Cellular Plasticity in Response to Suppression of Storage Proteins in the Brassica napus Embryo.

The tradeoff between protein and oil storage in oilseed crops has been tested here in oilseed rape (Brassica napus) by analyzing the effect of suppressing key genes encoding protein storage products (napin and cruciferin). The phenotypic outcomes were assessed using NMR and mass spectrometry imaging, microscopy, transcriptomics, proteomics, metabolomics, lipidomics, immunological assays, and flux balance analysis. Surprisingly, the profile of storage products was only moderately changed in RNA interference transgenics. However, embryonic cells had undergone remarkable architectural rearrangements. The suppression of storage proteins led to the elaboration of membrane stacks enriched with oleosin (sixfold higher protein abundance) and novel endoplasmic reticulum morphology. Protein rebalancing and amino acid metabolism were focal points of the metabolic adjustments to maintain embryonic carbon/nitrogen homeostasis. Flux balance analysis indicated a rather minor additional demand for cofactors (ATP and NADPH). Thus, cellular plasticity in seeds protects against perturbations to its storage capabilities and, hence, contributes materially to homeostasis. This study provides mechanistic insights into the intriguing link between lipid and protein storage, which have implications for biotechnological strategies directed at improving oilseed crops.

Ectopic overexpression of a membrane-tethered transcription factor gene NAC60 from oilseed rape positively modulates programmed cell death and age-triggered leaf senescence.

Senescence is an integrative final stage of plant development that is governed by internal and external cues. The NAM, ATAF1/2, CUC2 (NAC) transcription factor (TF) family is specific to plants and membrane-tethered NAC TFs (MTTFs) constitute a unique and sophisticated mechanism in stress responses and development. However, the function of MTTFs in oilseed rape (Brassica napus L.) remains unknown. Here, we report that BnaNAC60 is an MTTF associated with the endoplasmic reticulum (ER) membrane. Expression of BnaNAC60 was induced during the progression of leaf senescence. Translocation of BnaNAC60 into nuclei was induced by ER stress and oxidative stress treatments. It binds to the NTLBS motif, rather than the canonical NAC recognition site. Overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, induces significant reactive oxygen species (ROS) accumulation and hypersensitive response-like cell death in both tobacco (Nicotiana benthamiana) and oilseed rape protoplasts. Moreover, ectopic overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, in Arabidopsis also induces precocious leaf senescence. Furthermore, screening and expression profiling identified an array of functional genes that are significantly induced by BnaNAC60 expression. Further it was found that BnaNAC60 can activate the promoter activities of BnaNYC1, BnaRbohD, BnaBFN1, BnaZAT12, and multiple BnaVPEs in a dual-luciferase reporter assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative PCR assays revealed that BnaNAC60 directly binds to the promoter regions of these downstream target genes. To summarize, our data show that BnaNAC60 is an MTTF that modulates cell death, ROS accumulation, and leaf senescence.

Trehalose 6-Phosphate Positively Regulates Fatty Acid Synthesis by Stabilizing WRINKLED1.

WRINKLED1 (WRI1), the transcriptional activator of fatty acid synthesis, was recently identified as a target of KIN10, a catalytic α-subunit of the SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1). We tested the hypothesis that trehalose 6-phosphate (T6P), a signal of cellular sucrose status, can regulate fatty acid synthesis by inhibiting SnRK1. Incubation of Brassica napus suspension cells in medium containing T6P, or overexpression of the Escherichia coli T6P synthase, OtsA, in Nicotiana benthamiana, significantly increased T6P levels, WRI1 levels, and fatty acid synthesis rates. T6P directly bound to purified recombinant KIN10 with an equilibrium dissociation constant (K d) of 32 ± 6 µM based on microscale thermophoresis. GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1) bound to KIN10 (K d 19 ± 3 µM) and activated it by phosphorylation. In the presence of T6P, the GRIK1-KIN10 association was weakened by more than 3-fold (K d 68 ± 9.8 µM), which reduced both the phosphorylation of KIN10 and its activity. T6P-dependent inhibition of SnRK1 activity was reduced in extracts of individual Arabidopsis thaliana grik1 and grik2 mutants relative to the wild type, while SnRK1 activity in grik1 grik2 extracts was enhanced by T6P. These results indicate that the T6P sensitivity of SnRK1 in vivo is GRIK1/GRIK2 dependent. Based on our findings, we propose a mechanistic model that links sugar signaling and fatty acid homeostasis.

