Biomedical subjects

Chandrashekhar P Joshi

Publications and source records attributed to Chandrashekhar P Joshi.

8 recordsLinked to original sources

PubMed2006

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees.

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.

Arabidopsis Proteins↗

PubMed2004

Differential expression patterns of two cellulose synthase genes are associated with primary and secondary cell wall development in aspen trees.

The quality and quantity of cellulose deposited in the primary and secondary cell walls of plants vary in accordance with their biological function. However, the molecular basis of such cellulose heterogeneity has so far remained unclear. Since enrichment of better-quality cellulose, in terms of increased degree of polymerization and crystallinity, is one of the goals of forest biotechnology, our main objective is to decipher the roles of distinct cellulose synthase (CesA) genes in tree development, with special reference to wood production. Here, we report two full-length CesA cDNAs, PtrCesA3 and PtrCesA4, from an economically important tree aspen (Populus tremuloides). PtrCesA3 is orthologous to the Arabidopsis AtCesA4 gene involved in secondary wall formation, whereas PtrCesA4 is orthologous to the Arabidopsis AtCesA1 gene involved in primary cell wall formation. To define the specific cell types expressing these CesA genes, we explored the natural distribution patterns of PtrCesA3 and PtrCesA4 transcripts in a variety of aspen organs, such as leaves, petiole, stem, and roots, using in situ hybridization with hypervariable region-specific antisense riboprobes. Such a side-by-side comparison suggested that PtrCesA3 is exclusively expressed in secondary-wall-forming cells of xylem and phloem fibers, whereas PtrCesA4 is predominantly expressed in primary-wall-forming expanding cells in all aspen organs examined. These findings suggest a functionally distinct role for each of these two types of PtrCesAs during primary and secondary wall biogenesis in aspen trees, and that such functional distinction appears to be conserved between annual herbaceous plants and perennial trees.

Cell Wall↗

PubMed2004

Differential expression patterns of two new primary cell wall-related cellulose synthase cDNAs, PtrCesA6 and PtrCesA7 from aspen trees.

Based on elegant molecular genetic analyses, distinct classes of cellulose synthase (CesA) genes have been associated with either primary or secondary cell wall development in Arabidopsis. Here, we report on cloning of two new CesA cDNAs, PtrCesA6 and PtrCesA7 involved in the primary cell wall development in aspen (Populus tremuloides) trees. Both these distinct cDNAs, isolated from a developing xylem cDNA library, share only 60-67% identities with each other as well as with five other previously known aspen CesA cDNAs. Interestingly, PtrCESA6 from aspen, a dicot species, shares maximum identity of 81-84% with three CESA isoforms from maize and rice, two monocot species. On the other hand, PtrCESA7 shares a maximum identity of 86% with AtCESA2, a primary wall-related CesA member from Arabidopsis, a dicot species. Gene expression analyses by reverse transcriptase-polymerase chain reactions (RT-PCRs) suggested that both these genes are expressed at a low level in all aspen tissues examined but PtrCesA7 is expressed at a higher level than PtrCesA6. While corroborating these results, in situ mRNA hybridization studies using three different aspen organs also suggested that PtrCesA6 and PtrCesA7 genes are expressed in all expanding cells depositing primary cell wall but PtrCesA7 is expressed at a higher level than PtrCesA6. These differential gene expression profiles suggest that each of these CesAs may be playing a specific role during primary cell wall development in aspen trees. Isolation of two primary wall related CesA genes from xylem tissues also suggest their importance during xylem development, which is traditionally considered to be enriched in secondary cell wall forming cells of economical significance.

Blotting, Southern↗

PubMed2004

Suppression subtractive hybridization-mediated transcriptome analysis from multiple tissues of aspen (Populus tremuloides) altered in phenylpropanoid metabolism.

A PCR-based suppression subtractive hybridization (SSH) technique was used to identify differentially expressed genes in developing tissues of control and transgenic aspen (Populus tremuloides Michx.) with down-regulated 4CL1 (4-coumarate:coenzyme A ligase) expression and enhanced growth. A total of 11,308 expressed sequence tags (ESTs) representing 5,028 non-redundant transcripts encoding 4,224 unique proteins was obtained from shoot apex, young stem, young leaf and root tip SSH libraries. Putative functions can be assigned to 60% of these transcripts. Approximately 14% of the ESTs are not represented among the 111,000 entries already present in Populus EST databases. In general, ESTs of the metabolism class occurred at a higher frequency in control- than transgenic-enriched libraries of all tissues, whereas protein synthesis and protein fate ESTs were over-represented in meristematic tissues of transgenics where 4CL1 was relatively strongly suppressed. Among all tissues, leaves yielded the highest percentage of ESTs with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. Of particular interest was a large number of ESTs (16%) associated with signal transduction in transgenic leaves. Among these were several leucine-rich-repeat receptor-like protein kinases with markedly elevated expression in transgenic leaves. We also identified homologs of transposable elements that were up-regulated in transgenic tissues, providing the first experimental data for active expression of DNA mobile elements in long-lived tree species.

DNA Transposable Elements↗

PubMed2004

Molecular cloning of ten distinct hypervariable regions from the cellulose synthase gene superfamily in aspen trees.

