Microautophagy Mediates Vacuolar Delivery of Storage Proteins in Maize Aleurone Cells.

The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles via an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment via microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in Arabidopsis leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.

Leaf Trichome Distribution Pattern in Arabidopsis Reveals Gene Expression Variation Associated with Environmental Adaptation.

Gene expression varies stochastically even in both heterogenous and homogeneous cell populations. This variation is not simply useless noise; rather, it is important for many biological processes. Unicellular organisms or cultured cell lines are useful for analyzing the variation in gene expression between cells; however, owing to technical challenges, the biological relevance of this variation in multicellular organisms such as higher plants remain unclear. Here, we addressed the biological relevance of this variation between cells by examining the genetic basis of trichome distribution patterns in Arabidopsis thaliana. The distribution pattern of a trichome on a leaf is stochastic and can be mathematically represented using Turing's reaction-diffusion (RD) model. We analyzed simulations based on the RD model and found that the variability in the trichome distribution pattern increased with the increase in stochastic variation in a particular gene expression. Moreover, differences in heat-dependent variability of the trichome distribution pattern between the accessions showed a strong correlation with environmental factors to which each accession was adapted. Taken together, we successfully visualized variations in gene expression by quantifying the variability in the Arabidopsis trichome distribution pattern. Thus, our data provide evidence for the biological importance of variations in gene expression for environmental adaptation.

Primed histone demethylation regulates shoot regenerative competency.

Acquisition of pluripotency by somatic cells is a striking process that enables multicellular organisms to regenerate organs. This process includes silencing of genes to erase original tissue memory and priming of additional cell type specification genes, which are then poised for activation by external signal inputs. Here, through analysis of genome-wide histone modifications and gene expression profiles, we show that a gene priming mechanism involving LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3) specifically eliminates H3K4me2 during formation of the intermediate pluripotent cell mass known as callus derived from Arabidopsis root cells. While LDL3-mediated H3K4me2 removal does not immediately affect gene expression, it does facilitate the later activation of genes that act to form shoot progenitors when external cues lead to shoot induction. These results give insights into the role of H3K4 methylation in plants, and into the primed state that provides plant cells with high regenerative competency.

Genome-wide analysis of MpBHLH12, a IIIf basic helix-loop-helix transcription factor of Marchantia polymorpha.

The evolution of plants on land required adaptation to UV radiation and dry environments, and involved the appearance and/or rewiring of genetic connections, known as gene regulatory networks (GRNs), which consist of one or more transcription factors (TFs). The liverwort, Marchantia polymorpha, is a basal land plant, with a recently sequenced genome. The number of genes encoding basic helix-loop-helix (bHLH) family members is considerably higher in M. polymorpha than in charophyte green algae, suggesting the contribution of bHLH proteins to the evolution of GRNs associated with the adaptation of plants to land. Although an understanding of the evolutionary aspects of GRNs is fundamental for elucidating the mechanisms of environmental adaptation, the evolution of GRNs that led to land adaptation in plants remains poorly understood. In this study, we isolated a single gene encoding a IIIf bHLH TF from M. polymorpha, MpBHLH12. Transgenic M. polymorpha constitutively overexpressing MpBHLH12 showed smaller and fewer gemma cups than wild type, suggesting that MpBHLH12 is involved in the regulation of morphological development. Transcriptomic analysis of MpBHLH12 overexpressor (MpBHLH12ox) lines revealed an overlap with the GRN of MpMYB14, which regulates the biosynthesis of anthocyanins and phenolic compounds. However, MpBHLH12ox did not show anthocyanin accumulation. Results of the transient reporter assay suggest that MpBHLH12 could function in repression rather than activation. Our findings suggest that although the IIIf bHLH MpBHLH12 shows highest amino acid similarity with IIIf bHLH clade and is involved in developmental process and partly biosynthesis of phenolic compounds in M. polymorpha like Arabidopsis IIIf bHLH, the GRN involving MpBHLH12 would be distinct one from those of the IIIf bHLH TFs of seed plants.

Arabidopsis JMJD5/JMJ30 Acts Independently of LUX ARRHYTHMO Within the Plant Circadian Clock to Enable Temperature Compensation.

