Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm.

Development of the cereal endosperm involves cell differentiation processes that enable nutrient uptake from the maternal plant, accumulation of storage products, and their utilization during germination. However, little is known about the regulatory mechanisms that link cell differentiation processes with those controlling storage product synthesis and deposition, including the activation of zein genes by the maize (Zea mays) bZIP transcription factor Opaque-2 (O2). Here, we mapped in vivo binding sites of O2 in B73 endosperm and compared the results with genes differentially expressed in B73 and B73o2 We identified 186 putative direct O2 targets and 1677 indirect targets, encoding a broad set of gene functionalities. Examination of the temporal expression patterns of O2 targets revealed at least two distinct modes of O2-mediated gene activation. Two O2-activated genes, bZIP17 and NAKED ENDOSPERM2 (NKD2), encode transcription factors, which can in turn coactivate other O2 network genes with O2. NKD2 (with its paralog NKD1) was previously shown to be involved in regulation of aleurone development. Collectively, our results provide insights into the complexity of the O2-regulated network and its role in regulation of endosperm cell differentiation and function.

FERTILIZATION-INDEPENDENT SEED-Polycomb Repressive Complex 2 Plays a Dual Role in Regulating Type I MADS-Box Genes in Early Endosperm Development.

Early endosperm development presents a unique system in which to uncover epigenetic regulatory mechanisms because the contributing maternal and paternal genomes possess differential epigenetic modifications. In Arabidopsis (Arabidopsis thaliana), the initiation of endosperm coenocytic growth upon fertilization and the transition to endosperm cellularization are regulated by the FERTILIZATION-INDEPENDENT SEED (FIS)-Polycomb Repressive Complex 2 (PRC2), a putative H3K27 methyltransferase. Here, we address the possible role of the FIS-PRC2 complex in regulating the type I MADS-box gene family, which has been shown previously to regulate early endosperm development. We show that a subclass of type I MADS-box genes (C2 genes) was expressed in distinct domains of the coenocytic endosperm in wild-type seeds. Furthermore, the C2 genes were mostly up-regulated biallelically during the extended coenocytic phase of endosperm development in the FIS-PRC2 mutant background. Using allele-specific expression analysis, we also identified a small subset of C2 genes subjected to FIS-PRC2-dependent maternal or FIS-PRC2-independent paternal imprinting. Our data support a dual role for the FIS-PRC2 complex in the regulation of C2 type I MADS-box genes, as evidenced by a generalized role in the repression of gene expression at both alleles associated with endosperm cellularization and a specialized role in silencing the maternal allele of imprinted genes.

Gain-of-function mutations in beet DODA2 identify key residues for betalain pigment evolution.

The key enzymatic step in betalain biosynthesis involves conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) to betalamic acid. One class of enzymes capable of this is 3,4-dihydroxyphenylalanine 4,5-dioxygenase (DODA). In betalain-producing species, multiple paralogs of this gene are maintained. This study demonstrates which paralogs function in the betalain pathway and determines the residue changes required to evolve a betalain-nonfunctional DODA into a betalain-functional DODA. Functionalities of two pairs of DODAs were tested by expression in beets, Arabidopsis and yeast, and gene silencing was performed by virus-induced gene silencing. Site-directed mutagenesis identified amino acid residues essential for betalamic acid production. Beta vulgaris and Mirabilis jalapa both possess a DODA1 lineage that functions in the betalain pathway and at least one other lineage, DODA2, that does not. Site-directed mutagenesis resulted in betalain biosynthesis by a previously nonfunctional DODA, revealing key residues required for evolution of the betalain pathway. Divergent functionality of DODA paralogs, one clade involved in betalain biosynthesis but others not, is present in various Caryophyllales species. A minimum of seven amino acid residue changes conferred betalain enzymatic activity to a betalain-nonfunctional DODA paralog, providing insight into the evolution of the betalain pigment pathway in plants.

SUPPRESSOR OF FRIGIDA (SUF4) Supports Gamete Fusion via Regulating Arabidopsis EC1 Gene Expression.

