Thermopriming triggers splicing memory in Arabidopsis.

Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat-shock memory and the role of priming in Arabidopsis thaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat-shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link 'splicing memory' to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat-stress responses in plants and other organisms as many of the key components are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

Ca(2+)/calmodulin regulates salicylic-acid-mediated plant immunity.

Intracellular calcium transients during plant-pathogen interactions are necessary early events leading to local and systemic acquired resistance. Salicylic acid, a critical messenger, is also required for both of these responses, but whether and how salicylic acid level is regulated by Ca(2+) signalling during plant-pathogen interaction is unclear. Here we report a mechanism connecting Ca(2+) signal to salicylic-acid-mediated immune response through calmodulin, AtSR1 (also known as CAMTA3), a Ca(2+)/calmodulin-binding transcription factor, and EDS1, an established regulator of salicylic acid level. Constitutive disease resistance and elevated levels of salicylic acid in loss-of-function alleles of Arabidopsis AtSR1 suggest that AtSR1 is a negative regulator of plant immunity. This was confirmed by epistasis analysis with mutants of compromised salicylic acid accumulation and disease resistance. We show that AtSR1 interacts with the promoter of EDS1 and represses its expression. Furthermore, Ca(2+)/calmodulin-binding to AtSR1 is required for suppression of plant defence, indicating a direct role for Ca(2+)/calmodulin in regulating the function of AtSR1. These results reveal a previously unknown regulatory mechanism linking Ca(2+) signalling to salicylic acid level.

The calmodulin-binding transcription factor SIGNAL RESPONSIVE1 is a novel regulator of glucosinolate metabolism and herbivory tolerance in Arabidopsis.

The Arabidopsis Ca(2+)/calmodulin (CaM)-binding transcription factor SIGNAL RESPONSIVE1 (AtSR1/CAMTA3) was previously identified as a key negative regulator of plant immune responses. Here, we report a new role for AtSR1 as a critical component of plant defense against insect herbivory. Loss of AtSR1 function impairs tolerance to feeding by the generalist herbivore Trichoplusia ni as well as wound-induced jasmonate accumulation. The susceptibility of the atsr1 mutant is associated with decreased total glucosinolate (GS) levels. The two key herbivory deterrents, indol-3-ylmethyl (I3M) and 4-methylsulfinylbutyl (4MSOB), showed the most significant reductions in atsr1 plants. Further, changes in AtSR1 transcript levels led to altered expression of several genes involved in GS metabolism including IQD1, MYB51 and AtST5a. Overall, our results establish AtSR1 as an important component of plant resistance to insect herbivory as well as one of only three described proteins involved in Ca(2+)/CaM-dependent signaling to function in the regulation of GS metabolism, providing a novel avenue for future investigations of plant-insect interactions.

Spatiotemporal organization of pre-mRNA splicing proteins in plants.

The general organization ofeukaryotic nuclei, including plant nuclei, into functional domains is now widely recognized. Conventional immunocytochemistry and visualization of proteins fused to fluorescent proteins (FP) have revealed that in plants, RNA and protein components of pre-mRNA splicing are spatially organized depending on the stage of cell cycle, development, and the cell's physiological state. Application of some of the latest microscopy techniques, which reveal biophysical properties such as diffusion and interaction properties of proteins, has begun to provide important insights into the functional organization of spliceosomal proteins in plants. Although some progress has been made in understanding the spatial and temporal organization of splicing machinery in plants, the mechanisms that regulate this organization and its functional consequences remain unresolved.

Regulation of alternative splicing of pre-mRNAs by stresses.

A substantial fraction (approximately 30%) of plant genes is alternatively spliced, but how alternative splicing is regulated remains unknown. Many plant genes undergo alternative splicing in response to a variety of stresses. Large-scale computational analyses and experimental approaches focused on select genes are beginning to reveal that alternative splicing constitutes an integral part of gene regulation in stress responses. Based on the studies discussed in this chapter, it appears that alternative splicing generates transcriptome/proteome complexity that is likely to be important for stress adaptation. However, the signaling pathways that relay stress conditions to splicing machinery and if and how the alternative spliced products confer adaptive advantages to plants are poorly understood.

Mitochondrial Neurogastrointestinal Encephalomyopathy: A Nonrenal Indication for Peritoneal Dialysis.

