Proteome Dynamics of the Developing Açaí Berry Pericarp (Euterpe oleracea Mart.).

Quantitative proteome analysis of four developmental stages of pericarp tissues of the açaí berry (Euterpe oleracea Mart.) was performed by the isobaric labeling of peptides with iTRAQ 4-plex, hydrophilic interaction liquid chromatography pre-fractionation of labeled peptides, and high-performance mass spectrometry analysis. This analysis resulted in the identification of 4286 proteins, of which 476 presented differential abundance between the stages. The differential abundance of these proteins was seen to be coordinated with the metabolic demands during cell division, lignification, and cell expansion at developmental stages 1 and 2 as well as phenolic acid accumulation and metabolic changes in the fruit maturation at developmental stages 3 and 4. The distinct accumulation of anthocyanins observed in the pericarp at developmental stage 4 was correlated with the increase in abundance of some key biosynthetic enzymes, such as leucoanthocyanidin dioxygenase, anthocyanidin O-3-glycosyltransferase, and UDP-glycosyltransferase. Here, evidence is also provided for the presence in the açaí berry of secondary metabolites not previously described in açaí, such as pterostilbene, matairesinol, and furaneol. Together, these results will pave the way for studies aimed at the genetic improvement of the nutritional properties of this important fruit crop.

Seed development of Jatropha curcas L. (Euphorbiaceae): integrating anatomical, ultrastructural and molecular studies.

KEY MESSAGE: This work provides a detailed histological analysis of the development of Jatropha curcas seeds, together with an assessment of the role of programmed cell death in this process. Seeds of Jatropha curcas are a potential source of raw material for the production of biodiesel, but very little is known about how the architecture of the seeds is shaped by the coordinated development of the embryo, endosperm and maternal tissues, namely integuments and nucellus. This study used standard anatomical and ultrastructural techniques to evaluate seed development and programmed cell death (PCD) in the inner integument was monitored by qPCR. In these studies, we found that the embryo sac formation is of the Polygonum type. We also found that embryogenesis is a slow process and the embryo is nourished by the suspensor at earlier stages and by nutrients remobilized from the lysis of the inner integument at later stages. Two types of programmed cell death contribute to the differentiation of the inner integument that begins at early stages of seed development. In addition, the mature embryo presents features of adaptation to dry environments such as the presence of four seminal roots, water absorbing stomata in the root zone and already differentiated protoxylem elements. The findings in this study fill in gaps related to the ontogeny of J. curcas seed development and provide novel insights regarding the types of PCD occurring in the inner integument.

Deep proteome analysis of gerontoplasts from the inner integument of developing seeds of Jatropha curcas.

UNLABELLED: The inner integument of Jatropha curcas seeds is a non-photosynthetic tissue that acts primarily as a conduit for the delivery of nutrients to the embryo and endosperm. In this study we performed a histological and transmission electron microscopy analysis of the inner integument in stages prior to fertilization to 25days after pollination, to establish the structural changes associated with the plastid to gerontoplast transition. This study showed that plastids are subjected to progressive changes, which include the dismantling of the internal membrane system, matrix degradation and the formation of stromule-derived vesicles. A proteome analysis of gerontoplasts isolated from the inner integument at 25days after pollination, resulted in the identification of 1923 proteins, which were involved in a myriad of metabolic functions, such as synthesis of amino acids and fatty acids. Among the identified proteins, were also a number of hydrolases (peptidases, lipases and carbohydrases), which presumably are involved in the ordered dismantling of this organelle to provide additional sources of nutrients for the growing embryo and endosperm. The dataset we provide here may provide a foundation for the study of the proteome changes associated with the plastid to gerontoplast transition in non-photosynthetic tissues. SIGNIFICANCE: We describe ultrastructural features of gerontoplasts isolated from the inner integument of developing seeds of Jatropha curcas, together with a deep proteome analysis of these gerontoplasts. This article explores a new aspect of the biology of plastids, namely the ultrastructural and proteome changes associated with the transition plastid to gerontoplast in a non-photosynthetic tissue.

Time-course proteome analysis of developing extrafloral nectaries of Ricinus communis.

Floral and extrafloral nectaries are unique organs that secrete energy rich chemical components, but their contribution for nectar production is largely unknown. Here, we present the first comparative proteome dataset of four developmental stages of the extrafloral nectaries from castor plant (Ricinus communis), an important biofuel crop. Respectively, from stage I-IV, we identified 626, 613, 449 and 356 proteins, respectively, summing up 882 nonredundant proteins. Surprisingly, we identified two isoforms of the potent toxin ricin, indicating that ricin expression is not limited to seeds, but it may serve a general defense purpose for the castor plant. To date, this is the most complete dataset of proteins either from floral or extrafloral nectaries, thus contributing to lay the foundations for investigations on their ecological and evolutionary importance.

Proteomic Analysis of the Endosperm Ontogeny of Jatropha curcas L. Seeds.

