Proteomic Analysis of Desiccation Tolerance and Its Re-Establishment in Different Embryo Axis Tissues of Germinated Pea Seeds.

The model of loss and re-establishment of desiccation tolerance (DT) in germinated seeds has been well developed to explore the mechanisms associated with DT, but little attention has been paid to the tissue variation in this model. Herein, we investigated DT in different embryo axis tissues of germinated pea seeds and its re-establishment by poly(ethylene glycol) (PEG) treatment and then employed an iTRAQ-based proteomic method to explore the underlying mechanisms. DT varied among the four embryo axis parts of germinated seeds: epicotyl > hypocotyl-E (hypocotyl part attached to the epicotyl) > hypocotyl-R (hypocotyl part attached to the radicle) > radicle. Meanwhile, PEG treatment of germinated seeds resulted in a differential extent of DT re-establishment in these tissues. Proteins involved in detoxification and stress response were enriched in desiccation-tolerant hypocotyls-E and epicotyls of germinated seeds, respectively. Upon rehydration, proteome change during dehydration was recovered in the hypocotyls-E but not in the radicles. PEG treatment of germinated seeds led to numerous changes in proteins, in abundance in desiccation-sensitive radicles and hypocotyls-R, of which many accumulated in the hypocotyls-E and epicotyls before the treatment. We hypothesized that accumulation of groups 1 and 5 LEA proteins and proteins related to detoxification, ABA, ethylene, and calcium signaling contributed mainly to the variation of DT in different tissues and its re-establishment.

Changes in the mitochondrial proteome of developing maize seed embryos.

Mitochondria are required for seed development, but little information is available about their function and role during this process. We isolated the mitochondria from developing maize (Zea mays L. cv. Nongda 108) embryos and investigated the mitochondrial membrane integrity and respiration as well as the mitochondrial proteome using two proteomic methods, the two-dimensional gel electrophoresis (2-DE) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH). Mitochondrial membrane integrity and respiration were maintained at a high level up to 21 days after pollination (DAP) and decreased thereafter, while total mitochondrial number, cytochrome c oxidase activity and respiration per embryo exhibited a bell-shaped change with peaks at 35-45 DAP. A total of 286 mitochondrial proteins changed in abundance during embryo development. During early stages of seed development (up to 21 DAP), proteins involved in energy production, basic metabolism, protein import and folding as well as removal of reactive oxygen species dominated, while during mid or late stages (35-70 DAP), some stress- and detoxification-related proteins increased in abundance. Our study, for the first time, depicted a relatively comprehensive map of energy production by mitochondria during embryo development. The results revealed that mitochondria were very active during the early stages of maize embryo development, while at the late stages of development, the mitochondria became more quiescent, but well-protected, presumably to ensure that the embryo passes through maturation, drying and long-term storage. These results advance our understanding of seed development at the organelle level.

Proteome analysis reveals an energy-dependent central process for Populus×canadensis seed germination.

Poplar (Populus×canadensis) seeds rapidly germinated in darkness at 10, 15, and 20°C and reached 50% seed germination after about 22, 4.5, and 3.5h, respectively. Germination of poplar seeds was markedly inhibited by abscisic acid (ABA) at 50µM and cycloheximide (CHX) at 100µM, and these inhibitive roles were temperature-dependent. In the present study, mature poplar seeds were used to investigate the differentially changed proteome of seeds germinating in water, ABA, and CHX. A total of 130 protein spots showed a significant change (1.5-fold increase/decrease, P<0.05) in abundance, and 101 protein spots were successfully identified. Most of the proteins were associated with cell defense and rescue (21%), storage proteins (21%), protein synthesis and destination (20%), metabolism (16%), and energy (14%). The germination of poplar seeds is closely related with the increase in those proteins involved in amino acid and lipid metabolism, the tricarboxylic acid cycle and pentose phosphate pathway, protein synthesis and destination, cell defense and rescue, and degradation of storage proteins. ABA and CHX inhibit the germination of poplar seeds by decreasing the protein abundance associated with protein proteolysis, protein folding, and storage proteins. We conclude that poplar seed germination is an energy-dependent active process, and is accompanied by increasing amino acid activation, protein synthesis and destination, as well as cell defense and rescue, and degradation of storage proteins.

