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Anthocyanin is an important pigment responsible for plant coloration and beneficial to human health. Kale (Brassica oleracea var. acephala), a primary cool-season flowers and vegetables, is an ideal material to study anthocyanin biosynthesis and regulation mechanisms due to its anthocyanin-rich leaves. However, the underlying molecular mechanism of anthocyanin accumulation in kale remains poorly understood. Previously, we demonstrated that BoDFR1 is a key gene controlling anthocyanin biosynthesis in kale. Here, we discovered a 369-bp InDel variation in the BoDFR1 promoter between the two kale inbred lines with different pink coloration, which resulted in reduced transcriptional activity of the BoDFR1 gene in the light-pink line. With the 369-bp insertion as a bait, an R2R3-MYB repressor BoMYB4b was identified using the yeast one-hybrid screening. Knockdown of the BoMYB4b gene led to increased BoDFR1 expression and anthocyanin accumulation. An E3 ubiquitin ligase, BoMIEL1, was found to mediate the degradation of BoMYB4b, thereby promoting anthocyanin biosynthesis. Furthermore, the expression level of BoMYB4b was significantly reduced by light signals, which was attributed to the direct repression of the light-signaling factor BoMYB1R1 on the BoMYB4b promoter. Our study revealed that a novel regulatory module comprising BoMYB1R1, BoMIEL1, BoMYB4b, and BoDFR1 finely regulates anthocyanin accumulation in kale. The findings aim to establish a scientific foundation for genetic improvement of leaf color traits in kale, meanwhile, providing a reference for plant coloration studies., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Self-incompatibility in the Brassicaceae family is governed by SRK and SCR, which are two highly polymorphic genes located at the S-locus. Previously, the Arabidopsis lyrata SRK and SCR genes were introduced into Arabidopsis thaliana to generate self-incompatible lines. However, there are no reports showing that Brassica SRK and SCR genes confer self-incompatibility in A. thaliana. Doing so would further advance the mechanistic understanding of self-incompatibility in Brassicaceae. Therefore, we attempted to generate A. thaliana transformants showing the self-recognition activity of Brassica rapa by introducing BrSCR along with a chimeric BrSRK (BrSRK chimera, in which the kinase domain of BrSRK was replaced with that of AlSKR-b). We found that the BrSRK chimera and BrSCR of B. rapa S-9 and S-46 haplotypes, but not those of S-29, S-44, and S-60 haplotypes, conferred self-recognition activity in A. thaliana. Analyses of A. thaliana transformants expressing mutant variants of the BrSRK-9 chimera and BrSCR-9 revealed that mutations at the amino acid residues involved in BrSRK9-BrSCR9 interaction caused defects in the self-incompatibility response. The method developed in this study for generating self-incompatible A. thaliana transformants showing B. rapa self-recognition activity will be useful for analysis of self-recognition mechanisms in Brassicaceae., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Leaf heading is an important and economically valuable horticultural trait in many vegetables. The formation of a leafy head is a specialized leaf morphogenesis characterized by the emergence of the enlarged incurving leaves. However, the transcriptional regulation mechanisms underlying the transition to leaf heading remain unclear. We carried out large-scale time-series transcriptome assays covering the major vegetative growth phases of two headingBrassica crops, Chinese cabbage and cabbage, with the non-heading morphotype Taicai as the control. A regulatory transition stage that initiated the heading process is identified, accompanied by a developmental switch from rosette leaf to heading leaf in Chinese cabbages. This transition did not exist in the non-heading control. Moreover, we reveal that the heading transition stage is also conserved in the cabbage clade. Chinese cabbage acquired through domestication a leafy head independently from the origins of heading in other cabbages; phylogenetics supports that the ancestor of all cabbages is non-heading. The launch of the transition stage is closely associated with the ambient temperature. In addition, examination of the biological activities in the transition stage identified the ethylene pathway as particularly active, and we hypothesize that this pathway was targeted for selection for domestication to form the heading trait specifically in Chinese cabbage. In conclusion, our findings on the transcriptome transition that initiated the leaf heading in Chinese cabbage and cabbage provide a new perspective for future studies of leafy head crops., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Successful pollination in Brassica brings together the mature pollen grain and stigma papilla, initiating an intricate series of molecular processes meant to eventually enable sperm cell delivery for fertilization and reproduction. At maturity, the pollen and stigma cells have acquired proteomes, comprising the primary molecular effectors required upon their meeting. Knowledge of the roles and global composition of these proteomes in Brassica species is largely lacking. To address this gap, gel-free shotgun proteomics was performed on the mature pollen and stigma of Brassica carinata, a representative of the Brassica family and its many crop species (e.g. Brassica napus, Brassica oleracea and Brassica rapa) that holds considerable potential as a bio-industrial crop. A total of 5608 and 7703 B. carinata mature pollen and stigma proteins were identified, respectively. The pollen and stigma proteomes were found to reflect not only their many common functional and developmental objectives, but also the important differences underlying their cellular specialization. Isobaric tag for relative and