Mitochondrial neurogastrointestinal encephalomyopathy is a rare autosomal recessive disorder characterized by severe muscle wasting, gastrointestinal dysmotility, leukoencephalopathy, peripheral neuropathy, and ophthalmoplegia. The pathogenesis involves the accumulation of very high concentrations of nucleosides dThd and dUrd along with depletion of nucleotide dCTP. One of the treatment measures is the removal of nucleosides dThd and dUrd by hemodialysis and peritoneal dialysis. Only a few patient reports of dialysis as a measure to remove nucleosides had been reported.

Expression of a cyanobacterial delta 6-desaturase gene results in gamma-linolenic acid production in transgenic plants.

Gamma-linolenic acid (GLA), a nutritionally important fatty acid in human and animal diets, is not produced in oil seed crops. Many oil seed plants, however, produce significant quantities of linoleic acid, a fatty acid that could be converted to GLA by the enzyme delta 6-desaturase if it were present. As a first step to producing GLA in oil seed crops, we have cloned a cyanobacterial delta 6-desaturase gene. Expression of this gene in transgenic tobacco resulted in GLA accumulation. Octadecatetraenoic acid, a highly unsaturated, industrially important fatty acid, was also found in transgenic tobacco plants expressing the cyanobacterial delta 6-desaturase. This is the first example of engineering the production of 'novel' polyunsaturated fatty acids in transgenic plants.

Sources of Resistance to Tobacco streak virus in Wild Arachis (Fabaceae: Papilionoidae) Germplasm.

Stem necrosis disease caused by Tobacco streak virus (TSV), first recognized in 2000, has emerged as a potential threat to peanut (Arachis hypogaea) in southern states of India. The virus induces severe necrosis of shoots leading to death of the plant, and plants that survive are malformed, with severe reduction in pod yield. All the currently grown peanut cultivars in India are highly susceptible to the virus. Therefore, wild relatives of peanut were evaluated to identify potential sources of resistance to TSV infection. In all, 56 germplasm accessions from 20 wild Arachis spp. in four sections (Arachis, Erectoides, Procumbente, and Rhizomatosae), along with susceptible peanut cultivars (JL 24 and K 1375), were evaluated for resistance to TSV under greenhouse conditions using mechanical sap inoculations. Systemic virus infection, determined by enzyme-linked immunosorbent assay (ELISA), in the test accessions ranged between 0 and 100%. Twenty-four accessions in section Arachis that had 0 to 35% systemically infected plants were retested, and systemic infection was not detected in eight of these accessions in repeated trials in the greenhouse. These are International Crops Research Institute for the Semi-Arid Tropics groundnut (ICG) accession nos. 8139, 8195, 8200, 8203, 8205, and 11550 belonging to A. duranensis; ICG 8144 belonging to A. villosa; and ICG 13210 belonging to A. stenosperma. Even though the resistant accessions had 0 to 100% TSV infection in inoculated leaves, TSV was not detected in the subsequently emerged leaves. This is the first report of TSV resistance in Arachis spp. The eight TSV resistant accessions are cross compatible with A. hypogaea for utilization in breeding for stem necrosis disease resistance.

Cloning of a family of cyclins from Arabidopsis thaliana.

Degenerate oligonucleotide primers corresponding to two conserved regions in animal cyclins were used to amplify cyclin sequences from Arabidopsis thaliana by polymerase chain reaction (PCR). Screening of a floral meristem cDNA library with two distinct PCR-generated cyclin sequences resulted in the isolation of four different cyclin cDNAs. Southern analysis of genomic DNA and sequence information from the isolated clones suggest that there is a family of cyclins in Arabidopsis.

Cloning of the cDNA for glutamyl-tRNA synthetase from Arabidopsis thaliana.

We cloned and characterized a full-length cDNA that encodes a glutamyl-tRNA synthetase (GluRSAt) from Arabidopsis. The GluRSAt is coded by a single gene. A transcript of about 2.3 kb hybridized with the cDNA. The deduced protein from the cDNA contained 719 amino acids with an estimated molecular mass of 81 kDa. Expression of the GluRSAt in E. coli resulted in a protein of the expected size. Comparison of the amino acid sequence GluRSAt to other glutamyl-tRNA synthetases showed strong sequence similarity to cytoplasmic GluRS proteins.

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

Molecular motors and their functions in plants.