The EGG CELL1 (EC1) gene family of Arabidopsis (Arabidopsis thaliana) comprises five members that are specifically expressed in the egg cell and redundantly control gamete fusion during double fertilization. We investigated the activity of all five EC1 promoters in promoter-deletion studies and identified SUF4 (SUPPRESSOR OF FRIGIDA4), a C2H2 transcription factor, as a direct regulator of the EC1 gene expression. In particular, we demonstrated that SUF4 binds to all five Arabidopsis EC1 promoters, thus regulating their expression. The down-regulation of SUF4 in homozygous suf4-1 ovules results in reduced EC1 expression and delayed sperm fusion, which can be rescued by expressing SUF4-ß-glucuronidase under the control of the SUF4 promoter. To identify more gene products able to regulate EC1 expression together with SUF4, we performed coexpression studies that led to the identification of MOM1 (MORPHEUS' MOLECULE1), a component of a silencing mechanism that is independent of DNA methylation marks. In mom1-3 ovules, both SUF4 and EC1 genes are down-regulated, and EC1 genes show higher levels of histone 3 lysine-9 acetylation, suggesting that MOM1 contributes to the regulation of SUF4 and EC1 gene expression.

RNA sequencing of laser-capture microdissected compartments of the maize kernel identifies regulatory modules associated with endosperm cell differentiation.

Endosperm is an absorptive structure that supports embryo development or seedling germination in angiosperms. The endosperm of cereals is a main source of food, feed, and industrial raw materials worldwide. However, the genetic networks that regulate endosperm cell differentiation remain largely unclear. As a first step toward characterizing these networks, we profiled the mRNAs in five major cell types of the differentiating endosperm and in the embryo and four maternal compartments of the maize (Zea mays) kernel. Comparisons of these mRNA populations revealed the diverged gene expression programs between filial and maternal compartments and an unexpected close correlation between embryo and the aleurone layer of endosperm. Gene coexpression network analysis identified coexpression modules associated with single or multiple kernel compartments including modules for the endosperm cell types, some of which showed enrichment of previously identified temporally activated and/or imprinted genes. Detailed analyses of a coexpression module highly correlated with the basal endosperm transfer layer (BETL) identified a regulatory module activated by MRP-1, a regulator of BETL differentiation and function. These results provide a high-resolution atlas of gene activity in the compartments of the maize kernel and help to uncover the regulatory modules associated with the differentiation of the major endosperm cell types.

TTG2 controls the developmental regulation of seed coat tannins in Arabidopsis by regulating vacuolar transport steps in the proanthocyanidin pathway.

The brown color of Arabidopsis seeds is caused by the deposition of proanthocyanidins (PAs or condensed tannins) in their inner testa layer. A transcription factor complex consisting of TT2, TT8 and TTG1 controls expression of PA biosynthetic genes, just as similar TTG1-dependent complexes have been shown to control flavonoid pigment pathway gene expression in general. However, PA synthesis is controlled by at least one other gene. TTG2 mutants lack the pigmentation found in wild-type seeds, but produce other flavonoid compounds, such as anthocyanins in the shoot, suggesting that TTG2 regulates genes in the PA biosynthetic branch of the flavonoid pathway. We analyzed the expression of PA biosynthetic genes within the developing seeds of ttg2-1 and wild-type plants for potential TTG2 regulatory targets. We found that expression of TT12, encoding a MATE type transporter, is dependent on TTG2 and that TTG2 can bind to the upstream regulatory region of TT12 suggesting that TTG2 directly regulates TT12. Ectopic expression of TT12 in ttg2-1 plants partially restores seed coat pigmentation. Moreover, we show that TTG2 regulation of TT12 is dependent on TTG1 and that TTG1 and TTG2 physically interact. The observation that TTG1 interacts with TTG2, a WRKY type transcription factor, proposes the existence of a novel TTG1-containing complex, and an addendum to the existing paradigm of flavonoid pathway regulation.

Advances in the MYB-bHLH-WD Repeat (MBW) Pigment Regulatory Model: Addition of a WRKY Factor and Co-option of an Anthocyanin MYB for Betalain Regulation.