absolute quantification (iTRAQ) was exploited in the first analysis of a developing Brassicaceae stigma, and revealed 251 B. carinata proteins that were differentially abundant during stigma maturation, providing insight into proteins involved in the initial phases of pollination. Corresponding pollen and stigma transcriptomes were also generated, highlighting functional divergences between the proteome and transcriptome during different stages of pollen-stigma interaction. This study illustrates the investigative potential of combining the most comprehensive Brassicaceae pollen and stigma proteomes to date with iTRAQ and transcriptome data to provide a unique global perspective of pollen and stigma development and interaction., (© 2021 Her Majesty the Queen in Right of Canada The Plant Journal © 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Mitochondria serve as major sites of ATP production and play key roles in many other metabolic processes that are critical to the cell. As relicts of an ancient bacterial endosymbiont, mitochondria contain their own hereditary material (i.e. mtDNA, or mitogenome) and a machinery for protein biosynthesis. The expression of the mtDNA in plants is complex, particularly at the post-transcriptional level. Following transcription, the polycistronic pre-RNAs undergo extensive modifications, including trimming, splicing and editing, before being translated by organellar ribosomes. Our study focuses on N 6 -methylation of adenosine ribonucleotides (m 6 A-RNA) in plant mitochondria. m 6 A is a prevalent modification in nuclear-encoded mRNAs. The biological significance of this dynamic modification is under investigation, but it is widely accepted that m 6 A mediates structural switches that affect RNA stability and/or activity. Using m 6 A-pulldown/RNA-seq (m 6 A-RIP-seq) assays of Arabidopsis and cauliflower mitochondria, we provide information on the m 6 A-RNA landscapes in Arabidopsis thaliana and Brassica oleracea mitochondria. The results show that m 6 A targets different types of mitochondrial transcripts, including known genes, mtORFs, as well as non-coding (transcribed intergenic) RNA species. While ncRNAs undergo multiple m 6 A modifications, N 6 -methylation of adenosine residues with mRNAs seem preferably positioned near start codons and may modulate their translatability., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Grafting-induced variations have been observed in many plant species, but the heritability of variation in progeny is not well understood. In our study, adventitious shoots from the C cell lineage of shoot apical meristem (SAM) grafting chimera TCC (where the origin of the outmost, middle and innermost cell layers, respectively, of SAM is designated by 'T' for tuber mustard and 'C' for red cabbage) were induced and identified as r-CCC (r = regenerated). To investigate the maintenance of grafting variations during cell propagation and regeneration, different generations of asexual progeny (r-CCCn, n = generation) were established through successive regeneration of axillary shoots from r-CCC. The fourth generation of r-CCC (r-CCC4) was selected to perform whole genome bisulfite sequencing for comparative analysis of hetero-grafting-induced global methylation changes relative to r-s-CCC4 (s = self-grafting). Increased CHH methylation levels and proportions were observed in r-CCC4, with substantial changes occurring in the repeat elements. Small RNA sequencing revealed 1135 specific small interfering RNA (siRNA) tags that were typically expressed in r-CCC, r-CCC2 and r-CCC4. Notably, 65% of these specific siRNAs were associated with repeat elements, termed RE siRNAs. Subsequent analysis revealed that the CHH methylation of RE siRNA-overlapping regions was mainly hypermethylation in r-CCC4, indicating that they were responsible for directing and maintaining grafting-induced CHH methylation. Moreover, the expression of 13 differentially methylated genes (DMGs) correlated with the phenotypic variation, showing differential expression levels between r-CCC4 and r-s-CCC4. These DMGs were predominantly CG hypermethylated, their methylation modifications corresponded to the transcription of relative methyltransferase., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Arabinogalactan proteins (AGPs) are extensively glycosylated hydroxyproline-rich glycoproteins ubiquitous in all plant tissues and cells. AtAGP6 and AtAGP11, the only two functionally known pollen-specific classical AGP encoding genes in Arabidopsis, are reported to have redundant functions in microspore development. BcMF18 and BcMF8 isolated from Brassica campestris are the orthologues of AtAGP6 and AtAGP11, respectively. In contrast to the functional redundancy of AtAGP6 and AtAGP11, single-gene disruption of BcMF8 led to deformed pollen grains with abnormal intine development and ectopic aperture formation in B. campestris. Here, we further explored the action of BcMF18 and its relationship with BcMF8. BcMF18 was specifically expressed in pollen during the late stages of microspore development. Antisense RNA transgenic lines with BcMF18 reduction resulted in aberrant pollen grains with abnormal cellulose distribution, lacking intine, cytoplasm and nuclei. Transgenic plants with repressive expression of both BcMF8 and BcMF18 showed a hybrid phenotype, expressing a mixture of the phenotypes of the single gene knockdown plant lines. In addition, we identified functional diversity between BcMF18/BcMF8 and AtAGP6/AtAGP11, mainly reflected by the specific contribution of BcMF18 and BcMF8 to pollen wall formation. These results suggest that, unlike the orthologous genes AtAGP6 and AtAGP11 in Arabidopsis, BcMF18 and BcMF8 are both integral to pollen biogenesis in B. campestris, acting through independent pathways during microspore development., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

