Mapping Transcriptional Networks in Plants: Data-Driven Discovery of Novel Biological Mechanisms

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Annu. Rev. Plant Biol. 2016.67:575-594. Downloaded from www.annualreviews.org
Novel Biological Mechanisms

Allison Gaudinier and Siobhan M. Brady
Department of Plant Biology and Genome Center, University of California, Davis,
California 95616; email: sbrady@ucdavis.edu
Annu. Rev. Plant Biol. 2016. 67:575–94 Keywords

First published online as a Review in Advance on transcription factor, systems biology, regulation, chromatin, genetics
January 25, 2016
The Annual Review of Plant Biology is online at Abstract

plant.annualreviews.org
In plants, systems biology approaches have led to the generation of a variety
This article’s doi: of large data sets. Many of these data are created to elucidate gene expres-
10.1146/annurev-arplant-043015-112205
sion profiles and their corresponding transcriptional regulatory mechanisms
Copyright c 2016 by Annual Reviews. across a range of tissue types, organs, and environmental conditions. In an ef-
All rights reserved
fort to map the complexity of this transcriptional regulatory control, several
types of experimental assays have been used to map transcriptional regu-
latory networks. In this review, we discuss how these methods can be best
used to identify novel biological mechanisms by focusing on the appropriate
biological context. Translating network biology back to gene function in
the plant, however, remains a challenge. We emphasize the need for vali-
dation and insight into the underlying biological processes to successfully
exploit systems approaches in an effort to determine the emergent properties
revealed by network analyses.
575
Contents
BIGGER IS NOT NECESSARILY BETTER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
TRANSCRIPTION AND THE CENTRAL DOGMA
OF MOLECULAR BIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
TRANSCRIPTION FACTOR–CENTERED APPROACHES . . . . . . . . . . . . . . . . . . . . . 578
GENE-CENTERED APPROACHES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
AN UNBIASED APPROACH: MAPPING OPEN CHROMATIN . . . . . . . . . . . . . . . . . 581
IN PLANTA VALIDATION OF TRANSCRIPTIONAL REGULATORY
INTERACTIONS AND THEIR BIOLOGICAL SIGNIFICANCE . . . . . . . . . . . . . 581
LITERATURE SUPPORT FOR GENERATING HYPOTHESES
FROM NETWORKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
FOCUSED FUNCTIONAL PHENOTYPING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

EMERGENT PROPERTIES REVEALED:

LINKING BIOLOGICAL PROCESSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
LEVERAGING NETWORK TOPOLOGY AND METRICS . . . . . . . . . . . . . . . . . . . . . 585
DATA INTEGRATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
TEMPORAL DYNAMICS: FROM TRANSCRIPTION FACTOR
BINDING TO CHROMATIN ACCESSIBILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
PERSPECTIVES FOR THE FUTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
BIGGER IS NOT NECESSARILY BETTER

The term big data is used in popular culture to refer to the collection of massive amounts of data
in business, medicine, marketing, and science. It is thought that such data can be transformative
by, for example, improving hedge fund investments, delivering a commercial product to a cus-
tomer more quickly and cheaply, targeting medicine to diverse populations, or changing the face
of agriculture. With respect to biology, the big data generated by genomic and systems biology
approaches have increased the speed and depth at which biologists can ask and answer biological
questions. Ideally, big data in biology allows researchers to step back from studying single genes to
instead study biological processes and the organism as a whole. However, this idea has not come to
fruition equally across all areas of plant biology. These efforts are limited, in part, by researchers’
ability to test gene regulation and gene function in a high-throughput manner. Furthermore, an
overwhelming amount of data is available, but there is a lack of methods to appropriately interro-
gate these data in a way that takes into account the biological context of the question being asked.
Plant growth and survival rely on coordinating biomass production sufficient for reproduc-
tion while responding and adapting to a diversity of environmental conditions. Whether a plant
is sensing and responding to the number of hours of light or temperature during the day or
combatting pests, it integrates and processes a large number of signals, often with distinct devel-
opmental outcomes. The integration of internal and environmental inputs into organized growth
and development is in large part coordinated through controlled regulation of gene expression.
High-throughput transcriptomic and sequencing technologies have helped elucidate plants’ tran-
scriptional signatures during development and in response to a variety of environmental stimuli,
often at spatiotemporal resolution (8, 11, 35, 38, 66). Additional approaches have mapped genetic
or physical interactions between genes with the goal of making sense of these big data from a
network-based perspective (2, 12, 36, 51).
576 Gaudinier · Brady

In a gene network, the nodes represent genes and the edges represent relationships or in-
teractions between genes. These edges can describe a variety of interactions, including mRNA
or metabolite coexpression, physical interactions between proteins, and physical interactions be-
Node: a variable in a
tween transcription factors (TFs) and regulatory regions of DNA. In this review, we argue that gene network; in a gene
networks that incorporate regulatory information within the appropriate biological context (the regulatory network,
biological circumstances that frame the network being mapped or analyzed, taking into account the nodes are genes
the spatial, temporal, and environmental setting) are ultimately the most useful and potentially Edge: a relationship
transformative with respect to our understanding of plant biology. A significant proportion of between two nodes in
such mapped networks reported in the literature are transcriptional regulatory networks (1, 4, 12, a network; in a gene
regulatory network,
14, 16, 21, 22, 30, 32, 37, 39, 42, 45, 49, 55, 57, 58, 60–62, 64, 69, 70, 72–74, 76, 78, 81, 85, 86),
the edges represent
and these networks are therefore the focus of this review. Accounting for the correct spatial, tem- genetic, binding, or
poral, or environmental context has enabled transformative discoveries regarding the connection physical interactions
of previously unlinked pathways via hormone signaling or genes with multiple or moonlighting between genes
functions. We provide further perspective on how a contextual approach can help elucidate the Transcription factor
integration of the diverse signals that allow plants to adapt and survive in often complex and hostile (TF): a protein that
environments. binds to DNA and
has the capacity to
regulate the expression
level of the genes it
TRANSCRIPTION AND THE CENTRAL DOGMA binds to
OF MOLECULAR BIOLOGY
Transcriptional
The central dogma of molecular biology describes transcription of a DNA template into mRNA regulatory network:
as the first step in the conversion of genetic information to a functional product. Therefore, a network comprising
determining how gene expression is regulated within an appropriate context is an important step in transcription factors
and the genes they
understanding organismal growth and development. One mechanism to regulate gene expression
bind and regulate
is through TFs. TFs are proteins that enhance or impair the activity of transcriptional machinery
(including RNA polymerase II and the Mediator complex) at a gene (Figure 1). They can act in
complexes or alone to influence the expression of a target gene. The experimental tractability of
profiling mRNA abundance with either microarrays or next-generation sequencing technologies
has resulted in thousands of gene expression patterns in distinct cell types, tissues, and organs and
in response to a variety of abiotic and biotic stresses. In order to make sense of these patterns,
predictive networks have been generated based on similarities in gene expression (reviewed in 5,
a b
Mediator Mediator
complex complex
Pol II Pol II
Activation Inhibition
TF TF
Distal promoter Core Distal promoter Core

promoter promoter
Figure 1
Transcriptional regulation via regions of open chromatin. (a) A transcription factor (TF) acts as an activator
by recruiting core transcriptional machinery to commence gene transcription. (b) A TF acts as a repressor by
blocking or inhibiting the core transcriptional machinery from transcribing the gene. Additional
abbreviation: Pol II, RNA polymerase II.
www.annualreviews.org • Mapping Transcriptional Networks in Plants 577

