Histone modification-dependent production of peptide hormones facilitates acquisition of pluripotency during leaf-to-callus transition in Arabidopsis.

Chromatin configuration is critical for establishing tissue identity and changes substantially during tissue identity transitions. The crucial scientific and agricultural technology of in vitro tissue culture exploits callus formation from diverse tissue explants and tissue regeneration via de novo organogenesis. We investigated the dynamic changes in H3ac and H3K4me3 histone modifications during leaf-to-callus transition in Arabidopsis thaliana. We analyzed changes in the global distribution of H3ac and H3K4me3 during the leaf-to-callus transition, focusing on transcriptionally active regions in calli relative to leaf explants, defined by increased accumulation of both H3ac and H3K4me3. Peptide signaling was particularly activated during callus formation; the peptide hormones RGF3, RGF8, PIP1 and PIPL3 were upregulated, promoting callus proliferation and conferring competence for de novo shoot organogenesis. The corresponding peptide receptors were also implicated in peptide-regulated callus proliferation and regeneration capacity. The effect of peptide hormones in plant regeneration is likely at least partly conserved in crop plants. Our results indicate that chromatin-dependent regulation of peptide hormone production not only stimulates callus proliferation but also establishes pluripotency, improving the overall efficiency of two-step regeneration in plant systems.

N6-methyladenosine-modified RNA acts as a molecular glue that drives liquid-liquid phase separation in plants.

Liquid-like condensates are organized by multivalent intrinsically disordered proteins and RNA molecules. We here demonstrate that N6-methyladenosine (m6A)-modified RNA is widespread in establishing diverse plant cell condensates. Several m6A-reader proteins contain putative prion-like domains, and the ect2/3/4 mutant exhibited reduced formation of key nuclear and cytoplasmic condensates in Arabidopsis.

Peptide Signaling during Plant Reproduction.

Plant signaling peptides are involved in cell-cell communication networks and coordinate a wide range of plant growth and developmental processes. Signaling peptides generally bind to receptor-like kinases, inducing their dimerization with co-receptors for signaling activation to trigger cellular signaling and biological responses. Fertilization is an important life event in flowering plants, involving precise control of cell-cell communications between male and female tissues. Peptide-receptor-like kinase-mediated signaling plays an important role in male-female interactions for successful fertilization in flowering plants. Here, we describe the recent findings on the functions and signaling pathways of peptides and receptors involved in plant reproduction processes including pollen germination, pollen tube growth, pollen tube guidance to the embryo sac, and sperm cell reception in female tissues.

Recent advances in peptide signaling during Arabidopsis root development.

Roots provide the plant with water and nutrients and anchor it in a substrate. Root development is controlled by plant hormones and various sets of transcription factors. Recently, various small peptides and their cognate receptors have been identified as controlling root development. Small peptides bind to membrane-localized receptor-like kinases, inducing their dimerization with co-receptor proteins for signaling activation and giving rise to cellular signaling outputs. Small peptides function as local and long-distance signaling molecules involved in cell-to-cell communication networks, coordinating root development. In this review, we survey recent advances in the peptide ligand-mediated signaling pathways involved in the control of root development in Arabidopsis. We describe the interconnection between peptide signaling and conventional phytohormone signaling. Additionally, we discuss the diversity of identified peptide-receptor interactions during plant root development.

The Circadian Clock Sets the Time of DNA Replication Licensing to Regulate Growth in Arabidopsis.

The circadian clock and cell cycle as separate pathways have been well documented in plants. Elucidating whether these two oscillators are connected is critical for understanding plant growth. We found that a slow-running circadian clock decelerates the cell cycle and, conversely, a fast clock speeds it up. The clock component TOC1 safeguards the G1-to-S transition and controls the timing of the mitotic cycle at early stages of leaf development. TOC1 also regulates somatic ploidy at later stages of leaf development and in hypocotyl cells. The S-phase is shorter and delayed in TOC1 overexpressing plants, which correlates with the diurnal repression of the DNA replication licensing gene CDC6 through binding of TOC1 to the CDC6 promoter. The slow cell-cycle pace in TOC1-ox also results in delayed tumor progression in inflorescence stalks. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment.

Coordination of matrix attachment and ATP-dependent chromatin remodeling regulate auxin biosynthesis and Arabidopsis hypocotyl elongation.