Proteome changes associated with dormancy release of Dongxiang wild rice seeds.

Seed dormancy provides optimum timing for seed germination and subsequent seedling growth, but the mechanism of seed dormancy is still poorly understood. Here, we used Dongxiang wild rice (DXWR) seeds to investigate the dormancy behavior and the differentially changed proteome in embryo and endosperm during dormancy release. DXWR seed dormancy was caused by interaction of embryo and its surrounding structure, and was an intermediate physiological dormancy. During seed dormancy release, a total of 109 and 97 protein spots showed significant change in abundance and were successfully identified in embryo and endosperm, respectively. As a result of dormancy release, the abundance of nine proteins involved in storage protein, cell defense and rescue and energy changed in the same way in both embryo and endosperm, while 67 and 49 protein spots changed differentially in embryo and endosperm, respectively. Dormancy release of DXWR seeds was closely associated with degradation of storage proteins in both embryo and endosperm. At the same time, the abundance of proteins involved in metabolism, glycolysis and TCA cycle, cell growth and division, protein synthesis and destination and signal transduction increased in embryos while staying constant or decreasing in endosperms.

Proteomic Analysis Reveals Different Involvement of Embryo and Endosperm Proteins during Aging of Yliangyou 2 Hybrid Rice Seeds.

Seed aging is a process that results in a delayed germination, a decreased germination percentage, and finally a total loss of seed viability. However, the mechanism of seed aging is poorly understood. In the present study, Yliangyou 2 hybrid rice (Oryza sativa L.) seeds were artificially aged at 100% relative humidity and 40°C, and the effect of artificial aging on germination, germination time course and the change in protein profiles of embryo and endosperm was studied to understand the molecular mechanism behind seed aging. With an increasing duration of artificial aging, the germination percentage and germination rate of hybrid rice seeds decreased. By comparing the protein profiles from the seeds aged for 0, 10 and 25 days, a total of 91 and 100 protein spots were found to show a significant change of more than 2-fold (P < 0.05) in abundance, and 71 and 79 protein spots were identified, in embryos and endosperms, respectively. The great majority of these proteins increased in abundance in embryos (95%) and decreased in abundance in endosperms (99%). In embryos, most of the identified proteins were associated with energy (30%), with cell defense and rescue (28%), and with storage protein (18%). In endosperms, most of the identified proteins were involved in metabolism (37%), in energy (27%), and in protein synthesis and destination (11%). The most marked change was the increased abundance of many glycolytic enzymes together with the two fermentation enzymes pyruvate decarboxylase and alcohol dehydrogenase in the embryos during aging. We hypothesize that the decreased viability of hybrid rice seeds during artificial aging is caused by the development of hypoxic conditions in the embryos followed by ethanol accumulation.

Proteomics analysis reveals distinct involvement of embryo and endosperm proteins during seed germination in dormant and non-dormant rice seeds.

Seed germination is a complex trait which is influenced by many genetic, endogenous and environmental factors, but the key event(s) associated with seed germination are still poorly understood. In present study, the non-dormant cultivated rice Yannong S and the dormant Dongxiang wild rice seeds were used as experimental materials, we comparatively investigated the water uptake, germination time course, and the differential proteome of the effect of embryo and endosperm on germination of these two types of seeds. A total of 231 and 180 protein spots in embryo and endosperm, respectively, showed a significant change in abundance during germination. We observed that the important proteins associated with seed germination included those involved in metabolism, energy production, protein synthesis and destination, storage protein, cell growth and division, signal transduction, cell defense and rescue. The contribution of embryo and endosperm to seed germination is different. In embryo, the proteins involved in amino acid activation, sucrose cleavage, glycolysis, fermentation and protein synthesis increased; in endosperm, the proteins involved in sucrose cleavage and glycolysis decreased, and those with ATP and CoQ synthesis and proteolysis increased. Our results provide some new knowledge to understand further the mechanism of seed germination.

Identification of embryo proteins associated with seed germination and seedling establishment in germinating rice seeds.

