Ethylene acts as a negative regulator of glucose induced lateral root emergence in Arabidopsis

Plants, being sessile organisms, are more exposed to the hazards of constantly changing environmental conditions globally. During the lifetime of a plant, the root system encounters various challenges such as obstacles, pathogens, high salinity, water logging, nutrient scarcity etc. The developmental plasticity of the root system provides brilliant adaptability to plants to counter the changes exerted by both external as well as internal cues and achieve an optimized growth status. Phytohormones are one of the major intrinsic factors regulating all aspects of plant growth and development both independently as well as through complex signal integrations at multiple levels. We have previously shown that glucose (Glc) and brassinosteroid (BR) signalings interact extensively to regulate lateral root (LR) development in Arabidopsis.¹ Auxin efflux as well as influx and downstream signaling components are also involved in Glc-BR regulation of LR emergence. Here, we provide evidence for involvement of ethylene signaling machinery downstream to Glc and BR in regulation of LR emergence.     

The type-A response regulator,ARR15, acts as a negative regulator in the cytokinin-mediated signal transduction in Arabidopsis thaliana

The Arabidopsis thaliana AHK4 histidine kinase (also known as CRE1 or WOL) acts as a cytokinin signal transducer, presumably, in concert with downstream components, such as histidine-containing phosphotransfer factors (AHPs) and response regulators (ARRs), through the histidine-to-aspartate (His-->Asp) phosphorelay. Among 10 members of the type-A ARR family, the cytokinin-induced expression of ARR15 in roots is selectively impaired in the cre1-1 mutant, which carries a mutation in the AHK4 gene, suggesting a link between this type-A response regulator and the AHK4-mediated cytokinin signal transduction in roots. To address this issue further, we characterized a T-DNA insertion mutant of ARR15, and also constructed transgenic lines (referred to as ARR15-ox) that overexpress the ARR15 gene in a manner independent of cytokinin. While the T-DNA insertion mutant (arr15-1) showed no apparent phenotype, the cytokinin-independent overexpression of ARR15 in ARR15-ox plants resulted in a reduced sensitivity toward exogenously applied cytokinin, not only in elongation of roots in plants, but also in green callus formation (or shoot formation) in explants. Cytokinin-induced expressions of certain type-A ARRs were also down-regulated in ARR15-ox plants. These results support the view that ARR15 acts as a repressor that mediates a negative feedback loop in the cytokinin and AHK4-mediated His-->Asp phosphorelay.     

Bfl-1/A1 acts as a negative regulator of autophagy in mycobacteria infected macrophages

Expression of Bcl-2 family protein, Bfl-1/A1 has been found to differ considerably amongst macrophages infected with virulent Mycobacterium tuberculosis H37Rv or with avirulent M. tuberculosis H37Ra. Present work was undertaken to deduce the significance of differential expression of Bfl-1/A1 in the outcome of mycobacterial infection. We have studied the role of Bfl-1/A1 particularly in autophagy formation in tubercle bacilli infected cells since autophagy has been recognized as a component of innate immunity against pathogenic mycobacteria. First, we have confirmed that upon infection virulent strain H37Rv retain Bfl-1/A1 for longer period and impose autophagosome maturation block within infected cells as evident from confocal microscopy. Moreover, down regulation of Bfl-1/A1 by siRNA induced autophagy formation and reduced bacterial growth. Furthermore, even the avirulent strain H37Ra resist autophagosome maturation and survive if the cellular level of Bfl-1 is maintained in THP-1 cells by stable transfection (Bfl-1 overexpressing cells). No noteworthy difference in mTOR expression was observed between normal THP-1 and Bfl-1 overexpressing THP-1 cells infected with either strain of mycobacteria. Interestingly, we found that not only mTOR but also Bfl-1/A1 is involved in rapamycin induced autophagy in mycobacteria infected macrophages. We have found that Bfl-1 physically interacts with Beclin 1 in Bfl-1 overexpressing THP-1 as well as in H37Rv infected THP-1 cells as they co-precipitated. Taken together, our results clearly demonstrated that Bfl-1/A1 negatively regulates autophagy and expression of Bfl-1/A1 in H37Rv infected macrophages provides the bacteria a survival strategy to overcome host defense.     

HRS1 acts as a negative regulator of abscisic acid signaling to promote timely germination of Arabidopsis seeds

In this work, we conducted functional analysis of Arabidopsis HRS1 gene in order to provide new insights into the mechanisms governing seed germination. Compared with wild type (WT) control, HRS1 knockout mutant (hrs1-1) exhibited significant germination delays on either normal medium or those supplemented with abscisic acid (ABA) or sodium chloride (NaCl), with the magnitude of the delay being substantially larger on the latter media. The hypersensitivity of hrs1-1 germination to ABA and NaCl required ABI3, ABI4 and ABI5, and was aggravated in the double mutant hrs1-1abi1-2 and triple mutant hrs1-1hab1-1abi1-2, indicating that HRS1 acts as a negative regulator of ABA signaling during seed germination. Consistent with this notion, HRS1 expression was found in the embryo axis, and was regulated both temporally and spatially, during seed germination. Further analysis showed that the delay of hrs1-1 germination under normal conditions was associated with reduction in the elongation of the cells located in the lower hypocotyl (LH) and transition zone (TZ) of embryo axis. Interestingly, the germination rate of hrs1-1 was more severely reduced by the inhibitor of cell elongation, and more significantly decreased by the suppressors of plasmalemma H(+)-ATPase activity, than that of WT control. The plasmalemma H(+)-ATPase activity in the germinating seeds of hrs1-1 was substantially lower than that exhibited by WT control, and fusicoccin, an activator of this pump, corrected the transient germination delay of hrs1-1. Together, our data suggest that HRS1 may be needed for suppressing ABA signaling in germinating embryo axis, which promotes the timely germination of Arabidopsis seeds probably by facilitating the proper function of plasmalemma H(+)-ATPase and the efficient elongation of LH and TZ cells.     