Hypocotyl elongation is extensively controlled by hormone signaling networks. In particular, auxin metabolism and signaling play key roles in light-dependent hypocotyl growth. The nuclear matrix facilitates organization of DNA within the nucleus, and dynamic interactions between nuclear matrix and DNA are related to gene regulation. Conserved scaffold/matrix attachment regions (S/MARs) are anchored to the nuclear matrix by the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) proteins in Arabidopsis. Here, we found that ESCAROLA (ESC)/AHL27 and SUPPRESSOR OF PHYTOCHROME B-4 #3 (SOB3)/AHL29 redundantly regulate auxin biosynthesis in the control of hypocotyl elongation. The light-inducible AHL proteins bind directly to an S/MAR region of the YUCCA 9 (YUC9) promoter and suppress its expression to inhibit hypocotyl growth in light-grown seedlings. In addition, they recruit the SWI2/SNF2-RELATED 1 (SWR1) complex and promote exchange of H2A with the histone variant H2A.Z at the YUC9 locus to further elaborately control auxin biosynthesis. Consistent with these results, the long hypocotyl phenotypes of light-grown genetic mutants of the AHLs and H2A.Z-exchanging components were suppressed by potent chemical inhibitors of auxin transport and YUC enzymes. These results suggest that the coordination of matrix attachment and chromatin modification underlies auxin biosynthesis in light-dependent hypocotyl growth.

The E3 ubiquitin ligase HOS1 is involved in ethylene regulation of leaf expansion in Arabidopsis.

Ethylene regulates a variety of physiological processes, such as flowering, senescence, abscission, and fruit ripening. In particular, leaf expansion is also controlled by ethylene in Arabidopsis. Exogenous treatment with ethylene inhibits leaf expansion, and consistently, ethylene insensitive mutants show increased leaf area. Here, we report that the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) regulates leaf expansion in an ethylene signaling pathway. The HOS1-deficient mutant showed reduced leaf area and was insensitive to ethylene perception inhibitor, silver thiosulfate (STS). Accordingly, genes encoding ethylene signaling components were significantly up-regulated in hos1-3. This study demonstrates that the HOS1 protein is involved in ethylene signal transduction for the proper regulation of leaf expansion possibly under environmentally stressful conditions.

Natural variation in floral nectar proteins of two Nicotiana attenuata accessions.

BACKGROUND: Floral nectar (FN) contains not only energy-rich compounds to attract pollinators, but also defense chemicals and several proteins. However, proteomic analysis of FN has been hampered by the lack of publically available sequence information from nectar-producing plants. Here we used next-generation sequencing and advanced proteomics to profile FN proteins in the opportunistic outcrossing wild tobacco, Nicotiana attenuata. RESULTS: We constructed a transcriptome database of N. attenuata and characterized its nectar proteome using LC-MS/MS. The FN proteins of N. attenuata included nectarins, sugar-cleaving enzymes (glucosidase, galactosidase, and xylosidase), RNases, pathogen-related proteins, and lipid transfer proteins. Natural variation in FN proteins of eleven N. attenuata accessions revealed a negative relationship between the accumulation of two abundant proteins, nectarin1b and nectarin5. In addition, microarray analysis of nectary tissues revealed that protein accumulation in FN is not simply correlated with the accumulation of transcripts encoding FN proteins and identified a group of genes that were specifically expressed in the nectary. CONCLUSIONS: Natural variation of identified FN proteins in the ecological model plant N. attenuata suggests that nectar chemistry may have a complex function in plant-pollinator-microbe interactions.

Alternative splicing of transcription factors in plant responses to low temperature stress: mechanisms and functions.