Seed germination is a critical phase in the plant life cycle, but the mechanism of seed germination is still poorly understood. In the present study, rice (Oryza sativa L. cv. Peiai 64S) seeds were sampled individually when they reached different germination stages, quiescent, germinated sensu stricto, germinated completely and seedling, and were used to study the changes in the embryo proteome. A total of 88 protein spots showed a significant change in abundance during germination in water, and the results showed an activation of metabolic processes. Cell division, cell wall synthesis, and secondary metabolism were activated at late seed germination and during preparation for subsequent seedling establishment. Cycloheximide (CHX) at 70µM inhibited seedling establishment without an apparent negative effect on seed germination, while CHX at 500µM completely blocked seed germination. We used this observation to identify the potentially important proteins involved in seed germination (coleoptile protrusion) and seedling establishment (coleoptile and radicle protrusion). Twenty-six protein spots, mainly associated with sugar/polysaccharide metabolism and energy production, showed a significant difference in abundance during seed germination. Forty-nine protein spots, mainly involved in cell wall biosynthesis, proteolysis as well as cell defense and rescue, were required for seedling establishment. The results help improve our understanding of the key events (proteins) involved in germination and seedling development.

A proteomic analysis of seeds from Bt-transgenic Brassica napus and hybrids with wild B. juncea.

Transgene insertions might have unintended side effects on the transgenic host, both crop and hybrids with wild relatives that harbor transgenes. We employed proteomic approaches to assess protein abundance changes in seeds from Bt-transgenic oilseed rape (Brassica napus) and its hybrids with wild mustard (B. juncea). A total of 24, 15 and 34 protein spots matching to 23, 13 and 31 unique genes were identified that changed at least 1.5 fold (p < 0.05, Student's t-test) in abundance between transgenic (tBN) and non-transgenic (BN) oilseed rape, between hybrids of B. juncea (BJ) × tBN (BJtBN) and BJ × BN (BJBN) and between BJBN and BJ, respectively. Eight proteins had higher abundance in tBN than in BN. None of these proteins was toxic or nutritionally harmful to human health, which is not surprising since the seeds are not known to produce toxic proteins. Protein spots varying in abundance between BJtBN and BJBN seeds were the same or homologous to those in the respective parents. None of the differentially-accumulated proteins between BJtBN and BJBN were identical to those between tBN and BN. Results indicated that unintended effects resulted from transgene flow fell within the range of natural variability of hybridization and those found in the native host proteomes.

De novo assembly and characterization of germinating lettuce seed transcriptome using Illumina paired-end sequencing.

At supraoptimal temperature, germination of lettuce (Lactuca sativa L.) seeds exhibits a typical germination thermoinhibition, which can be alleviated by sodium nitroprusside (SNP) in a nitric oxide-dependent manner. However, the molecular mechanism of seed germination thermoinhibition and its alleviation by SNP are poorly understood. In the present study, the lettuce seeds imbibed at optimal temperature in water or at supraoptimal temperature with or without 100 µM SNP for different periods of time were used as experimental materials, the total RNA was extracted and sequenced, we gained 147,271,347 raw reads using Illumina paired-end sequencing technique and assembled the transcriptome of germinating lettuce seeds. A total of 51,792 unigenes with a mean length of 849 nucleotides were obtained. Of these unigenes, a total of 29,542 unigenes were annotated by sequence similarity searching in four databases, NCBI non-redundant protein database, SwissProt protein database, euKaryotic Ortholog Groups database, and NCBI nucleotide database. Among the annotated unigenes, 22,276 unigenes were assigned to Gene Ontology database. When all the annotated unigenes were searched against the Kyoto Encyclopedia of Genes and Genomes Pathway database, a total of 8,810 unigenes were mapped to 5 main categories including 260 pathways. We first obtained a lot of unigenes encoding proteins involved in abscisic acid (ABA) signaling in lettuce, including 11 ABA receptors, 94 protein phosphatase 2Cs and 16 sucrose non-fermenting 1-related protein kinases. These results will help us to better understand the molecular mechanism of seed germination, thermoinhibition of seed germination and its alleviation by SNP.

Proteome Analysis of Poplar Seed Vigor.