Recent molecular genetic data suggest that cellulose synthase (CesA) genes coding for the enzymes that catalyze cellulose biosynthesis (CESAs) in Arabidopsis and other herbaceous plants belong to a large gene family. Much less is known about CesA genes from forest trees. To isolate new CesA genes from tree species, discriminative but easily obtainable homologous DNA probes are required. Hypervariable regions (HVRII) of CesA genes represent highly divergent DNA sequences that can be used to examine structural, expressional and functional relationships among CesA genes. We used a reverse transcriptase-polymerase chain reaction (RT-PCR)-based technique to identify HVRII regions from eight types of CesA genes and two types of CesA-like D (CslD) genes in quaking aspen (Populus tremuloides Michx.). Comparison of these aspen CESA/CSLD HVRII regions with the predicted proteins from eight full-length CesA/CslD cDNAs available in our laboratory and with searches for aspen CesA/CslD homologs in the recently released Populus trichocarpa Torr. & A. Gray. genome confirmed the utility of this approach in identifying several CesA/CslD gene members from the Populus genome. Phylogenetic analysis of 56 HVRII domains from a variety of plant species suggested that at least six distinct classes of CESAs exist in plants, supporting a previous proposal for renaming HVRII regions as class-specific regions (CSR). This method of CSR cloning could be applied to other crop plants and tree species, especially softwoods, for which the whole genome sequence is unlikely to become available in the near future because of the large size of these genomes.

Cloning, Molecular↗

PubMed2003

Isolation and characterization of a new, full-length cellulose synthase cDNA, PtrCesA5 from developing xylem of aspen trees.

Cellulose synthases (CesA) represent a group of beta-1,4 glycosyltransferases involved in cellulose biosynthesis. Recent molecular studies confirmed that a large CesA gene family encoding various CesA isoforms exists in plants. The isolation and characterization of a new, full-length CesA cDNA, PtrCesA5 from an economically important tree, quaking aspen (Populus tremuloides) is reported here. The predicted PtrCesA5 protein is highly similar to Arabidopsis AtCesA3 (88%) involved in primary cell wall synthesis. However, gene expression studies here suggest that PtrCesA5 transcripts are greatly enhanced in developing xylem tissues enriched in secondary cell wall synthesis as compared with leaf tissues actively undergoing primary cell wall synthesis. Availability of this new, dual-function CesA from a woody species will assist in a better understanding of the mechanism of cellulose biosynthesis during wood development.

DNA, Complementary↗

PubMed2003

Xylem-specific and tension stress-responsive expression of cellulose synthase genes from aspen trees.

Genetic improvement of cellulose biosynthesis in woody trees is one of the major goals of tree biotechnology research. Yet, progress in this field has been slow owing to (1) unavailability of key genes from tree genomes, (2) the inability to isolate active and intact cellulose synthase complexes and, (3) the limited understanding of the mechanistic processes involved in the wood cellulose development. Here I report on the recent advances in molecular genetics of cellulose synthases (CesA) from aspen trees. Two different types of cellulose synthases appear to be involved in cellulose deposition in primary and secondary walls in aspen xylem. The three distinct secondary CesAs from aspen- PtrCesA1, PtrCesA2, and PtrCesA3-appear to be aspen homologs of Arabidopsis secondary CesAs AtCesA8, AtCesA7, and AtCesA4, respectively, based on their high identity/similarity (>80%). These aspen CesA proteins share the transmembrane domain (TMD) structure that is typical of all known "true" CesA proteins: two TMDs toward the N-terminal and six TMDs toward the C-terminal. The putative catalytic domain is present between TMDs 2 and 3. All signature motifs of processive glycosyltransferases are also present in this catalytic domain. In a phylogenetic tree based on various predicted CesA proteins from Arabidopsis and aspen, aspen CesAs fall into families similar to those seen with Arabidopsis CesAs, suggesting their functional similarity. The coordinate expression of three aspen secondary CesAs in xylem and phloem fibers, along with their simultaneous tension stress-responsive upregulation, suggests that these three CesAs may play a pivotal role in biosynthesis of better-quality cellulose in secondary cell walls of plants. These results are likely to have a direct impact on genetic manipulation of trees in the future.

Arabidopsis↗

PubMed2002

A new cellulose synthase gene (PtrCesA2) from aspen xylem is orthologous to Arabidopsis AtCesA7 (irx3) gene associated with secondary cell wall synthesis.

We report here the molecular cloning and characterization of a new full-length cellulose synthase (CesA) cDNA, PtrCesA2 from aspen (Populus tremuloides) trees. The predicted PtrCesA2 protein shows a high degree of identity/similarity (87%/91%) to the predicted gene product of Arabidopsis AtCesA7 gene that has been associated with secondary cell wall development. Previously, a mutation in AtCesA7 gene (irx3) was correlated with a significant decrease in the amount of cellulose synthesized (about 70%) and genetic complementation of irx3 mutant with a wild-type AtCesA7 gene restored the normal phenotype. This is the first report of a full-length AtCesA7 ortholog from any non-Arabidopsis species. Interestingly, PtrCesA2 shares only 64% identity with our earlier reported PtrCesA1 from aspen suggesting its structural distinctness from the only other known CesA member from the aspen genome. PtrCesA1 is a xylem-specific and tension stress responsive gene that is highly similar to another Arabidopsis gene, AtCesA8 which also has been associated with secondary wall development. Moreover, AtCesA7 and AtCesA8 are suggested to be part of the same cellulose synthase complex. Isolation of PtrCesA2 from a xylem library enriched in cells with active secondary wall synthesis, PtrCesA2 expression levels similar to PtrCesA1 and high similarity of PtrCesA1 and PtrCesA2 to AtCesA8 and AtCesA7, respectively, suggest that both these aspen genes might be involved in the secondary wall development in aspen woody tissues. Availability of two aspen CesA orthologs will now enable us to examine if PtrCesA1 and PtrCesA2 functionally interact during aspen wood development that has long-term implications on genetic improvement of forest trees.

Arabidopsis Proteins↗