Transcription factors play a central role in the gene regulatory networks that mediate various aspects of plant developmental processes and responses to environmental changes. Therefore, their activities are elaborately regulated at multiple steps. In particular, accumulating evidence illustrates that post-transcriptional control of mRNA metabolism is a key molecular scheme that modulates the transcription factor activities in plant responses to temperature fluctuations. Transcription factors have a modular structure consisting of distinct protein domains essential for DNA binding, dimerization, and transcriptional regulation. Alternative splicing produces multiple proteins having different structural domain compositions from a single transcription factor gene. Recent studies have shown that alternative splicing of some transcription factor genes generates small interfering peptides (siPEPs) that negatively regulate the target transcription factors via peptide interference (PEPi), constituting self-regulatory circuits in plant cold stress response. A number of splicing factors, which are involved in RNA binding, splice site selection, and spliceosome assembly, are also affected by temperature fluctuations, supporting the close association of alternative splicing of transcription factors with plant responses to low temperatures. In this review, we summarize recent progress on the temperature-responsive alternative splicing of transcription factors in plants with emphasis on the siPEP-mediated PEPi mechanism.

Identification and characterization of circadian clock genes in a native tobacco, Nicotiana attenuata.

BACKGROUND: A plant's endogenous clock (circadian clock) entrains physiological processes to light/dark and temperature cycles. Forward and reverse genetic approaches in Arabidopsis have revealed the mechanisms of the circadian clock and its components in the genome. Similar approaches have been used to characterize conserved clock elements in several plant species. A wild tobacco, Nicotiana attenuata has been studied extensively to understand responses to biotic or abiotic stress in the glasshouse and also in their native habitat. During two decades of field experiment, we observed several diurnal rhythmic traits of N. attenuata in nature. To expand our knowledge of circadian clock function into the entrainment of traits important for ecological processes, we here report three core clock components in N. attenuata. RESULTS: Protein similarity and transcript accumulation allowed us to isolate orthologous genes of the core circadian clock components, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1), and ZEITLUPE (ZTL). Transcript accumulation of NaLHY peaked at dawn and NaTOC1 peaked at dusk in plants grown under long day conditions. Ectopic expression of NaLHY and NaZTL in Arabidopsis resulted in elongated hypocotyl and late-flowering phenotypes. Protein interactions between NaTOC1 and NaZTL were confirmed by yeast two-hybrid assays. Finally, when NaTOC1 was silenced in N. attenuata, late-flowering phenotypes under long day conditions were clearly observed. CONCLUSIONS: We identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.

Targeted inactivation of transcription factors by overexpression of their truncated forms in plants.

Transcription factors are central constituents of gene regulatory networks that control diverse aspects of plant development and environmental adaptability. Therefore they have been explored for decades as primary targets for agricultural biotechnology. A gene of interest can readily be introduced into many crop plants, whereas targeted gene inactivation is practically difficult in many cases. Here, we developed an artificial small interfering peptide (a-siPEP) approach, which is based on overexpression of specific protein domains, and evaluated its application for the targeted inactivation of transcription factors in the dicot model, Arabidopsis, and monocot model, Brachypodium. We designed potential a-siPEPs of two representative MADS box transcription factors, SUPPRESSOR OF OVEREXPRESSOR OF CONSTANS 1 (SOC1) and AGAMOUS (AG), and a MYB transcription factor, LATE ELONGATED HYPOCOTYL (LHY). Transgenic plants overproducing the a-siPEPs displayed phenotypes comparable to those of gene-deficient mutants. The a-siPEPs attenuate nuclear import and DNA-binding of target transcription factors. Our data demonstrate that the a-siPEP tool is an efficient genetic means of inactivating specific transcription factors in plants.

A Golgi-localized MATE transporter mediates iron homoeostasis under osmotic stress in Arabidopsis.

Iron is an essential micronutrient that acts as a cofactor in a wide variety of pivotal metabolic processes, such as the electron transport chain of respiration, photosynthesis and redox reactions, in plants. However, its overload exceeding the cellular capacity of iron binding and storage is potentially toxic to plant cells by causing oxidative stress and cell death. Consequently, plants have developed versatile mechanisms to maintain iron homoeostasis. Organismal iron content is tightly regulated at the steps of uptake, translocation and compartmentalization. Whereas iron uptake is fairly well understood at the cellular and organismal levels, intracellular and intercellular transport is only poorly understood. In the present study, we show that a MATE (multidrug and toxic compound extrusion) transporter, designated BCD1 (BUSH-AND-CHLOROTIC-DWARF 1), contributes to iron homoeostasis during stress responses and senescence in Arabidopsis. The BCD1 gene is induced by excessive iron, but repressed by iron deficiency. It is also induced by cellular and tissue damage occurring under osmotic stress. The activation-tagged mutant bcd1-1D exhibits leaf chlorosis, a typical symptom of iron deficiency. The chlorotic lesion of the mutant was partially recovered by iron feeding. Whereas the bcd1-1D mutant accumulated a lower amount of iron, the iron level was elevated in the knockout mutant bcd1-1. The BCD1 protein is localized to the Golgi complex. We propose that the BCD1 transporter plays a role in sustaining iron homoeostasis by reallocating excess iron released from stress-induced cellular damage.