28, 63). These networks, referred to as coexpression networks, provide a platform for hypothesis
generation and in planta testing. However, it is important to note the limitations of the hypotheses
that can be generated via coexpression networks.
The intent of coexpression network approaches is to group genes with similar expression pro-
files, on the assumption that a common TF or group of TFs is responsible for generating their
expression profile or pattern. More recent efforts have attempted to use computational methods
to identify putative TFs responsible for these patterns (3, 77). These hypotheses, however, need to
be further confirmed by direct experimental evidence showing a direct transcriptional regulatory
interaction. Researchers have also embraced more direct genome-wide experimental approaches
to map physical binding of TFs to promoters, including chromatin immunoprecipitation (ChIP)
coupled with sequencing (ChIP-seq), mapping of DNase I–hypersensitive sites, and heterologous
systems such as yeast one-hybrid (Y1H). However, these methods indicate only physical interac-
tions or regions of active chromatin, not gene regulation. Thus, placing the mapped interactions
in the context of transcriptional regulatory networks requires further experimental evidence. In

the next section, we describe experimental methods for detecting physical interactions between
TFs and their targets and more recent efforts to observe these interactions within an appropriate
context.
TRANSCRIPTION FACTOR–CENTERED APPROACHES

One approach to studying transcriptional regulation is to focus on the downstream targets of TFs
of interest. Researchers use this approach when they are trying to more thoroughly understand
a particular TF by identifying the genes that it directly targets. Generally, researchers select
these TFs because they are central or master regulators of a biological process and have well-
characterized mutant phenotypes. Knowing which genes are directly regulated by a TF of interest
can elucidate, in a more holistic manner, the regulatory functions of that TF, leading to a better
understanding of how and why a gene can function in one or more pathways related to hormone
signaling, development, or environmental responses.
ChIP is the most common in vivo technique for determining TF-DNA binding and provides
the most direct evidence for this binding. In this approach, proteins are cross-linked to chromatin,
the TF of interest is immunoprecipitated, and the DNA segments to which the TF is bound are
characterized, generally by a microarray or next-generation sequencing approach (Figure 2a).
ChIP is often most successful when a TF is expressed abundantly and across many cell types.
However, most TFs are often expressed at low levels or only in very specific cell types, and in
these cases, ChIP can be technically challenging. In addition, a TF may be expressed abundantly
but may regulate different targets depending on the cell type, tissue, organ, or environmental
stimulus to which the plant is exposed, and current ChIP protocols cannot capture the cell type
or context specificity of TF binding. Despite these limitations, ChIP has been used successfully
to identify the targets of many key regulators. Below, we describe a traditional ChIP experiment
along with two modified approaches that can alleviate some of these limitations and allow a better
assessment of TF targets in the appropriate, biologically relevant context.
Chang et al. (16) used ChIP-seq to characterize ethylene-dependent targets of ETHYLENE
INSENSITIVE 3 (EIN3), a master ethylene response transcriptional regulator. To link these
binding events to expression regulation, they generated a matching RNA sequencing (RNA-seq)
data set in the presence of ethylene at each time point. After ethylene treatment, EIN3 is highly
expressed in whole seedlings, which makes it an ideal TF for traditional ChIP assays. However,
the characteristics of EIN3 and its effectiveness in ChIP assays are not typical of TFs, many of
which have highly specific expression patterns and/or low abundance.

Assay Readout Network Requirements
a
TF Gene A Gene A
TF
TF antibody or
TF tagged translational fusion
Gene B Gene B
TF A B C
Gene C Gene C
b TF2 TF3
1
2 TF1 TF4 Promoter clone and
TF
TF library
Promoter Reporter 3
4
GOI
c
TF Gene A ARF GATA
Gene A
TGTCTC TGTCTC
Known TF binding sites and
TF tagged nuclei
Gene B Gene B
TGATAG TGATAG
A B
Figure 2
Three experimental approaches to identify transcriptional interactions and map components of transcriptional regulatory networks.
The Assay column shows the type of transcriptional interaction obtained in each experimental assay, the Readout column shows the
result of the experimental assay that will be interpreted, the Network column shows the type of transcription factor (TF)–promoter
interaction obtained and its directionality, and the Requirements column shows the resources needed to perform these assays.
(a) Chromatin immunoprecipitation (ChIP). The TF is cross-linked to chromatin, and DNA physically associated with the TF is
isolated and sequenced. This is a TF-centered approach designed to identify target regulatory regions of a gene of interest (GOI). An
antibody to the TF or a tagged translational fusion is required. (b) Yeast one-hybrid (Y1H). Promoters are cloned and screened against
a TF library in yeast. If a TF interacts with a promoter, a positive interaction is indicated by blue colonies (LacZ) or growth on media
containing a competitive growth inhibitor (not shown). This is a gene-centered approach designed to identify upstream TFs that
interact with the promoter of a GOI. A clone of the promoter and a collection of TFs are required. (c) DNase I hypersensitivity assay.
Regions of open chromatin that are likely bound by TFs and transcriptional machinery are digested by DNase I and sequenced.
Linking footprinting assays to known TF binding sites can indicate which TFs are binding to the regions of open chromatin. Databases
of known TF binding sites are required, and nucleus-tagging approaches are needed to increase the efficiency of the process.
In contrast to the central hormone regulator, EIN3, SPEECHLESS (SPCH) is a transcriptional

regulator of a critical asymmetric cell division in the stomatal development lineage (46). Stomata
are the pores on the leaf epidermis that regulate gas exchange. Because stomata represent only a
fraction of cells in leaves, enrichment methods are necessary to increase the efficiency and repro-
ducibility of SPCH ChIP and reliably determine downstream targets. To address this issue, Lau
et al. (42) increased the amount of input tissue 16-fold. They then divided this tissue into smaller
aliquots for cross-linking of the protein to DNA, nuclear isolation, and DNA shearing and pooled
the aliquots to generate one biological replicate. To ensure statistically significant identification of
SPCH target DNA sequence, they analyzed six biological replicates. Using this altered approach,
named maximized objects for better enrichment (MOBE)–ChIP, they were able to determine that
SPCH binds to other known genes integral for stomatal development and, surprisingly, genes
essential for brassinosteroid biosynthesis. RNA-seq profiling after SPCH induction determined