It has been well documented that most nuclear protein-coding genes in organisms can be classified into two categories: positively selected genes (PSGs) and negatively selected genes (NSGs). The characteristics and evolutionary fates of different types of genes, however, have been poorly understood. In this study, the rates of nonsynonymous substitution (K a ) and the rates of synonymous substitution (K s ) were investigated by comparing the orthologs between the two sequenced Brassica species, Brassica rapa and Brassica oleracea, and the evolutionary rates, gene structures, expression patterns, and codon bias were compared between PSGs and NSGs. The resulting data show that PSGs have higher protein evolutionary rates, lower synonymous substitution rates, shorter gene length, fewer exons, higher functional specificity, lower expression level, higher tissue-specific expression and stronger codon bias than NSGs. Although the quantities and values are different, the relative features of PSGs and NSGs have been largely verified in the model species Arabidopsis. These data suggest that PSGs and NSGs differ not only under selective pressure (K a /K s ), but also in their evolutionary, structural and functional properties, indicating that selective modes may serve as a determinant factor for measuring evolutionary rates, gene compactness and expression patterns in Brassica., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

The plant mitochondrial DNA-binding protein ODB1 was identified from a mitochondrial extract after DNA-affinity purification. ODB1 (organellar DNA-binding protein 1) co-purified with WHY2, a mitochondrial member of the WHIRLY family of plant-specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52-1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo-protein complexes. Both proteins co-immunoprecipitated in a DNA-dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single-stranded than for double-stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co-immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52-like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild-type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology-mediated recombination. These observations identify ODB1 as a further component of homologous recombination-dependent DNA repair in plant mitochondria., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Here, we describe the characteristics of a Brassica napus male sterile mutant 7365A with loss of the BnMs3 gene, which exhibits abnormal enlargement of the tapetal cells during meiosis. Later in development, the absence of the BnMs3 gene in the mutant results in a loss of the secretory function of the tapetum, as suggested by abortive callose dissolution and retarded tapetal degradation. The BnaC.Tic40 gene (equivalent to BnMs3) was isolated by a map-based cloning approach and was confirmed by genetic complementation. Sequence analyses suggested that BnaC.Tic40 originated from BolC.Tic40 on the Brassica oleracea linkage group C9, whereas its allele Bnms3 was derived from BraA.Tic40 on the Brassica rapa linkage group A10. The BnaC.Tic40 gene is highly expressed in the tapetum and encodes a putative plastid inner envelope membrane translocon, Tic40, which is localized into the chloroplast. Transmission electron microscopy (TEM) and lipid staining analyses suggested that BnaC.Tic40 is a key factor in controlling lipid accumulation in the tapetal plastids. These data indicate that BnaC.Tic40 participates in specific protein translocation across the inner envelope membrane in the tapetal plastid, which is required for tapetal development and function., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

