Development of Pusa 5SD for seed dressing and Pusa Biopellet 10G for soil application formulations of Trichoderma harzianum and their evaluation for integrated management of dry root rot of mungbean (Vigna radiata)

Various seed dressing and soil application formulations were developed from Trichoderma viride, T. virens and T. harzianum to increase the shelf life of bio-formulations used to manage dry root rot (Rhizoctonia bataticola) of mungbean (Vigna radiata), a major yield limiting factor in mungbean production. The shelf life of the formulations developed in the present study was monitored by counting colony forming units (cfu) up to 25 months of storage at room temperature (26 ± 8 °C). A newly developed seed dressing formulation, Pusa 5SD based on peat powder (47.5%), Sabudana powder (Manihot esculenta) (47.5%) and carboxymethyl cellulose (5%) and a newly developed soil application formulation, Pusa Biopellet (PBP) based on sodium alginate, aluminium silicate, Sabudana powder and tap water (1:5:5:100 w/w/w/v) exhibited longer shelf life. Another formulation Pusa Biogranule (PBG) based on wheat and pulse brans varied in cfu counts during different periods of storage. Pusa 5SD could be used up to 25 months of storage while PBP 10G and PBG 5 could be used up to 15 months of storage (>10⁵ cfu). The efficacy of the formulations was evaluated in pot experiments against the disease. In these experiments, T. harzianum based PBP 10G and PBG 5 for soil application, and Pusa 5SD for seed treatment were found to be superior to others in reducing the dry root rot incidence, and increasing the seed germination and shoot and root lengths. However, a combination of soil application of PBP 10G (T. harzianum) and seed treatment with T. harzianum based Pusa 5SD + carboxin was found superior to the use of any of these formulations alone in reducing the dry root rot incidence (87.2%) and increasing the seed germination (43.0%), shoot length (40.3%), root length (37.0%) and grain yield (54.6%) of mungbean crop over those of untreated control under sick field conditions.     

Changes in ubiquitin and ubiquitin-protein conjugates during seed formation and germination

Changes in both free ubiquitin and ubiquitin-protein conjugateswere followed in cotyledons of lupin (Lupinus albus L.) duringthe course of seed formation, from the flower to the dry seed,and during germination and seedling growth, from the dry seedto the senescing cotyledons. The observed levels of ubiquitinconjugates, detected by immunoblotting using antiubiquitin antibodiesand by autoradiography using ¹²⁵I-labelled ubiquitin, suggestan intense involvement of the ubiquitin-mediated proteolyticpathway during the highly regulated phases of seed formationand germination. High amounts of free ubiquitin are presentat all stages in all tissues examined. With the exception ofthe dry seed, the high molecular mass ubiquitin-protein conjugatesare also present at all stages. Higher amounts of these conjugateswere found during the initial stages of pod development andseed germination and during the most active phases of storageprotein deposition and degradation. Germination and seedlinggrowth in total darkness not only delays the degradation ofthe storage proteins, but also extends the period characterizedby the presence of a high amount of these conjugates. No suchconjugates were detected in the dry seeds, probably reflectingthe extremely low metabolic activity observed in these organs.A number of smaller molecular mass polypeptides were also detectedat different stages of seed development, germination and seedlinggrowth. Of particular interest is the abrupt accumulation ofan abundant 20 kDa polypeptide in the cotyledons during the4th day after imbibition, which is maintained in high amountsin these organs, rapidly declining after about 12–14 d.The pattern of accumulation of the 20 kDa polypeptide is controlledneither by light nor by the embryo axes, and large variationsin its concentration are observed during heat shock. Key words: Ubiquitin, ubiquitin-protein conjugates, seed storage proteins, protein synthesis, protein degradation     

Variability in reproductive traits in Jatropha curcas L. accessions during early developmental stages under warm subtropical conditions

Variability in floral, fruit, and seed characteristics, and oil content of 15 accession of Jatropha curcas during early development were assessed during two flowering periods in south Florida subtropical climate. The two flowering periods had leaf flushing in March. Field evaluation using 18 quantitative traits showed significant variation among accessions. The number of female flowers and female : male flower ratio ranged from 1 to 15 and 1 : 8.8 to 1 : 67.8, respectively. Fruit set by natural pollination was 89 and 66% during the first (1st) and second (2nd) flowering periods, respectively. A higher number of female‐type inflorescences were observed during summer. There were significant differences in seed traits, except for number of seeds per fruit. Accession TREC 31 had the highest individual seed dry weight and 100‐seed weight (0.83 g and 79.7 g, respectively). The oil content varied from 19.30% to 35.62%. Seed dry weight had positive correlation with seed fresh weight, seed length, seed thickness, seed width, and 100‐seed weight, but negative correlation with oil content. Based on the cluster analysis using 15 morphological traits, jatropha accessions were grouped into five main clusters and accessions from different geographic regions grouped together in a cluster. Principal component analyses (PCA) revealed morphological variation. The first three components explained 73.5% of the total variation and seed dry weight, 100‐seed weight, total flowers per inflorescence, male flowers per inflorescence and fruit set can be used to distinguish accessions. The PCA also indicated that flowering traits were more influenced by seed origin while seed traits were affected by flowering spans. Although evaluations were performed in plants during the juvenile phase, accessions TREC 31 and TREC 55 had superior averages for almost all characters evaluated. These results provide a preliminary assessment of the high variability in jatropha accessions evaluated and their potential for use in breeding and genetic improvement programs.     

Biosynthesis of storage proteins in developing rice seeds

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10- to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76- and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.

The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with [¹⁴C]leucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kilodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.

In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.

