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1.  Combinatorial Complexity in a Transcriptionally Centered Signaling Hub in Arabidopsis  
Molecular Plant  2014;7(11):1598-1618.
SUMMARY
Integrated ChIP-seq and RNA-seq analysis of multiple mutant combinations identifies a diverse network of genes that are direct targets of transcriptional regulation by a quartet of closely related phytochrome-interacting bHLH transcription factors (PIFs) in promoting seedling skotomorphogenesis. The evidence unveils an intriguing dual-layered mechanism of regulation whereby both the level of promoter binding-site occupancy and in situ modulation of bound transcription-factor intrinsic activity combine to generate a complex matrix of shared, but quantitatively differential, gene expression patterns, under the collective control of these PIFs across the network.
A subfamily of four Phytochrome (phy)-Interacting bHLH transcription Factors (PIFs) collectively promote skotomorphogenic development in dark-grown seedlings. This activity is reversed upon exposure to light, by photoactivated phy molecules that induce degradation of the PIFs, thereby triggering the transcriptional changes that drive a transition to photomorphogenesis. The PIFs function both redundantly and partially differentially at the morphogenic level in this process. To identify the direct targets of PIF transcriptional regulation genome-wide, we analyzed the DNA-binding sites for all four PIFs by ChIP-seq analysis, and defined the genes transcriptionally regulated by each PIF, using RNA-seq analysis of pif mutants. Despite the absence of detectable differences in DNA-binding-motif recognition between the PIFs, the data show a spectrum of regulatory patterns, ranging from single PIF dominance to equal contributions by all four. Similarly, a broad array of promoter architectures was found, ranging from single PIF-binding sites, containing single sequence motifs, through multiple PIF-binding sites, each containing one or more motifs, with each site occupied preferentially by one to multiple PIFs. Quantitative analysis of the promoter occupancy and expression level induced by each PIF revealed an intriguing pattern. Although there is no robust correlation broadly across the target-gene population, examination of individual genes that are shared targets of multiple PIFs shows a gradation in correlation from strongly positive, through uncorrelated, to negative. This finding suggests a dual-layered mechanism of transcriptional regulation, comprising both a continuum of binding-site occupancy by each PIF and a superimposed layer of local regulation that acts differentially on each PIF, to modulate its intrinsic transcriptional activation capacity at each site, in a quantitative pattern that varies between the individual PIFs from gene to gene. These findings provide a framework for probing the mechanisms by which transcription factors with overlapping direct-target genes integrate and selectively transduce signals to their target networks.
doi:10.1093/mp/ssu087
PMCID: PMC4587546  PMID: 25122696
phytochromes; light-signaling; PIFs; bHLH transcription factors; promoter occupancy; Arabidopsis; transcriptional regulation; ChIP-seq; RNA-seq.
3.  Arabidopsis VIM Proteins Regulate Epigenetic Silencing by Modulating DNA Methylation and Histone Modification in Cooperation with MET1 
Molecular Plant  2014;7(9):1470-1485.
SUMMARY
Arabidopsis VIM/ORTH family proteins, which have SRA-domain methylcytosine-binding activity, are required for the maintenance of DNA methylation and epigenetic gene silencing in heterochromatic regions. In this study, endogenous targets of the VIM proteins in epigenetic silencing were identified through a genome-wide transcript profiling analysis in a vim1/2/3 triple mutant. We furthermore revealed the molecular mechanisms by which the VIM proteins regulate epigenetic silencing by modulating DNA methylation and histone modification in cooperation with MET1.
Methylcytosine-binding proteins containing SRA (SET- and RING-Associated) domain are required for the establishment and/or maintenance of DNA methylation in both plants and animals. We previously proposed that Arabidopsis VIM/ORTH proteins with an SRA domain maintain DNA methylation and epigenetic gene silencing in heterochromatic regions. However, their endogenous targets of epigenetic gene silencing have not been analyzed globally and the mechanisms by which VIM proteins coordinate DNA methylation and epigenetic silencing are largely unknown. In this study, a genome-wide transcript profiling analysis revealed 544 derepressed genes in a vim1/2/3 triple mutant, including 133 known genes. VIM1 bound to promoter and transcribed regions of the up-regulated genes in vim1/2/3 and VIM deficiency caused severe DNA hypomethylation in all sequence contexts at direct VIM1 targets. We found a drastic loss of H3K9me2 at heterochromatic chromocenters in vim1/2/3 nuclei. Furthermore, aberrant changes in transcriptionally active and repressive histone modifications were observed at VIM1 targets in vim1/2/3. VIM1-binding capacity to target genes was significantly reduced in the met1 background, indicating that VIM1 primarily recognizes CG methylation deposited by MET1. Overall, our data indicate that VIM proteins regulate genome-wide epigenetic gene silencing through coordinated modulation of DNA methylation and histone modification status in collaboration with MET1.