We report on the structural characterization of a functional U3 snoRNA ribonucleoprotein complex isolated from Brassica oleracea. The BoU3 snoRNP complex (formerly NF D) binds ribosomal DNA (rDNA), specifically cleaves pre-rRNA at the primary cleavage site in vitro and probably links transcription to early pre-rRNA processing in vivo. Using a proteomic approach we have identified 62 proteins in the purified BoU3 snoRNP fraction, including small RNA associated proteins (Fibrillarin, NOP5/Nop58p, Diskerin/Cbf5p, SUS2/PRP8 and CLO/GFA1/sn114p) and 40S ribosomal associated proteins (22 RPS and four ARCA-like proteins). Another major protein group is composed of chaperones/chaperonins (HSP81/TCP-1) and at least one proteasome subunit (RPN1a). Remarkably, RNA-dependent RNA polymerase (RdRP) and Tudor staphylococcal nuclease (TSN) proteins, which have RNA- and/or DNA-associated activities, were also revealed in the complex. Furthermore, three U3 snoRNA variants were identified in the BoU3 snoRNP fraction, notably an evolutionarily conserved and variable stem loop structure located just downstream from the C-box domain of the U3 sequence structures. We conclude that the BoU3 snoRNP complex is mainly required for 40S pre-ribosome synthesis. It is also expected that U3 snoRNA variants and interacting proteins might play a major role in BoU3 snoRNP complex assembly and/or function. This study provides a basis for further investigation of these novel ribonucleoprotein factors and their role in plant ribosome biogenesis.

Telomeres define the ends of linear eukaryotic chromosomes and are required for genome maintenance and continued cell proliferation. The extreme ends of telomeres terminate in a single-strand protrusion, termed the G-overhang, which, in vertebrates and fission yeast, is bound by evolutionarily conserved members of the POT1 (protection of telomeres) protein family. Unlike most other model organisms, the flowering plant Arabidopsis thaliana encodes two divergent POT1-like proteins. Here we show that the single-strand telomeric DNA binding activity present in A. thaliana nuclear extracts is not dependent on POT1a or POT1b proteins. Furthermore, in contrast to POT1 proteins from yeast and vertebrates, recombinant POT1a and POT1b proteins from A. thaliana, and from two additional Brassicaceae species, Arabidopsis lyrata and Brassica oleracea (cauliflower), fail to bind single-strand telomeric DNA in vitro under the conditions tested. Finally, although we detected four single-strand telomeric DNA binding activities in nuclear extracts from B. oleracea, partial purification and DNA cross-linking analysis of these complexes identified proteins that are smaller than the predicted sizes of BoPOT1a or BoPOT1b. Taken together, these data suggest that POT1 proteins are not the major single-strand telomeric DNA binding activities in A. thaliana and its close relatives, underscoring the remarkable functional divergence of POT1 proteins from plants and other eukaryotes.

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.