that 23% of the target genes are indeed regulated by SPCH. This link between stomatal devel-
opment and hormone biosynthesis is a powerful connection that has changed our understanding
of the genetic and signaling pathways that integrate to form this specialized cell type (42).
Transient assay reporting genome-wide effects of transcription factors (TARGET) is an al-
ternative and rapid approach to assay the direct regulation of TFs and overcome problems with
transient binding by TFs that are missed in traditional ChIP experiments (4). In this system, TFs
are expressed as a glucocorticoid receptor fusion, which allows their conditional nuclear import
upon the addition of dexamethasone. The addition of cycloheximide allows the monitoring of
direct transcriptional interactions because it impairs translation and thus de novo biosynthesis
of TFs and indirect transcriptional regulation. The cells are protoplasted, and gene expression
is profiled in the presence and absence of dexamethasone and in the presence and absence of
cycloheximide. Protoplasting allows the cells to be rapidly exposed to these chemicals and enables
reporting of the fast and transient TF-DNA interactions that cannot be detected by traditional
ChIP. An additional advantage of this approach is that cells can be subjected to a variety of treat-
ments, which allows researchers to observe condition-dependent binding and regulatory events
(58) and thereby the study the dynamics of transcriptional regulation (see Temporal Dynamics:
From Transcription Factor Binding to Chromatin Accessibility, below).
GENE-CENTERED APPROACHES
Gene-centered approaches represent an additional and complementary way to build transcrip-
tional regulatory networks. These approaches focus on target genes in an effort to identify up-
stream transcriptional regulators. Y1H is the most common technique to determine which TFs
potentially bind to a DNA segment of interest in vitro (Figure 2b). Y1H has been adapted to
high-throughput technology for efficient screening of thousands of potential interactions (23, 60).
Additionally, the development of complete gold-standard TF collections in Arabidopsis and maize
has made this approach feasible at a genome-wide scale in multiple plant species (13, 60).
Multiple research groups have used Y1H to map putative regulatory networks. From the per-
spective of understanding a biological process, this procedure casts a wider net by choosing target
genes based on expression data and known function. Taylor-Teeples et al. (74) described a net-
work characterizing xylem cell specification and secondary cell wall differentiation. Secondary cell
walls provide structural support and the waterproofing needed for xylem cells to transport water
and nutrients throughout the plant. In this study, the authors screened promoters of genes in-
volved in lignin, cellulose, and hemicellulose biosynthesis expressed in xylem cells and previously
characterized TFs known to play a role in xylem development. The resulting network displayed
highly interconnected binding by xylem-expressed TFs. Interestingly, it also showed a link be-
tween two previously characterized pathways involved in xylem specification—one mediated by
VASCULAR NAC DOMAIN 6 (VND6) and VND7 TFs (41, 82–84) and one mediated by
the five class III HOMEODOMAIN–LEUCINE ZIPPER (HD-ZIP III) TFs (15). The authors
determined that VND7 is an upstream regulator of at least one of the HD-ZIP III TF genes,
REVOLUTA (REV). In xylem development, REV is expressed early to organize cell patterning
within vascular tissue and is subsequently repressed by VND7 as xylem differentiation is initi-
ated. Taylor-Teeples et al. (74) determined that REV binds to the promoters and represses the
expression of enzymes associated with lignin biosynthesis. Thus, upon activation of VND7, REV
expression is repressed, and lignin biosynthesis is able to occur after cell patterning has occurred.
This heterologous yeast system allows researchers to identify testable hypotheses in planta in order
to gain a more complete understanding of xylem development.

An additional example of how the Y1H approach can enable researchers to use prior expression
knowledge to infer biology is a screen of the regulatory regions of the circadian clock genes
CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY )
against a collection of TFs known to be circadian regulated (61). This focused approach of using
known circadian-regulated TFs generates a defined scope for biological validation. In this study,
Pruneda-Paz et al. (61) identified CCA1 HIKING EXPEDITION (CHE) as a novel player in the
regulation of the circadian clock. CHE, in an antagonistic relationship with TIMING OF CAB
EXPRESSION 1 (TOC1), represses CCA1 and controls the period length in the circadian rhythm.
Circadian rhythms ensure that the plant can adjust its metabolic cycles, immunity responses, and
physiological changes in response to day and night cycles (48, 79).
AN UNBIASED APPROACH: MAPPING OPEN CHROMATIN

The third approach to mapping transcriptional regulatory networks is an unbiased, genome-wide

study of putative TF binding sites via regions of open chromatin using DNase I hypersensi-
tivity assays. The DNase I enzyme cleaves accessible chromatin regions referred to as DNase
I–hypersensitive sites. Open chromatin regions are typically associated with active transcriptional
regulation (i.e., regions that are actively engaged with RNA polymerase II and associated transcrip-
tional machinery, or regions that have bound TFs). The resulting DNA products of this cleavage
are sequenced to map the regions of open chromatin (75). Sullivan et al. (72) used this approach
to identify regions of open chromatin in a time course for dark-grown seedlings transferred to a
light treatment and a time course for seedlings exposed to heat shock. Heat shock can result in
oxidative stress and protein damage, which leads to poor plant growth and decreased yield.
If sequencing after DNase I cleavage is performed at sufficient depth, the precise nucleotide
sequence to which a TF is bound, or its footprint, can be identified, thus providing a snapshot
of the TF occupancy of DNA (Figure 2c). Combining this footprinting data with known TF
binding sites leads to the generation of a putative transcriptional regulatory network without
initially focusing on a specific TF or targets. The networks generated by Sullivan et al. (72) were
filtered by treatment and additionally by clustering of similar binding in the time course profiles
or known gene family function.
This technique, however, is limited by the knowledge of binding sites, which has greatly
improved in recent years but remains limited (80). TF consensus sequences have been determined
using protein-binding microarrays that were queried by probing with DNA-binding domains of
Arabidopsis TFs. These data have been expanded to include indirect evidence for untested TFs by
inferring that binding sites remain evolutionarily similar across TF families with related DNA-
binding domains (80). Thus, the size and hierarchy of networks generated through this approach
are limited mainly by binding site knowledge and sequencing depth.
IN PLANTA VALIDATION OF TRANSCRIPTIONAL REGULATORY

INTERACTIONS AND THEIR BIOLOGICAL SIGNIFICANCE
We have outlined a variety of experimental approaches that can be routinely used in plants to map
different components of transcriptional networks. The generation of such large data sets inherently
yields false positive results, and thus interactions to be pursued for biological significance should
be validated in planta. The next step in plant network biology is validating and analyzing the
biological significance of mapped interactions—specifically, the ability of a TF to regulate its
target gene in the appropriate biological context.

