Analysis of expressed sequence tags from biodiesel plant Jatropha curcas embryos at different developmental stages

Jatropha curcas is considered a potential biodiesel feedstock plant whose seeds contain up to 40% oil. However, little is currently known about the seed biology of Jatropha. Therefore, it would be valuable to understand the mechanisms of development and lipid metabolism in Jatropha seeds. In the present study, three cDNA libraries were constructed with mRNA from Jatropha embryos at different stages of seed development. A total of 9844 expressed sequence tags (ESTs) were produced from these libraries, from which 1070 contigs and 3595 singletons were obtained. One hundred and seven unigenes were found to be differentially expressed in the three cDNA libraries of Jatropha embryos, indicating that these genes may play key roles in seed development. We have identified 59 and 61 unigenes that might be involved in the development and lipid metabolism in Jatropha seeds, respectively. Some of these genes may also play important roles in embryogenesis, morphogenesis, defense response and adaptive mechanisms in plants.     

Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum

Jatropha curcas L. is an excellent biofuel crop, which displays a high efficiency of carbon absorption, and seed oil of Jatropha can be efficiently processed to produce high-quality biodiesel. Plant phosphoenolpyruvate carboxylases (PEPCs) play important roles not only in initial fixation of atmospheric CO₂ in C₄ and Crassulacean acid metabolism (CAM) plants, but also in fatty acid biosynthesis in seeds of oil plants by regulating carbon partitioning. Here, we identified JcPEPC1 from J. curcas L. by homology cloning, and alignment analysis of protein sequence revealed JcPEPC1 was a plant C₃-type PEPC, and shared high similarity to PEPC of castor oil plant Ricinus communis. We implemented detailed functional characterization of JcPEPC1 by expression analysis and transgenic tobacco. JcPEPC1 gene expressed in the leaves and seeds of J. curcas L., and remarkable increase of expression level was also detected at seed oil-accumulating stages. We overexpressed JcPEPC1 in tobacco, and showed the enzymatic activity of PEPC in transgenic plants was notably higher than wild type. Gas chromatography (GC) analysis elucidated the composition and total content of fatty acids were also altered. This study indicated JcPEPC1 played a fundamental role in fatty acid biosynthesis in Jatropha seeds. Our results proposed enhanced PEPC activity of Jatropha could improve biosynthesis of fatty acid, which implied critical functions in primary metabolism of non-photosynthetic PEPC.     

Expression and interaction of the CBLs and CIPKs from immature seeds of kidney bean (Phaseolus vulgaris L.)

Protein phosphorylation plays a key regulatory role in a variety of cellular processes. To better understand the function of protein phosphorylation in seed maturation, a PCR-based cloning method was employed and five cDNA clones (pvcipk1-5) for protein kinases were isolated from a cDNA library prepared from immature seeds of kidney bean (Phaseolus vulgaris L.). The deduced amino acid sequences showed that the five protein kinases (PvCIPK1-5) are members of the sucrose non-fermenting 1-related protein kinase type 3 (SnRK3) family, which interacts with calcineurin B-like proteins (CBLs). Two cDNA clones (pvcbl1 and 2) for CBLs were further isolated from the cDNA library. The predicted primary sequences of the proteins (PvCBL1 and 2) displayed significant identity (more than 90%) with those of other plant CBLs. Semi-quantitative RT-PCR analysis showed that the isolated genes, except pvcbl1, are expressed in leaves and early maturing seeds, whereas pvcbl1 is constitutively expressed during seed development. Yeast two-hybrid assay indicated that among the five PvCIPKs, only PvCIPK1 interacts with both PvCBL1 and PvCBL2. These results suggest that calcium-dependent protein phosphorylation-signaling via CBL-CIPK complexes occurs during seed development.     

