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1.
Anal Biochem ; 434(1): 60-6, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23149232

ABSTRACT

MicroRNAs (miRNAs) affect fundamental processes of development. In plants miRNAs regulate organ development, transition to flowering, and responses to abiotic/biotic stresses. To understand the biological role of miRNAs, in addition to identifying their targeted transcripts, it is necessary to characterize the spatiotemporal regulation of their expression. Many methods have been used to define the set of organ-specific miRNAs by tissue dissection and miRNA profiling but none of them can describe their tissue and cellular distribution at the high resolution provided by in situ hybridization (ISH). This article describes the setup and optimization of a whole-mount ISH protocol to target endogenous miRNAs on intact Arabidopsis seedlings using DIG-labeled Zip Nucleic Acid (ZNA) oligonucleotide probes. Automation of the main steps of the procedure by robotized liquid handling has also been implemented in the protocol for best reproducibility of results, enabling running of ISH experiments at high throughput.


Subject(s)
Arabidopsis/genetics , In Situ Hybridization , MicroRNAs/analysis , Oligonucleotide Probes/metabolism , Arabidopsis/growth & development , Automation , Seedlings/genetics
2.
New Phytol ; 192(2): 338-52, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21762167

ABSTRACT

Given the importance of nitrogen for plant growth and the environmental costs of intense fertilization, an understanding of the molecular mechanisms underlying the root adaptation to nitrogen fluctuations is a primary goal for the development of biotechnological tools for sustainable agriculture. This research aimed to identify the molecular factors involved in the response of maize roots to nitrate. cDNA-amplified fragment length polymorphism was exploited for comprehensive transcript profiling of maize (Zea mays) seedling roots grown with varied nitrate availabilities; 336 primer combinations were tested and 661 differentially regulated transcripts were identified. The expression of selected genes was studied in depth through quantitative real-time polymerase chain reaction and in situ hybridization. Over 50% of the genes identified responded to prolonged nitrate starvation and a few were identified as putatively involved in the early nitrate signaling mechanisms. Real-time results and in situ localization analyses demonstrated co-regulated transcriptional patterns in root epidermal cells for genes putatively involved in nitric oxide synthesis/scavenging. Our findings, in addition to strengthening already known mechanisms, revealed the existence of a new complex signaling framework in which brassinosteroids (BRI1), the module MKK2-MAPK6 and the fine regulation of nitric oxide homeostasis via the co-expression of synthetic (nitrate reductase) and scavenging (hemoglobin) components may play key functions in maize responses to nitrate.


Subject(s)
Nitrate Reductase/genetics , Nitrate Reductase/metabolism , Nitrates/metabolism , Zea mays/genetics , Zea mays/metabolism , Amplified Fragment Length Polymorphism Analysis , DNA, Complementary/genetics , DNA, Complementary/metabolism , Gene Expression Profiling , Gene Expression Regulation, Plant , Genes, Plant , Hemoglobins , Plant Roots/genetics , Plant Roots/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Zea mays/enzymology
3.
Plant Biol (Stuttg) ; 12 Suppl 1: 3-14, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20712616

ABSTRACT

Auxin interacts with its own polar transport to influence cell polarity and tissue patterning. Research over the past decade has started to deliver new insights into the molecular mechanisms that drive and regulate polar auxin transport. The most prominent auxin efflux protein, PIN1, has subsequently become a crucial component of auxin transport models because it is now known to direct auxin flow and maintain local auxin gradients. Recent molecular and genetic experiments have allowed the formulation of conceptual models that are able to interpret the role of (i) auxin, (ii) its transport, and (iii) the dynamics of PIN1 in generating temporal and spatial patterns. Here we review the current mathematical models of patterning in two specific developmental contexts: lateral shoot and vein formation, focusing on how these models can help to untangle the details of auxin transport-mediated patterning.


Subject(s)
Indoleacetic Acids/metabolism , Membrane Transport Proteins/metabolism , Models, Biological , Plants/metabolism , Biological Transport , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Plant Development , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Shoots/growth & development , Plant Shoots/metabolism
4.
Plant Biol (Stuttg) ; 12(4): 604-14, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20636903

