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The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana  [PDF]
Junhua Li, Yingying Han, Qingzhen Zhao, Chunhua Li, Qi Xie, Kang Chong, Yunyuan Xu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0071078
Abstract: Ubiquitination is an important post-translational protein modification that is known to play critical roles in diverse biological processes in eukaryotes. The RING E3 ligases function in ubiquitination pathways, and are involved in a large diversity of physiological processes in higher plants. The RING domain-containing E3 ligase AtRDUF1 was previously identified as a positive regulator of ABA-mediated dehydration stress response in Arabidopsis. In this study, we report that AtRDUF1 is involved in plant responses to salt stress. AtRDUF1 expression is upregulated by salt treatment. Overexpression of AtRDUF1 in Arabidopsis results in an insensitivity to salt and osmotic stresses during germination and seedling growth. A double knock-out mutant of AtRDUF1 and its close homolog AtRDUF2 (atrduf1atrduf2) was hypersensitive to salt treatment. The expression levels of the stress-response genes RD29B, RD22, and KIN1 are more sensitive to salt treatment in AtRDUF1 overexpression plants. In summary, our data show that AtRDUF1 positively regulates responses to salt stress in Arabidopsis.
PRL1, an RNA-Binding Protein, Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis  [PDF]
Shuxin Zhang,Yuhui Liu ,Bin Yu
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004841
Abstract: The evolutionary conserved WD-40 protein PRL1 plays important roles in immunity and development. Here we show that PRL1 is required for the accumulation of microRNAs (miRNAs) and small interfering RNAs (siRNAs). PRL1 positively influences the processing of miRNA primary transcripts (pri-miRNAs) and double-stranded RNAs (dsRNAs). Furthermore, PRL1 interacts with the pri-miRNA processor, DCL1, and the dsRNA processors (DCL3 and DCL4). These results suggest that PRL1 may function as a general factor to promote the production of miRNAs and siRNAs. We also show that PRL1 is an RNA-binding protein and associates with pri-miRNAs in vivo. In addition, prl1 reduces pri-miRNA levels without affecting pri-miRNA transcription. These results suggest that PRL1 may stabilize pri-miRNAs and function as a co-factor to enhance DCL1 activity. We further reveal the genetic interaction of PRL1 with CDC5, which interacts with PRL1 and regulates transcription and processing of pri-miRNAs. Both miRNA and pri-miRNA levels are lower in cdc5 prl1 than those in either cdc5 or prl1. However, the processing efficiency of pri-miRNAs in cdc5 prl1 is similar to that in cdc5 and slightly lower than that in prl1. Based on these results, we propose that CDC5 and PRL1 cooperatively regulate pri-miRNA levels, which results in their synergistic effects on miRNA accumulation, while they function together as a complex to enhance DCL1 activity.
MMS21/HPY2 and SIZ1, Two Arabidopsis SUMO E3 Ligases, Have Distinct Functions in Development  [PDF]
Takashi Ishida, Mika Yoshimura, Kenji Miura, Keiko Sugimoto
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0046897
Abstract: The small ubiquitin related modifier (SUMO)-mediated posttranslational protein modification is widely conserved among eukaryotes. Similar to ubiquitination, SUMO modifications are attached to the substrate protein through three reaction steps by the E1, E2 and E3 enzymes. To date, multiple families of SUMO E3 ligases have been reported in yeast and animals, but only two types of E3 ligases have been identified in Arabidopsis: SAP and Miz 1 (SIZ1) and Methyl Methanesulfonate-Sensitivity protein 21 (MMS21)/HIGH PLOIDY 2 (HPY2), hereafter referred to as HPY2. Both proteins possess characteristic motifs termed Siz/PIAS RING (SP-RING) domains, and these motifs are conserved throughout the plant kingdom. Previous studies have shown that loss-of-function mutations in HPY2 or SIZ1 cause dwarf phenotypes and that the phenotype of siz1-2 is caused by the accumulation of salicylic acid (SA). However, we demonstrate here that the phenotype of hpy2-1 does not depend on SA accumulation. Consistently, the expression of SIZ1 driven by the HPY2 promoter does not complement the hpy2-1 phenotypes, indicating that they are not functional homologs. Lastly, we show that the siz1-2 and hpy2-1 double mutant results in embryonic lethality, supporting the hypothesis that they have non-overlapping roles during embryogenesis. Together, these results suggest that SIZ1 and HPY2 function independently and that their combined SUMOylation is essential for plant development.
Mutation in SUMO E3 ligase, SIZ1, Disrupts the Mature Female Gametophyte in Arabidopsis  [PDF]
Yu Ling, Chunyu Zhang, Tong Chen, Huaiqing Hao, Peng Liu, Ray A. Bressan, Paul M. Hasegawa, Jing Bo Jin, Jinxing Lin
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0029470
Abstract: Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance.