The circadian system ensures that plants respond appropriately to environmental change by predicting regular transitions that occur during diel cycles. In order to be most useful, the circadian system needs to be compensated against daily and seasonal changes in temperature that would otherwise alter the pace of this biological oscillator. We demonstrate that an evening-phased protein, the putative histone demethylase JMJD5, contributes to temperature compensation. JMJD5 is co-expressed with components of the Evening Complex, an agglomeration of proteins including EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHYMO (LUX), which also integrates temperature changes into the molecular clockwork. One role of the Evening Complex is to regulate expression of PSEUDORESPONSE REGULATOR9 (PRR9) and PRR7, important components of the temperature compensation mechanism. Surprisingly we find that LUX, but not other Evening Complex components, is dispensable for clock function at low temperatures. Further genetic analysis suggests JMJD5 acts in a parallel pathway to LUX within the circadian system. Although an intact JMJD5 catalytic domain is required for its function within the clock, our findings suggest JMJD5 does not directly regulate H3K36 methylation at circadian loci. Such data refine our understanding of how JMDJ5 acts within the Arabidopsis circadian system.

Transcriptome-wide effects of expansin gene manipulation in etiolated Arabidopsis seedling.

Expansin is a non-enzymatic protein which plays a pivotal role in cell wall loosening by inducing stress relaxation and extension in the plant cell wall. Previous studies on Arabidopsis, Petunia × hybrida, and tomato demonstrated that the suppression of expansin gene expression reduced plant growth but expansin overexpression does not necessarily promotes growth. In this study, both expansin gene suppression and overexpression in dark-grown transgenic Arabidopsis seedlings resulted in reduced hypocotyl length at late growth stages with a more pronounced effect for the overexpression. This defect in hypocotyl elongation raises questions about the molecular effect of expansin gene manipulation. RNA-seq analysis of the transcriptomic changes between day 3 and day 5 seedlings for both transgenic lines found numerous differentially expressed genes (DEGs) including transcription factors and hormone-related genes involved in different aspects of cell wall development. These DEGs imply that the observed hypocotyl growth retardation is a consequence of the concerted effect of regulatory factors and multiple cell-wall related genes, which are important for cell wall remodelling during rapid hypocotyl elongation. This is further supported by co-expression analysis through network-centric approach of differential network cluster analysis. This first transcriptome-wide study of expansin manipulation explains why the effect of expansin overexpression is greater than suppression and provides insights into the dynamic nature of molecular regulation during etiolation.

GTL1 and DF1 regulate root hair growth through transcriptional repression of ROOT HAIR DEFECTIVE 6-LIKE 4 in Arabidopsis.

How plants determine the final size of growing cells is an important, yet unresolved, issue. Root hairs provide an excellent model system with which to study this as their final cell size is remarkably constant under constant environmental conditions. Previous studies have demonstrated that a basic helix-loop helix transcription factor ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes root hair growth, but how hair growth is terminated is not known. In this study, we demonstrate that a trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 repress root hair growth in Arabidopsis Our transcriptional data, combined with genome-wide chromatin-binding data, show that GTL1 and DF1 directly bind the RSL4 promoter and regulate its expression to repress root hair growth. Our data further show that GTL1 and RSL4 regulate each other, as well as a set of common downstream genes, many of which have previously been implicated in root hair growth. This study therefore uncovers a core regulatory module that fine-tunes the extent of root hair growth by the orchestrated actions of opposing transcription factors.

A Novel Regulatory Pathway for K+ Uptake in the Legume Symbiont Azorhizobium caulinodans in Which TrkJ Represses the kdpFABC Operon at High Extracellular K+ Concentrations.