Seeds of Jatropha curcas L. represent a potential source of raw material for the production of biodiesel. However, this use is hampered by the lack of basic information on the biosynthetic pathways associated with synthesis of toxic diterpenes, fatty acids, and triacylglycerols, as well as the pattern of deposition of storage proteins during seed development. In this study, we performed an in-depth proteome analysis of the endosperm isolated from five developmental stages which resulted in the identification of 1517, 1256, 1033, 752, and 307 proteins, respectively, summing up 1760 different proteins. Proteins with similar label free quantitation expression pattern were grouped into five clusters. The biological significance of these identifications is discussed with special focus on the analysis of seed storage proteins, proteins involved in the metabolism of fatty acids, carbohydrates, toxic components and proteolytic processing. Although several enzymes belonging to the biosynthesis of diterpenoid precursors were identified, we were unable to find any terpene synthase/cyclase, indicating that the synthesis of phorbol esters, the main toxic diterpenes, does not occur in seeds. The strategy used enabled us to provide a first in depth proteome analysis of the developing endosperm of this biodiesel plant, providing an important glimpse into the enzymatic machinery devoted to the production of C and N sources to sustain seed development.

Proteome analysis of the inner integument from developing Jatropha curcas L. seeds.

In this study, we performed a systematic proteomic analysis of the inner integument from developing seeds of Jatropha curcas and further explored the protein machinery responsible for generating the carbon and nitrogen sources to feed the growing embryo and endosperm. The inner integument of developing seeds was dissected into two sections called distal and proximal, and proteins were extracted from these sections and from the whole integument and analyzed using an EASY-nanoLC system coupled to an ESI-LTQ-Orbitrap Velos mass spectrometer. We identified 1526, 1192, and 1062 proteins from the proximal, distal, and whole inner integuments, respectively. The identifications include those of peptidases and other hydrolytic enzymes that play a key role in developmental programmed cell death and proteins associated with the cell-wall architecture and modification. Because many of these proteins are differentially expressed within the integument cell layers, these findings suggest that the cells mobilize an array of hydrolases to produce carbon and nitrogen sources from proteins, carbohydrates, and lipids available within the cells. Not least, the identification of several classes of seed storage proteins in the inner integument provides additional evidence of the role of the seed coat as a transient source of reserves for the growing embryo and endosperm.

Differential expression of cysteine peptidase genes in the inner integument and endosperm of developing seeds of Jatropha curcas L. (Euphorbiaceae).

In several plant tissues, programmed cell death (PCD) is mediated by the combined action of cysteine peptidases, namely KDEL-tailed cysteine peptidases (KDEL-CysEP) and vacuolar processing enzymes (VPE). Here, we performed a search of the draft genome of Jatropha curcas L. (Euphorbiaceae) and identified 2 genes for KDEL-CysEP (Jc-CysEP1 and Jc-CysEP2) and 3 genes for VPE (Jc-ßVPE, Jc-γVPE and Jc-δVPE) and determined the expression patterns of these genes by RT-qPCR in integument and cellular endosperm of seeds collected at seven different developmental stages. We were able to demonstrate that the expression of Jc-CysEP1, Jc-CysEP2, Jc-ßVPE and Jc-γVPE proceeded rapidly from Stage IV, with Jc-CysEP2 displaying the highest relative expression; expression of Jc-δVPE could not be detected in any of the tissues/developmental stages analyzed. Additionally, we showed that the expression pattern of these peptidases correlates with anatomical changes in integument and cellular endosperm, thus suggesting a role for both classes of peptidases in PCD and in protein processing, both of which occur simultaneously in each of these tissues.

Proteome analysis of plastids from developing seeds of Jatropha curcas L.

In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled inline to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 proteins were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in PPDB, SUBA, or PlProt databases while homologues for 13 proteins were not found in any of the three plastid proteins databases but were marked as plastidial by at least one of the three prediction programs used. Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in the plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, our study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level.

Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor.

The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-ß-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, α-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor.

Proteomic profile of the nucellus of castor bean (Ricinus communis L.) seeds during development.

In this study, we performed a proteomic analysis of nucellus from two developmental stages of Ricinus communis seeds by a GeLC-MS/MS approach, using of a high resolution orbitrap mass spectrometer, which resulted in the identification of a total of 766 proteins that were grouped into 553 protein groups. The distribution of the identified proteins in stages III and IV into different Gene Ontology categories was similar, with a remarkable abundance of proteins associated with the protein synthesis machinery of cells, as well as several classes of proteins involved in protein degradation, particularly of peptidases associated with programmed cell death. Consistent with the role of the nucellus in mediating nutrient transfer from maternal tissues to the endosperm and embryo, a significant proportion of the identified proteins are related to amino acid metabolism, but none of the identified proteins are known to have a role as storage proteins. Moreover for the first time, ricin isoforms were identified in tissues other than seed endosperm. Results are discussed in the context of the spatial and temporal distribution of the identified proteins within the nucellar cell layers.