Seed vigor is a complex property that determines the seed's potential for rapid uniform emergence and subsequent growth. However, the mechanism for change in seed vigor is poorly understood. The seeds of poplar (Populus × Canadensis Moench), which are short-lived, were stored at 30 °C and 75 ± 5% relative humidity for different periods of time (0-90 days) to obtain different vigor seeds (from 95 to 0% germination). With decreasing seed vigor, the temperature range of seed germination became narrower; the respiration rate of the seeds decreased markedly, while the relative electrolyte leakage increased markedly, both levelling off after 45 days. A total of 81 protein spots showed a significant change in abundance (≥ 1.5-fold, P < 0.05) when comparing the proteomes among seeds with different vigor. Of the identified 65 proteins, most belonged to the groups involved in metabolism (23%), protein synthesis and destination (22%), energy (18%), cell defense and rescue (17%), and storage protein (15%). These proteins accounted for 95% of all the identified proteins. During seed aging, 53 and 6 identified proteins consistently increased and decreased in abundance, respectively, and they were associated with metabolism (22%), protein synthesis and destination (22%), energy (19%), cell defense and rescue (19%), storage proteins (15%), and cell growth and structure (3%). These data show that the decrease in seed vigor (aging) is an energy-dependent process, which requires protein synthesis and degradation as well as cellular defense and rescue.

Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P<0.05) in abundance in at least one germination treatment. Nineteen proteins increasing and one protein decreasing in abundance during germination had higher abundance in germinated 15°C, 15°C after imbibition at 25°C for 48 h, and 25°C in KNO3 seeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition.

Proteomics of seed development, desiccation tolerance, germination and vigor.

Proteomics, the large-scale study of the total complement of proteins in a given sample, has been applied to all aspects of seed biology mainly using model species such as Arabidopsis or important agricultural crops such as corn and rice. Proteins extracted from the sample have typically been separated and quantified by 2-dimensional polyacrylamide gel electrophoresis followed by liquid chromatography and mass spectrometry to identify the proteins in the gel spots. In this way, qualitative and quantitative changes in the proteome during seed development, desiccation tolerance, germination, dormancy release, vigor alteration and responses to environmental factors have all been studied. Many proteins or biological processes potentially important for each seed process have been highlighted by these studies, which greatly expands our knowledge of seed biology. Proteins that have been identified to be particularly important for at least two of the seed processes are involved in detoxification of reactive oxygen species, the cytoskeleton, glycolysis, protein biosynthesis, post-translational modifications, methionine metabolism, and late embryogenesis-abundant (LEA) proteins. It will be useful for molecular biologists and molecular plant breeders to identify and study genes encoding particularly interesting target proteins with the aim to improve the yield, stress tolerance or other critical properties of our crop species.

A proteomic analysis of rice seed germination as affected by high temperature and ABA treatment.

Seed germination is a critical phase in the plant life cycle, but the specific events associated with seed germination are still not fully understood. In this study, we used two-dimensional gel electrophoresis followed by mass spectrometry to investigate the changes in the proteome during imbibition of Oryza sativa seeds at optimal temperature with or without abscisic acid (ABA) and high temperature (germination thermoinhibition) to further identify and quantify key proteins required for seed germination. A total of 121 protein spots showed a significant change in abundance (1.5-fold increase/decrease) during germination under all conditions. Among these proteins, we found seven proteins specifically associated with seed germination including glycosyl hydrolases family 38 protein, granule-bound starch synthase 1, Os03g0842900 (putative steroleosin-B), N-carbamoylputrescine amidase, spermidine synthase 1, tubulin α-1 chain and glutelin type-A; and a total of 20 imbibition response proteins involved in energy metabolism, cell growth, cell defense and storage proteins. High temperature inhibited seed germination by decreasing the abundance of proteins involved in methionine metabolism, amino acid biosynthesis, energy metabolism, reserve degradation, protein folding and stress responses. ABA treatment inhibited germination and decreased the abundance of proteins associated with methionine metabolism, energy production and cell division. Our results show that changes in many biological processes including energy metabolism, protein synthesis and cell defense and rescue occurred as a result of all treatments, while enzymes involved in methionine metabolism and weakening of cell wall specifically accumulated when the seeds germinated at the optimal temperature.