doi:10.1093/mp/ssu079
PMCID: PMC4207863  PMID: 25009302
VIM/ORTH; SRA; MET1; epigenetic silencing; DNA methylation; histone modification.
4.  Growth–Defense Tradeoffs in Plants: A Balancing Act to Optimize Fitness 
Molecular Plant  2014;7(8):1267-1287.
SUMMARY
Growth–defense tradeoffs are thought to occur in plants due to resource limitations to optimize plant fitness. Hormone crosstalk appears to be the primary means for plant modulation of growth and defense. Understanding the molecular processes governing plant prioritization and diversion of resources towards growth or defense may enable genetic tailoring of plants to harness this natural plasticity for optimization of both growth and defense under variable environmental conditions.
Growth–defense tradeoffs are thought to occur in plants due to resource restrictions, which demand prioritization towards either growth or defense, depending on external and internal factors. These tradeoffs have profound implications in agriculture and natural ecosystems, as both processes are vital for plant survival, reproduction, and, ultimately, plant fitness. While many of the molecular mechanisms underlying growth and defense tradeoffs remain to be elucidated, hormone crosstalk has emerged as a major player in regulating tradeoffs needed to achieve a balance. In this review, we cover recent advances in understanding growth–defense tradeoffs in plants as well as what is known regarding the underlying molecular mechanisms. Specifically, we address evidence supporting the growth–defense tradeoff concept, as well as known interactions between defense signaling and growth signaling. Understanding the molecular basis of these tradeoffs in plants should provide a foundation for the development of breeding strategies that optimize the growth–defense balance to maximize crop yield to meet rising global food and biofuel demands.
doi:10.1093/mp/ssu049
PMCID: PMC4168297  PMID: 24777989
plant immunity; plant hormone; salicylic acid; jasmonate; PAMP; plant growth.
5.  Salicylic Acid Signaling Controls the Maturation and Localization of the Arabidopsis Defense Protein ACCELERATED CELL DEATH6 
Molecular Plant  2014;7(8):1365-1383.
SUMMARY
The multipass membrane protein ACD6 forms large protein complexes and is subject to constitutive ER-associated degradation. Salicylic acid signaling increases the size of ACD6 complexes and the plasma membrane levels of ACD6 and the ACD6-associated pattern receptor FLS2. ACD6 is necessary for promoting increased levels of FLS2 and its co-receptor BAK1 specifically at the plasma membrane during salicylic acid (SA) signaling, suggesting that ACD6 maturation and plasma membrane levels are tightly controlled as a mechanism for regulating these pattern receptors.
ACCELERATED CELL DEATH6 (ACD6) is a multipass membrane protein with an ankyrin domain that acts in a positive feedback loop with the defense signal salicylic acid (SA). This study implemented biochemical approaches to infer changes in ACD6 complexes and localization. In addition to forming endoplasmic reticulum (ER)- and plasma membrane (PM)-localized complexes, ACD6 forms soluble complexes, where it is bound to cytosolic HSP70, ubiquitinated, and degraded via the proteasome. Thus, ACD6 constitutively undergoes ER-associated degradation. During SA signaling, the soluble ACD6 pool decreases, whereas the PM pool increases. Similarly, ACD6-1, an activated version of ACD6 that induces SA, is present at low levels in the soluble fraction and high levels in the PM. However, ACD6 variants with amino acid substitutions in the ankyrin domain form aberrant, inactive complexes, are induced by a SA agonist, but show no PM localization. SA signaling also increases the PM pools of FLAGELLIN SENSING2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). FLS2 forms complexes ACD6; both FLS2 and BAK1 require ACD6 for maximal accumulation at the PM in response to SA signaling. A plausible scenario is that SA increases the efficiency of productive folding and/or complex formation in the ER, such that ACD6, together with FLS2 and BAK1, reaches the cell surface to more effectively promote immune responses.