On approach to validate the occurrence of transcriptional regulation is genetic analysis. Two

main categories of phenotypes should be considered for interaction validation: the morphological
phenotype and the molecular phenotype. A morphological phenotype describes a quantifiable
change in some aspect of plant growth or architecture resulting from perturbation of the TF of
interest that is somehow associated with regulation of the target gene. A molecular phenotype
indicates a change in the transcript levels of a target gene of interest in a mutant background of a
TF relative to the wild type.
SHORT-ROOT (SHR) provides an example of both a morphological and molecular phe-
notype. This TF regulates root development and has several downstream functions, including
regulation of xylem development, endodermis development, and maintenance of the cells within
the quiescent center (6, 15, 18, 29, 44, 47, 54, 71). With respect to regulation of endodermis devel-
opment, the shr mutant root lacks an endodermal layer (6), which is considered a morphological
phenotype and indicates that SHR has an important role because its loss of function is sufficient
to cause a change in root morphology. SHR binds to the promoter of the TF gene SCARECROW
(SCR) and regulates its expression (71). In the shr mutant, SCR expression is nearly undetectable
(29), which can be determined by monitoring gene expression—for instance, by using real-time
quantitative polymerase chain reaction (qPCR)—or by monitoring the expression pattern of the
SCR promoter fused to a reporter gene (18, 67, 71). Monitoring the impact of SHR loss on its
target gene expression is considered validation of a molecular phenotype. However, the presence
of both a morphological phenotype and an associated molecular phenotype does not necessarily
indicate that SHR directly binds to the SCR promoter or that this binding is directly translated into
regulation of SCR gene expression. A ChIP-qPCR assay with an antibody that recognizes the SHR
protein indicated that SHR physically interacts with the SCR promoter (18). Conditional induction
assays provide additional evidence of regulation; in this example, induction of the SHR protein
fused to the glucocorticoid receptor (SHR::SHR-GR) in an shr mutant line containing an SCR
transcriptional reporter rescues both the morphological and molecular phenotypes (71). Rescue of
the molecular phenotype can be observed by the visualization of SCR expression via an SCR tran-
scriptional reporter and by rescue of the mutant layer phenotype—that is, visualization of a mor-
phological phenotype by a cell division in the mutant layer to produce an endodermis cell layer (71).
The network mapping methodologies described above can map hundreds or thousands
of potential interactions, but it is not feasible to test all interactions through this type of in
planta genetic analysis (Table 1). It is also not feasible for a single study to provide a thorough
understanding of all genes in a network. Furthermore, an expectation that a single TF will
Table 1 Techniques for biological validation to prove transcription factor (TF)–DNA binding, TF regulatory role, or both
Experimental
technique Evaluates System Confirms Appropriate network type
Misexpression of Change in target gene In planta or TF regulatory role ChIP, Y1H, or DNase I
TF expression heterologous
Misexpression of Change in target gene In planta TF regulatory role ChIP, Y1H, or DNase I
TF + pro- expression level or
moter:GFP/GUS domain
Transient assay Change in target gene Protoplasted cells or TF regulatory role ChIP, Y1H, or DNase I
expression tobacco cells
ChIP TF-DNA binding In planta TF-DNA binding Y1H or DNase I
Gel shift assay TF-DNA binding In vitro TF-DNA binding DNase I
Abbreviations: ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; GUS, β-glucuronidase; Y1H, yeast one-hybrid.

yield both a morphological and a molecular phenotype is not reasonable given the extensive
genetic redundancy in plants. This means that genetic validation requires higher-order mutant
combinations and transformation of transcriptional reporters to identify both a morphological
and a molecular phenotype. In a Y1H study, Brady et al. (12) mapped a network to determine the
upstream transcriptional control of TFs in root stele development. From this mapped network,
the authors and others analyzed mutants of 30 TFs in the network for altered root morphology.
Of these mutants, 5 (17%) displayed morphological phenotypes, likely because of functional
redundancy within the network. At a molecular level, however, the authors were able to detect
altered levels of target gene expression for 35 (54%) of the tested Y1H TF-DNA interactions.
Heterologous approaches such as transient assays in tobacco or protoplast-based systems pro-
vide an alternative to the approaches described above. Here, a promoter of a target gene is fused to
a reporter such as luciferase (Luc), green fluorescent protein (GFP), or β-glucuronidase (GUS),
activation of which shows that a TF can bind and regulate the expression of a target. Although
these types of assays do not provide genetic evidence or screen for regulatory interactions in the
correct biological context, they can be performed in a high-throughput manner and thus allow
for rapid screening. Ideally, interactions obtained using these assays will be followed up with
experiments that provide genetic evidence.
LITERATURE SUPPORT FOR GENERATING HYPOTHESES

FROM NETWORKS
It can be useful to do an in-depth literature analysis before performing genetic analysis or exper-
iments using high-throughput transient assays. Mining the literature for known interactions or
biological functions potentially allows researchers to draw powerful conclusions without perform-
ing large-scale validation experiments. However, networks are hypothesis-generating tools, and
using the literature to identify known interactions or other biological processes may lead inves-
tigators to ignore genes that were not the focus of previous published research—which, in many
ways, can generate the most exciting hypotheses and lead to the discovery of novel gene function.
Still, previous studies can provide insight and a quick explanation for network interactions. For
example, no previous studies had made the connection between the VND6/VND7 and HD-ZIP
III families that was found by Taylor-Teeples et al. (74). However, both classes of mutants have
published xylem phenotypes, and the previous results on the VND6/VND7 and HD-ZIP III gene
expression patterns and function clarified the link between the pathways of xylem patterning and
differentiation (15, 40, 82–84). This literature evidence supported prioritizing the validation of
this interaction in planta.
Sullivan et al. (72) used extensive literature-based knowledge about heat shock factor (HSF) TFs
as a basis for their network clustering analysis. Before these regions of chromatin were mapped,
three major HSFs—HSFA1a, HSFA1b, and HSF2A—had been reported to be important for a
quick response to heat shock (56). Sullivan et al. (72) used this knowledge to generate a subnetwork
from the DNase I–hypersensitive site mapping to determine which genes are differentially regu-
lated in response to heat stress and putatively dependent on these HSFs. Using this subnetwork,
they showed that many HSF TFs are responsive to heat shock and are potential regulators of
both common and unique target genes. Together, these data indicate that there is a core group
of HSF TFs involved in the heat shock response, whereas others function in separable modules.
This prior knowledge of HSF gene family function allowed the researchers to focus their analysis
on a subset of genes differentially bound in the DNase I hypersensitivity assay. Furthermore, the
integration of these previous findings assisted in elucidating the potential function of HSF family
members whose role is less well understood.