Competitive inhibition of transcription factors by small interfering peptides.

Combinatorial assortment by dynamic dimer formation diversifies gene transcriptional specificities of transcription factors. A similar but biochemically distinct mechanism is competitive inhibition in which small proteins act as negative regulators by competitively forming nonfunctional heterodimers with specific transcription factors. The most extensively studied is the negative regulation of auxin response factors by AUXIN/INDOLE-3-ACETIC ACID repressors. Similarly, Arabidopsis thaliana (Arabidopsis) little zipper and mini finger proteins act as competitive inhibitors of target transcription factors. Competitive inhibitors are also generated by alternative splicing and controlled proteolytic processing. Because they provide a way of attenuating transcription factors we propose to call them small interfering peptides (siPEPs). The siPEP-mediated strategy could be applied to deactivate specific transcription factors in crop plants.

Expression of Arabidopsis pathogenesis-related genes during nematode infection.

The expression pattern of pathogenesis-related genes PR-1 to PR-5 was examined in the roots and leaves of Arabidopsis thaliana plants on infection with beet-cyst (Heterodera schachtii) and root-knot (Meloidogyne incognita) nematodes. During H. schachtii parasitism of Arabidopsis, the expression of PR-1, PR-2 and PR-5, which are considered to be markers for salicylic acid (SA)-dependent systemic acquired resistance (SAR), was induced in both roots and leaves of infected plants. In addition, the expression of PR-3 and PR-4, which are used as markers for jasmonic acid (JA)-dependent SAR, was not altered in roots, but in the leaves of H. schachtii-infected plants, the expression PR-3 was induced, whereas the expression of PR-4 was down-regulated. During M. incognita infection of Arabidopsis, the expression of PR-1, PR-2 and PR-5 was highly induced in roots, as was PR-3 to a lesser extent, but the expression of PR-4 was not altered, indicating that infection with M. incognita activated both SA- and JA-dependent SAR in roots. However, all PRgenes examined (PR-1 to PR-5) were down-regulated in the leaves of M. incognita-infected plants, suggesting the suppression of both SA- and JA-dependent SAR. Furthermore, constitutive expression of a single PR in Arabidopsis altered the transcription patterns of other PR genes, and the over-expression of PR-1 reduced successful infection by both H. schachtii and M. incognita, whereas the over-expression of PR-3 reduced host susceptibility to M. incognita but had no effect on H. schachtii parasitism. The results suggest that fundamental differences in the mechanisms of infection by beet-cyst and root-knot nematodes differentially regulate PR protein production and mobilization within susceptible host plants.

Activation of a mitochondrial ATPase gene induces abnormal seed development in Arabidopsis.

The ATPases associated with various cellular activities (AAA) proteins are widespread in living organisms. Some of the AAA-type ATPases possess metalloprotease activities. Other members constitute the 26S proteasome complexes. In recent years, a few AAA members have been implicated in vesicle-mediated secretion, membrane fusion, cellular organelle biogenesis, and hypersensitive responses (HR) in plants. However, the physiological roles and biochemical activities of plant AAA proteins have not yet been defined at the molecular level, and regulatory mechanisms underlying their functions are largely unknown. In this study, we showed that overexpression of an Arabidopsis gene encoding a mitochondrial AAA protein, ATPase-in-Seed-Development (ASD), induces morphological and anatomical defects in seed maturation. The ASD gene is expressed at a high level during the seed maturation process and in mature seeds but is repressed rapidly in germinating seeds. Transgenic plants overexpressing the ASD gene are morphologically normal. However, seed formation is severely disrupted in the transgenic plants. The ASD gene is induced by abiotic stresses, such as low temperatures and high salinity, in an abscisic acid (ABA)-dependent manner. The ASD protein possesses ATPase activity and is localized into the mitochondria. Our observations suggest that ASD may play a role in seed maturation by influencing mitochondrial function under abiotic stress.

miR172 signals are incorporated into the miR156 signaling pathway at the SPL3/4/5 genes in Arabidopsis developmental transitions.

