Biomedical subjects

Rishikesh P Bhalerao

Publications and source records attributed to Rishikesh P Bhalerao.

10 recordsLinked to original sources

PubMed2005

Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana.

BACKGROUND: Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific. RESULTS: We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes. CONCLUSIONS: The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21,000 Arabidopsis transcripts.

Arabidopsis↗

PubMed2004

A Populus EST resource for plant functional genomics.

Trees present a life form of paramount importance for terrestrial ecosystems and human societies because of their ecological structure and physiological function and provision of energy and industrial materials. The genus Populus is the internationally accepted model for molecular tree biology. We have analyzed 102,019 Populus ESTs that clustered into 11,885 clusters and 12,759 singletons. We also provide >4,000 assembled full clone sequences to serve as a basis for the upcoming annotation of the Populus genome sequence. A public web-based EST database (POPULUSDB) provides digital expression profiles for 18 tissues that comprise the majority of differentiated organs. The coding content of Populus and Arabidopsis genomes shows very high similarity, indicating that differences between these annual and perennial angiosperm life forms result primarily from differences in gene regulation. The high similarity between Populus and Arabidopsis will allow studies of Populus to directly benefit from the detailed functional genomic information generated for Arabidopsis, enabling detailed insights into tree development and adaptation. These data will also valuable for functional genomic efforts in Arabidopsis.

Ecosystem↗

PubMed2004

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Arabidopsis↗

PubMed2004

Organellar gene transcription and early seedling development are affected in the rpoT;2 mutant of Arabidopsis.

An Arabidopsis mutant that exhibited reduced root length was isolated from a population of activation-tagged T-DNA insertion lines in a screen for aberrant root growth. This mutant also exhibited reduced hypocotyl length as well as a delay in greening and altered leaf shape. Molecular genetic analysis of the mutant indicated a single T-DNA insertion in the gene RpoT;2 encoding a homolog of the phage-type RNA polymerase (RNAP), that is targeted to both mitochondria and plastids. A second T-DNA-tagged allele also showed a similar phenotype. The mutation in RpoT;2 affected the light-induced accumulation of several plastid mRNAs and proteins and resulted in a lower photosynthetic efficiency. In contrast to the alterations in the plastid gene expression, no major effect of the rpoT;2 mutation on the accumulation of examined mitochondrial gene transcripts and proteins was observed. The rpoT;2 mutant exhibited tissue-specific alterations in the transcript levels of two other organelle-directed nuclear-encoded RNAPs, RpoT;1 and RpoT;3. This suggests the existence of cross-talk between the regulatory pathways of the three RNAPs through organelle to nucleus communication. These data provide an important information on a role of RpoT;2 in plastid gene expression and early plant development.

Arabidopsis↗

PubMed2004

Differential stage-specific regulation of cyclin-dependent kinases during cambial dormancy in hybrid aspen.

The cambium of woody plants cycles between active and dormant states. Dormancy can be subdivided into eco- and endodormant stages. Ecodormant trees resume growth upon exposure to growth-promotive signals, while the establishment of endodormant state results in loss of the ability to respond to these signals. In this paper, we analysed the regulation of cyclin-dependent kinases (CDKs) to understand the differential response of cell division machinery to growth-promotive signals during the distinct stages of dormancy in hybrid aspen. We show that 4 weeks of short-day (SD) treatment causes termination of the cambial cell division and establishment of the ecodormant state. This coincides with a steady decline in the histone H1 kinase activity of the PSTAIRE-type poplar CDKA (PttCDKA) and the PPTTLRE-type PttCDKB kinase complexes. However, neither the transcript nor the polypeptide levels of PttCDKA and PttCDKB are reduced during ecodormancy. In contrast, 6 weeks of SD treatment establishes endodormancy, which is marked by the reduction and disappearance of the PttCDKA and PttCDKB protein levels and the PttCDKB transcript levels. The transition to endodormancy is preceded by an elevated E2F (adenosine E2 promoter binding factor) phosphorylation activity of the PttCDKA kinase that reduces the DNA-binding activity of E2F in vitro. The transition to endodormancy is followed by a reduction of retinoblastoma (Rb) phosphorylation activity of PttCDKA protein complexes. Both phosphorylation events could contribute to block the G1 to S phase transition upon the establishment of endodormancy. Our results indicate that eco- and endodormant stages of cambial dormancy involve a stage-specific regulation of the cell cycle effectors at multiple levels.