FOCUSED FUNCTIONAL PHENOTYPING

Translating network interactions to plant phenotypes requires insight into the context of how the
network was mapped. Experimental approaches that use plant tissue to determine or infer TF-
DNA binding (ChIP or DNase I hypersensitivity) make it straightforward for researchers to deter-
mine where they should focus their phenotyping: the same conditions that were sampled for net-
work mapping. For networks developed using heterologous systems, such as Y1H, networks are not
mapped from in planta experiments. This creates an interesting challenge in validation because the
network interactions could potentially take place under a variety of conditions and/or in specific cell
types. Focusing on predicted phenotypes, however, makes this issue manageable. Using this bio-
logical context to drive analysis and validation of the network increases the probability of success.
Predicted phenotypes are typically based on the annotation of genes involved in the network
and do not necessarily require determination of transcriptional regulation. An example of this is
a Y1H network that mapped transcriptional control of aliphatic glucosinolate biosynthesis (45).
The authors chose a subset of TFs for functional validation based on the availability of TF mutants
and high connectivity within the network. Of the TF mutants tested, 75% were validated with
targeted metabolite analysis (aliphatic and indole glucosinolates) in two different organs (seeds
and leaves) and in two different environmental conditions relative to the wild type (45). Although
the TFs that were screened were not chosen based on their organ or environmental specificity
of expression, determination of glucosinolate phenotypes revealed likely organ or environmental
context specificity. This focused functional validation revealed that glucosinolate biosynthesis can
be transcriptionally regulated by some TFs independent of environment and cell type, whereas
other TFs have a more narrow scope of regulatory control: a specific tissue, a specific environmental
stress, or a combination of both.
Similarly, in their Y1H studies that identified putative transcriptional regulators of CCA1,
Pruneda-Paz et al. (60, 61) used circadian clock transcriptional readout as their phenotype of
interest. Overexpression of either CHE or FLOWERING BHLH 1 (FBH1) in a CCA1:Luc
background changed the control circadian readout of the Luc reporter. This molecular phenotype
provides direct evidence that the TFs CHE and FBH1 affect the expression level of CCA1 and
subsequently the rhythm of the circadian clock—an indication of impaired circadian clock function.
This well-established and focused assay was an efficient and direct confirmation of CHE and FBH1
function in the circadian clock.
Focused phenotyping from predictive networks can also lead to increased validation rates. Lee
et al. (43) linked novel genes to known pathways through integrative predictive network mapping
using the AraNet algorithm. As a biological test of the method, their phenotyping focused on
the function of genes in seed pigmentation. The validation of mutants of genes associated in
the network with known pigmentation genes was 16%, which represents a tenfold increase in
validation compared with screening a random T-DNA mutant pool. This focused approach to
gene discovery can be more successful in terms of validation rate and can reduce effort relative to
classical genetic screens.
EMERGENT PROPERTIES REVEALED:

LINKING BIOLOGICAL PROCESSES
One powerful aspect of analysis of mapped transcriptional networks is the ability to reveal emergent
properties of the system (31). Emergent properties cannot be determined when focusing on the
function of individual genes. One such emergent property is previously undefined connections
between biological processes.

Plant biologists have been aware of hormone crosstalk for decades. Genes identified as be-
ing involved in multiple hormone signaling pathways show phenotypes in response to multiple
hormones (7, 10). Thus, it was understood that these pathways are coregulated, but the exact
mechanism by which this occurs was unknown. Chang et al. (16) found that the proportion of
hormone-related genes among EIN3 targets (46%) was more than twice the overall proportion of
hormone-related genes in the Arabidopsis genome (21%), and many of these genes were involved in
more than one hormone response. Identification of EIN3 targets could partly elucidate the mech-
anism by which this hormone crosstalk occurs. EIN3 targets include genes involved in responses
to auxin, jasmonic acid, gibberellic acid, brassinosteroid, abscisic acid, cytokinin, and salicylic
acid. Some mechanisms can, in part, be directly revealed. For instance, Chang et al. (16) showed
that EIN3 transcriptionally regulates the PINOID auxin efflux transporter and transcriptionally
regulates type A and type B Arabidopsis response regulator (ARR) TFs, which are considered the
direct output of cytokinin signaling, thus providing a direct link and explanation of how hormone
crosstalk can be mediated.

An additional example of an emergent property revealed by these studies is a Y1H analysis of
glucosinolate regulation that linked a classic developmental gene with glucosinolate production
(45). AINTEGUMENTA (ANT ) is known for its role in controlling ovule development, lateral
organs, and plant organ size (20). ANT binds to four promoters of aliphatic glucosinolate enzymes
in the network, and metabolite analysis of ant showed the largest effect for mutants analyzed
in both the glucosinolate profiles and altered levels of glucosinolate metabolites (45). Previous
research on ANT had focused almost exclusively on its role in plant development. Glucosinolates
are central defense molecules in the Brassicaceae, and discovering a novel role in plant defense
for this critical regulator of plant development was possible only through this gene-centered
approach to network mapping.
LEVERAGING NETWORK TOPOLOGY AND METRICS

Independent of gene identity, properties of the network can inform researchers about potential
gene functionality. Network topology refers to specific quantifiable characteristics or patterns
of nodes (TFs or target genes) and their connections with one another. A commonly accepted
biologically relevant attribute of networks is the essentiality of hub nodes, as determined by
characterizing the phenotypes of mutations in hub proteins relative to non-hubs in a protein-
protein interaction network (34). Independent of the type of interactions in a given network, a
hub is a node with a large number of interactions relative to other nodes. Hub genes can also be
integral connectors between groups of genes.
Additional characterization of node properties within a protein-protein interaction network—
specifically, gene expression in space and time—resulted in the identification of so-called party
and date hubs (27) (Figure 3). Party hubs are nodes that act within a module (a region of highly
interconnected genes within the network) and function with all their interactors simultaneously to
regulate a biological process (27); date hubs are nodes that have many interactions across multiple
biological processes and potentially in different contexts. Misregulation or mutation of a hub
gene, especially a date hub, significantly alters the network structure and communication between
biological processes (27). Although the concept of date and party hubs has not been linked in the
literature to transcriptional regulatory networks, the EIN3-dependent gene regulatory network
illustrates that EIN3 is an example of a date hub, as it regulates the response of most hormone
signaling pathways in an ethylene-dependent and temporally distinct fashion. Mutants in EIN3 do
not display the classical seedling triple response when exposed to ethylene (26, 65), a phenotype that
likely reflects the complexity of the hormone crosstalk and regulation of its downstream pathways.

Z
A
B
Figure 3
Example of how network topology can reveal transcription factor (TF) function. The nodes (TFs and target
genes) are shown as circles, and the edges (interactions between TFs and DNA) are shown as arrows. The
colored circles indicate a node with a high out-degree (orange), a node with a high in-degree bound by
partially redundant TFs A and B (blue), a date hub ( green), and a party hub ( purple). The red arrows show a
feed-forward loop with nodes X, Y, and Z.
In transcriptional regulatory networks, researchers must consider both in-degree and out-
degree distribution when defining a hub. The node in-degree refers to the number of TFs bind-
ing to a target gene’s promoter, and the out-degree refers to the number of targets of a given
TF (Figure 3). EIN3, which has hundreds of regulated targets, is an example of a TF with a
high out-degree (16). Two Y1H studies identified upstream transcriptional regulators for three of
the five HD-ZIP III TFs (12, 23). A significantly larger number of TFs bind to their promoters,
making these HD-ZIP III TFs hubs based on their in-degree, or number of upstream TFs. HD-
ZIP III genes are critical in plant development, especially vascular patterning. Thus, using node
in-degree as a network property can help researchers generate hypotheses about the expected
biological functions of genes. A prediction following from this node feature is that critical devel-
opmental regulators will be in-degree hubs in a network mapped using Y1H. When Brady et al.
(12) tested this prediction in stele-enriched gene regulatory networks, they found a significant
correlation between a morphological (root) phenotype and the promoters of genes that are bound
by a relatively large number of TFs (in-degree hubs), but no significant correlation between this
phenotype and TFs that bind a relatively large number of promoters (out-degree hubs).
Given the apparent importance of in-degree hub genes in development, their putative
redundant regulation is not necessarily intuitive. If a positive regulator TF of an in-degree hub
gene developed a mutation, another positive regulator TF would act as a backup to ensure the
proper expression and function of that same hub gene (see TFs A and B in Figure 3). Single