doi:10.1093/mp/ssu072
PMCID: PMC4168298  PMID: 24923602
ACD6; protein trafficking; protein quality control; salicylic acid
6.  Unfolding the Mysteries of Strigolactone Signaling 
Molecular Plant  2014;7(6):934-936.
doi:10.1093/mp/ssu021
PMCID: PMC4040469  PMID: 24623790
7.  New Insights into Aluminum Tolerance in Rice: The ASR5 Protein Binds the STAR1 Promoter and Other Aluminum-Responsive Genes 
Molecular Plant  2013;7(4):709-721.
SUMMARY
Acidic soils comprise a large portion of the Earth’s crust. In this environment, aluminum becomes soluble to plants affecting directly plant development. Among crops, rice is the most Al-resistant but the base of this tolerance is far from being elucidated. In this work, we showed a large-scale profile of Al-responsive genes in rice. Besides, we extended the study in relation to ASR5, a protein previously found by our group as having an important function in Al resistance.
Aluminum (Al) toxicity in plants is one of the primary constraints in crop production. Al3+, the most toxic form of Al, is released into soil under acidic conditions and causes extensive damage to plants, especially in the roots. In rice, Al tolerance requires the ASR5 gene, but the molecular function of ASR5 has remained unknown. Here, we perform genome-wide analyses to identify ASR5-dependent Al-responsive genes in rice. Based on ASR5_RNAi silencing in plants, a global transcriptome analysis identified a total of 961 genes that were responsive to Al treatment in wild-type rice roots. Of these genes, 909 did not respond to Al in the ASR5_RNAi plants, indicating a central role for ASR5 in Al-responsive gene expression. Under normal conditions, without Al treatment, the ASR5_RNAi plants expressed 1.756 genes differentially compared to the wild-type plants, and 446 of these genes responded to Al treatment in the wild-type plants. Chromatin immunoprecipitation followed by deep sequencing identified 104 putative target genes that were directly regulated by ASR5 binding to their promoters, including the STAR1 gene, which encodes an ABC transporter required for Al tolerance. Motif analysis of the binding peak sequences revealed the binding motif for ASR5, which was confirmed via in vitro DNA-binding assays using the STAR1 promoter. These results demonstrate that ASR5 acts as a key transcription factor that is essential for Al-responsive gene expression and Al tolerance in rice.
doi:10.1093/mp/sst160
PMCID: PMC3973494  PMID: 24253199
Aluminum; ChIP-Seq; RNA-Seq; rice; ASR.
8.  Open and Closed: The Roles of Linker Histones in Plants and Animals 
Molecular Plant  2013;7(3):481-491.
SUMMARY
Linker histones play key roles alongside core histones in the regulation and maintenance of chromatin. Here, we illustrate our current understanding of the contributions of linker histones to the cell cycle, development, and chromatin structure in plants and animals.
Histones package DNA in all eukaryotes and play key roles in regulating gene expression. Approximately 150 base pairs of DNA wraps around an octamer of core histones to form the nucleosome, the basic unit of chromatin. Linker histones compact chromatin further by binding to and neutralizing the charge of the DNA between nucleosomes. It is well established that chromatin packing is regulated by a complex pattern of posttranslational modifications (PTMs) to core histones, but linker histone function is less well understood. In this review, we describe the current understanding of the many roles that linker histones play in cellular processes, including gene regulation, cell division, and development, while putting the linker histone in the context of other nuclear proteins. Although intriguing roles for plant linker histones are beginning to emerge, much of our current understanding comes from work in animal systems. Many unanswered questions remain and additional work is required to fully elucidate the complex processes mediated by linker histones in plants.
doi:10.1093/mp/sst164
PMCID: PMC3941478  PMID: 24270504
linker histone; histone H1; chromatin; gene regulation; development; differentiation; imprinting; posttranslational modifications; DNA methylation; high mobility group proteins.
9.  Covering Your Bases: Inheritance of DNA Methylation in Plant Genomes 
Molecular Plant  2013;7(3):472-480.