Amino Acid Sequence↗

PubMed2004

Cambial meristem dormancy in trees involves extensive remodelling of the transcriptome.

The establishment of the dormant state in meristems involves considerable physiological and metabolic alterations necessary for surviving unfavourable growth conditions. However, a global molecular analysis of dormancy in meristems has been hampered by the difficulty in isolating meristem cells. We used cryosectioning to isolate purified cambial meristem cells from the woody plant Populus tremula during active growth and dormancy. These samples were used to generate meristem-specific cDNA libraries and for cDNA microarray experiments to define the global transcriptional changes underlying cambial dormancy. The results indicate a significant reduction in the complexity of the cambial transcriptome in the dormant state. Although cell division is terminated in the dormant cambium, the cell cycle machinery appears to be maintained in a skeletal state as suggested by the continued presence of transcripts for several cell cycle regulators. The downregulation of PttPIN1 and PttPIN2 transcripts explains the reduced basipetal polar auxin transport during dormancy. The induction of a member of the SINA family of ubiquitin ligases implicated in auxin signalling indicates a potential mechanism for modulation of auxin sensitivity during cambial dormancy. The metabolic alterations during dormancy are mirrored in the induction of genes involved in starch breakdown and the glyoxysomal cycle. Interestingly, the induction of RGA1 like gene suggests modification of gibberellin signalling in cambial dormancy. The induction of genes such as poplar orthologues of FIE and HAP2 indicates a potential role for these global regulators of transcription in orchestrating extensive changes in gene expression during dormancy.

Cell Cycle↗

PubMed2003

Gravity-regulated differential auxin transport from columella to lateral root cap cells.

Gravity-induced root curvature has long been considered to be regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients, and the transport mechanisms involved, remain to be identified. Here, we describe a GFP-based auxin biosensor to monitor auxin during Arabidopsis root gravitropism at cellular resolution. We identify elevated auxin levels at the root apex in columella cells, the site of gravity perception, and an asymmetric auxin flux from these cells to the lateral root cap (LRC) and toward the elongation zone after gravistimulation. We differentiate between an efflux-dependent lateral auxin transport from columella to LRC cells, and an efflux- and influx-dependent basipetal transport from the LRC to the elongation zone. We further demonstrate that endogenous gravitropic auxin gradients develop even in the presence of an exogenous source of auxin. Live-cell auxin imaging provides unprecedented insights into gravity-regulated auxin flux at cellular resolution, and strongly suggests that this flux is a prerequisite for root gravitropism.

Arabidopsis↗

PubMed2003

The case for morphogens in plants.

Plants and animals have evolved as multicellular organisms independently of one another. This raises the intriguing question of whether plants and animals have developed similar or distinct patterning strategies to establish their body plans. Animals use concentration gradients of signals termed morphogens for tissue patterning, but whether they are also used by plants is unclear. Here we compare and contrast the plant hormone auxin with animal morphogens, and speculate as to whether plants have independently evolved similar mechanisms to regulate pattern formation.

Animals↗

PubMed2002

Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings.

Lateral root formation is profoundly affected by auxins. Here we present data which indicate that light influences the formation of indole-3-acetic acid (IAA) in germinating Arabidopsis seedlings. IAA transported from the developing leaves to the root system is detectable as a short-lived pulse in the roots and is required for the emergence of the lateral root primordia (LRP) during early seedling development. LRP emergence is inhibited by the removal of apical tissues prior to detection of the IAA pulse in the root, but this treatment has minimal effects on LRP initiation. Our results identify the first developing true leaves as the most likely source for the IAA required for the first emergence of the LRP, as removal of cotyledons has only a minor effect on LRP emergence in contrast to removal of the leaves. A basipetal IAA concentration gradient with high levels of IAA in the root tip appears to control LRP initiation, in contrast to their emergence. A significant increase in the ability of the root system to synthesize IAA is observed 10 days after germination, and this in turn is reflected in the reduced dependence of the lateral root emergence on aerial tissue-derived auxin at this stage. We propose a model for lateral root formation during early seedling development that can be divided into two phases: (i) an LRP initiation phase dependent on a root tip-localized IAA source, and (ii) an LRP emergence phase dependent on leaf-derived IAA up to 10 days after germination.

Arabidopsis↗

PubMed2002

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen.

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.

Amino Acid Sequence↗