Organelle genome composition and candidate gene identification for Nsa cytoplasmic male sterility in Brassica napus.

BACKGROUND: Nsa cytoplasmic male sterility (CMS) is a novel alloplasmic male sterility system derived from somatic hybridization between Brassica napus and Sinapis arvensis. Identification of the CMS-associated gene is a prerequisite for a better understanding of the origin and molecular mechanism of this CMS. With the development of genome sequencing technology, organelle genomes of Nsa CMS line and its maintainer line were sequenced by pyro-sequencing technology, and comparative analysis of the organelle genomes was carried out to characterize the organelle genome composition of Nsa CMS as well as to identify the candidate Nsa CMS-associated genes. RESULTS: Nsa CMS mitochondrial genome showed a higher collinearity with that of S. arvensis than B. napus, indicating that Nsa CMS mitochondrial genome was mainly derived from S. arvensis. However, mitochondrial genome recombination of parental lines was clearly detected. In contrast, the chloroplast genome of Nsa CMS was highly collinear with its B. napus parent, without any evidence of recombination of the two parental chloroplast genomes or integration from S. arvensis. There were 16 open reading frames (ORFs) specifically existed in Nsa CMS mitochondrial genome, which could not be identified in the maintainer line. Among them, three ORFs (orf224, orf309, orf346) possessing chimeric and transmembrane structure are most likely to be the candidate CMS genes. Sequences of all three candidate CMS genes in Nsa CMS line were found to be 100% identical with those from S. arvensis mitochondrial genome. Phylogenetic and homologous analysis showed that all the mitochondrial genes were highly conserved during evolution. CONCLUSIONS: Nsa CMS contains a recombined mitochondrial genome of its two parental species with the majority form S. arvensis. Three candidate Nsa CMS genes were identified and proven to be derived from S. arvensis other than recombination of its two parental species. Further functional study of the candidate genes will help to identify the gene responsible for the CMS and the underlying molecular mechanism.

Induction of telomere-mediated chromosomal truncation and behavior of truncated chromosomes in Brassica napus.

Engineered minichromosomes could be stably inherited and serve as a platform for simultaneously transferring and stably expressing multiple genes. Chromosomal truncation mediated by repeats of telomeric sequences is a promising approach for the generation of minichromosomes. In the present work, direct repetitive sequences of Arabidopsis telomere were used to study telomere-mediated truncation of chromosomes in Brassica napus. Transgenes containing alien Arabidopsis telomere were successfully obtained, and Southern blotting and fluorescence in situ hybridization (FISH) results show that the transgenes resulted in successful chromosomal truncation in B. napus. In addition, truncated chromosomes were inherited at rates lower than that predicted by Mendelian rules. To determine the potential manipulations and applications of the engineered chromosomes, such as the stacking of multiple transgenes and the Cre/lox and FRT/FLP recombination systems, both amenable to genetic manipulations through site-specific recombination in somatic cells, were tested for their ability to undergo recombination in B. napus. These results demonstrate that alien Arabidopsis telomere is able to mediate chromosomal truncation in B. napus. This technology would be feasible for chromosomal engineering and for studies on chromosome structure and function in B. napus.

A Novel NAC-Type Transcription Factor, NAC87, from Oilseed Rape Modulates Reactive Oxygen Species Accumulation and Cell Death.

Reactive oxygen species (ROS) are thought to play a dual role in plants by functioning as signaling molecules and toxic by-products of aerobic metabolism. The hypersensitive response (HR) is a typical feature of immune responses in plants and also a type of programmed cell death (PCD). How these two processes are regulated in oilseed rape (Brassica napus L.) at the transcriptional level remains largely unknown. In this study, we report that an oilseed rape (Brassica napus L.) NAM-ATAF-CUC (NAC)-type transcription factor NAC87 modulates ROS and cell death accompanied by typical changes at the morphological and cellular levels. The BnaNAC87 gene was induced by multiple stress and hormone treatments and was highly expressed in senescent leaves by quantitative reverse transcription-PCR (qRT-PCR). BnaNAC87 is located in nuclei and has transcriptional activation activity. Expression of BnaNAC87 promoted significant ROS production, cell death as well as death of protoplasts, as indicated by histological staining. In addition, putative downstream target genes of NAC87 were identified through both qRT-PCR and dual luciferase reporter assays. We found that genes implicated in ROS generation (RbohB), cell death (VPE1a, ZEN1), leaf senescence (WRKY6, ZAT12) and defense (PR2, PR5 and HIN1) were significantly induced. Through an electrophoretic mobility shift assay (EMSA), we confirmed that BnaNAC87 directly binds to the NACRS-containing promoter fragments of ZEN1, ZAT12, HIN1 and PR5 genes. From these results, we conclude that oilseed rape NAC87 is a novel NAC transcription factor that acts as a positive regulator of ROS metabolism and cell death.

