann A, Dieterle M, Genschik P (2005) Plant CULLIN-based
Bot 57:3007–3018 E3s: phytohormones come first. FEBS Lett 579:3239–3245
Murashige T, Skoog F (1962) A revised medium for rapid growth and Tjaden J, Möhlmann T, Kampfenkel K, Henrichs G, Neuhaus HE
bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497 (1998) Altered plastidic ATP/ADP-transporter activity influences
Nakano T, Fujisawa M, Shima Y, Ito Y (2013) Expression profiling of potato (Solanum tuberosum L.) tuber morphology, yield and com-
tomato pre-abscission pedicels provides insights into abscission position of tuber starch. Plant J 16:531–540
zone properties including competence to respond to abscission Tohge T, Nishiyama Y, Hirai MY, Yano M, Nakajima J, Awazuhara
signals. BMC Plant Biol 13:40 M, Inoue E, Takahashi H, Goodenowe DB, Kitayama M, Noji M,
Nakazawa M, Yabe N, Ichikawa T, Yamamoto YY, Yoshizumi T, Yamazaki M, Saito K (2005) Functional genomics by integrated
Hasunuma K, Matsui M (2001) DFL1, an auxin-responsive GH3 analysis of metabolome and transcriptome of Arabidopsis plants
gene homologue, negatively regulates shoot cell elongation and over-expressing an MYB transcription factor. Plant J 42:218–235
lateral root formation, and positively regulates the light response Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice
of hypocotyl length. Plant J 25:213–221 junctions with RNA-Seq. Bioinformatics 25:1105–1111
Noh YS, Amasino RM (1999) Identification of a promoter region Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren
responsible for the senescence-specific expression of SAG12. MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly
Plant Mol Biol 41:181–194 and quantification by RNA-Seq reveals unannotated transcripts
Perez-Torrado R, Yamada D, Defossez PA (2006) Born to bind: and isoform switching during cell differentiation. Nat Biotechnol
the BTB protein–protein interaction domain. BioEssays 28:511–515
28:1194–1202 Tucker ML, Whitelaw CA, Lyssenko NN, Nath P (2002) Functional
Qin G, Gu H, Zhao Y, Ma Z, Shi G, Yang Y, Pichersky E, Chen H, analysis of regulatory elements in the gene promoter for an
Liu M, Chen Z, Qu LJ (2005) An indole-3-acetic acid carboxyl abscission-specific cellulase from bean and isolation, expression,
methyltransferase regulates Arabidopsis leaf development. Plant and binding affinity of three TGA-type basic leucine zipper tran-
Cell 17:2693–2704 scription factors. Plant Physiol 130:1487–1496
Qu N, Gan W, Bi D, Xia S, Li X, Zhang Y (2010) Two BTB proteins Usadel B, Poree F, Nagel A, Lohse M, Czedik-Eysenberg A, Stitt
function redundantly as negative regulators of defense against M (2009) A guide to using MapMan to visualize and compare
pathogens in Arabidopsis. Botany 88:953–960 Omics data in plants: a case study in the crop species, maize.
Radouani A (2003) Effect of plant growth regulators, plantlet attrib- Plant Cell Environ 32:1211–1229
utes and nutrient medium on in vivo and in vitro potato tuberiza- van der Knaap E, Kim JH, Kende H (2000) A novel gibberellin-
tion. In: Agronomiques Science (ed) Rabat. Institut Agronomique induced gene from rice and its potential regulatory role in stem
& Vétérinaire Hassan II, Morocco growth. Plant Physiol 122:695–704
Regan S, Gustafson V, Mallubhotla S, Chakravarty B, Bagchi M, van Eck HJ, Jacobs JM, Stam P, Ton J, Stiekema WJ, Jacobsen E
Siahbazi M, Rothwell C, Sardana R, Goyer C, Audy P, Li X-Q, (1994) Multiple alleles for tuber shape in diploid potato detected
Wang-Pruski G, De Koeyer D, Flinn B (2006) Finding the perfect by qualitative and quantitative genetic analysis using RFLPs.
potato: using functional genomics to improve disease resistance Genetics 137:303–309
and tuber quality traits. Can J Plant Path 28:S247–S255 Wan S, Wu J, Zhang Z, Sun X, Lv Y, Gao C, Ning Y, Ma J, Guo Y,
Sadanandom A, Mesmar J, Yang C, Ewan R, Carr C, O’ Donnell E Zhang Q, Zheng X, Zhang C, Ma Z, Lu T (2009) Activation tag-
(2008) The cell death regulator AtPUB17 directly interacts with ging, an efficient tool for functional analysis of the rice genome.
the BTB/POZ domain transcriptional repressor, AtBTB1 to con- Plant Mol Biol 69:69–80
trol disease resistance in plants. Comp Biochem Physiol A Mo Weigel D, Ahn JH, Blázquez MA, Borevitz JO, Christensen SK,
Integr Physiol 150:S178–S179 Fankhauser C, Ferrándiz C, Kardailsky I, Malancharuvil EJ, Neff
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory MM, Nguyen JT, Sato S, Wang ZY, Xia Y, Dixon RA, Harrison
manual. Cold Spring Harbour Laboratory Press, Cold Spring MJ, Lamb CJ, Yanofsky MF, Chory J (2000) Activation tagging
Harbour in Arabidopsis. Plant Physiol 122:1003–1013

13
Author's personal copy

658 Plant Mol Biol (2014) 84:635–658

Xu X, van Lammeren AA, Vermeer E, Vreugdenhil D (1998) The Zubko E, Adams CJ, Machaekova I, Malbeck J, Scollan C, Meyer P
role of gibberellin, abscisic acid, and sucrose in the regulation of (2002) Activation tagging identifies a gene from Petunia hybrida
potato tuber formation in vitro. Plant Physiol 117:575–584 responsible for the production of active cytokinins in plants. Plant
Yuasa S, Onizuka T, Shimoji K, Ohno Y, Hara M, Ogawa S, Fukuda J 29:797–808
K (2008) The Novel BTB/POZ protein, Rhobtb3, regulates mito-
chondrial function via the ubiquitin proteasome system. Circul
Res 103:E53–E54

13