The interactions between pollen and stigma are essential for plant reproduction and are made possible by compounds, such as proteins and lipids, located on their surfaces. The pollen coat is formed in part by compounds synthesized in, and released from, the tapetum, which become transferred to the pollen coat late in pollen development. In the Brassicaceae the predominant proteins of the mature pollen coat are the tapetal oleosin-like proteins, which are highly expressed in, and ultimately transferred from, the tapetum. Here we report the modification of the protein composition of the pollen coat by the addition of an active enzyme which was synthesized in the tapetum. The marker enzyme beta-glucuronidase (GUS) was successfully targeted to the pollen coat in transgenic Brassica carinata plants expressing GUS translationally fused to a B. napus tapetal oleosin-like protein (BnOlnB;4). To our knowledge this is the first demonstration of the targeting of an enzyme to the pollen coat.

Previous studies on cold-triggered events leading to Ca2+ influx during cold acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of cold-induced genes as end-point markers. Whether the results of these studies are valid for intact plants or their organs is not known. Here we examine cold signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold-inducible BN115 gene, the beta-glucuronidase (GUS) gene placed under control of the BN115 promoter. The activity of BN115 promoter was monitored at the transcriptional and translational levels by determining accumulation of BN115 transcripts and by histochemical assay of GUS activity. Cold-activation of BN115 was strongly inhibited by the membrane fluidizer benzyl alcohol, but mimicked at 25 degrees C by the membrane rigidifier dimethylsulfoxide (DMSO). The cold induction of BN115 was also inhibited by stabilizers of microtubules and actin microfilaments, taxol and jasplakinolide, respectively, but was mimicked at 25 degrees C by microtubule destabilizer oryzalin or colchicine, or by microfilament destabilizer latrunculin B. Gd3+ or ruthenium red prevented the cold activation of BN115, but Ca2+ ionophore A23187 or cyclic ADP-ribose activated it at 25 degrees C. Inhibitors of tyrosine kinases, protein kinase C and phosphoinositide kinases prevented the cold activation of BN115, but inhibitors of protein phosphatases (PP) 1 and 2 A activated BN115 at 25 degrees C. Constitutively expressed GUS activity in another transgenic line of the same cultivar of B. napus, was not affected by cold or any of the chemical treatments used in the experimentation. Activation of BN115 at 25 degrees C by DMSO, Ca2+ ionophore, cADPR, and by inhibitors of PP1 and 2A was accompanied by an increased freezing tolerance. It was concluded that the cold-activation of BN115 requires membrane rigidification, cytoskeleton reorganization, Ca2+ influx and action of several types of protein kinases.

The Or gene of cauliflower (Brassica oleracea var. botrytis) causes many tissues of the plant to accumulate carotenoids and turn orange, which is suggestive of a perturbation of the normal regulation of carotenogenesis. A series of experiments to explore the cellular basis of the carotenoid accumulation induced by the Or gene was completed. The Or gene causes obvious carotenoid accumulation in weakly or unpigmented tissues such as the curd, pith, leaf bases and shoot meristems, and cryptically in some cells of other organs, including the roots and developing fruits. The dominant carotenoid accumulated is beta-carotene, which can reach levels that are several hundred-fold higher than those in comparable wild-type tissues. The beta-carotene accumulates in plastids mainly as a component of massive, highly ordered sheets. The Or gene does not affect carotenoid composition of leaves, nor does it alter color and chromoplast appearance in flower petals. Interestingly, mRNA from carotenogenic and other isoprenoid biosynthetic genes upstream of the carotenoid pathway was detected both in orange tissues of the mutant, and in comparable unpigmented wild-type tissues. Thus the unpigmented wild-type tissues are likely to be competent to synthesize carotenoids, but this process is suppressed by an unidentified mechanism. Our results suggest that the Or gene may induce carotenoid accumulation by initiating the synthesis of a carotenoid deposition sink in the form of the large carotenoid-sequestering sheets.