SUMMARY
Whole-genome bisulfite sequencing has rapidly enabled the identification of DNA methylation variants within and between plant species. How these DNA methylation variants form, how they are inherited, and how stable they are over time is the topic of this review.
Cytosine methylation is an important base modification that is inherited across mitotic and meiotic cell divisions in plant genomes. Heritable methylation variants can contribute to within-species phenotypic variation. Few methylation variants were known until recently, making it possible to begin to address major unanswered questions: the extent of natural methylation variation within plant genomes, its effects on phenotypic variation, its degree of dependence on genotype, and how it fits into an evolutionary context. Techniques like whole-genome bisulfite sequencing (WGBS) make it possible to determine cytosine methylation states at single-base resolution across entire genomes and populations. Application of this method to natural and novel experimental populations is revealing answers to these long-standing questions about the role of DNA methylation in plant genomes.
doi:10.1093/mp/sst165
PMCID: PMC3941479  PMID: 24270503
DNA methylation; epigenetics; epiallele; whole-genome bisulfite sequencing.
10.  Enhancement of Indole-3-Acetic Acid Photodegradation by Vitamin B6 
Molecular Plant  2013;6(6):1992-1995.
doi:10.1093/mp/sst089
PMCID: PMC3834970  PMID: 23723155
11.  Application of the CRISPR–Cas System for Efficient Genome Engineering in Plants 
Molecular Plant  2013;6(6):2008-2011.
doi:10.1093/mp/sst121
PMCID: PMC3916745  PMID: 23963532
12.  Age-Triggered and Dark-Induced Leaf Senescence Require the bHLH Transcription Factors PIF3, 4, and 5 
Molecular Plant  2014;7(12):1776-1787.
SUMMARY
Numerous lines of evidence have suggested an involvement of phytochromes in the regulation of leaf senescence, but the related signaling pathway and physiological mechanisms are poorly understood. In this study, we demonstrated that PIF3, 4, and 5, the master regulators of light signaling, modulate age-triggered and dark-induced senescence and particularly PIF4 positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and chloroplast activity maintaining as well as ethylene biosynthesis.
Leaf senescence can be triggered and promoted by a large number of developmental and environmental factors. Numerous lines of evidence have suggested an involvement of phytochromes in the regulation of leaf senescence, but the related signaling pathways and physiological mechanisms are poorly understood. In this study, we initially identified phytochrome-interacting factors (PIFs) 3, 4, and 5 as putative mediators of leaf senescence. Mutations of the PIF genes resulted in a significantly enhanced leaf longevity in age-triggered and dark-induced senescence, whereas overexpressions of these genes accelerated age-triggered and dark-induced senescence in Arabidopsis. Consistently, loss-of-function of PIF4 attenuated dark-induced transcriptional changes associated with chloroplast deterioration and reactive oxygen species (ROS) generation. ChIP–PCR and Dual-Luciferase assays demonstrated that PIF4 can activate chlorophyll degradation regulatory gene NYE1 and repress chloroplast activity maintainer gene GLK2 by binding to their promoter regions. Finally, dark-induced ethylene biosynthesis and ethylene-induced senescence were both dampened in pif4, suggesting the involvement of PIF4 in both ethylene biosynthesis and signaling pathway. Our study provides evidence that PIF3, 4, and 5 are novel positive senescence mediators and gains an insight into the mechanism of light signaling involved in the regulation of leaf senescence.
doi:10.1093/mp/ssu109
PMCID: PMC4261840  PMID: 25296857
phytochrome-interacting factor (PIF); leaf senescence; chloroplast deterioration; NYE1/SGR1; GLK2; ethylene.
13.  Splicing of Receptor-Like Kinase-Encoding SNC4 and CERK1 is Regulated by Two Conserved Splicing Factors that Are Required for Plant Immunity 
Molecular Plant  2014;7(12):1766-1775.
SUMMARY
Two conserved splicing factors, SUA and RSN2, were identified from a suppressor screen of snc4-1D. Both are required for alternative splicing of receptor-like kinase (RLK)-encoding SNC4 and CERK1 and their functions, suggesting that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1.
Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1.
doi:10.1093/mp/ssu103
PMCID: PMC4261838  PMID: 25267732
plant immunity; receptor-like kinase; alternative splicing; SUA; RSN2; SNC4; CERK1.