Redox regulation of a guard cell SNF1-related protein kinase in Brassica napus, an oilseed crop.

Kinase-mediated phosphorylation is a pivotal regulatory process in stomatal responses to stresses. Through a redox proteomics study, a sucrose non-fermenting 1-related protein kinase (SnRK2.4) was identified to be redox-regulated in Brassica napus guard cells upon abscisic acid treatment. There are six genes encoding SnRK2.4 paralogs in B. napus Here, we show that recombinant BnSnRK2.4-1C exhibited autophosphorylation activity and preferentially phosphorylated the N-terminal region of B. napus slow anion channel (BnSLAC1-NT) over generic substrates. The in vitro activity of BnSnRK2.4-1C requires the presence of manganese (Mn2+). Phosphorylation sites of autophosphorylated BnSnRK2.4-1C were mapped, including serine and threonine residues in the activation loop. In vitro BnSnRK2.4-1C autophosphorylation activity was inhibited by oxidants such as H2O2 and recovered by active thioredoxin isoforms, indicating redox regulation of BnSnRK2.4-1C. Thiol-specific isotope tagging followed by mass spectrometry analysis revealed specific cysteine residues responsive to oxidant treatments. The in vivo activity of BnSnRK2.4-1C is inhibited by 15 min of H2O2 treatment. Taken together, these data indicate that BnSnRK2.4-1C, an SnRK preferentially expressed in guard cells, is redox-regulated with potential roles in guard cell signal transduction.

Genetic effects and genotype × environment interactions govern seed oil content in Brassica napus L.

BACKGROUND: As seed oil content (OC) is a key measure of rapeseed quality, better understanding the genetic basis of OC would greatly facilitate the breeding of high-oil cultivars. Here, we investigated the components of genetic effects and genotype × environment interactions (GE) that govern OC using a full diallel set of nine parents, which represented a wide range of the Chinese rapeseed cultivars and pure lines with various OCs. RESULTS: Our results from an embryo-cytoplasm-maternal (GoCGm) model for diploid seeds showed that OC was primarily determined by genetic effects (VG) and GE (VGE), which together accounted for 86.19% of the phenotypic variance (VP). GE (VGE) alone accounted for 51.68% of the total genetic variance, indicating the importance of GE interaction for OC. Furthermore, maternal variance explained 75.03% of the total genetic variance, embryo and cytoplasmic effects accounted for 21.02% and 3.95%, respectively. We also found that the OC of F1 seeds was mainly determined by maternal effect and slightly affected by xenia. Thus, the OC of rapeseed was simultaneously affected by various genetic components, including maternal, embryo, cytoplasm, xenia and GE effects. In addition, general combining ability (GCA), specific combining ability (SCA), and maternal variance had significant influence on OC. The lines H2 and H1 were good general combiners, suggesting that they would be the best parental candidates for OC improvement. Crosses H3 × M2 and H1 × M3 exhibited significant SCA, suggesting their potentials in hybrid development. CONCLUSIONS: Our study thoroughly investigated and reliably quantified various genetic factors associated with OC of rapeseed by using a full diallel and backcross and reciprocal backcross. This findings lay a foundation for future genetic studies of OC and provide guidance for breeding of high-oil rapeseed cultivars.

Genome-wide identification and homeolog-specific expression analysis of the SnRK2 genes in Brassica napus guard cells.