The roles of aldehyde dehydrogenases (ALDHs) in the PDH bypass of Arabidopsis
Yanling Wei, Ming Lin, David J Oliver, Patrick S Schnable
BMC Biochemistry , 2009, DOI: 10.1186/1471-2091-10-7
Abstract: To test for the presence of the PDH bypass in the sporophytic tissue of plants, Arabidopsis plants homozygous for mutant alleles of all three Family 2 ALDH genes were fed with 14C-ethanol along with wild type controls. Comparisons of the incorporation rates of 14C-ethanol into fatty acids in mutants and wild type controls provided direct evidence for the presence of the PDH bypass in sporophytic tissue. Among the three Family 2 ALDHs, one of the two mitochondrial ALDHs (ALDH2B4) appears to be the primary contributor to this pathway. Surprisingly, single, double and triple ALDH mutants of Arabidopsis did not exhibit detectable phenotypes, even though a Family 2 ALDH gene is required for normal anther development in maize.The PDH bypass is active in sporophytic tissue of plants. Blocking this pathway via triple ALDH mutants does not uncover obvious visible phenotypes.Aldehydes vary in length and in characteristics of their alkyl chains but all are usually deleterious to biological systems due to their chemical reactivity. Aldehyde dehydrogenases (ALDHs, EC 1.2.1) oxidize aldehydes into carboxylic acids, using NAD+ or NADP+ as a co-factor. As such ALDHs play an important role in detoxifying aldehydes that are generated endogenously or introduced from the environment. ALDHs are very diverse in that some only use either NAD+ or NADP+ as the co-factor, while others can use both, some oxidize only a limited number of aldehydes, while others have broader substrate spectra, and ALDHs exist in various subcellular compartments, including the cytosol, mitochondria, plastids and microsomes.Over 550 ALDH genes have been identified across virtually all species, and those from eukaryotes have been classified into more than 20 families [1]. Family 2 ALDHs are mitochondrial or cytosolic homotetrameric enzymes. The well studied human mitochondrial Family 2 ALDH, ALDH2, detoxifies acetaldehyde generated via alcohol intake [2]. Family 2 ALDHs in plants have gained attention since the cl
AaERF1 Positively Regulates the Resistance to Botrytis cinerea in Artemisia annua  [PDF]
Xu Lu, Weimin Jiang, Ling Zhang, Fei Zhang, Fangyuan Zhang, Qian Shen, Guofeng Wang, Kexuan Tang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057657
Abstract: Plants are sessile organisms, and they can not move away under abiotic or biotic stresses. Thus plants have evolved a set of genes that response to adverse environment to modulate gene expression. In this study, we characterized and functionally studied an ERF transcription factor from Artemisia annua, AaERF1, which plays an important role in biotic stress responses. The AaERF1 promoter had been cloned and GUS staining results of AaERF1 promoter-GUS transgenic A. annua showed that AaERF1 is expressed ubiquitiously in all organs. Several putative cis-acting elements such as W-box, TGA-box and Py-rich element, which are involved in defense responsiveness, are present in the promoter. The expression of AaERF1 can be induced vigorously by methyl jasmonate as well as by ethephon and wounding, implying that AaERF1 may activate some of the defense genes via the jasmonic acid and ethylene signaling pathways of A. annua. The results of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid experiments showed that AaERF1 was able to bind to the GCC box cis-acting element in vitro and in yeast. Ectopic expression of AaERF1 could enhance the expression levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2) and BASIC CHITINASE (ChiB), and increase the resistance to Botrytis cinerea in the 35S::AaERF1 transgenic Arabidopsis. The down-regulated expression level of AaERF1 evidently reduced the resistance to B. cinerea in A. annua. The overall results showed that AaERF1 positively regulated the resistance to B. cinerea in A. annua.
Erk1 Positively Regulates Osteoclast Differentiation and Bone Resorptive Activity  [PDF]
Yongzheng He, Karl Staser, Steven D. Rhodes, Yaling Liu, Xiaohua Wu, Su-Jung Park, Jin Yuan, Xianlin Yang, Xiaohong Li, Li Jiang, Shi Chen, Feng-Chun Yang
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0024780
Abstract: The extracellular signal-regulated kinases (ERK1 and 2) are widely-expressed and they modulate proliferation, survival, differentiation, and protein synthesis in multiple cell lineages. Altered ERK1/2 signaling is found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, Neurofibromatosis type 1, and Cardio-facio-cutaneous syndrome, suggesting that MEK-ERK signals regulate human skeletal development. Here, we examine the consequence of Erk1 and Erk2 disruption in multiple functions of osteoclasts, specialized macrophage/monocyte lineage-derived cells that resorb bone. We demonstrate that Erk1 positively regulates osteoclast development and bone resorptive activity, as genetic disruption of Erk1 reduced osteoclast progenitor cell numbers, compromised pit formation, and diminished M-CSF-mediated adhesion and migration. Moreover, WT mice reconstituted long-term with Erk1?/? bone marrow mononuclear cells (BMMNCs) demonstrated increased bone mineral density as compared to recipients transplanted with WT and Erk2?/? BMMNCs, implicating marrow autonomous, Erk1-dependent osteoclast function. These data demonstrate Erk1 plays an important role in osteoclast functions while providing rationale for the development of Erk1-specific inhibitors for experimental investigation and/or therapeutic modulation of aberrant osteoclast function.