mutants of either TF would likely not show a mutant phenotype because the TFs serve genetically
redundant roles. At first glance, this redundancy could be interpreted functionally as a TF being
less important relative to a mutant phenotype of a gene demonstrating a striking morphological
Feed-forward loop
phenotype. However, these TFs play integral roles in regulating these critical hub genes, and can (FFL): a specific
be considered “guards at the gate.” Thus, genetic redundancy can mask the true functional role of relationship between
a gene. Mutants of genes from TF families, some of which include 100 or more members, show three genes in a
no phenotypes until higher-order combinations of mutants are analyzed, either with partners in transcriptional
regulatory network
their respective family or with TFs in other families. However, elucidating their regulatory logic
that is significantly
is critical in understanding key biological processes. overrepresented
Identifying TF-promoter interactions relevant to a particular biological process using the Y1H relative to other genes
approach allows researchers to create an appropriate biological context by selecting a large set of (see Figure 3) (68)
promoters that are known (from either genetic evidence or expression profiling) to be involved
in a particular biological process. Additionally, by including promoters of TFs themselves, they
can begin to map a hierarchical network and identify network motifs such as feed-forward loops
(FFLs). FFLs have a structure in which one TF (TF X in Figure 3) binds to the promoters of
both another TF gene (TF Y) and a downstream gene (Z), while TF Y also binds to the promoter
of the Z gene (68). Mathematical modeling of the dynamics of FFLs has demonstrated that they
can generate specific and discrete examples of network behavior, such as a pulse of expression of
the downstream gene (25). In the xylem development and secondary cell wall synthesis regulatory
network, Taylor-Teeples et al. (74) identified a relatively large number of FFLs, suggesting that
these loops may play an important role in regulating xylem cell development.
DATA INTEGRATION
Researchers can leverage the data generated from transcriptional regulatory networks by using
complementary information from other large-scale experimental approaches to reveal novel bio-
logical mechanisms. In the past 15 years, thousands of studies have generated transcriptome data
across many cell types and growth conditions (8, 9, 38, 66). These data clearly demonstrate that the
suites of TFs expressed can vary widely in response to different environmental stimuli and even in
different cell types. Thus, investigators can select TF-target pairs for further exploration based on
particular developmental or environmental contexts. This approach has been used, for example,
to determine how the xylem cell secondary cell wall synthesis network changes in response to iron
deprivation or salt stress. In order to characterize how distinct developmental stresses may influ-
ence xylem cell development or secondary cell wall synthesis, tissue-resolution gene expression
profiling data sets were used in conjunction with the network mapped using a Y1H approach (19,
24, 33). The results showed that genes in the xylem and secondary cell wall synthesis network
are enriched for responsiveness to sulfur stress, salt stress, and iron deprivation. For each stress,
Taylor-Teeples et al. (74) then reduced the subnetwork based on the tissue-specific differential
expression and identified the critical TFs that act in response to each stress based on their number
of upstream regulators. They found that different stresses perturb the expression of TFs, leading
to distinct functional outcomes. For example, iron deprivation stress perturbs the expression of
REV, resulting in altered phenylpropanoid or lignin deposition, whereas high salt stress perturbs
the expression of VND7, resulting in ectopic and precocious xylem cell differentiation (74).
Other data sets can also be integrated with these networks, but they have not been consistently
employed with respect to transcriptional regulatory networks. In 2011, the Arabidopsis Interactome
Mapping Consortium (2) published a protein-protein interaction network with 6,200 physical
(protein-protein) interactions among 2,700 proteins. When coupled with a transcriptional reg-
ulatory network, these protein-protein interaction data could be used to identify TF-TF dimer

interactions and TFs that potentially act within complexes. This information could also lead to a
more thorough understanding of the regulatory logic underlying plant growth and development.
Additional proteomic, metabolomic, and translatomic data sets are also available but have not been
extensively utilized (for an example of matched data sets, see 50, 52, 59). Successfully integrating
different levels of information will require taking into account how each data set was generated
and the inherent biases of each experimental approach (53). Many such approaches have been
elucidated for yeast genome-level data, enabling the assembly of highly predictive networks, and
this type of systematic approach will be essential to integrate similar data in plants.
TEMPORAL DYNAMICS: FROM TRANSCRIPTION FACTOR

BINDING TO CHROMATIN ACCESSIBILITY
A majority of the published mapped transcriptional regulatory networks represent a static repre-
sentation of TF-target interactions. However, it is well understood that many biological processes
are dynamic in nature; they change over developmental time and with the tissue they are expressed
or function in, and may be specifically regulated in response to environmental stimuli. Therefore,
a critical binding and regulatory event may be transient relative to the lifetime of a plant.
Time course ChIP experiments allow for temporal analysis of TF-target binding and, when
coupled with RNA-seq, enable the inference of regulation (16). The EIN3 study demonstrated
the complexity of transcriptional regulation that can be employed by a single TF over time (16).
EIN3 binds to and regulates the expression of genes in four distinct waves of transcriptional
response after exposure to ethylene. One of these waves represents EIN3 targets associated with
a quick ethylene response, and other waves comprise genes (including many receptors) associated
with other hormone pathways. This temporal information enables the determination of short-
term, long-term, or continuous regulatory effects of EIN3. This study is a good example of how
capturing a TF regulatory network at a single time point will miss the full complexity of events that
drive the growth of a plant in response to ethylene. Essentially, interactions within the network
are rewired from time point to time point as environmental signals are processed and regulatory
changes are made.
Mapping networks using DNase I hypersensitivity can reveal the extent to which this complexity
is determined by chromatin availability. Each open chromatin region revealed over time can
represent the binding of one or more TFs and subsequent regulatory events, whereas a newly closed
chromatin region can indicate the completion of a binding event and consequent transcriptional
regulatory changes that are likely indirect. In their DNase I hypersensitivity assay that profiled
changes in chromatin availability over time after exposure to light or heat, Sullivan et al. (72) also
determined the connectivity profile of important TFs. First, they linked changes in chromatin
accessibility to changes in gene expression. The patterns in chromatin accessibility dynamics
mirrored differential gene expression patterns with respect to photomorphogenesis. Examples of
binding events inferred using these methods include the potential for ARR10 to bind chromatin
in the dark, for ZINC-DEPENDENT ACTIVATOR PROTEIN-1 (ZAP1) to bind chromatin
30 min after light exposure, and for HY5 to bind chromatin continuously.
A ChIP analysis of BASIC LEUCINE ZIPPER 1 (bZIP1) using the TARGET system illus-
trates the transient nature of TF binding and its importance in regulating gene expression (58).
In addition to the glucocorticoid receptor fusion of bZIP1 and the addition of cycloheximide,
nitrogen was added to the protoplasts to determine the nitrogen dependence of regulation by
bZIP1. Many critical nitrogen response genes that bZIP1 binds and regulates were bound by the
TF only after 5 min of bZIP1 induction. Therefore, the mode of action of this TF is to quickly
bind and regulate gene expression. The dynamics and regulatory role of bZIP1 determined by