14.  SPINDLY, ERECTA, and Its Ligand STOMAGEN Have a Role in Redox-Mediated Cortex Proliferation in the Arabidopsis Root 
Molecular Plant  2014;7(12):1727-1739.
SUMMARY
Reactive oxygen species (ROS) are signaling molecules, but how they are perceived in plants remains unclear. This study showed that cortex proliferation in the Arabidopsis root can be induced by hydrogen peroxide and that the receptor kinase ERECTA and one of its ligands, STOMAGEN, are involved in a signaling pathway that couples ROS sensing with redox-mediated cortex proliferation. This study also revealed a new role for SPINDLY (SPY), a putative O-GlcNAc transferase, in cellular redox homeostasis.
Reactive oxygen species (ROS) are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mechanisms of ROS perception and developmental response in plants. We here report that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY (SPY), an O-linked glucosamine acetyltransferase, regulates cortex proliferation by maintaining cellular redox homeostasis. We also found that mutation in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling. SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. We therefore have not only gained insight into the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signaling in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed.
doi:10.1093/mp/ssu106
PMCID: PMC4261839  PMID: 25267734
SPY; ERECTA; STOMAGEN; redox homeostasis; ROS signaling; abiotic stress; cortex proliferation; Arabidopsis thaliana.
15.  Genome-Wide Analysis of Histone Modifications: H3K4me2, H3K4me3, H3K9ac, and H3K27ac in Oryza sativa L. Japonica 
Molecular Plant  2013;6(5):1463-1472.
SUMMARY
H3K4me2/3, H3K9ac, and H3K27ac investigated by ChIP-Seq showed enrichment in generic regions and transcription start sites, and associated with active transcription in rice. They were used to discover unannotated genes and to predict transcription factor binding sites together with DNase-Seq data.
While previous studies have shown that histone modifications could influence plant growth and development by regulating gene transcription, knowledge about the relationships between these modifications and gene expression is still limited. This study used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), to investigate the genome-wide distribution of four histone modifications: di and trimethylation of H3K4 (H3K4me2 and H3K4me3) and acylation of H3K9 and H3K27 (H3K9ac and H3K27ac) in Oryza sativa L. japonica. By analyzing published DNase-Seq data, this study explored DNase-Hypersensitive (DH) sites along the rice genome. The histone marks appeared mainly in generic regions and were enriched around the transcription start sites (TSSs) of genes. This analysis demonstrated that the four histone modifications and the DH sites were all associated with active transcription. Furthermore, the four histone modifications were highly concurrent with transcript regions—a promising feature that was used to predict missing genes in the rice gene annotation. The predictions were further validated by experimentally confirming the transcription of two predicted missing genes. Moreover, a sequence motif analysis was constructed in order to identify the DH sites and many putative transcription factor binding sites.
doi:10.1093/mp/sst018
PMCID: PMC3842134  PMID: 23355544
bioinformatics; chromatin structure and remodeling; epigenetics; gene regulation; genomics; rice.
16.  1O2-Mediated and EXECUTER-Dependent Retrograde Plastid-to-Nucleus Signaling in Norflurazon-Treated Seedlings of Arabidopsis thaliana  
Molecular Plant  2013;6(5):1580-1591.
SUMMARY
Seedlings of Arabidopsis have been exposed to norflurazon. Depending on the developmental stage at which seedlings were first exposed to the inhibitor, enhanced production of reactive oxygen species occurred and, among others, 1O2-mediated and EXECUTER-dependent retrograde signaling was induced.
Chloroplast development depends on the synthesis and import of a large number of nuclear-encoded proteins. The synthesis of some of these proteins is affected by the functional state of the plastid via a process known as retrograde signaling. Retrograde plastid-to-nucleus signaling has been often characterized in seedlings of Arabidopsis thaliana exposed to norflurazon (NF), an inhibitor of carotenoid biosynthesis. Results of this work suggested that, throughout seedling development, a factor is released from the plastid to the cytoplasm that indicates a perturbation of plastid homeostasis and represses nuclear genes required for normal chloroplast development. The identity of this factor is still under debate. Reactive oxygen species (ROS) were among the candidates discussed as possible retrograde signals in NF-treated plants. In the present work, this proposed role of ROS has been analyzed. In seedlings grown from the very beginning in the presence of NF, ROS-dependent signaling was not detectable, whereas, in seedlings first exposed to NF after light-dependent chloroplast formation had been completed, enhanced ROS production occurred and, among others, 1O2-mediated and EXECUTER-dependent retrograde signaling was induced. Hence, depending on the developmental stage at which plants are exposed to NF, different retrograde signaling pathways may be activated, some of which are also active in non-treated plants under light stress.
doi:10.1093/mp/sst020
PMCID: PMC3842135  PMID: 23376773
retrograde signaling; singlet oxygen; Executer; norflurazon; programmed cell death; photo-oxidative stress.