An endogenous F-box protein regulates ARGONAUTE1 in Arabidopsis thaliana
K Earley, MR Smith, R Weber, BD Gregory, RS Poethig
Silence , 2010, DOI: 10.1186/1758-907x-1-15
Abstract: Argonaute proteins are core components of the RNA-induced silencing complex (RISC) [1-3]. These proteins use microRNAs (miRNAs) and/or small interfering RNAs (siRNAs) as guides to direct RISC to a specific site in target mRNAs, resulting in the cleavage or translational repression of these target mRNAs. Some Argonaute proteins also promote transcriptional repression through their effect on chromatin structure [2-4].ARGONAUTE1 (AGO1) is one of 10 Argonaute proteins in Arabidopsis thaliana [2,5]. Genetic analyses [6-9], as well as the identification of the small RNAs that co-purify with AGO1 [10,11], indicate that AGO1 plays a central role in both miRNA and siRNA-mediated RNA silencing. Arabidopsis is exquisitely sensitive to the level of AGO1 activity, as evident from the broad range of phenotypes displayed by hypomorphic mutations of this gene [5,6,8,12]. In wild-type plants, the expression of AGO1 is maintained at a constant level by a negative feedback loop involving miR168. AGO1 is a target of miR168 and negatively regulates its own activity by promoting the activity and stability of miR168 [9,13] and by promoting the activity of siRNAs derived from the AGO1 transcript [14]. AGO1 activity is negatively regulated by PNH/ZLL/AGO10 [15] and positively regulated by SQUINT (SQN), the Arabidopsis orthologue of the protein chaperone, Cyclophilin-40 [12].Null alleles of SQN have a morphological phenotype that is nearly identical to the phenotype of weak loss-of-function alleles of AGO1 [12]. In order to identify genes involved in AGO1-mediated processes, we screened for mutations that suppress the phenotype of sqn-1. This screen yielded several alleles of the F-box gene FBW2. Here we show FBW2 is a negative regulator of AGO1 and controls the sensitivity of plants to the hormone abscisic acid.Previously we found that SQN directly or indirectly promotes AGO1 activity [12]. In particular, we showed that the phenotype of loss-of-function alleles of SQN can be largely, if not
The Circadian Clock Regulates Auxin Signaling and Responses in Arabidopsis  [PDF]
Michael F. Covington,Stacey L. Harmer
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.0050222
Abstract: The circadian clock plays a pervasive role in the temporal regulation of plant physiology, environmental responsiveness, and development. In contrast, the phytohormone auxin plays a similarly far-reaching role in the spatial regulation of plant growth and development. Went and Thimann noted 70 years ago that plant sensitivity to auxin varied according to the time of day, an observation that they could not explain. Here we present work that explains this puzzle, demonstrating that the circadian clock regulates auxin signal transduction. Using genome-wide transcriptional profiling, we found many auxin-induced genes are under clock regulation. We verified that endogenous auxin signaling is clock regulated with a luciferase-based assay. Exogenous auxin has only modest effects on the plant clock, but the clock controls plant sensitivity to applied auxin. Notably, we found both transcriptional and growth responses to exogenous auxin are gated by the clock. Thus the circadian clock regulates some, and perhaps all, auxin responses. Consequently, many aspects of plant physiology not previously thought to be under circadian control may show time-of-day–specific sensitivity, with likely important consequences for plant growth and environmental responses.
The Circadian Clock Regulates Auxin Signaling and Responses in Arabidopsis  [PDF]
Michael F Covington,Stacey L Harmer
PLOS Biology , 2007, DOI: 10.1371/journal.pbio.0050222
Abstract: The circadian clock plays a pervasive role in the temporal regulation of plant physiology, environmental responsiveness, and development. In contrast, the phytohormone auxin plays a similarly far-reaching role in the spatial regulation of plant growth and development. Went and Thimann noted 70 years ago that plant sensitivity to auxin varied according to the time of day, an observation that they could not explain. Here we present work that explains this puzzle, demonstrating that the circadian clock regulates auxin signal transduction. Using genome-wide transcriptional profiling, we found many auxin-induced genes are under clock regulation. We verified that endogenous auxin signaling is clock regulated with a luciferase-based assay. Exogenous auxin has only modest effects on the plant clock, but the clock controls plant sensitivity to applied auxin. Notably, we found both transcriptional and growth responses to exogenous auxin are gated by the clock. Thus the circadian clock regulates some, and perhaps all, auxin responses. Consequently, many aspects of plant physiology not previously thought to be under circadian control may show time-of-day–specific sensitivity, with likely important consequences for plant growth and environmental responses.
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