In plants, developmental timing is coordinately regulated by a complex signaling network that integrates diverse intrinsic and extrinsic signals. miR172 promotes photoperiodic flowering. It also regulates adult development along with miR156, although the molecular mechanisms underlying this regulation are not fully understood. Here, we demonstrate that miR172 modulates the developmental transitions by regulating the expression of a subset of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are also regulated by miR156. The SPL3/4/5 genes were upregulated in the miR172-overproducing plants (35S:172) and its target gene mutants that exhibit early flowering. In contrast, expression of other SPL genes was not altered to a discernible level. Kinetic measurements of miR172 abundance in the transgenic plants expressing the MIR156a gene driven by a ß-estradiol-inducible promoter revealed that expressions of miR172 and miR156 are not directly interrelated. Instead, the 2 miRNA signals are integrated at the SPL3/4/5 genes. Notably, analysis of developmental patterns in the 156 × 172 plants overproducing both miR172 and miR156 showed that whereas vegetative phase change was delayed as observed in the miR156-overproducing plants (35S:156), flowering initiation was accelerated as observed in the 35S:172 transgenic plants. Together, these observations indicate that although miR172 and miR156 play distinct roles in the timing of developmental phase transitions, there is a signaling crosstalk mediated by the SPL3/4/5 genes.

Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR.

BACKGROUND: The wild grass species Brachypodium distachyon (Brachypodium hereafter) is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data. RESULTS: A systematic exploration of suitable reference genes in Brachypodium is presented here. Nine reference gene candidates were chosen, and their gene sequences were obtained from the Brachypodium expressed sequence tag (EST) databases. Their expression levels were examined by quantitative real-time PCR (qRT-PCR) using 21 different Brachypodium plant samples, including those from different plant tissues and grown under various growth conditions. Effects of plant growth hormones were also visualized in the assays. The expression stability of the candidate genes was evaluated using two analysis software packages, geNorm and NormFinder. In conclusion, the ubiquitin-conjugating enzyme 18 gene (UBC18) was validated as a suitable reference gene across all the plant samples examined. While the expression of the polyubiquitin genes (Ubi4 and Ubi10) was most stable in different plant tissues and growth hormone-treated plant samples, the expression of the S-adenosylmethionine decarboxylase gene (SamDC) ranked was most stable in plants grown under various environmental stresses. CONCLUSION: This study identified the reference genes that are most suitable for normalizing the gene expression data in Brachypodium. These reference genes will be particularly useful when stress-responsive genes are analyzed in order to produce transgenic plants that exhibit enhanced stress resistance.

The GIGANTEA-regulated microRNA172 mediates photoperiodic flowering independent of CONSTANS in Arabidopsis.

Regulated RNA metabolism appears to be a critical component of molecular mechanisms directing flowering initiation in plants. A group of RNA binding proteins exerts their roles through the autonomous flowering pathway. Posttranscriptional mechanisms regulated by microRNAs (miRNAs) also play a key role in flowering-time control. Here, we demonstrate that the GIGANTEA (GI)-regulated miR172 defines a unique genetic pathway that regulates photoperiodic flowering by inducing FLOWERING LOCUS T (FT) independent of CONSTANS (CO). A late-flowering mutant in which a miR172 target gene, TARGET OF EAT1, is constitutively activated by the nearby insertion of the cauliflower mosaic virus 35S enhancer normally responded to vernalization and gibberellic acid treatments. By contrast, its response to daylength changes was severely disrupted. In the mutant, FT was significantly repressed, but other flowering genes were unaffected. Notably, miR172 abundance is regulated by photoperiod via GI-mediated miRNA processing. Accordingly, miR172-overproducing plants exhibit early flowering under both long days and short days, even in the absence of functional CO, indicating that miR172 promotes photoperiodic flowering through a CO-independent genetic pathway. Therefore, it appears that GI-mediated photoperiodic flowering is governed by the coordinated interaction of two distinct genetic pathways: one mediated via CO and the other mediated via miR172 and its targets.