Proteomic comparison between maturation drying and prematurely imposed drying of Zea mays seeds reveals a potential role of maturation drying in preparing proteins for seed germination, seedling vigor, and pathogen resistance.

We have studied the role(s) of maturation drying in the acquisition of germinability, seedling vigor and pathogen resistance by comparing the proteome changes in maize embryo and endosperm during mature and prematurely imposed drying. Prematurely imposed dried seeds at 40 days after pollination (DAP) germinated almost as well as mature seeds (at 65 DAP), but their seedling growth was slower and they were seriously infected by fungi. A total of 80 and 114 proteins were identified to change at least two-fold (p < 0.05) in abundance during maturation drying in embryo and endosperm, respectively. Fewer proteins (48 and 59 in embryo and endosperm, respectively) changed in abundance during prematurely imposed drying. A number of proteins, 33 and 38 in embryo and endosperm, respectively, changed similarly in abundance during both maturation and prematurely imposed drying. Storage proteins were abundant in this group and may contribute to the acquisition of seed germinability. However, a relatively large number of proteins changed in the embryo (47 spots) and endosperm (76 spots) specifically during maturation drying. Among these proteins, storage proteins in the embryo and defense proteins in the endosperm may be particularly important for seedling vigor and resistance to fungal infection, respectively.

Development of a threshold model to predict germination of Populus tomentosa seeds after harvest and storage under ambient condition.

Effects of temperature, storage time and their combination on germination of aspen (Populus tomentosa) seeds were investigated. Aspen seeds were germinated at 5 to 30°C at 5°C intervals after storage for a period of time under 28°C and 75% relative humidity. The effect of temperature on aspen seed germination could not be effectively described by the thermal time (TT) model, which underestimated the germination rate at 5°C and poorly predicted the time courses of germination at 10, 20, 25 and 30°C. A modified TT model (MTT) which assumed a two-phased linear relationship between germination rate and temperature was more accurate in predicting the germination rate and percentage and had a higher likelihood of being correct than the TT model. The maximum lifetime threshold (MLT) model accurately described the effect of storage time on seed germination across all the germination temperatures. An aging thermal time (ATT) model combining both the TT and MLT models was developed to describe the effect of both temperature and storage time on seed germination. When the ATT model was applied to germination data across all the temperatures and storage times, it produced a relatively poor fit. Adjusting the ATT model to separately fit germination data at low and high temperatures in the suboptimal range increased the models accuracy for predicting seed germination. Both the MLT and ATT models indicate that germination of aspen seeds have distinct physiological responses to temperature within a suboptimal range.

Proteomic analysis of embryonic axis of Pisum sativum seeds during germination and identification of proteins associated with loss of desiccation tolerance.

Seed germination is an important stage in life cycle of higher plants. The germination processes and its associated loss of desiccation tolerance, however, are still poorly understood. In present study, pea seeds were used to study changes in embryonic axis proteome during germination by 2-DE and mass spectrometry. We identified a total of 139 protein spots showing a significant (>2-fold) change during germination. The results show that seed germination is not only the activation of a series of metabolic processes, but also involves reorganization of cellular structure and activation of protective systems. To uncouple the physiological processes of germination and its associated loss of desiccation tolerance, we used the fact that pea seeds have different desiccation tolerance when imbibed in water, CaCl(2) and methylviologen at the same germination stage. We compared the proteome amongst these seeds to identify the candidate proteins associated with the loss of desiccation tolerance and found a total of seven proteins - tubulin alpha-1 chain, seed biotin-containing protein SBP65, P54 protein, vicilin, vicilin-like antimicrobial peptides 2-3, convicilin and TCP-1/cpn60 chaperonin family protein. The metabolic function of these proteins indicates that seed desiccation tolerance is related to pathogen defense, protein conformation conservation and cell structure stabilization.

The role of recovery of mitochondrial structure and function in desiccation tolerance of pea seeds.