this study indicate that TARGET is a suitable platform for studying the importance of binding
time on direct transcriptional regulation.
FFLs are a type of network motif that has been enriched in the context of the xylem devel-
opment and secondary cell wall synthesis network using Y1H assays. Mathematical modeling of
the dynamic transcriptional regulation conferred by FFLs has been elucidated in the single-cell
organism Escherichia coli (68). Mathematically modeling the dynamics of FFL loops requires first
determining the TF binding and type of regulation (activating or repressing). Two types of FFLs
are overrepresented in E. coli transcriptional regulatory networks: coherent FFLs and incoherent
FFLs. An FFL is coherent if the direct effect of the general TF (TF X in Figure 3) on the gene (Z)
has the same regulatory effect (negative or positive) as its net indirect effect through the specific
TF Y. Simple mathematical modeling of a coherent FFL demonstrated that the Z target gene can
be transcribed only in the presence of persistent activation of TF X, and any rapid fluctuations of
the level of TF X or its activity will be rejected. By contrast, mathematical modeling of a specific
type of incoherent FFL demonstrated that this type of network motif can produce a pulse of
expression of the Z gene.
PERSPECTIVES FOR THE FUTURE

Are big data really better? Absolutely, but with certain caveats. The generation of large amounts
of data and leveraging of existing genome-scale data to interpret the functional significance of
interconnected regulatory interactions require a well-designed biological question and a solid
understanding of biological context. Is there a best way to determine transcriptional regulatory
interactions? No: The methods described in this review have their own advantages and disadvan-
tages and are truly complementary in the information they provide.
What emergent properties have been revealed by systems analyses of these networks, and how
have these changed our understanding of the molecular mechanisms underlying plant growth,
development, and responses to the environment? First, and most dramatically, the determination
that critical TF binding and regulatory events can occur within 5 min of exposure to a given stimu-
lus should radically change our understanding of how quickly transcriptional regulation can occur.
We propose that the speed of this regulatory response may in fact be more common than previ-
ously accepted, and that the design of ChIP experiments should change when considering how
a TF is able to translate exposure to an abiotic or biotic stimulus. Although TARGET assays do
not necessarily provide an authentic biological context because they are performed in protoplasts,
they are an ideal experimental approach for identifying these rapid regulatory interactions.
Second, the dynamic and rapid change of open chromatin regions can also provide clues to
the effective regulatory regions that are needed to determine transcriptional regulatory cascades
during growth, development, and responses to the environment. This can reduce the vast amounts
of intergenic and non-protein-coding DNA into a precise set of regulatory units. Furthermore,
these regulatory units can be used as bait in Y1H assays and screened against precisely defined TF
collections that contain TFs expressed in the specific biological context that was used for DNase I
hypersensitivity assays. These TF sets can be defined by querying spatial- and temporal-resolution
mRNA transcriptome or translatome profiling data or (if available) proteomic data. Identifying
putative regulatory TFs in this manner can help direct the next generation of ChIP experiments
that will expand our understanding of physical TF-DNA interactions. Generating TF resources
across diverse plant species will also help researchers understand how these regulatory networks
are conserved across evolution.
Finally, the dynamics of particular types of regulatory interactions identified as being more
prevalent in a network (such as FFLs) should be mathematically modeled. Such modeling will
help determine whether properties observed in E. coli are valid in multicellular plants.
The functional validation of regulatory interactions remains a complicated matter. Clearly,

the “best” way to functionally validate a transcriptional regulatory interaction is not feasible at a
high throughput. Focused functional phenotyping, such as the determination of metabolite levels
or specific aspects of a plant phenotype in multiple mutants, is an additional approach that is
amenable to a moderate throughput. In this context, we would also like to draw attention to a
concept discussed in a recent review that describes cryptotypes (17). A cryptotype (or “hidden
type”) is a latent combination of traits that maximizes the separation of a priori groups either
developmentally or in response to the environment. The concept of a cryptotype was proposed as
a way to discriminate quantitative trait locus effects. An individual trait that is measured would show
no distinguishable influence on genetic regulation of a biological process; however, combining
multiple, often subtle traits together can enable researchers to visualize a clear genetic influence
and identify putative regulatory loci (17).
We propose that the cryptotype concept can also be applied to morphological phenotypic
analysis of TF mutants in order to validate regulatory interactions within a transcriptional regu-

latory network. The results of phenotyping multiple traits in a pool of mutants in TFs identified
(via network mapping) as playing a role in a particular biological process can be compared with
the results of phenotyping traits in a randomly chosen pool (of equivalent size) of TF mutants.
Combining these traits in a linear discriminant analysis may indicate a clear role of these genes in
regulating a particular biological process. This approach may provide insight into subtle but im-
portant influences of these TFs in plant growth, development, and responses to the environment,
and could be important as researchers begin to untangle the genetic redundancy built into these
regulatory networks and find emergent properties of the system.
SUMMARY POINTS
1. Mapping transcriptional regulatory networks is a powerful way to understand gene reg-
ulation and generate hypotheses about the functions and biological roles of transcription
factors.
2. Various experimental approaches can be used to map transcriptional regulatory networks,
each with strengths and weaknesses.
3. Emphasis needs to be placed on in planta functional validation of interactions from
transcriptional regulatory network mapping approaches.
4. Recent work has highlighted the need to study the transcriptional network regulatory
dynamics and how it controls biological function.
5. Integration of multiple data types with the appropriate biological context in mapping
networks can add power to the hypotheses generated.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank Gina Turco and Erin Sparks for their suggestions and assistance. S.M.B. is funded by
National Science Foundation Division of Molecular and Cellular Biosciences grant 1330337.

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PP67-FrontMatter ARI 12 March 2016 14:40
Annual Review of
Plant Biology
Contents Volume 67, 2016
The Path to Thioredoxin and Redox Regulation in Chloroplasts

Bob B. Buchanan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Learning the Languages of the Chloroplast: Retrograde Signaling
and Beyond
Kai Xun Chan, Su Yin Phua, Peter Crisp, Ryan McQuinn, and Barry J. Pogson p p p p p p p p25
NDH-1 and NDH-2 Plastoquinone Reductases in Oxygenic
Photosynthesis
Gilles Peltier, Eva-Mari Aro, and Toshiharu Shikanai p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p55
Physiological Functions of Cyclic Electron Transport Around
Photosystem I in Sustaining Photosynthesis and Plant Growth
Wataru Yamori and Toshiharu Shikanai p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p81
The Costs of Photorespiration to Food Production Now
and in the Future
Berkley J. Walker, Andy VanLoocke, Carl J. Bernacchi, and Donald R. Ort p p p p p p p p p p p 107
Metabolite Damage and Metabolite Damage Control in Plants
Andrew D. Hanson, Christopher S. Henry, Oliver Fiehn,
and Valérie de Crécy-Lagard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 131
The Regulation of Essential Amino Acid Synthesis and Accumulation
in Plants
Gad Galili, Rachel Amir, and Alisdair R. Fernie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 153
Triacylglycerol Metabolism, Function, and Accumulation in Plant
Vegetative Tissues
Changcheng Xu and John Shanklin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 179
The Plant Polyester Cutin: Biosynthesis, Structure, and Biological
Roles
Eric A. Fich, Nicholas A. Segerson, and Jocelyn K.C. Rose p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 207
Biosynthesis of the Plant Cell Wall Matrix Polysaccharide Xyloglucan
Markus Pauly and Kenneth Keegstra p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 235
TOR Signaling and Nutrient Sensing
Thomas Dobrenel, Camila Caldana, Johannes Hanson, Christophe Robaglia,
Michel Vincentz, Bruce Veit, and Christian Meyer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 261
v
Rapid, Long-Distance Electrical and Calcium Signaling in Plants