Bacteria have multiple K+ uptake systems. Escherichia coli, for example, has three types of K+ uptake systems, which include the low-K+-inducible KdpFABC system and two constitutive systems, Trk (TrkAG and TrkAH) and Kup. Azorhizobium caulinodans ORS571, a rhizobium that forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, also has three types of K+ uptake systems. Through phylogenetic analysis, we found that A. caulinodans has two genes homologous to trkG and trkH, designated trkI and trkJ We also found that trkI is adjacent to trkA in the genome and these two genes are transcribed as an operon; however, trkJ is present at a distinct locus. Our results demonstrated that trkAI, trkJ, and kup were expressed in the wild-type stem nodules, whereas kdpFABC was not. Interestingly, Δkup and Δkup ΔkdpA mutants formed Fix- nodules, while the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant formed Fix+ nodules, suggesting that with the additional deletion of Trk system genes in the Δkup mutant, Fix+ nodule phenotypes were recovered. kdpFABC of the Δkup ΔtrkJ mutant was expressed in stem nodules, but not in the free-living state, under high-K+ conditions. However, kdpFABC of the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant was highly expressed even under high-K+ conditions. The cytoplasmic K+ levels in the Δkup ΔtrkA ΔtrkI mutant, which did not express kdpFABC under high-K+ conditions, were markedly lower than those in the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant. Taking all these results into consideration, we propose that TrkJ is involved in the repression of kdpFABC in response to high external K+ concentrations and that the TrkAI system is unable to function in stem nodules.IMPORTANCE K+ is a major cytoplasmic cation in prokaryotic and eukaryotic cells. Bacteria have multiple K+ uptake systems to control the cytoplasmic K+ levels. In many bacteria, the K+ uptake system KdpFABC is expressed under low-K+ conditions. For years, many researchers have argued over how bacteria sense K+ concentrations. Although KdpD of Escherichia coli is known to sense both cytoplasmic and extracellular K+ concentrations, the detailed mechanism of K+ sensing is still unclear. In this study, we propose that the transmembrane TrkJ protein of Azorhizobium caulinodans acts as a sensor for the extracellular K+ concentration and that high extracellular K+ concentrations repress the expression of KdpFABC via TrkJ.

Stringent Expression Control of Pathogenic R-body Production in Legume Symbiont Azorhizobium caulinodans.

R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than CaedibacterAzorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.IMPORTANCECaedibacter species, which are obligate endosymbiotic bacteria of paramecia, produce R bodies, and R-body-producing endosymbionts that are released from their hosts are pathogenic to symbiont-free paramecia. Besides Caedibacter species, R bodies have also been observed in a few free-living bacteria, but the significance of R-body production in these bacteria is still unknown. Recent advances in genome sequencing technologies revealed that many Gram-negative bacteria possess reb genes encoding R-body components, and interestingly, many of them are animal and plant pathogens. Azorhizobium caulinodans, a microsymbiont of the tropical legume Sesbania rostrata, also possesses reb genes. In this study, we demonstrate that A. caulinodans has ability to kill the host plant cells by producing R bodies, suggesting that pathogenicity conferred by an R body might be universal in bacteria possessing reb genes. Furthermore, we provide the first insight into the molecular mechanism underlying the expression of R-body production in response to environmental factors, such as temperature and 2-oxoglutarate.

CBF2A-CBF4B genomic region copy numbers alongside the circadian clock play key regulatory mechanisms driving expression of FR-H2 CBFs.

The C-Repeat Binding Factors (CBFs) are DNA-binding transcriptional activators that were identified using Arabidopsis thaliana. In barley, Hordeum vulgare, a cluster of CBF genes reside at FROST RESISTANCE-H2, one of two loci having major effects on winter-hardiness. FR-H2 was revealed in a population derived from the winter barley 'Nure' and the spring barley 'Trèmois'. 'Nure' harbors two to three copies of CBF2A and CBF4B as a consequence of tandem iteration of the genomic region encompassing these genes whereas 'Trèmois' harbors single copies, and these copy number differences are associated with their transcript level differences. Here we explore further the relationship between FR-H2 CBF gene copy number and transcript levels using 'Admire', a winter barley accumulating FR-H2 CBF gene transcripts to very high levels, and a group of lines related to 'Admire' through descent. DNA blot hybridization indicated the CBF2A-CBF4B genomic region is present in 7-8 copies in 'Admire' and is highly variable in copy number across the lines related to 'Admire'. At normal growth temperatures transcript levels of CBF12, CBF14, and CBF16 were higher in lines having greater CBF2A-CBF4B genomic region copy numbers than in lines having fewer copy numbers at peak expression level time points controlled by the circadian clock. Chromatin immunoprecipitation indicated CBF2 was at the CBF12 and CBF16 promoters at normal growth temperatures. These data support a scenario in which CBF2A-CBF4B genomic region copy numbers affect expression of other FR-H2 CBFs through a mechansim in which these other FR-H2 CBFs are activated by those in the copy number variable unit.