Self-incompatibility (SI) in Brassica is sporophytically controlled by the multi-allelic S locus. SI phenotypes of the stigma and pollen in an S heterozygote are determined by the two S haplotypes it carries; the two haplotypes may be co-dominant or exhibit a dominant/recessive relationship. Because the S receptor kinase (SRK) gene of the S locus was recently shown to determine the S haplotype specificity of the stigma, we wished to investigate whether SRK also plays a role in the dominance relationships between S haplotypes. We crossed plants carrying an SRK28 transgene with plants homozygous for one of five S haplotypes that are either co-dominant with, or recessive to, S28 haplotype in the stigma, and analyzed the SI phenotypes of the progeny. In all cases, the SI phenotype of the stigma of plants carrying the SRK28 transgene could be predicted by the known dominance relationships between the S haplotype(s) and the S28 haplotype. Moreover, in the S43 homozygote carrying the SRK28 transgene where the S43 phenotype in the stigma was masked by the presence of the SRK28, the transcript level of SRK28 was found to be much lower than that of SRK43. All these results suggest that the dominance relationships between S haplotypes in the stigma are determined by SRK, but not by virtue of its relative expression level.

Sulfate uptake and ATP sulfurylase activity in the roots of Arabidopsis thaliana and Brassica napus were enhanced by S deprivation and reduced following resupply of SO4(2-). Similar responses occurred in split-root experiments where only a portion of the root system was S-deprived, suggesting that the regulation involves inter-organ signaling. Phloem-translocated glutathione (GSH) was identified as the likely transducing molecule responsible for regulating SO4(2-) uptake rate and ATP sulfurylase activity in roots. The regulatory role of GSH was confirmed by the finding that ATP sulfurylase activity was inhibited by supplying Cys except in the presence of buthionine sulfoximine, an inhibitor of GSH synthesis. In direct and remote (split-root) exposures, levels of protein detected by antibodies against the Arabidopsis APS3 ATP sulfurylase increased in the roots of A. thaliana and B. napus during S starvation, decreased after SO4(2-) restoration, and declined after feeding GSH. RNA blot analysis revealed that the transcript level of APS1, which codes for ATP sulfurylase, was reduced by direct and remote GSH treatments. The abundance of AST68 (a gene encoding an SO4(2-) transporter) was similarly affected by altered sulfur status. This report presents the first evidence for the regulation of root genes involved in nutrient acquisition and assimilation by a signal that is translocated from shoot to root.

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.

In Brassica anthers during microsporogenesis, the tapetum cells contain two abundant lipid-rich organelles, the tapetosomes possessing oleosins and triacylglycerols (TAGs), and the elaioplasts having unique polypeptides and neutral esters. B. campestris, for its simplicity of possessing only the AA genome and one predominant oleosin of 45 kDa, was studied. In the developing anthers, the lipids and proteins of the tapetosomes and elaioplasts were concomitantly accumulated but selectively degraded or retained. Upon incubation of isolated tapetosomes in a pH-5 medium, the predominant 45 kDa oleosin underwent selective enzymatic proteolysis to a 37 kDa fragment, which was not further hydrolyzed upon prolonged incubation. The unreacted 45 kDa oleosin was retained in the organelles, whereas the 37 kDa fragment was released to the exterior. The fragment would become the predominant 37 kDa polypeptide in the pollen coat. Isolated tapetosomes did not undergo hydrolysis of the TAGs upon incubation in media of diverse pHs. An alkaline lipase in the soluble fraction of the anther extract was presumed to be the enzyme that would hydrolyze the tapetosome TAGs, which disappeared in the anthers during development. The tapetum elaioplasts contained several unique polypeptides of 31-36 kDa. The gene encoding a 32 kDa polypeptide was cloned, and its deduced amino acid sequence was homologous to those of two proteins known to be present on the surface of fibrils in chromoplasts. Upon incubation of isolated elaioplasts in media of diverse pHs, the organelle polypeptides were degraded completely and most rapidly at pH 5, whereas the neutral esters remained unchanged; these neutral esters would become the major lipid components of the pollen coat. The findings show that the constituents of the two major tapetum organelles underwent very different paths of degradation, or modification, and transfer to the pollen surface.