Molecular motors that hydrolyze ATP and use the derived energy to generate force are involved in a variety of diverse cellular functions. Genetic, biochemical, and cellular localization data have implicated motors in a variety of functions such as vesicle and organelle transport, cytoskeleton dynamics, morphogenesis, polarized growth, cell movements, spindle formation, chromosome movement, nuclear fusion, and signal transduction. In non-plant systems three families of molecular motors (kinesins, dyneins, and myosins) have been well characterized. These motors use microtubules (in the case of kinesines and dyneins) or actin filaments (in the case of myosins) as tracks to transport cargo materials intracellularly. During the last decade tremendous progress has been made in understanding the structure and function of various motors in animals. These studies are yielding interesting insights into the functions of molecular motors and the origin of different families of motors. Furthermore, the paradigm that motors bind cargo and move along cytoskeletal tracks does not explain the functions of some of the motors. Relatively little is known about the molecular motors and their roles in plants. In recent years, by using biochemical, cell biological, molecular, and genetic approaches a few molecular motors have been isolated and characterized from plants. These studies indicate that some of the motors in plants have novel features and regulatory mechanisms. The role of molecular motors in plant cell division, cell expansion, cytoplasmic streaming, cell-to-cell communication, membrane trafficking, and morphogenesis is beginning to be understood. Analyses of the Arabidopsis genome sequence database (51% of genome) with conserved motor domains of kinesin and myosin families indicates the presence of a large number (about 40) of molecular motors and the functions of many of these motors remain to be discovered. It is likely that many more motors with novel regulatory mechanisms that perform plant-specific functions are yet to be discovered. Although the identification of motors in plants, especially in Arabidopsis, is progressing at a rapid pace because of the ongoing plant genome sequencing projects, only a few plant motors have been characterized in any detail. Elucidation of function and regulation of this multitude of motors in a given species is going to be a challenging and exciting area of research in plant cell biology. Structural features of some plant motors suggest calcium, through calmodulin, is likely to play a key role in regulating the function of both microtubule- and actin-based motors in plants.

Environmental stresses modulate abundance and timing of alternatively spliced circadian transcripts in Arabidopsis.

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron-retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad-range thermal cycles triggered a sharp increase in the long IR CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or Pseudomonas syringae infection induced a similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts, whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out-of-phase oscillations of CCA1 IR transcripts. Taken together, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of nonsense-mediated mRNA decay (NMD)-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.

Inhibition of fungal and bacterial plant pathogens by synthetic peptides: in vitro growth inhibition, interaction between peptides and inhibition of disease progression.

Four synthetic cationic peptides, pep6, pep7, pep11 and pep20, were tested alone and in combinations for their antimicrobial activities against economically important plant pathogenic fungi (Phytophthora infestans and Alternaria solani) and bacteria (Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica). In in vitro studies, P. infestans and A. solani were inhibited by all four peptides, while E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica were inhibited only by pep11 and pep20. All peptides completely inhibited P. infestans and A. solani on potato leaves and P. infestans on tubers at concentrations comparable to the in vitro IC50 (effective concentration for 50% growth inhibition) values, suggesting that these peptides are more potent in preventing infection than in inhibiting hyphal growth in vitro. Microscopic observations of P. infestans and A. solani when treated with these peptides revealed hyphal anomalies. In tuber-infectivity assays, pep11 and pep20 reduced bacterial softrot symptoms by 50% at 2.0 to 2.30 microM and by 100% at 20 microM. In assays involving two-way combinations of these peptides, growth inhibitions of fungi and bacteria by the combinations were no more than the sum of growth inhibitions by each peptide when used alone, indicating that they act additively. pep11 and pep20 are not phytotoxic to potato plants at 200 microM. With strong and broad-spectrum antimicrobial activities of pep11 and pep20 against fungi and bacteria, and with no antagonistic activities, the expression of these peptides in transgenic potato plants could lead to enhanced disease resistance against these pathogens.

Kinesins in the Arabidopsis genome: a comparative analysis among eukaryotes.

BACKGROUND: Kinesins constitute a superfamily of microtubule motor proteins that are found in eukaryotic organisms. Members of the kinesin superfamily perform many diverse cellular functions such as transport of vesicles and organelles, spindle formation and elongation, chromosome segregation, microtubule dynamics and morphogenesis. Only a few kinesins have been characterized in plants including Arabidopsis thaliana. Because of the diverse cellular functions in which kinesins are involved, the number, types and characteristics of kinesins present in the Arabidopsis genome would provide valuable information for many researchers. RESULTS: Here we have analyzed the recently completed Arabidopsis genome sequence and identified sixty-one kinesin genes in the Arabidopsis genome. Among the five completed eukaryotic genomes the Arabidopsis genome has the highest percentage of kinesin genes. Further analyses of the kinesin gene products have resulted in identification of several interesting domains in Arabidopsis kinesins that provide clues in understanding their functions. A phylogenetic analysis of all Arabidopsis kinesin motor domain sequences with 113 motor domain sequences from other organisms has revealed that Arabidopsis has seven of the nine recognized subfamilies of kinesins whereas some kinesins do not fall into any known family. CONCLUSION: There are groups of Arabidopsis kinesins that are not present in yeast, Caenorhabditis elegans and Drosophila melanogaster that may, therefore, represent new subfamilies specific to plants. The domains present in different kinesins may provide clues about their functions in cellular processes. The comparative analysis presented here provides a framework for future functional studies with Arabidopsis kinesins.

