Phase In Arabidopsis Research Articles

Overexpression of ClWRKY48 from Cunninghamia lanceolata improves Arabidopsis phosphate uptake.

Our findings suggested that ClWRKY48 promoted the expression level of Arabidopsis phosphate transporter genes, enhanced phosphate uptake, and delayed the transition from the vegetative stage to the reproductive phase in Arabidopsis. Phosphorus (P) is an essential mineral for plants that influences their growth and development. ClWRKY48, one of the most highly expressed genes in the leaf, was identified by RT-PCR from Chinese fir [Cunninghamia lanceolata (Lamb.) Hook] (C. lanceolata). Furthermore, when treating C. lanceolata with increasing phosphate (Pi) concentration, the expression level of ClWRKY48 rose in leaves, the trends followed the increasing phosphate concentration treatment. ClWRKY48 is a transcription factor in C. lanceolata, according to the results of a yeast one hybridization experiment. Based on subcellular localization studies, ClWRKY48 is a nuclear-localized protein. Under Pi deficiency conditions, the phosphorus concentration of ClWRKY48 overexpressing Arabidopsis increased by 43.2-51.1% compared to the wild-type.Moreover, under Pi limiting conditions, the phosphate transporter genes AtPHT1;1 (Arabidopsis Phosphate transporter 1;1), AtPHT1;4, and AtPHO1 (Arabidopsis PHOSPHATE 1) were expressed 2.1-2.5, 2.2-2.7, and 6.7-7.3-fold greater than the wild-type in ClWRKY48 transgenic Arabidopsis, respectively. Under Pi-sufficient conditions, the phosphorus concentration and phosphate transporter genes of ClWRKY48 overexpression in Arabidopsis are not significantly different from the wild type. These findings indicated that ClWRKY48 increased phosphate absorption in transgenic Arabidopsis. Furthermore, compared to the wild type, the ClWRKY48 transgenic Arabidopsis not only had a delayed flowering time characteristic but also had lower expression of flowering-related genes AtFT (FLOWERING LOCUS T), AtFUL (FRUITFUL), and AtTSF (TWIN SISTER OF FT). Our findings show that ClWRKY48 enhances phosphate absorption and slows the transition from the vegetative to the reproductive stage in ClWRKY48 transgenic Arabidopsis.

Structural and functional studies of Arabidopsis thaliana triphosphate tunnel metalloenzymes reveal roles for additional domains

Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, and inorganic polyphosphate). TTMs in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here, we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Our tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane, mediated by the single C-terminal transmembrane domain, supporting earlier studies. In addition, we reveal from crystal structures of AtTTM1 in the presence and absence of a nonhydrolyzable ATP analog a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Our structural comparison indicates that a helical hairpin may facilitate movement of the TTM domain, thereby activating the kinase. Furthermore, we conducted genetic studies to show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. We demonstrate through rational design of mutations based on the 3D structure that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch. Together, our results provide insight into the structure and function of plant TTM domains.

Studies on the interactions of AFPs and bZIP transcription factor ABI5

AFP1 interacts with ABI5 and negatively regulates the abscisic acid (ABA) signaling by accelerating ABI5's degradation during the seed germination phase in Arabidopsis, but the underlying mechanism remains unclear. Moreover, the molecular basis of the interaction between AFP1 homologs and ABI5 has yet to be elucidated. In this study, the patterns of their interactions with ABI5 were investigated in detail. We found that AFP2/3/4 can bind two regions of ABI5, one is ABI51aa to 135aa and another is ABI5202aa to 213aa. However, AFP1 only interacts with the second region of ABI5, i.e. ABI5202aa to 213aa. Prior research has shown that ABI51aa to 135aa is related to the transcriptional activity of ABI5. Thus, our results suggest that AFPs may also modulate ABI5, by directly binding to its transcriptional activation domain, thereby influencing its transcriptional activity. Further, interactions between AFPs and ABI5 were not affected if the Ser42th in the ABI5-SnRK2 motif were mutated respectively to Glu or Ala. Nevertheless, interactions between AFPs and ABI5 were eliminated if the Thr47th and Thr206th of ABI5 were mutated respectively to Glu or Ala. Since the two residues of Thr47th and Thr206th were located in the phosphorylation motifs of CKII, AFPs might regulate the activities of ABI5 transcription factor through a CKII-dependent pathway.

Whole-Mount Immunostaining for the Identification of Histone Modifications in the S-Phase Nuclei of Arabidopsis Roots.