Maternally inherited chlorophyll deficiency, or albinism, is a standard marker in plant cytoplasmic genetics. Its stability is consistent with mutations in the plastid genome. Nuclear mutations inducing plastid ribosome deficiency (PRD) also lead to maternally inherited chlorophyll deficiency. Here we report that stable chlorophyll deficiency can be efficiently generated in cruciferous plants without mutagenesis by a short exposure to spectinomycin, an inhibitor of plastid protein synthesis. We show that the chlorophyll-deficient phenotype results from a deficiency in plastid ribosomes and plastid translation products. Loss of plastid ribosomes is irreversible. The data suggest that mutations are not essential for generating inheritable PRD. It allows the formulation of a more general model in which stable PRD can be induced by a variety of factors that prevent the formation of functional plastid ribosomes. A non-mutational mechanisms for generating inheritable chlorophyll deficiency has implications for the origin and inheritance of green-white variegation in nature.

The purpose of this study is to characterise the biosynthesis, targeting and processing of some of the major protein components of the pollen coat, or tryphine, of Brassica napus. The authors have N-terminally sequenced 11 of the most abundant pollen coat polypeptides, and nine of these sequences correspond to proteolytically cleaved products of seven oleosin-like genes, i.e. Oln B;1 to Oln B;6 and Oln B;11. The Oln B;11 gene product is co- or post-translationally targeted in vitro to canine microsomal membranes. This implies that the oleosin-like protein is targeted to the endoplasmic reticulum in tapetal cells in vivo. Affinity-purified antibodies raised against a 20-residue domain of Oln B;3 and B;4 gene products cross-reacted with full-length proteins of 45-48 kDa in early developing (< 2 mm to 5 mm) buds and anthers, but recognised truncated proteins of 32-38 kDa at later (4 mm to 7 mm) stages of development. The 45-48 kDa immunoreactive proteins were associated with a floating lipid body fraction obtained from a tapetal/locular fluid extract from maturing anthers and a major 48 kDa polypeptide from this fraction was confirmed by N-terminal sequencing to be a full length product of the Oln B;3 gene. Quantitative immunocytochemical studies showed that the full length 45-48 kDa oleosin-like proteins were specifically localised in the interior of tapetal cytoplasmic lipid bodies where they were associated with a regular hexagonal-like fibrous reticulum. No significant labelling of elaioplasts was observed. The same antibodies specifically labelled 32-38 kDa oleosin-like proteins on the extracellular pollen coat of maturing pollen grains. These results demonstrate for the first time that many of the major pollen coat proteins are derived from an endoproteolytic cleavage of precursor oleosin-like proteins that originally accumulate within the large cytoplasmic lipid bodies of tapetal cells.

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T2 seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS-PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10-1.14 g ml-1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studies of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bodies in vivo and of possible associated conformational/processing changes in the protein. Although oil-body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil-body size in desiccation-tolerant seeds.

mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15-residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS-PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of approximately 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N-terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of lowdensity organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule-filled plastids and lipid-containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low-density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil-body oleosins share common epitopes at the generally non-conserved C-terminal domain with the sporophytic tapetum oleosins.

The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6-8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino-acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene (PCP1) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea. Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F2 segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine-rich mature protein. The central part and C-terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther-specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S-locus glycoproteins, but does not appear to be genetically linked to the S-locus.

A large, heterogeneous, highly expressed gene family encoding oleosin-like proteins is described in the Brassicaceae. Seven related cDNA sequences were isolated from Brassica napus anther mRNA using RACE-PCR and compared with other recently described anther-specific oleosin-like genes from B. napus. The expression patterns of four representative members of this diverse gene family were analyzed by Northern blotting and in situ hybridization. In all cases, the genes were expressed specifically in the tapetum of 3-5 mm B. napus buds, which contained microspores at the late-vacuolate and bicellular stages of development. The predicted protein products are ordered into subclasses, each of which has a characteristic C-terminal domain, containing different amino acid motifs or repeated residues. Tryphine (pollen coat) fractions from mature B. napus pollen were found to be particularly enriched in polypeptides of apparent molecular weights 32-38 kDa, plus numerous less abundant polypeptides of less than 15 kDa. The N-terminal 15-20 residues of three of these polypeptides (12, 32 and 38 kDa) were found by microsequencing to be identical to parts of the predicted amino acid sequences of three of the tapetal-expressed oleosin-like genes. This indicates the possibility of post-translational modification of these proteins resulting in a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data imply that a large and diverse group of oleosin-like proteins is synthesized in the tapetum of B. napus anthers and that following tapetal degradation, these proteins, possibly in modified form, then relocate to the developing microspores where they eventually constitute some of the major components of the extracellular tryphine of mature pollen grains. These proteins share a conserved 70 amino acid residue hydrophobic domain and are related structurally to the seed-specific intracellular oleosins, although their biological function may be different.