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis.

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants.

A Maize Gene Regulatory Network for Phenolic Metabolism.

The translation of the genotype into phenotype, represented for example by the expression of genes encoding enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with the environment, is largely carried out by transcription factors (TFs) that recognize specific cis-regulatory elements in the genes that they control. TFs and their target genes are organized in gene regulatory networks (GRNs), and thus uncovering GRN architecture presents an important biological challenge necessary to explain gene regulation. Linking TFs to the genes they control, central to understanding GRNs, can be carried out using gene- or TF-centered approaches. In this study, we employed a gene-centered approach utilizing the yeast one-hybrid assay to generate a network of protein-DNA interactions that participate in the transcriptional control of genes involved in the biosynthesis of maize phenolic compounds including general phenylpropanoids, lignins, and flavonoids. We identified 1100 protein-DNA interactions involving 54 phenolic gene promoters and 568 TFs. A set of 11 TFs recognized 10 or more promoters, suggesting a role in coordinating pathway gene expression. The integration of the gene-centered network with information derived from TF-centered approaches provides a foundation for a phenolics GRN characterized by interlaced feed-forward loops that link developmental regulators with biosynthetic genes.

Ridaifen G, tamoxifen analog, is a potent anticancer drug working through a combinatorial association with multiple cellular factors.

Ridaifen-G (RID-G), a tamoxifen analog that we previously synthesized, has potent growth inhibitory activity against various cancer cell lines. Tamoxifen is an anticancer drug known to act on an estrogen receptor (ER) and other proteins. However, our previous studies interestingly suggested that the mechanism of action of RID-G was different from that of tamoxifen. In order to investigate the molecular mode of action of RID-G, we developed a novel chemical genetic approach that combined a phage display screen with a statistical analysis of drug potency and gene expression profiles in thirty-nine cancer cell lines. Application of this method to RID-G revealed that three proteins, calmodulin (CaM), heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), and zinc finger protein 638 (ZNF638) were the candidates of direct targets of RID-G. Moreover, cell lines susceptible to RID-G show similar expression profiles of RID-G target genes. These results suggest that RID-G involves CaM, hnRNP A2/B1, and ZNF638 in its growth inhibitory activity.

ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

Helix-loop-helix/basic helix-loop-helix transcription factor network represses cell elongation in Arabidopsis through an apparent incoherent feed-forward loop.

Cell elongation is promoted by different environmental and hormonal signals, involving light, temperature, brassinosteroid (BR), and gibberellin, that inhibit the atypical basic helix-loop-helix (bHLH) transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 (IBH1). Ectopic accumulation of IBH1 causes a severe dwarf phenotype, but the cell elongation suppression mechanism is still not well understood. Here, we identified a close homolog of IBH1, IBH1-LIKE1 (IBL1), that also antagonized BR responses and cell elongation. Genome-wide expression analyses showed that IBH1 and IBL1 act interdependently downstream of the BRASSINAZOLE-RESISTANT1 (BZR1)-PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)-DELLA module. Although characterized as non-DNA binding, IBH1 repressed direct IBL1 transcription, and they both acted in tandem to suppress the expression of a common downstream helix-loop-helix (HLH)/bHLH network, thus forming an incoherent feed-forward loop. IBH1 and IBL1 together repressed the expression of PIF4, known to stimulate skotomorphogenesis synergistically with BZR1. Strikingly, PIF4 bound all direct and down-regulated HLH/bHLH targets of IBH1 and IBL1. Additional genome-wide comparisons suggested a model in which IBH1 antagonized PIF4 but not the PIF4-BZR1 dimer.

Regulatory modules controlling maize inflorescence architecture.

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.