Flavonoids are secondary metabolites derived from the general phenylpropanoid pathway and are widespread throughout the plant kingdom. The functions of flavonoids are diverse, including defense against phytopathogens, protection against UV light damage and oxidative stress, regulation of auxin transport and allelopathy. One of the most conspicuous functions of flavonoids has long attracted the attention of pollinators and scientist alike: the vivid shades of red, pink, orange, blue and purple on display in the flowers of angiosperms. Thus, flavonoid pigments have perhaps been the most intensely studied phenylpropanoids. From Mendel to McClintock and up to the present, studies centered on flavonoid pigments have resulted in some of the most important scientific discoveries of the last 150 years, including the first examples of transcriptional regulation in plants. Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model for the regulation of the flavonoid pigment pathway. We will survey the history of the MBW model spanning the last three decades, highlighting the major findings that have contributed to our current understanding. In particular, recent discoveries regarding WRKY protein control of the flavonoid pigment pathway and its relationship to the MBW complex will be emphasized. In addition, we will discuss recent findings about the regulation of the beet betalain pigment pathway, and how a MYB member of the MBW complex was co-opted to regulate this chemically unrelated but functionally equivalent pathway.

Temporal patterns of gene expression in developing maize endosperm identified through transcriptome sequencing.

Endosperm is a filial structure resulting from a second fertilization event in angiosperms. As an absorptive storage organ, endosperm plays an essential role in support of embryo development and seedling germination. The accumulation of carbohydrate and protein storage products in cereal endosperm provides humanity with a major portion of its food, feed, and renewable resources. Little is known regarding the regulatory gene networks controlling endosperm proliferation and differentiation. As a first step toward understanding these networks, we profiled all mRNAs in the maize kernel and endosperm at eight successive stages during the first 12 d after pollination. Analysis of these gene sets identified temporal programs of gene expression, including hundreds of transcription-factor genes. We found a close correlation of the sequentially expressed gene sets with distinct cellular and metabolic programs in distinct compartments of the developing endosperm. The results constitute a preliminary atlas of spatiotemporal patterns of endosperm gene expression in support of future efforts for understanding the underlying mechanisms that control seed yield and quality.

The genetic architecture of constitutive and induced trichome density in two new recombinant inbred line populations of Arabidopsis thaliana: phenotypic plasticity, epistasis, and bidirectional leaf damage response.

BACKGROUND: Herbivory imposes an important selective pressure on plants. In Arabidopsis thaliana leaf trichomes provide a key defense against insect herbivory; however, trichome production incurs a fitness cost in the absence of herbivory. Previous work on A. thaliana has shown an increase in trichome density in response to leaf damage, suggesting a mechanism by which the cost associated with constitutively high trichome density might be mitigated; however, the genetic basis of trichome density induction has not been studied. RESULTS: Here, we describe the mapping of quantitative trait loci (QTL) for constitutive and damage induced trichome density in two new recombinant inbred line populations of A. thaliana; mapping for constitutive and induced trichome density also allowed for the investigation of damage response (plasticity) QTL. Both novel and previously identified QTL for constitutive trichome density and the first QTL for induced trichome density and response are identified. Interestingly, two of the four parental accessions and multiple RILs in each population exhibited lower trichome density following leaf damage, a response not previously described in A. thaliana. Importantly, a single QTL was mapped for the response phenotype and allelic variation at this locus appears to determine response trajectory in RILs. The data also show that epistatic interactions are a significant component of the genetic architecture of trichome density. CONCLUSIONS: Together, our results provide further insights into the genetic architecture of constitutive trichome density and new insights into induced trichome density in A. thaliana specifically and to our understanding of the genetic underpinnings of natural variation generally.

Natural allelic variation defines a role for ATMYC1: trichome cell fate determination.