SHORT HYPOCOTYL IN WHITE LIGHT1, a serine-arginine-aspartate-rich protein in Arabidopsis, acts as a negative regulator of photomorphogenic growth

Light is an important factor for plant growth and development. We have identified and functionally characterized a regulatory gene SHORT HYPOCOTYL IN WHITE LIGHT1 (SHW1) involved in Arabidopsis (Arabidopsis thaliana) seedling development. SHW1 encodes a unique serine-arginine-aspartate-rich protein, which is constitutively localized in the nucleus of hypocotyl cells. Transgenic analyses have revealed that the expression of SHW1 is developmentally regulated and is closely associated with the photosynthetically active tissues. Genetic and molecular analyses suggest that SHW1 acts as a negative regulator of light-mediated inhibition of hypocotyl elongation, however, plays a positive regulatory role in light-regulated gene expression. The shw1 mutants also display shorter hypocotyl in dark, and analyses of shw1 cop1 double mutants reveal that SHW1 acts nonredundantly with COP1 to control hypocotyl elongation in the darkness. Taken together, this study provides evidences that SHW1 is a regulatory protein that is functionally interrelated to COP1 and plays dual but opposite regulatory roles in photomorphogenesis.     

A stress-responsive caleosin-like protein, AtCLO4, acts as a negative regulator of ABA responses in Arabidopsis

Caleosins or related sequences have been found in a wide range of higher plants. In Arabidopsis, seed-specific caleosins are viewed as oil-body (OB)-associated proteins that possess Ca(2+)-dependent peroxygenase activity and are involved in processes of lipid degradation. Recent experimental evidence suggests that one of the Arabidopsis non-seed caleosins, AtCLO3, is involved in controlling stomatal aperture during the drought response; the roles of the other caleosin-like proteins in Arabidopsis remain largely uncharacterized. We have demonstrated that a novel stress-responsive and OB-associated Ca(2+)-binding caleosin-like protein, AtCLO4, is expressed in non-seed tissues of Arabidopsis, including guard cells, and down-regulated following exposure to exogenous ABA and salt stress. At the seed germination stage, a loss-of-function mutant (atclo4) was hypersensitive to ABA, salt and mannitol stresses, whereas AtCLO4-overexpressing (Ox) lines were more hyposensitive to those stresses than the wild type. In adult stage, atclo4 mutant and AtCLO4-Ox plants showed enhanced and decreased drought tolerance, respectively. Following exposure to exogenous ABA, the expression of key ABA-dependent regulatory genes, such as ABF3 and ABF4, was up-regulated in the atclo4 mutant, while it was down-regulated in AtCLO4-Ox lines. Based on these results, we propose that the OB-associated Ca(2+)-binding AtCLO4 protein acts as a negative regulator of ABA responses in Arabidopsis.     

FtMYB18 acts as a negative regulator of anthocyanin/proanthocyanidin biosynthesis in Tartary buckwheat

Plant Molecular Biology - FtMYB18 plays a role in the repression of anthocyanins and proanthocyanidins accumulation by strongly down-regulating the CHS and DFR genes in Tartary buckwheat, and the...     

The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.     

The t-SNARE AtVAM3p resides on the prevacuolar compartment in Arabidopsis root cells

Protein cargo is trafficked between the organelles of the endomembrane system inside transport vesicles, a process mediated by integral membrane proteins called SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) that reside on the surface of the vesicle (v-SNAREs) and target membrane (t-SNAREs). In examining transport of cargo between the trans-Golgi network and the vacuole in Arabidopsis, we have previously characterized AtPEP12p as a t-SNARE residing on the prevacuolar compartment and AtVTI1a as a v-SNARE that interacts with AtPEP12p. Recently, we have begun to characterize AtVAM3p, another Arabidopsis t-SNARE that shows high sequence homology to AtPEP12p. We have found that AtVTI1a also interacts with AtVAM3p, suggesting a role for this t-SNARE in post-Golgi trafficking. AtVAM3p has been suggested to localize to the vacuolar membrane in Arabidopsis cells; however, using specific antisera and expression of epitope-tagged versions of each t-SNARE, we have discovered that AtVAM3p is found on the same prevacuolar structure as AtPEP12p in Arabidopsis root cells.     

p-Nonylphenol acts as a promoter in the BALB/3T3 cell transformation

p-Nonylphenol (NP) has attracted attention as an estrogenic contaminant, and the environmental pollution by NP has been found to be extensive. NP is classified as a phenolic antioxidant based on the chemical activity and structure. Some phenolic antioxidants are known to induce and/or enhance carcinogenesis. We examined the effects of NP on the two-stage transformation of BALB/3T3 cells, a model of two-stage carcinogenesis. The treatment by NP in the promotion phase markedly enhanced the transformation of the cells pre-treated with a subthreshold dose of a carcinogen, 3-methylcholanthrene (MCA), but not that of non-pretreated cells. The promoting activity of NP was approximately one hundredth of that of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, in the cell transformation. The treatment by NP in the initiation phase did not induce cell transformation with and without post-treatment by TPA. These results indicate that NP acts as a pure promoter of cell transformation implying that it may cause the enhancement of carcinogenesis in vivo. The enhancement by NP of MCA-initiated transformation was suggested not to be mediated by estrogen receptors in BALB/3T3 cells because 17 beta-estradiol did not promote cell transformation in our experiments, and it has been reported that BALB/3T3 cells do not express estrogen receptors at a detectable level.     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.