Won-Gyu Choi, Richard Hilleary, Sarah J. Swanson, Su-Hwa Kim,
and Simon Gilroy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 287
Endocytosis and Endosomal Trafficking in Plants
Julio Paez Valencia, Kaija Goodman, and Marisa S. Otegui p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 309
Staying Tight: Plasmodesmal Membrane Contact Sites and the
Control of Cell-to-Cell Connectivity in Plants
Jens Tilsner, William Nicolas, Abel Rosado, and Emmanuelle M. Bayer p p p p p p p p p p p p p p p p 337
Pre-Meiotic Anther Development: Cell Fate Specification and
Differentiation
Virginia Walbot and Rachel L. Egger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 365
Plant Sex Chromosomes

Deborah Charlesworth p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 397
Haploidization via Chromosome Elimination: Means and Mechanisms
Takayoshi Ishii, Raheleh Karimi-Ashtiyani, and Andreas Houben p p p p p p p p p p p p p p p p p p p p p p p 421
Mechanisms Used by Plants to Cope with DNA Damage
Zhubing Hu, Toon Cools, and Lieven De Veylder p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 439
The Conservation and Function of RNA Secondary Structure in Plants
Lee E. Vandivier, Stephen J. Anderson, Shawn W. Foley, and Brian D. Gregory p p p p p p 463
Toxic Heavy Metal and Metalloid Accumulation in Crop Plants
and Foods
Stephan Clemens and Jian Feng Ma p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 489
Light-Mediated Hormonal Regulation of Plant Growth
and Development
Mieke de Wit, Vinicius Costa Galvão, and Christian Fankhauser p p p p p p p p p p p p p p p p p p p p p p p 513
Transcriptional Responses to the Auxin Hormone
Dolf Weijers and Doris Wagner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 539
Mapping Transcriptional Networks in Plants: Data-Driven Discovery
of Novel Biological Mechanisms
Allison Gaudinier and Siobhan M. Brady p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 575
The Intracellular Dynamics of Circadian Clocks Reach for the Light
of Ecology and Evolution
Andrew J. Millar p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 595
Environmental Control of Root System Biology
Rubén Rellán-Álvarez, Guillaume Lobet, and José R. Dinneny p p p p p p p p p p p p p p p p p p p p p p p p p p 619
The Haustorium, a Specialized Invasive Organ in Parasitic Plants
Satoko Yoshida, Songkui Cui, Yasunori Ichihashi, and Ken Shirasu p p p p p p p p p p p p p p p p p p p p p 643
vi Contents
Antibody Production in Plants and Green Algae

Vidadi Yusibov, Natasha Kushnir, and Stephen J. Streatfield p p p p p p p p p p p p p p p p p p p p p p p p p p p p 669
Perennial Grain and Oilseed Crops
Michael B. Kantar, Catrin E. Tyl, Kevin M. Dorn, Xiaofei Zhang,
Jacob M. Jungers, Joe M. Kaser, Rachel R. Schendel, James O. Eckberg,
Bryan C. Runck, Mirko Bunzel, Nick R. Jordan, Robert M. Stupar,
M. David Marks, James A. Anderson, Gregg A. Johnson, Craig C. Sheaffer,
Tonya C. Schoenfuss, Baraem Ismail, George E. Heimpel,
and Donald L. Wyse p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 703
Errata
An online log of corrections to Annual Review of Plant Biology articles may be found at
http://www.annualreviews.org/errata/arplant
Contents vii

Mapping Transcriptional Networks in Plants: Data-Driven Discovery of Novel Biological Mechanisms

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PP67CH23-Brady ARI 14 March 2016 13:6

Novel Biological Mechanisms

Annu. Rev. Plant Biol. 2016. 67:575–94 Keywords

The Annual Review of Plant Biology is online at Abstract

FOCUSED FUNCTIONAL PHENOTYPING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

EMERGENT PROPERTIES REVEALED:

BIGGER IS NOT NECESSARILY BETTER

576 Gaudinier · Brady

Distal promoter Core Distal promoter Core

www.annualreviews.org • Mapping Transcriptional Networks in Plants 577

in the context of transcriptional regulatory networks requires further experimental evidence. In

TRANSCRIPTION FACTOR–CENTERED APPROACHES

578 Gaudinier · Brady

Assay Readout Network Requirements

In contrast to the central hormone regulator, EIN3, SPEECHLESS (SPCH) is a transcriptional

www.annualreviews.org • Mapping Transcriptional Networks in Plants 579

580 Gaudinier · Brady

AN UNBIASED APPROACH: MAPPING OPEN CHROMATIN

The third approach to mapping transcriptional regulatory networks is an unbiased, genome-wide

IN PLANTA VALIDATION OF TRANSCRIPTIONAL REGULATORY

www.annualreviews.org • Mapping Transcriptional Networks in Plants 581

On approach to validate the occurrence of transcriptional regulation is genetic analysis. Two

582 Gaudinier · Brady

LITERATURE SUPPORT FOR GENERATING HYPOTHESES

www.annualreviews.org • Mapping Transcriptional Networks in Plants 583

FOCUSED FUNCTIONAL PHENOTYPING

EMERGENT PROPERTIES REVEALED:

584 Gaudinier · Brady

crosstalk can be mediated.

LEVERAGING NETWORK TOPOLOGY AND METRICS

www.annualreviews.org • Mapping Transcriptional Networks in Plants 585

586 Gaudinier · Brady

www.annualreviews.org • Mapping Transcriptional Networks in Plants 587

TEMPORAL DYNAMICS: FROM TRANSCRIPTION FACTOR

588 Gaudinier · Brady

PERSPECTIVES FOR THE FUTURE

The functional validation of regulatory interactions remains a complicated matter. Clearly,

analysis of TF mutants in order to validate regulatory interactions within a transcriptional regu-

590 Gaudinier · Brady

www.annualreviews.org • Mapping Transcriptional Networks in Plants 591

592 Gaudinier · Brady

www.annualreviews.org • Mapping Transcriptional Networks in Plants 593

inference of eukaryotic transcription factor sequence specificity. Cell 158:1431–43

594 Gaudinier · Brady

The Path to Thioredoxin and Redox Regulation in Chloroplasts

Rapid, Long-Distance Electrical and Calcium Signaling in Plants

Plant Sex Chromosomes

Antibody Production in Plants and Green Algae

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