Structural organization of a gene encoding a novel calmodulin-binding kinesin-like protein from Arabidopsis.

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified microtubule motor protein that appears to be unique to plants. Here we report isolation and sequence analysis of a gene encoding Arabidopsis KCBP. KCBP gene contains 21 exons and 20 introns. All exons except exon 3 are short (94-272 nt). Exons 1-9 code for the globular tail region whereas the coiled-coil region is coded by exons 10-15. The conserved motor domain is coded by exons 16-20. Calmodulin-binding domain that is present in the C-terminal region of the protein and unique to KCBP is coded by the last exon. The size of introns ranged from 71 (intron 17) to 320 (intron 19) nucleotides. As in most plant introns, the content of AT is very high in all introns (up to 76%). Phylogenetic analysis of KCBP using motor domain sequence grouped KCBP with other known C-terminal microtubule motor proteins. However, Arabidopsis KCBP together with its homologs from potato and tobacco constitute a distinct group within the C-terminal subfamily of motors which is consistent with structural and functional features of KCBP.

Novel antidiabetic and hypolipidemic agents. 5. Hydroxyl versus benzyloxy containing chroman derivatives.

Several thiazolidinediones having chroman moieties were synthesized and evaluated for their euglycemic and hypolipidemic activities. Some of the analogues having an aminoalkyl group as a linker between the chroman ring and 4-[5-(2,4-dioxo-1, 3-thiazolidinyl)methyl]phenoxy moiety seem to be better than troglitazone. In vitro transactivation assays of PPARgamma have been carried out with these glitazones to understand their molecular mechanism. For the first time we have found that some of the unsaturated thiazolidinediones are superior to their saturated counterpart in the in vivo assay. A more potent thiazolidinedione analogue than troglitazone is reported. Pharmacokinetic studies have shown that protection of the OH group in the chroman moiety leads to a decrease in metabolism, thereby resulting in a superior pharmacological profile.

Novel euglycemic and hypolipidemic agents. 4. Pyridyl- and quinolinyl-containing thiazolidinediones.

A series of substituted pyridyl- and quinolinyl-containing 2, 4-thiazolidinediones having interesting cyclic amine as a linker have been synthesized. Both unsaturated thiazolidinediones 5 and saturated thiazolidinediones 6 and their various salts were evaluated in db/db mice for euglycemic and hypolipidemic effects and compared with BRL compound 11 and BRL-49653, respectively. Some of the potent compounds were converted to various salts in order to obtain improved activities. Among all the salts evaluated, the maleate salt of unsaturated TZD 5a was found to be a very potent euglycemic and hypolipidemic compound. Some of the more interesting compounds have also been evaluated in ob/ob mice and compared with rosiglitazone (maleate salt of BRL-49653). Oral glucose tolerance tests were performed in both db/db and ob/ob mice. Pharmacokinetic studies of 5a maleate are also reported. Receptor binding studies of PPARgamma by 5a/5a maleate did not show any significant transactivation of PPARalpha or PPARgamma.

Inositol 1,4,5-trisphosphate induced calcium release from corn coleoptile microsomes.

The effect of inositol 1,4,5-trisphosphate (IP3) on Ca2+ release from microsomes of corn coleoptiles was investigated. Addition of micromolar concentrations of IP3 to Ca2+ loaded microsomes resulted in rapid release of 20-30% of sequestered Ca2+. Maximal and half maximal Ca2+ release occurred at 20 and 8 microM of IP3 respectively. Part of the Ca2+ released by IP3 was reaccumulated into microsomes within 4 min. The amount of Ca2+ released by IP3 was found to be dependent on free Ca2+ concentration in the incubation medium at the time of release. Maximum Ca2+ release was observed around 0.1 microM free Ca2+ concentration in the assay medium. These data suggest that IP3 might act as a second messenger in plants in a manner similar to animal systems by altering cytosolic levels of calcium.