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) proteins constitute a small plant-specific serine/threonine kinase family involved in abscisic acid (ABA) signaling and plant responses to biotic and abiotic stresses. Although SnRK2s have been well-studied in Arabidopsis thaliana, little is known about SnRK2s in Brassica napus. Here we identified 30 putative sequences encoding 10 SnRK2 proteins in the B. napus genome and the expression profiles of a subset of 14 SnRK2 genes in guard cells of B. napus. In agreement with its polyploid origin, B. napus maintains both homeologs from its diploid parents. The results of quantitative real-time PCR (qRT-PCR) and reanalysis of RNA-Seq data showed that certain BnSnRK2 genes were commonly expressed in leaf tissues in different varieties of B. napus. In particular, qRT-PCR results showed that 12 of the 14 BnSnRK2s responded to drought stress in leaves and in ABA-treated guard cells. Among them, BnSnRK2.4 and BnSnRK2.6 were of interest because of their robust responsiveness to ABA treatment and drought stress. Notably, BnSnRK2 genes exhibited up-regulation of different homeologs, particularly in response to abiotic stress. The homeolog expression bias in BnSnRK2 genes suggests that parental origin of genes might be responsible for efficient regulation of stress responses in polyploids. This work has laid a foundation for future functional characterization of the different BnSnKR2 homeologs in B. napus and its parents, especially their functions in guard cell signaling and stress responses.

Ectopic Expression of BnaC.CP20.1 Results in Premature Tapetal Programmed Cell Death in Arabidopsis.

Tapetal programmed cell death (PCD) is essential in pollen grain development, and cysteine proteases are ubiquitous enzymes participating in plant PCD. Although the major papain-like cysteine proteases (PLCPs) have been investigated, the exact functions of many PLCPs are still poorly understood in PCD. Here, we identified a PLCP gene, BnaC.CP20.1, which was closely related to XP_013596648.1 from Brassica oleracea. Quantitative real-time PCR analysis revealed that BnaC.CP20.1 expression was down-regulated in male-sterile lines in oilseed rape, suggesting a connection between this gene and male sterility. BnaC.CP20.1 is especially active in the tapetum and microspores in Brassica napus from the uninucleate stage until formation of mature pollen grains during anther development. On expression of BnaC.CP20.1 prior to the tetrad stage, BnA9::BnaC.CP20.1 transgenic lines in Arabidopsis thaliana showed a male-sterile phenotype with shortened siliques containing fewer or no seeds by self-crossing. Scanning electron microscopy indicated that the reticulate exine was defective in aborted microspores. Callose degradation was delayed and microspores were not released from the tetrad in a timely fashion. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay indicated that BnaC.CP20.1 ectopic expression led to premature tapetal PCD. Transmission electron microscopy analyses further demonstrated that the pollen abortion was due to the absence of tectum connections to the bacula in the transgenic anthers. These findings suggest that timely expression of BnaC.CP20.1 is necessary for tapetal degeneration and pollen wall formation.

BnPME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus.

BACKGROUND: Pectins are one of the main components of plant cell walls. They are secreted to the wall as highly methylesterified forms that can be de-esterified by pectin methylesterases (PMEs). The degree of methylesterification of pectins changes during development, PMEs are involved in the cell wall remodeling that occurs during diverse plant developmental processes. Nevertheless, the functional meaning of pectin-related wall remodeling in different cell types and processes remains unclear. In vivo, the microspore follows the gametophytic pathway and differentiates to form the pollen grain. In vitro, the microspore can be reprogrammed by stress treatments becoming a totipotent cell that starts to proliferate and follows the embryogenic pathway, a process known as microspore embryogenesis. RESULTS: To investigate if the change of developmental programme of the microspore towards embryogenesis involves changes in pectin esterification levels, which would cause the cell wall remodeling during the process, in the present study, dynamics of PME expression and degrees of pectin esterification have been analysed during microspore embryogenesis and compared with the gametophytic development, in Brassica napus. A multidisciplinary approach has been adopted including BnPME gene expression analysis by quantitative RT-PCR, fluorescence in situ hybridization, immuno-dot-blot and immunofluorescence with JIM5 and JIM7 antibodies to reveal low and highly-methylesterified pectins. The results showed that cell differentiation at advanced developmental stages involved induction of BnPME expression and pectin de-esterification, processes that were also detected in zygotic embryos, providing additional evidence that microspore embryogenesis mimics zygotic embryogenesis. By contrast, early microspore embryogenesis, totipotency and proliferation were associated with low expression of BnPME and high levels of esterified pectins. CONCLUSIONS: The results show that the change of developmental programme of the microspore involves changes in pectin esterification associated with proliferation and differentiation events, which may cause the cell wall remodeling during the process. The findings indicate pectin-related modifications in the cell wall during microspore embryogenesis, providing new insights into the role of pectin esterification and cell wall configuration in microspore totipotency, embryogenesis induction and progression.

Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox-sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard-cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis and isotope-coded affinity tagging. In total, 65 and 118 potential redox-responsive proteins were identified in ABA- and MeJA-treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of 'energy', 'stress and defense' and 'metabolism'. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA- and MeJA-treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non-fermenting 1-related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox-regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells.

Identification and characterization of plant-specific NAC gene family in canola (Brassica napus L.) reveal novel members involved in cell death.

NAC transcription factors are plant-specific and play important roles in plant development processes, response to biotic and abiotic cues and hormone signaling. However, to date, little is known about the NAC genes in canola (or oilseed rape, Brassica napus L.). In this study, a total of 60 NAC genes were identified from canola through a systematical analysis and mining of expressed sequence tags. Among these, the cDNA sequences of 41 NAC genes were successfully cloned. The translated protein sequences of canola NAC genes with the NAC genes from representative species were phylogenetically clustered into three major groups and multiple subgroups. The transcriptional activities of these BnaNAC proteins were assayed in yeast. In addition, by quantitative real-time RT-PCR, we further observed that some of these BnaNACs were regulated by different hormone stimuli or abiotic stresses. Interestingly, we successfully identified two novel BnaNACs, BnaNAC19 and BnaNAC82, which could elicit hypersensitive response-like cell death when expressed in Nicotiana benthamiana leaves, which was mediated by accumulation of reactive oxygen species. Overall, our work has laid a solid foundation for further characterization of this important NAC gene family in canola.

Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death.

MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of a series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death.

Verticillium infection triggers VASCULAR-RELATED NAC DOMAIN7-dependent de novo xylem formation and enhances drought tolerance in Arabidopsis.

The soilborne fungal plant pathogen Verticillium longisporum invades the roots of its Brassicaceae hosts and proliferates in the plant vascular system. Typical aboveground symptoms of Verticillium infection on Brassica napus and Arabidopsis thaliana are stunted growth, vein clearing, and leaf chloroses. Here, we provide evidence that vein clearing is caused by pathogen-induced transdifferentiation of chloroplast-containing bundle sheath cells to functional xylem elements. In addition, our findings suggest that reinitiation of cambial activity and transdifferentiation of xylem parenchyma cells results in xylem hyperplasia within the vasculature of Arabidopsis leaves, hypocotyls, and roots. The observed de novo xylem formation correlates with Verticillium-induced expression of the VASCULAR-RELATED NAC DOMAIN (VND) transcription factor gene VND7. Transgenic Arabidopsis plants expressing the chimeric repressor VND7-SRDX under control of a Verticillium infection-responsive promoter exhibit reduced de novo xylem formation. Interestingly, infected Arabidopsis wild-type plants show higher drought stress tolerance compared with noninfected plants, whereas this effect is attenuated by suppression of VND7 activity. Together, our results suggest that V. longisporum triggers a tissue-specific developmental plant program that compensates for compromised water transport and enhances the water storage capacity of infected Brassicaceae host plants. In conclusion, we provide evidence that this natural plant-fungus pathosystem has conditionally mutualistic features.

Brassica napus TT16 homologs with different genomic origins and expression levels encode proteins that regulate a broad range of endothelium-associated genes at the transcriptional level.

The transcription factor TRANSPARENT TESTA 16 (TT16) plays an important role in endothelial cell specification and proanthocyanidin (PA) accumulation. However, its precise regulatory function with regard to the expression of endothelial-associated genes in developing seeds, and especially in the PA-producing inner integument, remains largely unknown. Therefore, we endeavored to characterize four TT16 homologs from the allotetraploid oil crop species Brassica napus, and systematically explore their regulatory function in endothelial development. Our results indicated that all four BnTT16 genes were predominantly expressed in the early stages of seed development, but at distinct levels, and encoded functional proteins. Bntt16 RNA interference lines exhibited abnormal endothelial development and decreased PA content, while PA polymerization was not affected. In addition to the previously reported function of TT16 in the transcriptional regulation of anthocyanidin reductase (ANR) and dihydroflavonol reductase (TT3), we also determined that BnTT16 proteins played a significant role in the transcriptional regulation of five other genes involved in the PA biosynthetic pathway (P < 0.01). Moreover, we identified two genes involved in inner integument development that were strongly regulated by the BnTT16 proteins (TT2 and δ-vacuolar processing enzyme). These results will better our understanding of the precise role of TT16 in endothelial development in Brassicaceae species, and could potentially be used for the future improvement of oilseed crops.