This chapter describes a method used to analyze the behavior of histone modifications in S phase in Arabidopsis using a whole-mount immunostaining technique. Previous studies have demonstrated that dramatic changes in local chromatin structure are required for the initiation and progression of DNA replication, and that histone modifications play an essential role in the determination of chromatin structure in S phase. Since euchromatic and heterochromatic regions are replicated in distinct S-phase stages, it is important to identify histone modifications at each stage. Here, we introduce a protocol for whole-mount immunostaining combined with 5-ethynyl-2'-deoxyuridine (EdU) staining, which enables the visualization of spatial patterns in histone modifications in the early and late S-phase nuclei of Arabidopsis roots.

PcG Proteins MSI1 and BMI1 Function Upstream of miR156 to Regulate Aerial Tuber Formation in Potato.

Polycomb Repressive Complexes (PRC1 and PRC2) regulate developmental transitions in plants. AtBMI1, a PRC1 member, represses micro RNA156 (miR156) to trigger the onset of adult phase in Arabidopsis (Arabidopsis thaliana). miR156 overexpression (OE) reduces below-ground tuber yield, but stimulates aerial tubers in potato (Solanum tuberosum ssp andigena) under short-day (SD) photoperiodic conditions. Whether PRC members could govern tuber development through photoperiod-mediated regulation of miR156 is unknown. Here, we investigated the role of two PRC proteins, StMSI1 (PRC2 member) and StBMI1-1, in potato development. In wild-type andigena plants, StMSI1 and miR156 levels increased in stolon, whereas StBMI1-1 decreased under SD conditions. StMSI1-OE and StBMI1-1-antisense (AS) lines produced pleiotropic effects, including altered leaf architecture/compounding and reduced below-ground tuber yield. Notably, these lines showed enhanced miR156 accumulation accompanied by aerial stolons and tubers from axillary nodes, similar to miR156-OE lines. Further, grafting of StMSI1-OE or StBMI1-1-AS on wild-type stock resulted in reduced root biomass and showed increased accumulation of miR156a/b and -c precursors in the roots of wild-type stocks. RNA-sequencing of axillary nodes from StMSI1-OE and StBMI1-1-AS lines revealed downregulation of auxin and brassinosteroid genes, and upregulation of cytokinin transport/signaling genes, from 1,023 differentially expressed genes shared between the two lines. Moreover, we observed downregulation of genes encoding H2A-ubiquitin ligase and StBMI1-1/3, and upregulation of Trithorax group H3K4-methyl-transferases in StMSI1-OE Chromatin immunoprecipitation-quantitative PCR confirmed H3K27me3-mediated suppression of StBMI1-1/3, and H3K4me3-mediated activation of miR156 in StMSI1-OE plants. In summary, we show that cross talk between histone modifiers regulates miR156 and alters hormonal response during aerial tuber formation in potato under SD conditions.

Circadian Entrainment in Arabidopsis by the Sugar-Responsive Transcription Factor bZIP63

SummarySynchronization of circadian clocks to the day-night cycle ensures the correct timing of biological events. This entrainment process is essential to ensure that the phase of the circadian oscillator is synchronized with daily events within the environment [1], to permit accurate anticipation of environmental changes [2, 3]. Entrainment in plants requires phase changes in the circadian oscillator, through unidentified pathways, which alter circadian oscillator gene expression in response to light, temperature, and sugars [4, 5, 6]. To determine how circadian clocks respond to metabolic rhythms, we investigated the mechanisms by which sugars adjust the circadian phase in Arabidopsis [5]. We focused upon metabolic regulation because interactions occur between circadian oscillators and metabolism in several experimental systems [5, 7, 8, 9], but the molecular mechanisms are unidentified. Here, we demonstrate that the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) regulates the circadian oscillator gene PSEUDO RESPONSE REGULATOR7 (PRR7) to change the circadian phase in response to sugars. We find that SnRK1, a sugar-sensing kinase that regulates bZIP63 activity and circadian period [10, 11, 12, 13, 14] is required for sucrose-induced changes in circadian phase. Furthermore, TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1), which synthesizes the signaling sugar trehalose-6-phosphate, is required for circadian phase adjustment in response to sucrose. We demonstrate that daily rhythms of energy availability can entrain the circadian oscillator through the function of bZIP63, TPS1, and the KIN10 subunit of the SnRK1 energy sensor. This identifies a molecular mechanism that adjusts the circadian phase in response to sugars.

Effect of Constitutive miR164 Expression on Plant Morphology and Fruit Development in Arabidopsis and Tomato

Several miRNAs are conserved in different plant families, but their abundance and target genes vary between species, organs, and stages of development. Target genes of miRNAs are mostly transcription factors, involved in the control of many plant developmental processes, including fruit development. MiR164 is a conserved miRNA, highly expressed in fruits, and is validated to target a subset of genes of the NAC-domain transcription factor gene family. The objective of this work was to analyze the phenotypic effects of the constitutive expression of miR164 during the life cycle of Arabidopsis and tomato. MiR164 overexpression (164-OE) lines for Arabidopsis and tomato were generated and analyzed during plant development. The constitutive miR164 expression showed that miR164 affected the morphology of Arabidopsis and tomato, and it affected the transition from the vegetative to the reproductive phase in Arabidopsis. Moreover, the miR164 overexpression affected the time required for each developmental stage of tomato fruit. These results suggest that miR164 plays general and specific roles during development in Arabidopsis and tomato, including fruit development, which could be exploited for the improvement of traits of agronomic interest in important species.

Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis

Three-Amino-acid-Loop-Extension(TALE) homeodomain transcription factor BLH3 regulates timing of transition from vegetative to reproductive phase. Previous preliminary results obtained using large-scale yeast two-hybrids indicate that BLH3 protein possibly interact with Ovate Family Proteins(OFPs) transcription co-regulators. Nevertheless, it is uncertain whether OFP1–BLH3 complex is involved in regulation of timing of transition from vegetative to reproductive phase in Arabidopsis.The interaction between BLH3 and OFP1 was re-tested and verified by a yeast two-hybrid system. We found that the BLH3–OFP1 interaction was mainly mediated through the BLH3 homeodomain. Meanwhile, this interaction was further confirmed by bimolecular fluorescence complementation (BiFC) in vivo. Further, by establishing protoplast transient expression, we discovered that BLH3 acts as a transcriptional activator, whereas OFP1 functioned as a repressor. The interactions between OFP1 and BLH3 can reduce BLH3 transcriptional activity. The ofp1 mutant lines and blh3 mutant lines, OFP1 overexpress lines and BLH3 overexpress lines can both influence timing of transition from vegetative to reproductive phase. Furthermore, 35s:OFP1/blh3 plants exhibited flowering and leaf quantity similar to that of the wild-type controls. 35s:BLH3/ofp1 plants flowered earlier and had less leaves than wild-type controls, indicating that OFP1 protein might depend partially on BLH3 in its function to regulate the timing of transition from vegetative to reproductive phase. These results support our assumption that, by interacting with OFP1, BLH3 forms a functional protein complex that controls timing of progression from vegetative to reproductive phase, and OFP1 might negatively regulate BLH3 or the BLH-KNOX complex, an important interaction for sustaining the normal transition from vegetative to reproductive phase.

Imitation Switch chromatin remodeling factors and their interacting RINGLET proteins act together in controlling the plant vegetative phase in Arabidopsis

During their life cycle, flowering plants must experience a transition from vegetative to reproductive growth. Here, we report that double mutations in the Arabidopsis thaliana IMITATION SWITCH (AtISWI) genes, CHROMATIN REMODELING11 (CHR11) and CHR17, and the plant-specific DDT-domain containing genes, RINGLET1 (RLT1) and RLT2, resulted in plants with similar developmental defects, including the dramatically accelerated vegetative-to-reproductive transition. We demonstrated that AtISWI physically interacts with RLTs in preventing plants from activating the vegetative-to-reproductive transition early by regulating several key genes that contribute to flower timing. In particular, AtISWI and RLTs repress FT, SEP1, SEP3, FUL, and SOC1, but promote FLC in the leaf. Furthermore, AtISWI and RLTs may directly repress FT and SEP3 through associating with the FT and SEP3 loci. Our study reveals that AtISWI and RLTs represent a previously unrecognized genetic pathway that is required for the maintenance of the plant vegetative phase.

Broad-Spectrum Suppression of Innate Immunity Is Required for Colonization of Arabidopsis Roots by the FungusPiriformospora indica

Piriformospora indica is a root-colonizing basidiomycete that confers a wide range of beneficial traits to its host. The fungus shows a biotrophic growth phase in Arabidopsis (Arabidopsis thaliana) roots followed by a cell death-associated colonization phase, a colonization strategy that, to our knowledge, has not yet been reported for this plant. P. indica has evolved an extraordinary capacity for plant root colonization. Its broad host spectrum encompasses gymnosperms and monocotyledonous as well as dicotyledonous angiosperms, which suggests that it has an effective mechanism(s) for bypassing or suppressing host immunity. The results of our work argue that P. indica is confronted with a functional root immune system. Moreover, the fungus does not evade detection but rather suppresses immunity triggered by various microbe-associated molecular patterns. This ability to suppress host immunity is compromised in the jasmonate mutants jasmonate insensitive1-1 and jasmonate resistant1-1. A quintuple-DELLA mutant displaying constitutive gibberellin (GA) responses and the GA biosynthesis mutant ga1-6 (for GA requiring 1) showed higher and lower degrees of colonization, respectively, in the cell death-associated stage, suggesting that P. indica recruits GA signaling to help establish proapoptotic root cell colonization. Our study demonstrates that mutualists, like pathogens, are confronted with an effective innate immune system in roots and that colonization success essentially depends on the evolution of strategies for immunosuppression.