Dynamic distribution and the role of abscisic acid during seed development of a lady’s slipper orchid,Cypripedium formosanum

Background and Aims Although abscisic acid (ABA) is commonly recognized as a primary cause of seed dormancy, there is a lack of information on the role of ABA during orchid seed development. In order to address this issue, the localization and quantification of ABA were determined in developing seeds of Cypripedium formosanum.Methods The endogenous ABA profile of seeds was measured by enzyme-linked immunosorbent assay (ELISA). Temporal and spatial distributions of ABA in developing seeds were visualized by immunohistochemical staining with monoclonal ABA antibodies. Fluoridone was applied to test the causal relationship between ABA content and seed germinability.Key Results ABA content was low at the proembryo stage, then increased rapidly from 120 to 150 days after pollination (DAP), accompanied by a progressive decrease in water content and seed germination. Immunofluorescence signals indicated an increase in fluorescence over time from the proembryo stage to seed maturation. From immunogold labelling, gold particles could be seen within the cytoplasm of embryo-proper cells during the early stages of seed development. As seeds approached maturity, increased localization of gold particles was observed in the periplasmic space, the plasmalemma between embryo-proper cells, the surface wall of the embryo proper, and the inner walls of inner seed-coat cells. At maturity, gold particles were found mainly in the apoplast, such as the surface wall of the embryo proper, and the shrivelled inner and outer seed coats. Injection of fluoridone into capsules resulted in enhanced germination of mature seeds.Conclusions The results indicate that ABA is the key inhibitor of germination in C. formosanum. The distinct accumulation pattern of ABA suggests that it is synthesized in the cytosol of embryo cells during the early stages of seed development, and then exported to the apoplastic region of the cells for subsequent regulatory processes as seeds approach maturity.     

Molecular characterization and expression analysis of a gene encoding an isoamylase-type starch debranching enzyme 3 (ISA3) in grain amaranths

A cDNA clone from amaranth perisperm that encodes an isoamylase (ISA)-type starch debranching enzyme 3 was isolated and analyzed for the first time. The cDNA consisted of 2,715 bp with a single open reading frame of 2,346 bp, encoding a protein of 781 amino acid residues. The deduced amino acid sequence of CrISA3 shared 63–71 % identity with those of other plant ISA3s. We also investigated the genetic diversity of ISA3 in three species of grain amaranth. A comparison of their ISA3 coding sequences revealed an extremely high level of conservation and only 11 single nucleotide polymorphisms were detected. The expression of the CrISA3 gene in amaranth developmental seeds and several tissues was investigated by qRT-PCR analysis. The results showed that CrISA3 was rapidly expressed at the early stage during seed maturation. It was also expressed in non-storage tissues (leaf, petiole, stem, and root) as well as in storage tissue. This observation demonstrates that CrISA3 may play an important role in perisperm starch accumulation at the early developmental stages. In addition, our results indicate that CrISA3 plays important roles in the synthesis of storage and transitory starches. The characterization of the CrISA3 gene will contribute to further studies on starch biosynthesis in Amaranthus.     

EST-based in silico identification and in vitro test of antimicrobial peptides in Brassica napus

Background

Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression.

Results

In total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains.

Conclusion

The study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1849-x) contains supplementary material, which is available to authorized users.     

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.     

Efficacy of karanjin and phorbol ester fraction against termites (Odontotermes obesus)

The non-edible oil seeds of Jatropha curcas (physic nut) and Pongamia pinnata (karanja) contain some toxic components (phorbol esters in J. curcas and karanjin in P. pinnata), which may be used as biopesticides. In this study, the active components of J. curcas and P. pinnata oil were extracted and their efficacy against the termites Odontotermes obesus (Rambur), was tested. The phorbol ester fraction of J. curcas and karanjin of P. pinnata oil were found to be effective against termites. A mortality rate of 100% was achieved in 6 h with karanjin and in 12 h with phorbol ester fraction. The LC₅₀ levels of karanjin and phorbol esters fractions were 0.038 and 0.071 g ml⁻¹, respectively, after 24 h at a 95% (0.05) confidence limit.