18.  MADS-Box Transcription Factor AGL21 Regulates Lateral Root Development and Responds to Multiple External and Physiological Signals 
Molecular Plant  2014;7(11):1653-1669.
SUMMARY
MADS-box transcription factor AGL21 is responsive to several phytohormones as well as environmental cues and positively regulates auxin accumulation in lateral root primordia and lateral roots by enhancing local auxin biosynthesis, thus stimulating lateral root initiation and growth. Therefore, AGL21 may be involved in various environmental and physiological signals-mediated lateral root development.
Plant root system morphology is dramatically influenced by various environmental cues. The adaptation of root system architecture to environmental constraints, which mostly depends on the formation and growth of lateral roots, is an important agronomic trait. Lateral root development is regulated by the external signals coordinating closely with intrinsic signaling pathways. MADS-box transcription factors are known key regulators of the transition to flowering and flower development. However, their functions in root development are still poorly understood. Here we report that AGL21, an AGL17-clade MADS-box gene, plays a crucial role in lateral root development. AGL21 was highly expressed in root, particularly in the root central cylinder and lateral root primordia. AGL21 overexpression plants produced more and longer lateral roots while agl21 mutants showed impaired lateral root development, especially under nitrogen-deficient conditions. AGL21 was induced by many plant hormones and environmental stresses, suggesting a function of this gene in root system plasticity in response to various signals. Furthermore, AGL21 was found positively regulating auxin accumulation in lateral root primordia and lateral roots by enhancing local auxin biosynthesis, thus stimulating lateral root initiation and growth. We propose that AGL21 may be involved in various environmental and physiological signals-mediated lateral root development and growth.
doi:10.1093/mp/ssu088
PMCID: PMC4228986  PMID: 25122697
MADS; root system architecture; lateral root; AGL21; auxin; nitrate; sulfate.
19.  Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis  
Molecular Plant  2014;7(11):1637-1652.
SUMMARY
The Arabidopsis MAP kinase AtMPK10 has long been considered as a pseudo-gene without visible function for the plant. Here we show that AtMPK10 is functional only in a very narrow time window in leaves at sites of local auxin maxima where it regulates leaf venation complexity together with the upstream kinase AtMKK2.
Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2–AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency.
doi:10.1093/mp/ssu080
PMCID: PMC4228985  PMID: 25064848
Arabidopsis MAP kinase; leaf development; polar auxin transport; leaf venation pattern.
20.  Characterization and DNA-Binding Specificities of Ralstonia TAL-Like Effectors 
Molecular Plant  2013;6(4):1318-1330.
We report the characterization of three Ralstonia TAL-like effectors, which mediate DNA binding and can be used as customizable architectures for DNA targeting. We determined DNA-binding specificities of novel repeat variable di-residues (RVDs) and devised a repeat assembly approach for engineering Ralstonia solanacearum TALE-like proteins (RTLs).
Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.
doi:10.1093/mp/sst006
PMCID: PMC3716395  PMID: 23300258
Ralstonia solanacearum; genome engineering; TAL effectors; TALE activators and repressors; TALE nucleases (TALENs); targeted genome modifications
21.  Signaling in Pollen Tube Growth: Crosstalk, Feedback, and Missing Links 
Molecular Plant  2013;6(4):1053-1064.
SUMMARY
Pollen tubes represent an attractive model system to investigate polarized cell growth. We will discuss the current state of play of knowledge of the regulatory roles of signaling networks in the cellular activities of the pollen tube tip.