Ectopic over-expression of two apple Flowering Locus T homologues, MdFT1 and MdFT2, reduces juvenile phase in Arabidopsis

To get insight into mechanism by which apple tree (Malus domestica Borkh.) regulates flowering, two apple flowering locus T (FT) homologues, MdFT1 and MdFT2, were isolated from the leaf cDNAs of cultivar Gala. The open reading frames (ORFs) of two MdFTs encoded 174 amino acids. The deduced amino acid sequence of MdFT1 and MdFT2 showed 94.3 % similarity to each other, while 72.6 and 76.0 % to AtFT protein, respectively. Semi-quantitative RT-PCR indicated their specific expression in leaves. Visualization of MdFT2-GFP fusion protein demonstrated its localization on membrane. Ectopic overexpression of either MdFT1 or MdFT2 in Arabidopsis significantly induced early flowering by activating the downstream flowering-related genes.

Post-translational regulation of FLC is mediated by an E3 ubiquitin ligase activity of SINAT5 in Arabidopsis

Suppression of Flowering Locus C ( FLC) is critical for the transition from the vegetative to reproductive phase in Arabidopsis. FLC represses the expression of several genes involved in floral induction and its transcription is positively or negatively regulated by FRIGIDA or by vernalization, respectively. However, the regulatory mechanisms for the level and stability of FLC protein throughout development are unknown. Here, we show that SINAT5 colocalizes with FLC in the nuclear bodies and that its zinc finger motif interacts directly with the MADS-box domain of FLC. In addition, it is shown that SINAT5 has an E3 ubiquitin ligase activity towards FLC as a substrate, suggesting that SINAT5 participates in the regulation of flowering time through the ubiquitin-mediated proteolysis of FLC.

Expression of MADS-box genes during the embryonic phase in Arabidopsis

MADS domain factors play important roles as developmental regulators in plants. In Arabidopsis thaliana, MADS domain proteins have been shown to regulate various processes during the vegetative and reproductive phases. Relatively little is known, however, about family members expressed during the embryonic phase and their function. To determine which MADS-box genes are expressed during the embryonic phase in Arabidopsis, a family-wide survey involving gene-specific primers and RT-PCR was conducted. Transcripts corresponding to 64 (out of 109 total) family members could be detected in RNA samples isolated from embryonic culture tissue. Eight MADS-box genes that appear to be expressed at higher levels during the embryonic phase than in seedlings or in inflorescence apices were identified. The spatial pattern of expression in developing seeds was characterized for four MADS-box genes (FLOWERING LOCUS C, FLOWERING LOCUS M, AGAMOUS-LIKE 15, and AGAMOUS-LIKE 18) using reporter constructs encoding translational fusions to GUS. All four are expressed in cells throughout the endosperm and embryo. Finally, to test the hypothesis that AGAMOUS-LIKE15 (AGL15) and AGAMOUS-LIKE18 (AGL18) play essential roles during the embryonic phase, plants carrying T-DNA insertions that disrupt these genes were isolated. No embryo defects were observed in agl15 or agl18 single mutants or in agl15agl18 double mutants. These results indicate that multiple regulatory pathways that involve MADS domain factors are likely to operate in embryonic tissues, and that genetic and/or functional redundancy are likely to be as prevalent as in other phases of the life cycle.

EMF genes interact with late-flowering genes to regulate Arabidopsis shoot development.

To investigate the genetic mechanisms regulating the transition from vegetative to reproductive phase in Arabidopsis, double mutants between two embryonic flower (emf) and 12 different late-flowering mutants were constructed and analyzed. Double mutants in all combinations displayed the emf phenotypes without forming rosettes during early development; however, clear variations between different double mutants were observed during late development. fwa significantly enhanced the vegetative property of both emf mutants by producing a high number of sessile leaves without any further reproductive growth in emf1 fwa double mutants. It also produced numerous leaf-like flower structures similar to those in leafy ap1 double mutant in emf2 fwa double mutants. Nine late-flowering mutants, ft, fca, ld, fd, fpa, fe, fy, fha, and fve, caused different degrees of increase in the number of sessile leaves, the size of inflorescence, and the number of flowers only in weak emf1 and emf2 mutant alleles background. Two late-flowering mutants, co and gi, however, had no effect on either emf1 and emf2 mutant alleles in double mutants. Our results suggest that FWA function in distinct pathways from both EMF genes to regulate flower competence by activating genes which specify floral meristem identity. CO and GI negatively regulate both EMF genes, whereas the other nine late-flowering genes may interact with EMF genes directly or indirectly to regulate shoot maturation in Arabidopsis.