     

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Antarctic ice algae Chlamydomonas sp. ICE-L

A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length cDNA of CiHsp70 was 2,232 bp, consisting of a 5′-terminal untranslated region (UTR) of 76 bp, a 3′-terminal UTR of 203 bp with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17 amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or 93‰ NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment.     

Molecular analysis of the QM gene from <Emphasis Type="Italic">Penaeus monodon</Emphasis> and its expression on the different ovarian stages of development

In present study, a QM gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length black tiger prawn QM (PmQM) cDNA contained a 5′-UTR of 41 bp, an ORF of 663 bp encoding a polypeptide of 220 amino acids with molecular weight 25.5 kDa, and a 3′-UTR of 54 bp. Homology analysis of the deduced amino acid sequence of the PmQM with other known QM sequences by MatGAT software revealed that the PmQM was high homology with other invertebrates. A conserved signature sequence of the QM family was found in the PmQM deduced amino acid sequence. Analysis of the tissue expression pattern of the PmQM gene showed that the PmQM mRNA was expressed in all tissues tested, with highest levels in ovary. Furthermore, the PmQM expression was found to be different in three important ovarian stages of development. The results indicated PmQM might play an important role in ovarian development.     

Purification and characterization of Arabidopsis ornithine transcarbamoylase (OTCase), a member of a distinct and evolutionarily-conserved group of plant OTCases

We affinity-purified an ornithine transcarbamoylase (carbamoyl phosphate:L-ornithine carbamoyltransferase; EC 2.1.3.3) 676-fold to near homogeneity from leaf tissues of Arabidopsis thaliana L. cv. Columbia. The purified OTCase protein exhibited a molecular mass of 37 kDa on SDS-PAGE gels and exhibited a pI = 6.8. A 41-kDa polypeptide was immunoprecipitated from Arabidopsis leaf poly(A)⁺ RNA in vitro translation products by pea OTCase antiserum. This precursor OTCase (pOTCase) is the predicted size (41 170 Da) for a polypeptide encoded by an Arabidopsis OTCase cDNA. Characteristics of N-terminal residues of the deduced amino acid sequence of this pOTCase suggest that it is a chloroplast-targeted protein. The sequences of plant OTCases suggest that they represent a distinct and evolutionarily-conserved group of OTCases. No evidence was found for OTCase isoenzymes in Arabidopsis leaf tissues. The Arabidopsis pOTCase was poorly-expressed in Escherichia coli strain TB-2, an OTCase-deficient mutant, and did not complement the mutant on arginine-minus selection medium.     

Molecular cloning and characterization of a jasmonate biosynthetic pathway gene for allene oxide cyclase from <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Herein, we cloned a full-length cDNA encoding allene oxide cyclase (AOC, EC 5.3.99.6) that is a key enzyme in jasmonates (JAs) biosynthetic pathway from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels, named as JcAOC (GenBank accession no. FJ874630). The cDNA was 924 bp in length with a complete open reading frame of 750 bp, which encoded a polypeptide of 250 amino acids including a putative signal peptide of 65 amino acid residues and a mature protein of 185 amino acids with a predicted molecular mass of 20.7 kDa and a isoelectric point of 6.24. Phylogenetic analysis indicated that JcAOC belonged to the AOC superfamily. Semi-quantitative RT-PCR analysis revealed that JcAOC mRNA was expressed in roots, stems, leaves, young seeds, endosperms, and flowers, but that the expression level was highest in leaves and lowest in seeds, and mRNA expression of JcAOC could be induced by salt stress (300 mM NaCl) and low temperature (4°C). Furthermore, the full-length coding region of JcAOC excluding signal peptide sequence was inserted into pET-30a and was successfully expressed in Escherichia coli. Overexpression of JcAOC in E. coli conferred its resistance to salt stress and low temperature.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development

Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety “Red Kidney.”     

Characterization and expression profile analysis of a sucrose synthase gene from common bean (Phaseolus vulgaris L.) during seed development

A full-length cDNA encoding common bean (Phaseolus vulgaris L.) sucrose synthase (designated as Pv_BAT93 Sus), which catalyses the synthesis and cleavage of sucrose, was isolated from seeds at 15 days after pollination (DAP) by rapid amplification of cDNA ends (RACE). The full-length cDNA of Pv_BAT93 Sus had a 2,418 bp open reading frame (ORF) encoding a protein of 806 amino acid residues. Sequence comparison analysis showed that Pv_BAT93 Sus was very similar to several members of the sucrose synthase family of other plant species. Tissue expression pattern analysis showed that Pv_BAT93 Sus was expressed in leaves, flowers, stems, roots, cotyledons, and particularly during seed development. Expression studies using in situ hybridization revealed altered spatial and temporal patterns of Sus expression in the EMS mutant relative to wild-type and confirmed Sus expression in common bean developing seeds. The expression and accumulation of Sus mRNA was clearly shown in several tissues, such as the suspensor and embryo, but also in the transfer cells and endothelium. The results highlight the diverse roles that Sus might play during seed development in common bean.     

Cloning,characterization and expression of a cDNA encoding a granulin-like polypeptide in Ciona savignyi

Previous study in our laboratory confirmed that a novel polypeptide, CS5931 derived from Ciona savignyi possesses potent antitumor activity. In the present study, the full length cDNA of CS5931 precursor, termed Cs-pgrn-1 was cloned. The complete cDNA sequence of this gene consists of 685 bp containing an open reading frame (ORF) of 522 bp (173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin (GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares high homology with Ciona intestinalis GRN and is conserved during evolution. The polypeptide also shows high similarity with human GRN A, B, and C. Prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A. The CS5931 was expressed using a prokaryotic expression system and the purified polypeptide inhibited the growth of several tumor cell lines in vitro via apoptotic pathway. Our study revealed that CS5931 has the potential to be developed as a novel antitumor agent.