The molecular nature of biological variation is not well understood. Indeed, many questions persist regarding the types of molecular changes and the classes of genes that underlie morphological variation within and among species. Here we have taken a candidate gene approach based on previous mapping results to identify the gene and ultimately a polymorphism that underlies a trichome density QTL in Arabidopsis thaliana. Our results show that natural allelic variation in the transcription factor ATMYC1 alters trichome density in A. thaliana; this is the first reported function for ATMYC1. Using site-directed mutagenesis and yeast two-hybrid experiments, we demonstrate that a single amino acid replacement in ATMYC1, discovered in four ecotypes, eliminates known protein-protein interactions in the trichome initiation pathway. Additionally, in a broad screen for molecular variation at ATMYC1, including 72 A. thaliana ecotypes, a high-frequency block of variation was detected that results in >10% amino acid replacement within one of the eight exons of the gene. This sequence variation harbors a strong signal of divergent selection but has no measurable effect on trichome density. Homologs of ATMYC1 are pleiotropic, however, so this block of variation may be the result of natural selection having acted on another trait, while maintaining the trichome density role of the gene. These results show that ATMYC1 is an important source of variation for epidermal traits in A. thaliana and indicate that the transcription factors that make up the TTG1 genetic pathway generally may be important sources of epidermal variation in plants.

Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways.

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

Energy supplies and future engines for land, sea, and air.

The 2012 Critical Review Discussion complements Wilson, (2012), provides pointers to more detailed treatments of different topics and adds additional dimensions to the area of "energy". These include broader aspects of technologies driven by fuel resources and environmental issues, the concept of energy technology innovation, evolution in transportation resources, and complexities of energy policies addressing carbon taxes or carbon trading. National and global energy data bases are identified and evaluated and conversion factors are given to allow their comparability.

Mirabilis jalapa mottle virus: a new carlavirus infecting four o'clocks.

Analysis of a next-generation sequence dataset from Mirabilis jalapa resulted in the discovery of a novel virus in the genus Carlavirus (family Betaflexiviridae), mirabilis jalapa mottle virus (MjMV). The complete genome of MjMV was determined to consist of 8315 nucleotides (nt), with the six open reading frames indicative of carlaviruses. MjMV is most similar to kalanchoe latent virus (60% identity) and lily symptomless virus (59% identity). The virus can be transmitted mechanically to Mirabilis, but thus far MjMV has only been shown to infect Mirabilis jalapa, causing a slight leaf mottling and leaf wrinkling phenotype.

Genetic transformation technologies for the common dandelion, Taraxacum officinale.

BACKGROUND: Taraxacum officinale, or the common dandelion, is a widespread perennial species recognized worldwide as a common lawn and garden weed. Common dandelion is also cultivated for use in teas, as edible greens, and for use in traditional medicine. It produces latex and is closely related to the Russian dandelion, T. kok-saghyz, which is being developed as a rubber crop. Additionally, the vast majority of extant common dandelions reproduce asexually through apomictically derived seeds- an important goal for many major crops in modern agriculture. As such, there is increasing interest in the molecular control of important pathways as well as basic molecular biology and reproduction of common dandelion. RESULTS: Here we present an improved Agrobacterium-based genetic transformation and regeneration protocol, a protocol for generation and transformation of protoplasts using free DNA, and a protocol for leaf Agrobacterium infiltration for transient gene expression. These protocols use easily obtainable leaf explants from soil-grown plants and reagents common to most molecular plant laboratories. We show that common markers used in many plant transformation systems function as expected in common dandelion including fluorescent proteins, GUS, and anthocyanin regulation, as well as resistance to kanamycin, Basta, and hygromycin. CONCLUSION: Reproducible, stable and transient transformation methods are presented that will allow for needed molecular structure and function studies of genes and proteins in T. officinale.

TTG1 complex MYBs, MYB5 and TT2, control outer seed coat differentiation.

A suite of epidermal characters in Arabidopsis is under the transcriptional control of a combinatorial complex containing WD repeat, bHLH and MYB proteins. Many genetic, molecular and biochemical means have been employed to identify and characterize a complete minimal set of complex members required for the trichome initiation, root hair spacing, anthocyanin production and seed coat tannin production pathways. In addition, the WD and bHLH proteins required for outer seed coat differentiation have been identified. However, until now the MYB complex member(s) required for this last WD-bHLH-MYB complex-dependent character have remained elusive. Here we identify two MYBs, AtMYB5 and TT2, as partially redundant in regulating this outer seed coat developmental process with MYB5 having the major role. MYB5 and TT2 are shown to be expressed in this outer seed coat domain. We also show that MYB5 has weak pleiotropic control over trichome development and tannin production and is also expressed in the appropriate places for these functions. TT8 and the downstream GL2 and TTG2 regulators of seed coat development are found to be downregulated in the MYB mutants. Although the TTG1-dependent R2R3 MYBs are considered to be highly pathway specific, identification of MYBs responsible for outer seed coat development allowed for the elucidation of previously undetected novel developmental pleiotropy among these elements.

Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

The MYB98 subcircuit of the synergid gene regulatory network includes genes directly and indirectly regulated by MYB98.

The female gametophyte contains two synergid cells that play a role in many steps of the angiosperm reproductive process, including pollen tube guidance. At their micropylar poles, the synergid cells have a thickened and elaborated cell wall: the filiform apparatus that is thought to play a role in the secretion of the pollen tube attractant(s). MYB98 regulates an important subcircuit of the synergid gene regulatory network (GRN) that functions to activate the expression of genes required for pollen tube guidance and filiform apparatus formation. The MYB98 subcircuit comprises at least 83 downstream genes, including 48 genes within four gene families (CRP810, CRP3700, CRP3730 and CRP3740) that encode Cys-rich proteins. We show that the 11 CRP3700 genes, which include DD11 and DD18, are regulated by a common cis-element, GTAACNT, and that a multimer of this sequence confers MYB98-dependent synergid expression. The GTAACNT element contains the MYB98-binding site identified in vitro, suggesting that the 11 CRP3700 genes are direct targets of MYB98. We also show that five of the CRP810 genes, which include DD2, lack a functional GTAACNT element, suggesting that they are not directly regulated by MYB98. In addition, we show that the five CRP810 genes are regulated by the cis-element AACGT, and that a multimer of this sequence confers synergid expression. Together, these results suggest that the MYB98 branch of the synergid GRN is multi-tiered and, therefore, contains at least one additional downstream transcription factor.

Multiherbicide tolerance conferred by AtPgp1 and apyrase overexpression in Arabidopsis thaliana.

Herbicide resistance is an important trait often introduced into crop plants. Mechanisms of resistance can involve a mutant target protein that is unaffected by the herbicide, or metabolic detoxification or degradation of the herbicide. Recently, we showed that overexpression in Arabidopsis thaliana of either psNTP9, the garden pea apyrase gene, or AtPgp1, the A. thaliana homolog of the plant multidrug resistance (MDR) gene, enabled A. thaliana to germinate on the toxin cycloheximide and to grow better on toxic levels of the plant hormone N6-[2-isopentyl]adenine (2iP). Here we report that overexpression of either MDR or apyrase proteins resulted in increased resistance to herbicides from different chemical classes. Apyrase inhibition by small molecule inhibitors reversed this resistance. Treatment of untransformed plants with an apyrase inhibitor increased their sensitivity to the same herbicides. These results indicate that the genes may be involved in a resistance mechanism relating to decreased retention or increased active efflux of herbicide from the plant cell.

Mapping quantitative trait loci in multiple populations of Arabidopsis thaliana identifies natural allelic variation for trichome density.

The majority of biological traits are genetically complex. Mapping the quantitative trait loci (QTL) that determine these phenotypes is a powerful means for estimating many parameters of the genetic architecture for a trait and potentially identifying the genes responsible for natural variation. Typically, such experiments are conducted in a single mapping population and, therefore, have only the potential to reveal genomic regions that are polymorphic between the progenitors of the population. What remains unclear is how well the QTL identified in any one mapping experiment characterize the genetics that underlie natural variation in traits. Here we provide QTL mapping data for trichome density from four recombinant inbred mapping populations of Arabidopsis thaliana. By aligning the linkage maps for these four populations onto a common physical map, the results from each experiment were directly compared. Seven of the nine QTL identified are population specific while two were mapped in all four populations. Our results show that many lineage-specific alleles that either increase or decrease trichome density persist in natural populations and that most of this genetic variation is additive. More generally, these findings suggest that the use of multiple populations holds great promise for better understanding the genetic architecture of natural variation.