The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl-ACP thioesterase cDNA from C. hookeriana, designated Ch FatB2, has been identified, which, when expressed in Escherichia coli, provides thioesterase activity specific for 8:0- and 10:0-ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1, another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0-ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0-ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.

Self-incompatibility in Brassica is controlled by the S locus which contains at least two genes. SLG encodes a secreted S locus glycoprotein whilst SRK encodes a putative S locus receptor kinase which consists of three domains: an extracellular domain sharing extensive sequence identity with SLG, transmembrane region, and a cytoplasmic domain exhibiting a serine/threonine protein kinase activity. Here, the existence of truncated forms of the SRK protein corresponding to the extracellular domain of the putative receptor is reported. These proteins were detected by an antibody which recognizes the N-terminus of SRK3 and, in an F2 progeny segregating for the S3 haplotype, were only expressed in plants possessing the S3 haplotype. The truncated SRK proteins were expressed specifically in stigmas but, unlike the membrane-spanning SRK3 protein, were soluble and occurred as four different glycoforms sharing the same amino acid backbone as shown by deglycosylation experiments. Several SRK3 transcripts that may code for these truncated SRK3 proteins have been identified by RACE PCR, stigma cDNA library screening and RNA blot analysis. These transcripts are apparently generated by a combination of alternative splicing and the use of alternative polyadenylation signals. The existence of truncated forms of the S locus receptor kinase highlights some similarities between plant and animal receptor kinases. In animals, soluble extracellular domains of receptors have been described and, in some cases, have been shown to play a role in the modulation of signal transduction. By analogy, the soluble, truncated SRK proteins may play a similar role in the self-incompatibility response.

Two highly homologous Brassica napus flower cDNA clones, Sta 41-2 and Sta 41-9, were isolated and characterized. These clones were shown to correspond to genes expressed in the tapetum from the early uninucleate microspore stage to the dinucleate stage. The predicted Sta 41-2 and Sta 41-9 proteins possessed characteristics similar to oleosins such as a polar N-terminal domain, a large relatively conserved hydrophobic domain and a long C-terminal domain which consisted of four different groups of repeats. In addition, like oleosins, the Sta 41-2 and Sta 41-9 proteins have a basic pI, lack a signal peptide and are found in a tissue which accumulates lipids in small lipid bodies.

The expression and sequences of two related antherspecific cDNAs (I3 and C98) of Brassica napus (oilseed rape) were examined. These cDNAs were found to exhibit significant predicted amino acid sequence similarity with a group of seed-specific proteins, the oleosins, which are involved in oil body membrane structure. Pollen of B. napus also contains oil bodies, and the synthesis and accumulation of these organelles correlates with expression of the I3 and C98 transcripts. The protein content of purified pollen oil bodies was therefore examined in order to determine the presence or absence of possible oleosins. One major protein species of 14 kDa was identified and subjected to N-terminal amino acid sequence determination. The sequence of this pollen oil body associated protein is homologous to the predicted sequence of one of the anther-specific cDNs, I3, implying that this cDNA represents an anther-specific oleosin gene expressed in developing pollen. The expression of other genes active during seed oil body formation was also examined, and these were found to be specific to the sporophyte, inferring the existence of specific components of the pathway of lipid synthesis and storage in the male gametophyte. These possibilities and the relationship between different oleosin genes are discussed.