Epigenetic changes accompany developmental programmed cell death in tapetum cells.

The tapetum, the nursing tissue inside anthers, undergoes cellular degradation by programmed cell death (PCD) during late stages of microspore-early pollen development. Despite the key function of tapetum, little is known about the molecular mechanisms regulating this cell death process in which profound nuclear and chromatin changes occur. Epigenetic features (DNA methylation and histone modifications) have been revealed as hallmarks that establish the functional status of chromatin domains, but no evidence on the epigenetic regulation of PCD has been reported. DNA methylation is accomplished by DNA methyltransferases, among which DNA methyl transferase 1 (MET1) constitutes one of the CG maintenance methyltransferase in plants, also showing de novo methyltransferase activity. In this work, the changes in epigenetic marks during the PCD of tapetal cells have been investigated by a multidisciplinary approach to reveal the dynamics of DNA methylation and the pattern of expression of MET1 in relation to the main cellular changes of this PCD process which have also been characterized in two species, Brassica napus and Nicotiana tabacum. The results showed that tapetum PCD progresses with the increase in global DNA methylation and MET1 expression, epigenetic changes that accompanied the reorganization of the nuclear architecture and a high chromatin condensation, activity of caspase 3-like proteases and Cyt c release. The reported data indicate a relationship between the PCD process and the DNA methylation dynamics and MET1 expression in tapetal cells, suggesting a possible new role for the epigenetic marks in the nuclear events occurring during this cell death process and providing new insights into the epigenetic control of plant PCD.

Canola (Brassica napus L.) NAC103 transcription factor gene is a novel player inducing reactive oxygen species accumulation and cell death in plants.

NAC transcription factors are plant-specific and play important roles in many processes including plant development, response to biotic and abiotic stresses and hormone signaling. So far, only a few NAC genes have been identified to mediate cell death. In this study, we identified a novel NAC gene from canola (Brassica napus L.), BnaNAC103 which induces reactive oxygen species (ROS) accumulation and cell death in Nicotianabenthamiana leaves. We found that BnaNAC103 responded to multiple signalings, including cold, salicylic acid (SA) and a fungal pathogen Sclerotinia sclerotiorum. BnaNAC103 is located in the nucleus. Expression of full-length BnaNAC103, but not either the N-terminal NAC domain or C-terminal regulatory domain, was identified to induce hypersensitive response (HR)-like cell death when expressed in N. benthamiana. The cell death triggered by BnaNAC103 is preceded by accumulation of ROS, with diaminobenzidine (DAB) staining supporting this. Moreover, quantification of ion leakage and malondialdehyde (MDA) of leaf discs indicates significant cell membrane breakage and lipid peroxidation induced by BnaNAC103 expression. Taken together, our work has identified a novel NAC transcription factor gene modulating ROS level and cell death in plants.

Structural changes in senescing oilseed rape leaves at tissue and subcellular levels monitored by nuclear magnetic resonance relaxometry through water status.

Nitrogen use efficiency is relatively low in oilseed rape (Brassica napus) due to weak nitrogen remobilization during leaf senescence. Monitoring the kinetics of water distribution associated with the reorganization of cell structures, therefore, would be valuable to improve the characterization of nutrient recycling in leaf tissues and the associated senescence processes. In this study, nuclear magnetic resonance (NMR) relaxometry was used to describe water distribution and status at the cellular level in different leaf ranks of well-watered plants. It was shown to be able to detect slight variations in the evolution of senescence. The NMR results were linked to physiological characterization of the leaves and to light and electron micrographs. A relationship between cell hydration and leaf senescence was revealed and associated with changes in the NMR signal. The relative intensities and the transverse relaxation times of the NMR signal components associated with vacuole water were positively correlated with senescence, describing water uptake and vacuole and cell enlargement. Moreover, the relative intensity of the NMR signal that we assigned to the chloroplast water decreased during the senescence process, in agreement with the decrease in relative chloroplast volume estimated from micrographs. The results are discussed on the basis of water flux occurring at the cellular level during senescence. One of the main applications of this study would be for plant phenotyping, especially for plants under environmental stress such as nitrogen starvation.