Mitochondrial repair is of fundamental importance for seed germination. When mature orthodox seeds are imbibed and germinated, they lose their desiccation tolerance in parallel. To gain a better understanding of this process, we studied the recovery of mitochondrial structure and function in pea (Pisum sativum cv. Jizhuang) seeds with different tolerance to desiccation. Mitochondria were isolated and purified from the embryo axes of control and imbibed-dehydrated pea seeds after (re-)imbibition for various times. Recovery of mitochondrial structure and function occurred both in control and imbibed-dehydrated seed embryo axes, but at different rates and to different maximum levels. The integrity of the outer mitochondrial membrane reached 96% in all treatments. However, only the seeds imbibed for 12 h and then dehydrated recovered the integrity of the inner mitochondrial membrane (IMM) and State 3 (respiratory state in which substrate and ADP are present) respiration (with NADH and succinate as substrate) to the control level after re-imbibition. With increasing imbibition time, the degree to which each parameter recovered decreased in parallel with the decrease in desiccation tolerance. The tolerance of imbibed seeds to desiccation increased and decreased when imbibed in CaCl(2) and methylviologen solution, respectively, and the recovery of the IMM integrity similarly improved and weakened in these two treatments, respectively. Survival of seeds after imbibition-dehydration linearly increased with the increase in ability to recover the integrity of IMM and State 3 respiration, which indicates that recovery of mitochondrial structure and function during germination has an important role in seed desiccation tolerance.

Proteomics of desiccation tolerance during development and germination of maize embryos.

Maize seeds were used to identify the key embryo proteins involved in desiccation tolerance during development and germination. Immature maize embryos (28N) during development and mature embryos imbibed for 72 h (72HN) are desiccation sensitive. Mature maize embryos (52N) during development are desiccation tolerant. Thiobarbituric acid reactive substance and hydrogen peroxide contents decreased and increased with acquisition and loss of desiccation tolerance, respectively. A total of 111 protein spots changed significantly (1.5 fold increase/decrease) in desiccation-tolerant and -sensitive embryos before (28N, 52N and 72HN) and after (28D, 52D and 72HD) dehydration. Nine pre-dominantly proteins, 17.4 kDa Class I heat shock protein 3, late embryogenesis abundant protein EMB564, outer membrane protein, globulin 2, TPA:putative cystatin, NBS-LRR resistance-like protein RGC456, stress responsive protein, major allergen Bet v 1.01C and proteasome subunit alpha type 1, accumulated during embryo maturation, decreased during germination and increased in desiccation-tolerant embryos during desiccation. Two proteins, Rhd6-like 2 and low-molecular-weight heat shock protein precursor, showed the inverse pattern. We infer that these eleven proteins are involved in seed desiccation tolerance. We conclude that desiccation-tolerant embryos make more economical use of their resources to accumulate protective molecules and antioxidant systems to deal with maturation drying and desiccation treatment.

Identification and expression analysis of two small heat shock protein cDNAs from developing seeds of biodiesel feedstock plant Jatropha curcas.

Plant small heat shock proteins (sHSPs) are known to be important for environmental stress tolerance and involved in various developmental processes. In this study, two full-length cDNAs encoding sHSPs, designated JcHSP-1 and JcHSP-2, were identified and characterized from developing seeds of a promising biodiesel feedstock plant Jatropha curcas by expressed sequence tag (EST) sequencing of embryo cDNA libraries and rapid amplification of cDNA ends (RACE). JcHSP-1 and JcHSP-2 contained open-reading frames encoding sHSPs of 219 and 157 amino acids, with predicted molecular weights of 24.42kDa and 18.02kDa, respectively. Sequence alignment indicated that both JcHSP-1 and JcHSP-2 shared high similarity with other plant sHSPs. Real-time quantitative RT-PCR analysis showed that the transcriptional level of both JcHSP-1 and JcHSP-2 increased along with natural dehydration process during seed development. A sharp increase of JcHSP-2 transcripts occurred in response to water content dropping from 42% in mature seeds to 12% in dry seeds. Western blot analysis revealed that the accumulation profile of two cross-reacting proteins, whose molecular weight corresponding to the calculated size of JcHSP-1 and JcHSP-2, respectively, was well consistent with the mRNA expression pattern of JcHSP-1 and JcHSP-2 in jatropha seeds during maturation and natural dehydration. These results indicated that both JcHSPs might play an important role in cell protection and seed development during maturation of J. curcas seeds.

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.