ABSTRACT

Humic substances (HS) have positive effects on plant physiology, but the molecular mechanisms underlying these events are only partially understood. HS exert auxin-like activity, but data supporting this hypothesis are under debate. To investigate the auxin-like activity of HS, we studied their biological effect on lateral root initiation in Arabidopsis thaliana. To this aim we characterised HS by means of DRIFT and (13)C CP/MAS NMR spectroscopy, and measured their endogenous content of IAA. We then utilised a combination of genetic and molecular approaches to unravel HS auxin activity in the initiation of lateral roots. The data obtained using specific inhibitors of auxin transport or action showed that HS induce lateral root formation mostly through their 'auxin activity'. These findings were further supported by the fact that HS used in this study activated the auxin synthetic reporter DR5::GUS and enhanced transcription of the early auxin responsive gene IAA19.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Humic Substances/analysis , Plant Roots/growth & development , Arabidopsis/drug effects , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/metabolism , RNA, Plant/genetics , Spectrum Analysis , Transcription, Genetic
5.
Plant Biol (Stuttg) ; 10(4): 462-75, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18557906

ABSTRACT

A full-length cDNA encoding a putative high-affinity nitrate transporter (ZmNrt2.2) from maize was isolated and characterised, together with another previously identified transporter (ZmNrt2.1), in terms of phylogenesis, protein structure prediction and regulation of transcript accumulation in response to nitrate and sugar availability. The expression of both genes was evaluated by quantitative and semi-quantitative RT-PCR in response to nitrate and sugar supply and the in planta localisation of mRNA was studied by in situ hybridisation. Data obtained suggested similar genetic evolution and identical transmembrane structure prediction between the two deduced proteins, and differences in both regulation of their expression and mRNA localisation in response to nitrate, leading us to hypothesise a principal role for ZmNRT2.1 in the influx activity and the major involvement of ZmNRT2.2 in the xylem loading process. Our data suggest opposing sugar regulation by ZmNrt2.1 and ZmNrt2.2 transcription in the presence or absence of nitrate and the existence of both hexokinase-dependent and hexokinase-independent transduction mechanisms for the regulation of ZmNrt2.1 and ZmNrt2.2 expression by sugars.


Subject(s)
Anion Transport Proteins/genetics , Nitrates/pharmacology , Plant Proteins/genetics , Zea mays/metabolism , Anion Transport Proteins/chemistry , Anion Transport Proteins/metabolism , Gene Expression Regulation, Plant/drug effects , In Situ Hybridization , Nitrate Transporters , Phylogeny , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/classification , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Zea mays/genetics
6.
Mol Plant ; 1(2): 229-37, 2008 Mar.
Article in English | MEDLINE | ID: mdl-19825535

ABSTRACT

The regulation of plant growth responds to many stimuli. These responses allow environmental adaptation, thereby increasing fitness. In many cases, the relay of information about a plant's environment is through plant hormones. These messengers integrate environmental information into developmental pathways to determine plant shape. This review will use, as an example, auxin in the root of Arabidopsis thaliana to illustrate the complex nature of hormonal signal processing and transduction. It will then make the case that the application of a systems-biology approach is necessary, if the relationship between a plant's environment and its growth/developmental responses is to be properly understood.


Subject(s)
Hormones/physiology , Indoleacetic Acids/metabolism , Plant Growth Regulators/physiology , Plant Physiological Phenomena , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Base Sequence , Brassinosteroids , Cholestanols/metabolism , Cytokines/physiology , Cytokinins/physiology , Gene Expression Regulation, Plant , Genes, Plant , Gibberellins/physiology , Oligonucleotide Array Sequence Analysis/methods , Plant Development , Plant Roots/physiology , Plants/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/physiology , Signal Transduction , Steroid Hydroxylases/genetics , Steroids, Heterocyclic/metabolism , Transcription, Genetic
7.
New Microbiol ; 25(1): 83-8, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11837395

ABSTRACT

The early detection of mutations in the HIV-1 polymerase is a key point in the management of anti-retroviral therapy. While nucleotide substitutions and insertions have been well and frequently desribed in literature as linked to drug resistance, deletions have been rarely observed and desribed (ART67, Imamichi et al.). The aim of this study is to describe a case of deletion of three nucleotides in the RT gene (ART67) of a multi-treated HIV-1 infected patient. As this deletion has not been detected by the oligoprobe assay, the phenotyping test was used to support therapy but without an appreciable success in terms of viral load. Then a sequencing based genotyping system was used to analyse the viral polymerase and a novel deletion was found at codon 67 of RT gene.


Subject(s)
Drug Resistance, Viral/genetics , HIV Infections/drug therapy , HIV Reverse Transcriptase/genetics , Sequence Deletion , Adult , Base Sequence , DNA, Viral/analysis , Drug Therapy, Combination , Female , HIV Infections/diagnosis , Humans , Molecular Sequence Data , Point Mutation , Reverse Transcriptase Inhibitors/therapeutic use , Sequence Alignment , Sequence Analysis, DNA , Treatment Failure , Viral Load
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