Pollen tubes elongate rapidly at their tips through highly polarized cell growth known as tip growth. Tip growth requires intensive exocytosis at the tip, which is supported by a dynamic cytoskeleton and vesicle trafficking. Several signaling pathways have been demonstrated to coordinate pollen tube growth by regulating cellular activities such as actin dynamics, exocytosis, and endocytosis. These signaling pathways crosstalk to form a signaling network that coordinates the cellular processes required for tip growth. The homeostasis of key signaling molecules is critical for the proper elongation of the pollen tube tip, and is commonly fine-tuned by positive and negative regulations. In addition to the major signaling pathways, emerging evidence implies the roles of other signals in the regulation of pollen tube growth. Here we review and discuss how these signaling networks modulate the rapid growth of pollen tubes.
doi:10.1093/mp/sst070
PMCID: PMC3842152  PMID: 23873928
polarity; pollen development; reproductive biology; signal transduction.
22.  AtPRK2 Promotes ROP1 Activation via RopGEFs in the Control of Polarized Pollen Tube Growth 
Molecular Plant  2012;6(4):1187-1201.
The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors (RopGEFs) and in turn may be activated by an unknown factor through releasing RopGEF1’s auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprk1,2,5 triple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 (DN-PRK2) inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1(90–457), implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 (RopGEF1S460A and RopGEF1S480A) eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regulator of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.
doi:10.1093/mp/sss103
PMCID: PMC3888354  PMID: 23024212
AtPRK2; RopGEF1; ROP GTPase; auto-inhibition; polarity growth.
24.  Control of Cell Wall Extensibility during Pollen Tube Growth 
Molecular Plant  2013;6(4):998-1017.
SUMMARY
Tip-growing pollen tubes achieve rapid elongation while maintaining cell wall integrity by balancing local expansion, controlled by local changes in wall viscosity, against exocytosis, influenced by the activity of the actin cytoskeleton, cellular energetics, and calcium and proton physiology.
In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underlying processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma membrane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymerization in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity.
doi:10.1093/mp/sst103
PMCID: PMC4043104  PMID: 23770837
cell expansion; cell walls; cytoskeleton dynamics; polarity; pollen development.
25.  OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds 
Molecular Plant  2014;7(9):1455-1469.
SUMMARY
Long-distance communication between leaves and roots are key to properly regulate the uptake of trace metals from the soil. The molecular basis of this shoot-to-root signaling is currently unknown. In this manuscript, we describe the role of OPT3 in the shoot-to-root signaling of the iron status in Arabidopsis. We also show that reduced expression of OPT3 induces an over-accumulation of the toxic metal cadmium, but not other metals, in seeds.
Plants and seeds are the main dietary sources of zinc, iron, manganese, and copper, but are also the main entry point for toxic elements such as cadmium into the food chain. We report here that an Arabidopsis oligopeptide transporter mutant, opt3-2, over-accumulates cadmium (Cd) in seeds and roots but, unexpectedly, under-accumulates Cd in leaves. The cadmium distribution in opt3-2 differs from iron, zinc, and manganese, suggesting a metal-specific mechanism for metal partitioning within the plant. The opt3-2 mutant constitutively up-regulates the Fe/Zn/Cd transporter IRT1 and FRO2 in roots, indicative of an iron-deficiency response. No genetic mutants that impair the shoot-to-root signaling of iron status in leaves have been identified. Interestingly, shoot-specific expression of OPT3 rescues the Cd sensitivity and complements the aberrant expression of IRT1 in opt3-2 roots, suggesting that OPT3 is required to relay the iron status from leaves to roots. OPT3 expression was found in the vasculature with preferential expression in the phloem at the plasma membrane. Using radioisotope experiments, we found that mobilization of Fe from leaves is severely affected in opt3-2, suggesting that Fe mobilization out of leaves is required for proper trace-metal homeostasis. When expressed in yeast, OPT3 does not localize to the plasma membrane, precluding the identification of the OPT3 substrate. Our in planta results show that OPT3 is important for leaf phloem-loading of iron and plays a key role regulating Fe, Zn, and Cd distribution within the plant. Furthermore, ferric chelate reductase activity analyses provide evidence that iron is not the sole signal transferred from leaves to roots in leaf iron status signaling.
doi:10.1093/mp/ssu067
PMCID: PMC4153440  PMID: 24880337
phloem transport; seed loading; metal homeostasis; iron deficiency; ionomics.

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